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Plant Breeding In Post Genomics Era

Proceedings

Proceedings of

Second National Plant Breeding Congress


March 1-3, 2006

Jointly organized by

Indian Society of Plant Breeders


&

Tamil Nadu Agricultural University


Coimbatore 641 003, India

Plant Breeding in Post Genomics Era

Plant Breeding in Post Genomics Era

Proceedings of the

Second National Plant Breeding Congress


March 1-3, 2006 Coimbatore, INDIA

Jointly organized by

Indian Society of Plant Breeders & Tamil Nadu Agricultural University


Coimbatore 641 003, India

The organizers and publishers take no responsibility of the contents of papers presented and included in this publication

Publication No. 2

Published by the Indian Society of Plant Breeders Coimbatore 641 003

Editorial Committee

Convenor Dr. T.S. Raveendran Members Dr. S.R. Sree Rangasamy Dr. M. Kadambavanasundaram Dr. N. Nadarajan Dr. P. Vindhiya varman Dr. P.Sumathi Dr. J.R.Kannan Bapu Dr. S.Ganesh Ram Dr. M.Kumar Dr. K.K.Vinod

Printed at M/s. Laser Park, Coimbatore

Foreword
Agricultural research has made great strides in terms of innovations and development of viable, applicable and relevant technologies. These technological advancements were responsible for increasing productivity and production and made India an exporting country from the status of importing country. Nevertheless, we cannot be complacent and have to constantly work for enhancing the production to feed the population which increase every day. The estimated requirement of food grains for 2020 AD it is 300 million tonnes and by 2050 AD is 400 million tonnes as against the present production of 210 million tonnes with the rider of shrinking land and water resources. Among all the technologies responsible for overall agricultural production, improved varieties acclaim top most importance as they have a direct bearing on the production. From a simple procedure of mass selection during early 20th century, the crop improvement technologies have very steadily and rapidly evolved to the present stage of molecular breeding through the untiring efforts of Geneticists and Plant Breeders. During this transformation, a large volume of scientific data would also be generated, which on interpretation, provide the younger generation precise guidelines and directions on how to proceed the programmes in future. Such informations are constantly and periodically discussed in many scientific fora by scientists involved in crop improvement. The Indian Society of Plant Breeders a Forum registered under Societies Act, is striving hard for the scientific upliftment in the field of Plant Breeding and Genetics by organizing such Congresses, special lectures for the benefit of students and scientists and supporting meritorious students through fellowship programme and providing travel grant for attending seminars etc. This is the Second National Plant Breeding Congress organized by the Society to document the research findings and information generated after 1998, when it conducted the First National Plant Breeding Congress. Classifying the 305 papers contributed for the seminar, under six important titles such as, crop biodiversity, quantitative genetics, ploidy variations, hybrid breeding, in vitro breeding tools and genomics. The editors have chosen invited papers and presentations to cover the entire gamut of crop improvement and presented in a lucid form and assimilation of scientists particularly the younger group. I hope the reader will make the best of the information available in this book. I congratulate the editorial committee for bringing out this informative and useful publication for the benefit of researchers and students. Coimbatore
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Prof. C. Ramasamy Vice-Chancellor

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PREFACE
The science of plant breeding has great antiquity and is the most useful branch of science to the mankind. Though it was a simple procedure of selection of desirable plants for further perpetuation and utility to human community, the recent plant breeding procedures are technologically highly advanced and packed up with strong genetic base. Thus today, the methods are complicated but very efficient and precise to yield the desired results. The scientists engaged in crop improvement activities should also keep themselves abreast of the latest developments in Genetics, Cytogenetics, Genomics, Plant Breeding and Biotechnology. Besides, they should also listen to the socio-economic preferences and adjust to the IPR system. The Indian Society of Plant Breeders was started at the Tamil Nadu Agricultural University during 1998 with a view to promote the interest of Plant breeders and to provide a common platform for exchange, discuss and disseminate the latest knowledge and developments to the end-users. The society organized the First National Plant Breeding Congress in July 1998 with the primary objective of taking stock of the developments made during 20th century and to programme the crop improvement technologies during 21st century. Now, this second congress was organized jointly with the Tamil Nadu Agricultural University, Coimbatore to consolidate the research information generated during the last eight years in the field of crop diversity, heterosis breeding, ploidy breeding, biometrical and quantitative genetics and biotechnological approaches. There was overwhelming response from the scientists and more than 300 papers were received. The editorial committee carefully selected 46 articles including 11 invited papers for oral presentation and allotted 259 for poster presentations. There were 311 registered participants including scientists from SAUs, CSIR, ICAR and GOI institutes, International institutes and postgraduate and research scholars. A few represented private institutions too. There was also a special panel discussion on IPR issues which was valued by the participants. The Editorial Committee deem it a honour to publish all the oral presentation papers in this proceedings, the abstracts being printed and distributed to the participants on the inauguration day of the congress. The proceedings also contains the recommendations of the six technical sessions for easy follow up of the future program. The Editorial committee thank all the participants for their cooperation in sending the papers, revising them in the light of editors comments and sending back in time. The committee also thank the President, Secretary and Organizing Secretary of the Congress for their help. The committee also acknowledges the cooperation of the press M/s. Laser Park, Coimbatore in bringing out this publication in a nice way. The committee believes that this book will be very much useful to all the scientists engaged in crop improvement programmes including students and research scholars.

Coimbatore 09.02.07
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Editorial Committee

INDIAN SOCIETY OF PLANT BREEDERS


The commencement of crop breeding research work in Tamil Nadu dates back to 1870 when an exotic cotton variety was introduced in India from Mauritius. The first breeding station in Tamil Nadu was established in 1901 at Kovilpatti to take up breeding work in cotton and millets. Subsequently breeding stations for sugarcane (1912), paddy (1913), cotton (1922), millets (1923), oilseeds (1930) and pulses (1943) were established. A separate department for forage crops was started in 1976. During this period, the importance of crop breeding which formed the backbone activity of all the agricultural research stations and the institutes was well recognized. However, subsequently there was a change in this trend and the plant breeding science, started to lose its prime importance. Therefore, the plant breeders felt that a common forum, which can rejuvenate the interests of the breeders and revitalise the activities would be necessary. With the above idea in view, it was decided by a group of breeders headed by Dr. M. Rangaswamy, Director, School of Genetics (presently known as Centre for Plant Breeding and Genetics), Tamil Nadu Agricultural University, Coimbatore to start a forum for the plant breeders for encouraging the plant breeders serving in various capacities in different public and private sector institutions with the following objectives. 1. To promote brotherhood and progress among plant breeders 2. To encourage scientific and technological research on various aspects of plant breeding. 3. To provide a medium for the exchange, discussion and dissemination of current development in the field of plant breeding to its members. 4. To promote the general advancement of plant breeding science, to create a common platform to bring together and facilitate the exchange of Information and provide opportunities for its members to establish a firm link between the plant breeders in India and abroad. 5. To promote the profession of plant breeding and increase professional competence in developing improved varieties and hybrids in different crops. 6. Establishing a literature communication service to plant breeders.

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The plant breeders forum was inaugurated on February 26, 1995 by Dr. M.S. Swaminathan and the forum was registered as per S. No. 191 of 1995 on 6.11.1995. A total of 110 breeders from Tamil Nadu Agricultural University, Sugarcane Breeding Institute, Central Institute for Cotton Research (Regional Station), Forest Research Institute, Coimbatore scientists from private Companies and institutions and retired plant breeders joined the forum. Dr. J. Thuljaram Rao, Retired Director, Sugarcane Breeding Institute, Coimbatore delivered the keynote address. To extend the services of the forum from state level to national level, the members felt the need of changing its nomenclature as Indian Society of Plant Breeders (ISPB) and the society was reregistered as a national body. Now the society is having 200 members including 140 life members and 3 foreign scientists. The society is actively involved in organizing seminars, special lectures for the benefit of students and scientists, supporting meritorious students through fellowship programme and providing travel grant for attending seminars etc. The society is looking for the enrollment of scientists involved in crop improvement for strengthening its existence and activities in the years to come.

President Indian Society for Plant Breeders TNAU, Coimbatore 3.

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SECOND NATIONAL PLANT BREEDING CONGRESS PLANT BREEDING IN POST GENOMICS ERA CONTENTS I. II. III. IV. Inaugural address Presidential address Keynote address Valedictory address

Technical Session I - Evaluation and utilization of crop biodiversity


1. Advances in breeding of vegetables Peter, K.V. and K.R.M. Swamy 2. Advances in spices breeding Peter, K.V. and K. Nirmal Babu 3. Enhancing utilization of plant genetic resources in crop improvement Upadhyaya, H.D. and C.L.L. Gowda 4. Rice biodiversity and its utilization Subramanian, M. and S. Tirumeni 5. Genetic diversity of Robusta - Arabica hybrids of coffee and utilization in breeding Santa Ram, A., D. Ganesh, N. Sandhyarani, S.R. Mythrasree, C. Murugan, R.K. Sabir, K.P. Dinesh, A. Manoharan, M.K. Mishra and Jayarama 6. Evaluation and utilization of biodiversity in cassava (Manihot esculenta Crantz) Santha V. Pillai, R.R. Nair, M.S. Palaniswami, C.S. Ravindran, S.N. Moorthy, V. Ravi and S. Sree Lekha 7. Agro-morphological characterization and evaluation of rice germplasm for major biotic stress tolerance Subba Rao. L.V., T. Ram, N. Shobha Rani, V. Ravindra Babu, I.C.Pasalu, C.S. Reddy, A.S. Ram Prasad, B.C. Viraktmath and S.V. Subbaiah 8. Characterization of cotton (Gossypium hirsutum L.) genotypes and evaluation of genetic divergence Preetha-, S. and T.S. Raveendran
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9. Interfamily variation and family selection in intervarietal crosses in sugarcane under excess water stress condition Govindaraj, P. 10. Developing high yielding rice varieties for Kerala a new approach Chandrasekharan, P.
Technical Session II - Quantitative genetics and analysis of genotype x environment interaction

1. Quantitative genetics - Where are we today? Arunachalam, V. 2. Variability and association analysis for floral traits of coconut genotypes Augustine Jerard, B., V. Niral, V. Arunachalam and P.M. Kumaran 3. Breeding for improved yield and yellow mosaic virus disease resistance in black gram (Vigna mungo (L.) Hepper) Murugan, E. and N. Nadarajan 4. Complex inheritance in rice variety MR 1523 of resistance to gall midge biotypes Suneetha, K., J.S. Bentur, K. Hima Bindu, P. Vijaya Lakshmi, C. Cheeralu, P.Ram Mohan Rao 5. Leaf trichome density an indicator of jassid tolerance in cotton Kannan, S., R. Ravikesavan and M. Kumar 6. Variability for yield and quality attributes in interspecific progenies of Saccharum sp. Nagarajan, R., S. Alarmelu and R.M. Shanthi 7. Genetic studies on plant, maturity and physiological characters of maize (Zea mays L.) under rainfed and irrigated conditions Subba Rao, M. and R.D. Singh 8. Genetic analysis of leaf anatomical characters associated with jassid resistance in cotton (Gossypium spp.) Shimna Bhaskaran, R. Ravikesavan and T.S. Raveendran

Technical Session III - Utilization of ploidy breeding in crop improvement


1. Pre-breeding through ploidy manipulation to exploit alien genetic variability Amala J. Prabhakaran 2. Wheat polyploids as a model system for crop improvement ] Dalmir Singh and P.K.P. Meena
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3. Role of polyploidy in cotton Khadi, B.M. and Vinita P. Gotmare 4. Cryptic genomic exchange between cultivated safflower (Carthamus tinctorius L.) and wild species, C. glaucus M. Bieb. Subsp anatolicus (Bioss.) Anjani, K. and M. Pallavi 5. Morphological, biochemical and molecular characterization of ploidy variants in coffee for genetic improvement Mishra, M.K., M. Violet DSouza, N. Sandhyarani, S.B. Hareesh, Anil Kumar, S. R. Mythrasree, R.K. Sabir, A. SantaRam and Jayarama 6. Cytological studies on sugarcane intergeneric hybrids Babu, C., K.Koodalingam, U.S. Natarajan, R.M. Shanthi and S. Thangasamy 7. Cytological observations in colchicine induced hexaploids and their triploids of cross between Gossypium hirsutum [2n=4x=52, (AD1)] and Gossypium raimondii [2n = 2x = 26, D5] Saravanan, N.A., T.S. Raveendran and M. Kumar 8. Studies on the effect of preconditioning of pollen and dynamics of pollen tube growth in Gossypium sp. Gunasekaran, M. and T.S. Raveendran 9. Cytological analysis Vigna radiate x V. umbellata L. Hybrids Pandian, M., B. Subbalakshmi, AR. Muthiah and M. Kumar

Technical Session IV - Hybrid breeding in crops


1. Transgenic hybrid cotton technology and some genetic observations Narayanan, S.S. 2. Expression of Brix in tomato intervarietal hybrids Panagiotis A. Michalakopoulos and S.R. Sree Rangasamy 3. Development of male lines resistant to Fusarium wilt in castor (Ricinus communis L) Lavanya, C.and Raoof, M.A. 4. Development of superior inbreds and selection of efficient restorers for diverse CMS sources in sunflower Ranganatha, A.R.G., V. Vijay, C. Lavanya and K. Rukminidevi
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5. Restorer identification for CMS line IR 66707 A with O. perennis cytoplasm Banumathy, S., K. Thiyagarajan and K. Siddeswaran 6. Evaluation of isonuclear alloplasmic hybrids in chilli (Capsicum annuum L) Nanda, C., A. Mohan Rao, S. Ramesh and R.S. Kulkarni 7. Combining ability studies for quality traits in Indian mustard Mahak Singh and R.K.Dixit

Technical Session V - In vitro breeding tools in genetic enhancement of crops


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Combined expression of chitinase and -1,3-glucanase generates high levels of sheath blight resistance in homozygous transgenic rice lines Sridevi, G., C. Parameswari, N. Sabapathy and K. Veluthambi Transformation of three antioxidant genes from a highly salt tolerant gray mangrove, Aveicennia marina Forsk. (vierh.) in Indica rice Ajay Parida, S.R. Prashanth, M.N. Jithesh and K.R. Sivaprakash In vitro genetic transformation for the Helicoverpa resistance using Cry 1 A(B) in pigeonpea (Cajanus cajan L cv Maroti) Sandhyarani, N., Mukund Shiragur. Sumangala Bhat and M.S.Kuruvinshetti Direct organogenesis and somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp.) Josnamol Kurian, K. Ramakrishnan, R. Gnanam and A. Manickam Somatic embryogenesis and plant regeneration from immature inflorescence and leaf explants of sorghum cultivars Kumaravadivel, N., M.Umadevi and Susan Eapen Engineering sheath rot resistance in rice Rajesh, T., K. Kalpana, S. Maruthasalam, K. Poovannan, R. Samiyappan, D. Sudhakar and P. Balasubramanian

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3.

4.

5.

6.

Technical Session VI - Contributions of genomic tools in crop improvement


1. Molecular breeding for brown planthopper (BPH) and blast resistance in rice Kshirod K. Jena and David J. Mackill 2. Quantitative trait loci, DNA markers and candidate genes What do we do with these? Shashidhar, H.E.
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3. Microsatellite and isozyme based genetic diversity measures for deciding productive cross combinations in sugarcane improvement Hemaprabha, G., U.S. Natarajan, N. Balasundaram and N.K. Singh 4. Sequence characterized amplified region (SCAR) marker for the identification of cytoplasmic genic male sterile (CGMS) lines in pigeonpea (Cajanus cajan (L.) Millsp.) Souframanien, J., A. Joshi Saha, J.G. Manjaya and T. Gopalakrishna 5. Molecular tagging of fertility restorer gene in cotton Amudha, J., G.Balasubramani, Suman.B.Singh, P.Singh and B.M.Khadi 6. Assessment of genetic diversity and interrelationship among wild mulberry (Morus laevigata and M. serrata) collections of India through DNA marker analysis Girish Naik , M. V., B. Mathi Thumilan, Bhaskar Roy and S. B. Dandin 7. Use of SSR markers for the identification of interspecific and intergeneric hybrids of Saccharum Vijayan Nair, N., A. Selvi, S. Suresh Ramraj and K. Sundaravel Pandian 8. QTL pyramiding for rice root morphological traits and its effect on grain yield, roots and plant characters under submerged, aerobic and drought situations Shailaja Hittalmani, Grace Arul Selvi and Pavana J. Hiremath 9. Tracing quantitative trait loci the best and rest with reference to brown plant hopper resistance and nitrogen uptake in rice Maheswaran, M., S. Geethanjali, K.K. Vinod, P. Meenakshisundaram, T. Elaiyabharathi, P. Kadirvel, S. Senthilvel, P. Govindaraj, S. Arumugachamy, P. Shanmugasundaram, P. Malarvizhi and K. Gunathilagaraj

Sessionwise recommendations

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INAUGURAL ADDRESS Dr. C. Ramasamy. Vice Chancellor, Tamil Nadu Agricultural University, Coimbatore

The world population is expanding rapidly and may reach 7.75 billion by 2020 and 10 billion by 2050 from the current population of about 6.5 billion. In India, the population may increase from the current 1.025 billion to 1.334 billion by the year 2020. Currently 800 million people are chronically malnourished and 2 billion people lack physical and economic access to sufficient food to meet their dietary needs. Limited availability of additional aerable land and water resources, and the declining trend in crop yields globally make food security a major challenge in the 21st century. To meet the demand of increasing population, Indias food grain production must be increased from 200 m.t. in 2000 to about 300 m.t. by the year 2020. According to the projections, to achieve these targets, food grain production must increase at the rate of 5 m.t. per year over the next two decades to meet food demand of the growing world population. Agricultural production in India has made great strides during the post independent period. The food grain production has increased from 50 m.tonnes in 1950 to 220 m.tonnes during 200405. This was primarily due to the advent of high yielding varieties by various crop breeding strategies. Crop improvement is the introduction and adaptation of genetically improved crop varieties giving higher yields than the local varieties used by farmers. The discovery and successful transfer of dwarfing genes from Norin 10 in wheat and Dee gee woo gen in rice had opened a new chapter in the history of global agriculture. The new varieties supported by other inputs had resulted in a multifold increase in food grain production and saved millions of lives from starvation, providing sustainability to national food security. The crop breeding work in Tamil Nadu commenced as early as in 1870 by way of introduction of a foreign cotton variety from Mauritius. With the appointment of a separate economic botanist, in 1898, the crop breeding work was initiated in sugarcane. The first crop breeding station was established in the year 1901 at Kovilpatti for cotton and millets followed by a research station for paddy, sugarcane and groundnut at Palur in 1905. By establishment of full fledged breeding stations at Coimbatore in 1912 for sugarcane, 1912 for paddy, 1922 for cotton, 1923 for millets, 1930 for oilseeds, 1943 for pulses, 1976 for forage crops, the crop breeding work was intensified.
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Simultaneously, crop breeding stations were started for these crops in other centres of this state also. At present, there are 31 research stations which are actively engaged in crop breeding work for evolution of crop varieties and hybrids and for maintaining crop genetic resources. Concerted efforts by TNAU scientists through research programmes resulted in the release of 262 crop varieties in agriculture, 155 in horticulture, 9 varieties in mushroom and two tree species. o I am pleased to recollect the works rendered by our earlier breeders and genetists like Sir. T.S. Venkatraman (1912), famous Sugarcane Breeder who developed sugarcane varieties incorporating with biotic and abiotic stress and high biomass production gene complexes. Revolutionary changes in sugarcane cultivation and sugar industry with vastly improved yield and quality under nobilization programme by crossing among tropical S. officinarum, sub-tropical S.barberi and the wild S.spontaneum Dr. K.Ramaiah o o o o o o Started scientific career in 1914 in the Paddy Breeding Station, Coimbatore PBS is the oldest rice research station in India He was the founder Director of the CRRI, Cuttack In 1949, he led the International Rice Commission of the FAO Initiated the indica-japonica hybridization program in 1952 First and the only Rajya Sabha M.P. among Agricultural Scientists

Dr. G.N. Rangaswamy Ayyangar o o o o A great doyan among millet researchers Millet Breeding Station started in 1923 Set strong foundation to millet breeding in India Made land mark contributions in genetics and improvement of Sorghum and minor millets, particularly little and Italian Millets Dr. V.S. Ramans contribution to cytogenetics, Dr. Appadurais contribution to biometrics, Prof. A. Subramanians role in green revolution are note worthy. Tamil Nadu Agricultural University is the pioneer in release of first rice hybrid in India, in the identification of CGMS system in pearl millet and sesamum, in development of GMS based hybrids in Pigeon pea and leader in the development of photoinsensitive lab lab varieties. It is our pride to mention the contribution of GEB 24 and TKM 6 rice varieties as a donor of genes to many international rice varieties. SPV 462 (CO 26) Sorghum and PT 732A, the indigenous Bellary
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cytoplasm in Pearl Millet are important contributions from Millets. Cotton variety MCU 5 conforming to high fibre qualities required by mills is the only variety that can spin to 60s counts. TMV 2 and TMV 7 groundnut varieties highly demanded by groundnut growers even after so many decades of release are land marks in Plant Breeding. I am happy that the Plant Breeders of this prestigious institution have started a National Society called Indian Society of Plant Breeders in 1995 to promote the science of Plant Breeding and the society is effectively functioning by organizing special lectures honoring eminent Plant Breeders etc. It has organized First National Plant Breeding Congress during 1998. The growth rate of agricultural productivity is in declining trend and we need to intensify our efforts to enhance the rate of genetic upgradation in crops. We will have to look for newer genes, methodologies to transfer them at a much faster rate so that the variety developed with the required new trait in the already well adapted background can be transferred to the field without much loss of precious time. Biotechnology offers several advantages over classical breeding, in terms of precision, technology, gestation period, and gene transfer for specific traits even from the unrelated organisms. The conventional approach of breeding crops by itself may not be able to deliver the goods in the required time frame given the magnitude and urgency to feed the growing millions. In the context of a holistic agricultural development and ensuring household food security, role of biotechnology is going to be essentially much more important and vital than ever before. The conventional breeding methods will have to be complemented by an array of biotechnological tools and in a variety of ways such as tissue culture, DNA fingerprinting, molecular breeding, genomics, diagnostics, development of transgenics etc. Bioprospecting will have to essentially lay the foundation for effective mining and transfer of genes for specific traits. The first transgenic plants engineered for insect resistance in cotton, corn and soybean were released for commercial cultivation in 1996. In less than a decade (1996 to 2004), area under biotech crops has increased more than 47 times globally, from 1.7 million hectares in 1996 to 81.0 million hectares in 17 countries in 2004. Application of biotechnology in crop improvement programmes has started giving dividends. The area under Bt cotton has increased tremendously. Bt cotton and Bt corn are the important transgenic crops now under cultivation in India. Another exciting development in Biotechnology is the GM rice called golden rice, which is genetically engineered to produce beta-carotene, a
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substance which the body can convert to Vitamin A. The new rice could prove effective to overcome vitamin A deficiency, a condition which afflicts millions of people in developing countries, especially children and pregnant women. This rice is a product of genes transferred from a bacterium and a flower plant (daffodil). Tissue culture is yet another area with lot of scope for commercial exploitation. TNAU has developed protocols for successful dihaploid production in rice and micropropogation of banana, neem, jamun, pomegranate, rose, paulownia, orchid, Sthalavrisksha (trees) etc. which could be commercially exploited to benefit the community. While pursuing for higher productivity levels, we need to redesign the crop and to add value to the farm produce so as to make agriculture more rewarding to farmers. Also, the formation of harmful substances such as aflatoxin in groundnuts, neurotoxins in khesari dal, and cyanide in tapioca, besides several undesirable elements in chickpea, sweet pea, and potato, can be prevented by the use of modern biotechnological methods. There is no end to innovating the transformations in our future crop varieties/hybrids but it is important to look for our own indigenous gene constructs and promoters so as to be self-dependent and cost-effective in the wake of strong global IPR regimes. Incidentally, the onus lies on the public sector institutions, which undertake most of the transgenic research in India. Often referred to as Gene Revolution or Biorevolution, biotechnology - if judiciously harnessed, blended with traditional and conventional technologies and supported by policies - can lead to an ever-green revolution synergizing the sustainable pace of growth and development. The uncommon opportunities provided by fast developments in functional genomics, proteomics and DNA microchips must be brought to developing countries for progress in scientific research and development. It is high time to come up with the strategies for protecting our own varieties with new era of WTO and TRIPS. New varieties offered farmers a far higher yield and profit than traditional varieties. Naturally, the seeds of these varieties were in high demand. Seed saving and sharing by farmers met most of the demand, while the public and private seed supply systems met the rest. There was no demand for ownership on plant varieties during the days of the Green Revolution, when the seeds of many high yielding varieties evolved by scientists were in high demand. For agricultural sector, it was a kind of anathema, mainly because the Indian Patent Act
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1970 clearly prohibited patenting of methods of agriculture and horticulture. However, intellectual property protection has received enormous attention since 1986 when it was included in the Uruguay Round of Talks and particularly when Dunkels Proposal relating to GATT was published in 1991. TRIPS Provisions Relating to Agricultural Sector The provisions of the TRIPS Agreement have widened the scope of protection of intellectual property rights relating to agriculture through plant variety protection. A reference to Article 27 of the TRIPS will show that all inventions regardless of the field of technology are eligible for protection. Member countries will have to provide a legal framework for the protection of inventions relating to plant varieties. Indian Patent Act (1970) does not permit the patenting of plant varieties and animal breeds which are existing in nature. To protect the rights of the breeders and farmers, Govt. of India has enacted the Plant Varieties Protection and Farmers Rights (PVPFR ACT, 2001). Under the PPV&FR Act, Plant Breeders Right on a plant variety is established by registration of the variety. The PBR holder can be one person, a group or community or an institution. By registering a variety, the person or the institution becomes its PBR holder. The PBR holder alone has the exclusive right to produce, sell, market or distribute the seeds or planting material of that variety. Sensitizing agricultural scientists in IPR related issues will enhance the inventive capability of the agricultural research system, induce investment in agricultural research, strengthen domestic agricultural industries and generate confidence among domestic trade associations in our country. IPRs and Outlook for Scientific Research in Agriculture Out of the eight IPRs of the TRIPs Agreement, patents and plant variety protection will produce a marked change in the outlook for scientific research in agriculture. With a legal system in place for the protection of plant varieties, the scientists will try to come up with research and inventions of commercial value. Research especially in agriculture will not be carried out for the name sake of research. Agricultural scientists will endeavor to come up with inventions which can prove to be a commercial success. The provision for the protection of new plant varieties will have all pervasive effect in various fields of agriculture. In India, agricultural scientists have a unique orientation. Generally they develop varieties as they have to develop varieties for resource poor farmers. They do not visualize or anticipate any monetary reward to them forthcoming from their research. The protection available to them
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with Plant Breeders Rights will induce them to develop varieties which may command premium price in the market. In other words, the provision for the protection of new varieties in India will prove to be a great motivating force for the scientific community in agriculture. It will change their outlook for research. They should try to ensure before launching a research project that the products of their inventions are in demand in the market. IPRs and Inventive Capability of State Agricultural Universities Achieving self-sufficiency in food has been the cherished policy objective of our planners. As a result, a. reasonable infrastructure for agricultural research has come up. This infrastructure strives for developing varieties which can contribute to food production. However, the State Agricultural Universities and ICAR institutes may have to be necessarily active and vibrant. With a legal system of protection of inventions in place, the SAUs will be induced to prioritize research from the standpoint of the commercial value of the research. The SAUs will also be induced to catalogue indigenous germplasm and develop an inventory of the plant genetic resources. The inventories will enhance the bargaining power of our country. Our agricultural research system will thus experience many changes leading to their enhanced inventive capabilities. Our agricultural scientists may modify their approach from quantitative gains in crop yields to qualitative attributes of the crop products. They may gear up their research system to meet the quality requirements of the consumers, having high willingness to pay for the quality of the product. IPRs and Investment in Agriculture With increased inventive capability of SAUs and assured protection of new varieties and agricultural inventions, the level of investment in agriculture may increase. Assured protection of IPRs may induce the private sector to take up the protected varieties for commercial production. The domestic seed industry in India may expand and flourish. However, the prospects of enhanced investment in agricultural sector through IPRs will depend upon the configuration of the private sector, the level of involvement of public sector in agriculture and the size of the market of the new products. IPRs and Regulations of Access to Biological Resources The Biological Diversity Act (BOA) 2002 envisages regulation of access to biological resources. The biological resources have been defined as resources which include plants, animals,

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micro-organisms or plant thereof (excluding value added product) with actual or potential use but do not include human genetic material. Section 6 of the BDA-2002 stipulates that application for IPRs cannot be made without the prior approval of the National Biodiversity Authority (NBA) if the research is based on the use of biological material from India. The NBA may dispose such application for permission in 90 days and impose benefit sharing. All the IPR granting authorities will endorse a copy of the sanction issued by them in relevant cases to NBA. Thus IPRs will be used to regulate access to biological resources of India which is a very important for the economy of India. It would thus appear that new developments relating to IPRs in India have wide ranging implication for various sections in Indian economy. They will have implication for change in the outlook of scientists in agriculture, inventive capability of SAUs, investment in agriculture, trade association, growth of domestic industries, and regulation of access to biological resources. It is appropriate and worthy to take stock of the results achieved in each of the research area so far document and discuss them and based on the outcome, plan for the future. If we consider the plant breeding research early part of the 20th century was devoted to gaining basic information, cytogenetical and biometrical investigations during middle part, heterotic exploitation and germplasm conservation and utilization took place while during the current phase the beginning of biotechnological research, molecular biology and genetic transformation started. Now it is the blend of conventional and biotechnological investigations. It is therefore appropriate that the Congress will be useful to consolidate the research findings and plan for Plant Breeding activities in the 21st century so that the food and clothing needs of the growing population can be readily met without any shortage. I am happy to inaugurate the congress and wish that fruitful results should come out from the deliberations and the results should be transformed into action.

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PRESIDENTIAL ADDRESS Dr. K.V. Peter, Vice Chancellor, Kerala Agricultural University, Thrissur, Kerala

India was rich in biodiversity and home to a large numbers of medicinal plants. Indians had adopted agriculture as early as 2000 B.C., and the wisdom of plant breeders was tremendous, having accumulated over a period of 4000 years. Despite adequate food stocks in the country, a large section of the people did not have the purchasing power to buy what they needed for adequate nutrition.

Biotechnology is one of the answers, at least regarding micro propagation in cardamom, vanilla and pepper, where there have been success stories.

In 2006, the food production of crops such as rice, wheat, barley and millets was about 208 million tonnes. However, by nutritional standards, the country needs 260 million tonnes. India was likely to import rice. However, this was an unwise step, for Mahatma Gandhi himself had cautioned against it, saying that import of agriculture amounted to import of unemployment.

Planners and administrators had predicted that by 2015, India would require 400 million tones of food grain for its population of 120 crores.

Plant breeders would face marketing challenges to sustain production by the masses rather than mass production

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KEY-NOTE ADDRESS Dr. S. Prakash Tiwari, Director, National Academy of Agricultural Research Management (ICAR), Hyderabad It is my pleasure and privilege to be here at the TNAU, which lies in one of the most progressive states of India, i.e. Tamil Nadu, and is a leading agro-technology provider of India. Its graduates are recognized throughout the world. The University, since its genesis as an Agricultural School at Saidapet, Chennai, and its subsequent relocation at Coimbatore during 1906, has already completed its 100 years with laudable achievements. The science and practice of crop improvement has made great strides in the recent past. Truly, it is a post genomic era for plant breeding. I am happy to note that keeping in line with the great tradition of the TNAU, the Indian Society of Plant Breeders, Coimbatore has very timely organized this Second National Plant Breeding Congress on Plant Breeding in Post Genomic Era. The future of agriculture essentially lies in the new science-led agricultural growth towards farm prosperity. The whole biological world now belongs to a single gene pool. Gene of any organism can be transferred to any other organism. We can have designer plants. Crop improvement will benefit in an overall manner but mainly through the use of hybrid technology (used earlier as well) and agricultural applications of biotechnology, both being not mutually exclusive. The new tools of science, however, need deft handling in the interest of human welfare at large. The technologies have to be robust, farm-worthy, eco-friendly and to be made available to farmers at affordable cost in a scale-neutral manner. The farmers interests have to be protected. Farmers rights are primary rights and those should not be construed as secondary or concomitant benefits and privileges only. In the new era of the advent of GMOs / transgenics, bio safety of endemic variability riches such as that of Western Ghats are to be preserved. Our bio resources should be utilized on sustainable basis with equitable benefit sharing. We shall not replicate anything similar to what happened to maize land races in Mexico. Regulatory and operational bio safety regulations should be rigorously followed. There is a need for construction of an Integrated Database on Bio safety and use of GMOs in India.
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Any organism, including crop plants, can now be examined in terms of its whole hereditary organization through study of expression and interaction of genes a field that is broadly referred to as genomics. The genomics of Arabidopsis thaliana and rice has already provided a wealth of information. India has contributed in this endeavor as one of the global partners in the International Rice Genome Sequencing Project. The focus of genetic research has now shifted from highthroughput sequencing to elucidation of gene function i.e. from structural to functional genomics. There could be myriad positive implications of genomics with respect to food, nutrition and environmental security of the nation. The science of genomics offers tremendous opportunities for the humanity in the field of medicine, agriculture and industry alike. Novel genes and DNA markers linked to agriculturally important traits are being identified and these can be used for rapid variety improvement in a more precise and targeted manner using markers assisted selection (MAS). Genetically modified improved plant varieties or transgenics can be produced. Also, plants can be engineered to produce novel products including vaccines and nutraceuticals. Plants, thus, serve as bio-factories. The major challenge for decoding genomes of crop plants is their enormous size. For example, the size of maize genome is 6 times and that of wheat is 40 times bigger than the rice genome. Hence, so far sequencing of only two genomes of higher plant namely Arabidopsis (125 Mb) and rice (400 Mb) have been completed. International efforts are underway for the sequencing of banana, tomato, cotton and maize genomes and the gene-rich regions of wheat. Still bigger challenge is to understand the functions (functional genomics, proteomics) of each and every new gene. For example, scientists have predicted nearly 62,000 genes in rice. Each of these genes will also have several alternative forms (alleles) and their structure and function needs to be deciphered by allele mining. We shall start with developing mapping populations such as RILs, NILs etc. and undertake molecular characterization and systematic phenotyping. Eventually, QTL analysis, fine mapping and reducing the number of candidate genes would enable gene identification and validation. The old paradigm of looking for the phenotype is giving way to the new paradigm of looking for the genes. In India, several genes such as Am A1 and OXDC have been isolated,
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sequenced and used for transformation. Successful isolation of protease inhibitor and lectin genes and promoter sequences from indigenous legumes have been obtained. These genes are being mobilized in different crop species for developing transgenic crop plants. Genomic synteny and comparative genomics can help in gene discovery for desirable traits. Map-based cloning and allele mining is gaining importance (e.g. Rice blast resistance Pi-kh gene). Continuous gene and allele mining is needed for eventual gene deployment by (i) transgenics development, (ii) marker assisted selection, and (iii) gene pyramiding for (a) durable resistance for biotic stress and/or, (b) multiple stress tolerance. Thus, the research has to traverse the journey from gene discovery to trait synthesis for crop improvement. Innovative and Strategic Research in crop improvement is called for towards novel methods of gene transfer, marker-free selection of transformants, super promoters, tissue-specific expression and more insecticidal toxins.High power computing and a range of DNA analysis and data base management software along with the Internet revolution have played a crucial role in the wide spread genomic research. It has enabled scientists to work from anywhere in the world. Bioinformatics through orthologs identification and display, auto-pipeline and availability of gene expression data centralized to enable comparative analysis data mining would greatly help in plant breeding efforts. Gene Bank EST resources for crop plants are rapidly growing day by day. Use of the new tools of science is also enormous in biodiversity management viz. molecular characterization for biodiversity assessment, for IPR protection, for bio resource utilization, for building up core collections etc. Gene detection technologies can also help in minimising adventitious presence of transgenes in germplasm collections and farmers/traditional varieties and land races. We are in the new IPR-regime as well. We have to stake the claims of national sovereignty on our germplasm and varieties. A single biotech-generated product may have several IP-protections. Holder of one of them can block the commercialization of the product. This calls for partnership among public and private sectors to overcome IPR-encumbrances. The country is well-poised to benefit from the new approaches in crop improvement. The conventional plant breeding efforts should, however, have a desirable confluence with biotechnological applications and these two should not be taken as mutually exclusive approaches.

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VALEDICTORY ADDRESS V. Santhanam, FAO Expert President, Indian Society of Plant Breeders, Dr. T.S. Raveendran, President of this Session Dr. N.M. Ramaswamy, my esteemed colleagues, Dr. S.S. Narayanan, Secretary of ISPB Dr. N. Sivasamy, and distinguished participants of the Second National Plant Breeding Congress, I deem it honour and privilege to have this opportunity to address the galaxy of plant breeders and biotechnologists in the broader sense who have gathered at the Second National Plant Breeding Congress. Thanks to the dynamic efforts of the President, Dr. T.S. Raveendran, and all his colleagues of the organizing committee. I understand that you had a very hectic schedule during the last 3 days with comprehensive presentations and discussion on the widest range of topics covering the entire gamut of technological tools now available with the plant breeders before arriving at this closing session. Dr. Narayanan had very ably summarized the recommendations followed by the presidential address by Dr. Ramasamy and very critical review of the entire congress presented by Dr. Raveendran. I do not think therefore, that I should deliver a formal valedictory address which will add only to your fatigue at the end of the day. It is a very happy coincidence that this campus is in its centenary year, the function for an agricultural institute being laid for this very building in the year 1906. The institution which has grown around this main clock tower building in which we are meeting today during last 100 years, provides testimony to the vibrant growth of agricultural education, research and extension in this part of the country which have gained national and international recognition. The crop improvement and breeding sections established at the Coimbatore campus as a part of Agricultural College and Research Institute during early decades of 20th century have rendered yeoman service to the cause of agriculture and increasing crop production and quality. It may be pertinent to recall the names of the some of the early pioneers in plant breeding who built up the high traditions for the vibrant plant breeding programmes which are actively being continued by the present generation scientists in this campus and its regional stations. I see many known faces and distinguished scientists among the audience and it may be difficult for me to list out all of them. Among the older pioneers who are not here the names of Dr. K. Ramaiah, the eminent Rice breeder, who recognized the importance of rice quality with yield in varietal
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improvement, Dr. B.W.X. Ponniah, in millet breeding, Rao Baghadur Dr. Ramanatha Iyer and Prof. Balasubramanian in cotton breeding come to my mind. Some of the early pioneers or stalwarts like Dr. P. Madhava Menon in the millet breeding station in the early 1950s who was the first breeder to exploit hybrid vigour in the pearl millet improvement and Dr. P.V. Marappan, the former Director of School of Genetics, a predecessor to Dr. Raveendran, was responsible for milestone development of cotton variety MCU 5, the best ever hirsutum cotton released in India through introgressive hybridization. The worlds worst recorded food disaster happened in the year 1943 in Bengal of British India when an estimated 4 million people died of hunger. In a recent meeting held at CIRCOT, Bombay in December 2005, Mr. R.M. Lala, Chairman, Centre for Advancement of Philanthropy and also a trustee of MSSRF introduced Dr. Swaminathan, the main speaker at the function with the information that the Bengal catatrosphy in the year 1943 ignited a spark in young Swaminthan to choose an agriculture based career for himself. He joined Agricultural College, Coimbatore in the year 1944 and graduated in 1947. The latest history is too well known to be repeated to this august audience. The average Indian who was leading dependent life on food grain shipment in mid 1950s literally had a slip to mouth to existence, now proudly holds his head high in the international scenario due to Green Revolution. Food production has increased from 50 mt. in 1950 to over 200 mt. estimated for the current year with enough stock to feed over 1 billion people. It is the miracle of application of science and technology complimented with administrative support and political will. Dr. M.S. Swaminathan, as you all know, is now spreading a movement for an evergreen revolution to sustain the development. I started my professional career in the Cotton Breeding Station of this Institute in mid 1947 and I may therefore take the liberty of a couple of minutes saying specifically on the cotton breeding and varietal improvement scenario. The Indian cotton crop is the most diverse in the world in terms of botany and fibre quality range. A major landmark in the history of cotton breeding in India is the exploitation of hybrid technology with the release of the intra hirsutum hybrid in Gujarat by Dr. C.T. Patel in 1970 and the extension to commercial cultivation of first generation hybrid cottons. Subsequently a large number of hybrids both of intra and interspecific nature like Varalaxmi and DCH 32 from Karnataka and TCHB 213 from this Institute have all been extended in large scale cultivation. Currently, nearly 50% total cotton area is estimated to be covered by
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hybrids developed by the public sector as well as the dynamic private sector research and hybrid seed production contributing to over 50% of total cotton output in the country. Another significant milestone in the cotton breeding programme is the recent utilization of transgenic technology utilizing the Bt gene conferring resistance to Helicoverpa bollworms. I am sure this subject would have been dealt at length by my esteemed colleague Dr. S.S. Narayanan yesterday. To say, during the year 2005-2006, the transgenic Bt hybrid cotton is estimated to have covered about 18% of National cotton area and contribute about 25% of production. Genes for jeans is the slogan with target genes in mind. Insecticidal and herbicidal resistance, drought tolerance, seed oil and protein improvement, fibre modification and inducing male sterility are other avenues in biotechnological research. The phenomenal increase in cotton production to about 240 lakh bales of cotton lint in the current year 2005-2006 as against 26 lakh bales only in 1947 48 at the time of independence may well be considered a white revolution comparable to the praiseworthy green revolution in food crops. To commemorate this achievement, I may venture to suggest that the Indian Society of Plant Breeders consider their motto of breed and feed to be amended as breed, seed and feed the Nation. Perhaps seed alone in the broader sense includes agro industry also apart from alleviating hunger of billion mouths. Before I conclude, I wish to close with relevance to plant breeders. Dr. Norman Ernest Borlaug, the Nobel Laureate who is mainly responsible for high yielding varieties of Mexican dwarf wheat which seeded the green revolution in many parts of the world apart from India during 1970s used to observe in mock seriousness. I quote An ideal crop variety is an elusive to secure as an ideal wife. If the breeders were to wait to release an ideal variety combining in one cultivar of all desirable traits, he will retire from service, as a frustrated person without releasing any variety. Similarly the gentleman waiting for an ideal wife will remain unmarried for life. I would like to thank once again the Society and Organizing Committee for giving me this valuable opportunity to meet you all in this afternoon. I wish to congratulate one and all of you for the significant contributions made by you to breed and feed. May I close and wish you all good future. Thank you.
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TECHNICAL SESSION I EVALUATION AND UTILIZATION OF CROP BIODIVERSITY

ADVANCES IN BREEDING OF VEGETABLES


Peter, K.V1. and K.R.M. Swamy2

ABSTRACT
Vegetable crops are important sources of carbohydrates, vitamins, minerals and proteins. India is credited as the second largest producer of vegetables in the world next only to China. Because of varied agro-climatic conditions in India, a large number of vegetable crops are grown here and a great deal of research work conducted in the disciplines of vegetable breeding, production technology, plant protection, seed production and postharvest technology.

Advantages of vegetables are as follows: - Nutritional security. - Production of more biomass. - Reduction in malnutrition. - Digestible protein. - Economical to grow. - Well fitting in farming systems. - Suitable for mixed, companion and intercropping. - Maximum output and more income / unit area. - Suitable for small farmers. - Source of supplementary income. - Intensive employment. - Higher income. - Export potential. Quantity of vegetables produced / capita in India is much lower than what is recommended by dieticians. In India, per capita availability is around 135 g against minimum requirement of about 300 g for a balanced diet. Worlds per capita availability is 160 g/day as against 236 g/ capita/day in developed countries. In general, average/ capita / day availability of vegetables in South Asian region is only 96 g which is higher than only South-East Asia (63 g), sub-Saharan Africa and Latin America. In a few developed and developing countries, per capita /day consumption of vegetables is very high, e.g., Italy (593 g), Japan (523 g), USA (469 g), Canada (428 g), Australia (346 g), China (195 g), Philippines (167 g) and Thailand (163 g). India

has to go a long way for boosting vegetable production to meet minimum need for nutritional security of population. Scope for horizontal expansion of area under vegetable crops is much limited due to lack of suitable land and thus option is for vertical increase by enhancing productivity. Estimated area under vegetables in India is 8.0 million ha and production is 95 million tonnes with productivity of 13- 15 tonnes/ha. By 2020, area should be 12.5 million ha and production should be 200-250 million tonnes with productivity of 20 tonnes/ha. History of vegetable breeding in India Vegetable research in India is of recent origin. Major milestones of vegetable research * 1940 Successful attempt of seed production of temperate vegetables at Quetta (now in Pakistan). * 1947 Sanctioning of nucleus Plant Introduction Scheme at Indian agricultural Research Institute, New Delhi. * Simultaneous start of ad-hoc schemes by Indian Council of Agricultural Research in different states like Punjab, Uttar Pradesh, West Bengal, Maharashtra Himachal Pradesh, Jammu and Kashmir and Tamil * 1949 Establishment of Vegetable Breeding Station at Katrain in Kullu Valley,Himachal

1. Kerala Agricultural University, Thrissur, Kerala. 2. Division of Vegetable Crops, Indian Institute of Horticultural Research, Bangalore

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Pradesh for production of seeds of te mperate vegetables. * 1955 Transfer of Vegetable Breeding Station, Katrain to Indian Agricultural Research Institute, New Delhi to undertake research on temperate vegetable crops, standardization of seed production technology and to produce seeds of improved varieties of temp erate vegetable crops. * 1956 Creation of Division of Horticulture at Indian Agricultural Research Institute, New Delhi * 1960 Establishment of State Agricultural Universities (SAUs): The G.B.Pant University of Agriculture and Technology, formerly known as Uttar Pradesh Agricultural University (UPAU), Pantnagar was the first agricultural university to be established on land grant pattern in 1960. State agricultural universities establishment on the pattern of land grant colleges/ universities of United States of America had full-fledged and separate Departments of Horticulture and/or Vegetable Science started from 1960 onwards. These developments gave thrust to vegetable research. Twenty six state agricultural universities plus one central university on agriculture as given in Table-1 are now engaged in the conduct of research on vegetable improvement. In the past, vegetable improvement programmes were located in combined Departments of Horticulture. Lately, there has been a shift towards creation of separate and independent Departments of Vegetable Science after bifurcation/trifurcation of existing Departments of Horticulture to carry out vegetable breeding and production work more efficiently. Besides these 26 State Agricultural Universities conducting researches on
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vegetable improvement, a Central University on Agriculture with headquarters at Imphal, Manipur came into existence in 1993.This University has various colleges including a College of Horticulture with a separate Department of Vegetable Science. * 1968 - Establishment of Indian Institute of Horticultural Research (IIHR), Bangalore with a strong focus on vegetable improvement among other things. * 1970 Initiation of All India Co-ordinated Vegetable Improvement Project (AICRIP) with headquarters at Indian Agricultural Research Institute, New Delhi headed by a Project Co-ordinator. * 1984Recommendation of Quin quennial Review Team (QRT) of the Indian Council of Agricultural Research to upgrade the All India Co-ordinated Vegetable Improvement Project to the level of Project Directorate of Vegetable Research (PDVR). * 1987 Start of Project Directorate of Vegetable Research during the Seventh Five Year Plan by upgrading erstwhile All India Co-ordinated Vegetable Improvement Project, with head quarters at IARI, New Delhi. * 1992 Shifting of headquarters of PDVR from New Delhi to Varanasi. * 1994- Initiation of All India Co-ordinated Nadu Research Project under National Seed Project (NSP) for production of breeder seed of vegetable crops with a financial outlay of Rs.303.59 lakhs for 3 years spread over various centers engaged in vegetable research. * 1995 Initiation of ICAR research network on promotion of hybrid research in vegetable crops (ad-hoc project) for 3 years with total cost of Rs.330.38 lakhs spread over different vegetable research centers/

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State Agricultural Universities. The PDVR was later upgraded as an Institute, Indian Institute of Vegetable Research (IIVR). All India Co-ordinated Research Project on Vegetable Crops (AICRP-VC) has its headquarters at IIVR, Varanasi, and it includes seven main centers, 18 sub-centers, 19 voluntary centers and 34 private seed companies for conducting experiments/trials on vegetable crops. Achievements in breeding of vegetables Significant achievements were made in breeding of vegetable crops in India since 1950s by adopting different methods of breeding such as plant introduction, plant selection (individual plant selection, pure line selection, mass selection, line breeding, family breeding, selfing and massing, recurrent selection), hybridization and selection, back-cross method of breeding, mutation breeding, synthetic varieties, heterosis breeding etc. depending upon crops involved. Development of improved and high yielding varieties Tremendous progress was made in the development of improved and high yielding varieties of different vegetable crops. Over 400 varieties of different vegetable crops comprising solanaceous fruits, cole crops, bulb crops, peas and beans, cucurbits, root crops, leaf vegetables and others developed/identified by different ICAR institutes and agricultural universities by adopting breeding methods like introduction and acclimatization (Table-2), pure line selection (Table-3), mass selection (Table-4), line/ family breeding (Table-5), inbreeding/ inbred selection (Table-6), recurrent selection, hybridization and selection/ pedigree selection (Table 78), synthetic varieties development (Table 9), mutation breeding (Table 10), and back cross method of breeding (Table 11) are recommended for cultivation in various agro-climatic conditions based on multilocation and multidisciplinary
3

testing. At present, around 30 % of area under vegetable crops is covered by improved varieties. Non-availability of seeds of improved varieties is one of the major production constraints in India. Resistant varieties Vegetable crops are highly susceptible to a number of diseases. Breeding for disease resistance is given due importance to develop varieties against important diseases. Over 80 disease-resistant varieties/hybrids are developed in 13 vegetable crops (Table-11). Breeding methods depend on source of resistance and its inheritance. For simply inherited resistance, back-cross method of breeding is commonly employed to transfer resistance from donor parent to commercial variety. In certain cases, simple selection, pedigree methods and combination of backcross and pedigree method are employed in breeding. In polygenic control of resistance, mass selection, recurrent selection, controlled matings (among resistant progeny) in F2 and succeeding generations and other breeding methods involving gene pyramiding are employed. Biotechnological approaches like embryo rescue and protoplast fusion techniques need to be employed to overcome interspecific and even inter-generic barriers as shown by the crosses: (S.melongena x S.sisymbrifolium, S. gilo x S.integrifolium), (L.esculentum x L.peruvianum), (C.annum x C.baccatum var. pendulum), (Sinapis alba x Brassica oleracea var. botrytis), etc. Specific programmes need to be taken to integrate resistance breeding with heterosis breeding to develop promising disease-resistant hybrids. Parents resistant to indigenous pathogens or races of pathogens should be developed for their subsequent utilization to develop resistant hybrids. In India, resistance to diseases forms a significant objective in vegetable breeding programmes. Several resistant varieties were developed by simple selection and incorporation

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of resistance from donor parents. Interspecific hybridizations are successfully accomplished to develop resistant varieties. Yellow vein mosaic virus resistant varieties of okra (Arka Abhay, Arka Anamika etc.,) were developed employing resistant wild species Albemoschus manihot ssp. manihot and ssp. tetraphyllus. Two leaf curl resistant tomato varieties, Hisar Anmol and Hisar Gaurav were developed by transferring resistance from Lycopersicon hirsutum f.glabratum. Resistant varieties so far developed in India are presented in Table-11. Resistance in breeding should be viewed as a continuous process. Due attention must be paid to develop new varieties with higher level of resistance coupled with high quality attributes. In vegetable crops, resistant varieties would be of little use unless it possesses good horticultural characters. Resistance breeding must be integral part of any breeding programme. Hybrid varieties ICAR Research Institutes and Agricultural Universities contributed considerably to develop F1 hybrids. At present, over 80 F1 hybrid cultivars of 16 vegetable crops were developed by public sector organizations Table 12. Private seed companies did commendable work in popularizing hybrid varieties in India. Over 200 F1 hybrids in 15 vegetable crops are being sold by seed companies in India (Table 13). At present, there is competition among the private seed companies (both national and multi-national) in the present liberalization of seed policy. Most of hybrids released at national level were developed by public sector but their popularity among farmers is rather poor due to very weak seed production and marketing infrastructures at Government level. Private sector establishments are rather prompt and well planned in seed distribution. For this reason, most of the hybrids grown in India are of private sector

origin. Development of hybrid cultivars in various vegetable crops is receiving due and increasing attention by the All India Co-ordinated Vegetable Improvement Project. Importance being given to heterosis breeding in vegetable crops in India by Indian Council of Agricultural Research can be recognized from the fact that ICAR sanctioned a special adhoc research project on promotion of hybrid research in vegetable crops for a period of 3 years (1995-96 to 1997-98) with a total cost of Rs.330.38 lakhs. Vegetable crops included in this programme were tomato, chilli, capsicum, okra, onion, cucumber, bitter gourd, cabbage and brinjal. Future Thrusts * Emphasis needs to be given to introduce germplasm resistant to iotic and abiotic stresses, hybrids and varieties with high export potential (Table 14). * Development of highly stable resistant cultivars of okra to yellow vein mosaic virus which normally results in breakdown, besides resistance to other diseases, insects and nematodes. * Varieties suitable for processing purposes. * Varieties suitable for export purposes. * Okra seed contains good amount of oil (1820%) and crude protein (20- 23%) which needs commercial exploi tation. * Being sensitive to day length, ability to flowerthroughout the year, especially in tropics and sub-tropical regions, needs exploitation. * Short duration cultivars with branching habits, early flowering, more nodes, less inter-nodal distance need to be bred.

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Table 1. List of State Agricultural Universities showing combined Department of Horticulture/ independent Department of Vegetable Science. Sl. No. State Agricultural University Year of Establishment 1960 1962 1962 1963 1964 1965 1965 1969 1969 1969 1970 1971 1971 1972 1972 1972 Department

1. G.B.Pant University of Agriculture and Technology 2. Rajasthan Agricultural University, Bikaneer, Rajasthan 3. Orissa University of Agriculture and Technology, Bhubaneswar, Orissa 4. Punjab Agricultural University, Ludhiana, Punjab 5. Jawaharalal Nehru Krishi Viswa Vidyalaya, Jabalpur Madhya Pradesh 6. Andhra Pradesh Agricultural University, Rajendra Nagar, Hyderabad, Andhra Pradesh 7. University of Agricultural Sciences, Bangalore, Karnataka 8. Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra 9. Punjab Rao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra. 10. Assam Agricultural University, Jorhat, Assam 11. Chaudhury Charan Singh Haryana Agricultural University, Hissar, Haryana. 12. Tamil Nadu Agriculture University,Coimbatore, Tamil Nadu 13. Rajendra Agricultural University, Pusa, Samastipur, Bihar 14. Marathawada Agricultural University, Parbhani, Maharashtra 15. Konkan Krishi Vidyapeeth, Dapoli, Ratnagiri, Maharashtra 16. Kerala Agricultural University, Vellanikkara, Kerala. 17. Gujarat Agricultural University, Sardar, Krishinagar, Dantiwada, Gujarat (with Colleges of Agriculture at Anand, Navsari, Junnagadh, Sardar Krishinagar Vegetable science 18. Bidhan Chandra Krishi Viswa Vidyalaya,Kalyani, Nadia, West Bengal 19. Chandra Shekhar Azad University of Agriculture andTechnology, Kanpur, Uttar Pradesh [Vegetable Improvement under Economic Botanist (Veg.) at Kalyani] 20. Narendra Deo University of Agriculture and Technology, Narendranagar, Kumarganj, Faizabad, Uttar Pradesh 21. Himachal Pradesh Krishi Viswa Vidyalaya, Palampur, Himachal Pradesh 22. Birsa Agricultural University, Ranchi, Bihar 23. Sher-E-Kashmir University of Agriculture and Technology, Srinagar, Jammu & Kashmir 24. Y.S.Parmar University of Agriculture and Forestry, Solan, Himachal Pradesh. 25. University of Agricultural Sciences, Dharwar, Karnataka 26. Indira Gandhi Krishi Viswa Vidyalaya, Raipur, Madhya Pradesh.
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Vegetable science Horticulture, Horticulture Vegetable science Vegetable science Horticulture Horticulture Horticulture Horticulture Horticulture Vegetable science Vegetable science Vegetable science Horticulture Horticulture Vegetable science

1972 1974

Horticulture Horticulture

1975 1975 1978 1982 1982 1984 1986 1987

Horticulture Vegetable science Vegetable science Horticulture Horticulture Vegetable science Horticulture Horticulture

Table 2. Promising introductions in various vegetable crops Crop Tomato(9) Variety Roma Labonita Sioux Marvel Best of All Money Maker VC 48-1 NDT-10NDT-5 California Wonder Yolo Wonder World Beater Chinese Giant Golden Cal Wonder Bullnose Early Superb Meteor Arkel Little Marvel Early Badger Bonneville Lincon Alderman Perfection New Line Sylvia Contender Giant Stringless Kentucky Wonder Bountiful Masterpiece Jampa Philippines Early Improved Japanese D-96 Golden Acre Copenhagen Market Glory of Enkhuizen September Red Acre (Red cabbage) White Vienna
6

Sweet Pepper (6)

Pea (10)

French bean (6)

Cowpea (1) Cauliflower(2) Cabbage(5)

Knol-khol(3)

Introduced from USA USA USA USA USA USA Taiwan --USA USA USA USA USA USA UK UK UK UK USA USA USA USA USA USA Sweden USA USA USA USA USA Mexico Philippines Israel Israel Denmark Denmark The Netherlands Germany -Europe

Brussels sprouts(5)

Radish(3)

Carrot (3)

Garden beet (4)

Turnip (4)

Onion(3)

Watermelon(6)

Cucumber(4)

Summer squash(2) Bitter gourd (1)

Purple Vienna King of North Hilds Ideal Amager Market Catskill Danish Giant Danish Prize White Icicle Scarlet Globe Japanese White Nantes Chantney Danvers Detroit Dark Red Crimson Globe Crosby Egyptian Early Wonder Purple Top White Globe Golden Ball Snowball Early Millan Red Top Early Grano Barmuda Yellow Brown Spanish Asahi Yamato Sugar Baby New Hampshire Midget Improved Shipper Dixielee Fuken Japanese Long Green Straight Eight Poinsettee China Australian Green Patty Pan MD-4

Europe Europe Europe Europe Europe Denmark Denmark Europe Europe Japan Europe Europe Europe USA USA --Europe Europe Europe Europe USA Philippines Philippines USA USA USA USA USA -Japan USA USA -Australia USA --

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Table 3. Vegetable varieties developed by pure line selection

Crop
Tomato (15)

Variety
Improved Meeruti HS-110 Sonali Pant Bahar Arka Vikas Arka Saurabh Punjab Tropic Pusa-120 S-12 Arka Abha Arka Alok Arka Ahuti Pant-T-3 CO-1 CO-2 Pusa Purple Long Pusa Purple Cluster Pusa Purple Round Pant Samrat Arka Shirish Arka Kusumakar Arka Sheel Punjab Chamkila T-3 Krishnanagar Green Long Punjab Neelam Punjab Bahar G-2 G-3 K-1 CO-1 CO-2 GCA-154 Kaliayanpur Yellow Kaliyanpur Red Kaliyanpur Chaman Sabour Angar Sabour Arun Arka Lohit CA-960 Bhagyalakshmi Sindhur

Genetic stock
Indigenous Exotic Exotic Exotic Exotic (USA) Exotic(Canada) Exotic (USA) Exotic (USA) Exotic (USA) Exotic (Taiwan) Exotic (Taiwan) Exotic (Canada) Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Exotic (Portugal) Exotic (Sri Lanka) Exotic (C.A. 960)

Brinjal (12)

Chilli (15)

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Crop
Pea (2) French bean(4)

Variety
Asauji Harbhajan Pant Anupama VL Boni-1 Arka Komal Arka Bold Cowpea 263 Pusa Barsati Pusa Phalguni Sheetal RM-43 Durgapura Madhu Arka Rajhans Arka Jeet Pusa Madhuras Durgapura Meetha Durgapura Kesar CO-1 CO-2 CM-14 Arka Chandan` Punjab Chappan Kaddu-1 Early Yellow Prolific Arka Suryamukhi Coimbatore Long Pusa Do Mousami Arka Harit VK-1a-Priya CO-1 MC-23 Pusa Vishesh Punjab BG-14 NDB-1 Phule BG-6 Kaliyanpur Sona Pusa Nasdar CO-1 CO-2 Pusa Summer Prolific Long Punjab Long Arka Bahar Pusa Naveen
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Genetic stock
Indigenous Exotic Indigenous Indigenous Exotic(Australia) Exotic(Hungary) Indigenous Exotic (Philippines) Exotic (Canada) Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Exotic Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous

Cowpea (3)

Cucumber(1) Muskmelon(5)

Watermelon (2) Pumpkin (4)

Summer Squash(2) Winter Squash(1) Bitter gourd (11)

Ridge gourd (3)

Bottle gourd (7)

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Crop

Variety
Pusa Summer Prolific Round Punjab Round CO-1

Genetic stock
Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Exotic (Taiwan) Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous

Wax gourd (2) Snake gourd(3)

CO-1 KAU Local CO-1 CO-2 TA-19 Tinda S-48 Pusa Chikni Arka Sheetal Karnal Selection Badi Chaulai Kannara Local Pusa Kiran Chhoti Chaulai Pusa Kriti CO-1 CO-2 CO-3 Arka Suguna Arka Arunima Pusa Early Prolific JDL-79 JDL-53 K-6802 JDL-37 HD-18 HD-60 Deepaliwal Rajni CO-1 CO-8 Pusa Sadabahar Pusa Mausami PLG-850 CO-1 Perkins Long Green Punjab No.13 Pusa Makhmali Gujarat Bhendi-1
10

Indian Squash (Tinda) (1) Sponge gourd (1) Long melon(2) Amaranth(10)

Dolichos/ Hyacinth bean(11)

Cluster bean(3)

Okra (5)

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Table 4. Vegetable varieties developed by mass selection Crop


Tomato(1) Capsicum (3)

Variety
Arka Ashish Arka Mohini Arka Gourav Arka Basant

Genetic stock
Exotic Exotic Exotic Exotic Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Indigenous Exotic Indigenous

Cauliflower(1) Onion(16)

Pusa Katki Punjab Selection Pusa Red Arka Niketan Arka Kalyan Agrifound Dark Red CO-2 Nasik Red Arka Pragati Patna Red Pusa White Round N-53 Kaliyanpur Red Round Agrifound Light Red Hisar-2 Arka Bindu Pusa Madhavi

Radish (5)

Pusa Desi Punjab Safed Punjab Ageti Kaliyanpur-1 Arka Nishant

Palak (1)

HS-23

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Table 5. Vegetable varieties developed by Line/Family breeding Crop


Cauliflower (6)

Variety
Hisar -1 Pusa Himiyoti Snowball-16 Pusa Snowball K-1 Punjab Giant-26 Punjab Gant-35

Genetic stock
Exotic Exotic Exotic Exotic Exotic Exotic Exotic (Denmark) Exotic (USA) Indigenous Indigenous Indigenous Exotic

Cabbage (1) Onion (2) Radish (2) Turnip (1)

Pride of India Pusa Ratnar Hisar-2 Pusa Chetki CO-1 Pusa Sweti

Table 6. Vegetable varieties developed by Inbreeding/Inbred selection Crop


Cauliflower (3)

Variety
Pusa Deepali Dania Kalimpong Pusa Snowball-2

Genetic stock
Indigenous Exotic Exotic Indigenous Indigenous

Muskmelon (1) Palak(1)

Hara Madhu All Green

Table 7. Vegetable varieties developed by recurrent selection. Crop


Cauliflower (2)

Variety
Pant Gobhi-4 Pant Shubhra

Genetic stock
Indigenous Indigenous

Table 8. Hybridization and selection from advanced generations /Pedigree selection Crop
Tomato(17)

Variety
Pusa Early Dwarf Pusa Ruby HS-101 HS-102 Hisar Arun (Sel.7)
12

Parents involved
Improved Meeruti x Red Cloud Sioux x Improved Meeruti Sel.2-3 x Exotic cultivar Sel.12 x Pusa Early Dwarf Pusa Early Dwarf x K-2

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Crop

Variety
Punjab Chhuhara Hisar Lalima (Sel.18) Hisar Lalit Pusa Sheetal Sweet-72 Pusa Gaurav Punjab Kesri Marglobe Keck-Ruth Ageti Pusa Red Plum Sel.2 Arka Meghali Pusa Kranti PH-4 Hisar Shyamal (H-8) Hisar Jamuni (H-9) Pant Rituraj Pusa Anupam Punjab Barsati Sadabahar Baingan Pusa Uttam Pusa Bindu Pusa Upkar Arka Nidhi Arka Keshav Arka Neelkanth

Parents involved
Punjab Tropic x EC-55055 Pusa Early Dwarf x HS-101 Bangalore (resistant) x HS-101 Balkan (exotic, Bulgaria) x Jemnorrosnej (exotic, Russia) Pusa Red Plum x Sioux Glamour (exotic) x Watch (exotic) Punjab Tropic x EC-55055 Marvel x Globe Kachmethi x Rutgers L.esculentum x L.pimpinellifolium (HS-101 x Punjab Tropic) x (H-14 x Punjab Tropic) Arka Vikas x IIHR 554 (Pusa Purple Long x Hyderpur) x Wynad Giant Pusa Purple Long x Hyderpur Aushey x BR-112 Aushey x R-34 T-3 x Pusa Purple Cluster Pusa Kranti x Pusa Purple Cluster Pusa Purple Cluster x R-34 Japanese Long x R-34 GR x Pant Rituraj GR x Pant Rituraj GR x PB 91-1 Dingrass Multiple Purple x Arka Sheel Dingrass Multiple Purple x Arka Sheel Dingrass Multiple Purple x Arka Sheel B-70A x Sathur Samba Kalipeeth x Pusa Jwala Bhagyalakshmix Yellow anther mutant G-2 x B-31 Local x Puri Red NP 46-A x Puri Red Perennial chilli x Long Red NP-46-A x Kandhari (natural cross) G-3 x Huntaka (Exotic, Japan) Lavang Mirche x G-2 Indigenous
13

Brinjal (14)

Chilli (12)

K-2 Jawahar Mirch-218 X-235 (Bhaskar) G-5 NP 46A Pusa Jwala Punjab Lal Pant C-1 X-197 X-200 Arka Lohit

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Crop
Pea (13)

Variety
Arka Suphal Jawahar Matar-1 (GC-141) Jawahar Matar-2 (GC-477) Jawahar Matar-3 Jawahar Peas-54 Jawahar Peas-83 Hisar Harit Pusa-2 x Morrasis-55 Jawahar Peas-15 (JP-15) JM-6 (JP-4) VL-3 Matar Ageta-6 Arka Karthik Pusa Dofasli S-203 S-488 Pusa Komal Aseem Pusa Rituraj Narendra Lobia-1 BCKV-1 BCKV-2 Arka Suman Arka Samrudhi Hebbal Avare-1 Hebbal Avare-3 Hebbal Avare-4 Wal Konkan-1 CO-2 Pusa Naubahar P-28-1-1 Pusa Sawani Selection-2 Punjab Padmini Punjab-7 Parbhani Kranti Arka Anamika Arka Abhay Pusa Shubhra Pusa Snowball-1
14

Parents involved
Pant C1 x IIHR 517A T-19 x Greater Progress Greater Progress x Russian-2 T-19 x Little Marvel (Early December) (Arkel x JM-5) x (46C x JP-501) (JM-1 x JP-829) x (46C x JP-501) Bonneville x P-23 VL-7 (VL Ageti Matar-7) IP-3 x Arkel (JM-1 x R-98B) x JR-501 A/2 Local Yellow Batri x (6588 x 46C) Old SugarxEarly WrinkledDwarf-2-2-9 Massey Gem x Harabona Arka Ajit x IIHR 554 Pusa Phalguni x Philippines Bush Sel.2 x Virginia Virginia x Iron Grey (Pusa Dofasli x EC-26410) x P-426 Pusa Dofasli x Philippines Bush Pusa Dofasli x EC-26410 Pusa Komal x Varanasi Local EC-243954 (Unguiculata) x EC-305827 (Sesquipedalis) V-70(Biflora)xSel.TM-3 (Sesquipedlais) Pusa Komal x Arka Garima Arka Garima x P.Komal Local Avare x Red Typicus Hebbal Avare-1 x US 67-31 Hebbal Avare x CO-8 Wal-2-K2 x Wal 125-36 CO-8 x CO-1 Pusa Sadabahar x Pusa Mausami Pusa Naubahar x IC-11521 Pusa Makhmali x IC-1542 (Pusa Sawani x Best-1) x (Pusa Sawani x IC7-194) A.escuelntusx A.manihot ssp. Manihot A.escuelntusx A.manihot ssp. Manihot A.escuelntusx A.manihot ssp. Manihot A.esculentusxA.manihot ssp. Tetraphyllus A.esculentus x A.manihot ssp. Tetraphyllus (MGS-2-3 x 15-1-1) x D-96 EC-12012 x EC-12013

P-88

Cowpea (11)

Hyacinth bean (5)

Cluster Bean (2) Okra (7)

Cauliflower(2)

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Crop
Cabbage (2) Radish (2)

Variety
Pusa Mukta Pusa Drumhead Pusa Himani Pusa Safed Pusa Reshmi Imperator Selection-233 Pusa Kesar Pusa Meghali Pusa Yamadagni Pusa Chandrima Pusa Kanchan Pusa Swarnima Pusa Sharbati Punjab Sunheri Hisar Madhur Arka Manik Pusa Bedana (triploid) Punjab Komal Pusa Palak Pusa Harit Banarjees Giant Arka Arunima Arka Pitambar Arka Tinda Arka Sujat Arka Sumeet

Parents involved
EC-10109 x EC-24855 F1 hybrid from Japan Radish Black x Japanese White White-5 x Japanese White Green Top x Desi Type (Asiatic) Nantes x Chanteny Nantes x No.29 Local Red x Nantes Pusa Kesar x Nantes EC-9981 x Nantes Snowball x Japanese White Local Red Round x Golden Ball Golden Ball x Japanese White Kutana x PUR-6 (Cantaloupe) Hara Madhu x Edisto Pusa Sharbati x 75-34 IIHR-21 x Crimson Sweet Tetra-2 x Pusa Rasal LC-11 (inbred) x LC-5 (inbred) Swiss Chard x Local Palak Sugarbeet x Local Palak Local Palak x Beetroot IIHR 10 x IIHR 8 UD-102 (White) x IHR-396 (Red) T3(Raj) x T8 (Punjab) IIHR 54 x IIHR 18 IIHR 54 x IIHR 18

Carrot(5)

Turnip(3)

Muskmelon(3)

Watermelon(2) Bottle gourd(1) Palak (4)

Onion (1) Round Melon (1) Ridge gourd (2)

French Bean (1)

Arka Suvidha

Blue Crop X IIHR 909

Table 9. Development of synthetic varieties. Crop


Cauliflower(4)

Variety
Pusa Early Synthetic Synthetic 78-1 Pant Gobi-3 Pusa Synthetic

Number of inbred lines involved


6 8 7 -

Cabbage (1)

Pusa Synthetic

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Table 10. Vegetable varieties developed by mutation breeding Crop Tomato(4) Variety S-12 Maruthan (CO-3) PKM-1 Pusa Lal Meeruti Chilli (1) French bean (1) Hyacinth bean (1) Okra (1) Bitter gourd (1) Ridge gourd (1) Palak (1) MDU-1 Pusa Parbati CO-10 EMS-8 MDU-1 PKM-1 Jobner Green Mutant type X-ray mutant of Sioux Mutant of CO-1 Mutant of Annagi Gamma ray mutant of Meeruti Gamma ray mutant of K-1 X-ray mutant of Wax pod Gamma ray mutant of CO-6 EMS-treated mutant of Pusa Sawani Gamma ray mutant of MC-103 Spontaneous mutant from local cultivar

Table11. Vegetable varieties developed by backcross method of breeding / Disease Resistant varieties Crop Tomato(15) Disease Bacterial wilt (Pseudomonas solanacearum) Resistant or tolerant Variety developed Shakti (LE-79) Arka Alok, Arka Abhay VC-48-1 Utkal pallavi (BT-1), Utkal Deepali (BT-2), BT-10 Sonali TRB-1,TRB-2 Source* Kerala I.I.H.R. Assam Bhubaneswar Dapoli Ludhiana

Late blight (Phytophthora infestans) Verticillium wilt (Verticillium sp.) and Fusarium wilt (oxysporum f.lycopersici) Leaf curl virus

Pant Bahar

Pantnagar

Brinjal (13)

Bacterial wilt (Pseudomonas solanacearum)

Hisar Anmol (H-24), Hisar Gaurav (H-36), H-86, H-88 Arka Keshav, Arka Nidhi, Arka Neelkanth Pusa Purple Cluster Pusa Anupam Utkal Tarini (BB-7) Soorya (SM-6-6) ARU-2C Pant Rituraj JC-1, JC-2
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Hisar

I.I.H.R. I.A.R.I. Katrain

Bhubaneswar Kerala Almora Pantnagar Assam

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Crop

Disease

Resistant or tolerant Variety developed Pusa Bhairav Pant Samrat K-2 Pusa Jwala, Pusa Sadabahar Pant C-1 Punjab Lal, Punjab Surkh Utkal Rashmi, AAUM-1, AAUM-2 Arka Suphal Arka Anamika, Arka Abhay Sel-2 Parvani Kranti Punjab Padmini, Punjab-7 Varsha Upahar (HRB-9-2) Hisar Barsati (HRB-55) Utkal Gaurav (BO-2) KS-404 Arka Ajit (FC-1) KS-225, KS-245 JP-4, JP-83, JP-7L, JP-885 Pant P-5, PMR-21 DPP-62 VP-9003, VP:-8902 DMR-7 HFP-4, HFP-12 HUP-1 Pant Anupama Bean common mosaic Pusa Komal

Source*

Chilli (10)

Phomopsis blight (Phomopsis vexans) Bacterial wilt and Phomopsis Blight Fruit rot (Colletotrichum capsici) Leaf curl virus Leaf curl,CMV and TMV, wilt and die back Bacterial wilt Powdery Mildew Yellow vein mosaic Virus

I.A.R.I. Pantnagar Kovilpatti I.A.R.I. Pantnagar Ludhiana

Okra (10)

Bhubaneswar I.I.H.R. I.I.H.R. N.B.P.G.R. Parvani Ludhiana Hisar Bhubaneswar Kaliyanpur I.I.H.R. Kaliayanpur Jabalpur Pantnagar Palampur Almora I.A.R.I. Hisar BHU, Pantnagar I.A.R.I.

Pea (16)

Powdery mildew (Erysiphe polygoni)

French bean(1) Leaf spot (Cercospora cruenta), Cowpea(3) Bacterial blight (Xanthomonas vignicola) Golden mosaic virus Hyacinth(3) Dolichs Muskmelon(4) Yellow mosaic virus

Powdery mildew (Sphaerotheca fuliginea) Downy mildew (Pseudopernospora cubensis) Cucumber green Virus

BCKV-1 Arka Garima Wal Konkan-1 Arka Jay Arka Vijay Arka Rajhans

B.C.K.V. IIHR Dapoli IIHR IIHR I.I.H.R.

Punjab Rasila

Ludhiana

DVRM-1, DVRM-2
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Crop Watermelon(1)

Disease

Resistant or tolerant Variety developed

Source* I.I.H.R.

Onion (2)

Anthracnose Arka Manik (Colletotrichum lagenarium), Powdery Mildew (S.fuliginea) and Downy Mildew (P.cubensis) Purple blotch Arka Kalyan (Alternaria porri) Nasik Red Black rot (Xanthomonas campestris) and curd and inflorescence blight Alternaria brasicicola) Black rot Pusa Shubhra Pusa Snowball K-1

I.I.H.R. Rahuri I.A.R.I. Katrain

Cauliflower(2)

Cabbage(2) (X.campestris)

Pusa Mukta, Pusa Drumhead

Katrain

* Full name of the Agricultural Universities and ICAR Research Institutes have been mentioned in Annexure I Table12. Heterosis breeding- Public sector hybrids of vegetables Crop Tomato(24) Name of hybrid Source IARI Katrain IIHR Pantnagar Faizabad Rahuri Ludhiana IIHR IIVR IIHR IARI Kanpur Faizabad Rahuri Ludhiana Anand Pantnagar Ludhiana IIHR Solan Katrain Srinagar IARI Parbani PDVR, Varanasi Pusa Hybrid-1, Pusa Hybrid-2, DTH-4, DTH-8, Pusa Hybrid-4 KT-4 Arka Vishal, Arka Vardan, Arka Shreshta, Arka Abhijit Pant Hybrid-1, Pant Hybrid-2, Pant Hybrid-10, Pant Hybrid-11 NDTH-1, NDTH-2, NDTH-6, NDTH-4 Rajashree,Phule Hybrid-1, Hybrid-37 TH-2312 Arka Ananya Kashi Vishesh Brinjal(18) Arka Navneet, Arka Anand Pusa Anmol, Pusa Hybrid-5, Pusa Hybrid-6, Pusa Hybrid-9 Vijay Hybrid, Azad Hybrid NDBH-1, NDBH-6, NDBH-11, NDBH-7 Hybrid-2 Punjab Hybrid, BH-1 ABH-1, ABH-2 Pant Hybrid-2 Chilli (3) CH-1 Arka Meghana, Arka Sweta Sweet pepper(4) Solan Hybrid-1 KT-1 (Pusa Deepti), KT-2 Sel-2 Okra (5) DOH-3, DOH-4 JOH-5 DVR-1, DVR-2

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Crop Cauliflower(1) Cabbage(2) Watermelon(2) Muskmelon(6)

Cucumber(7)

Bottle gourd(7)

Pumpkin(7) Summer Squash(1) Bitter gourd(1) Onion (2) Carrot (1)

Name of hybrid Pusa Hybrid-2 (F1 hybrid) H-64 (Hybrid), BRH-5 Arka Jyoti RHRWH-2 Punjab Hybrid-1, MH-10 MHY-3, MHY-5 Pusa Rasaraj, DMH-4 Pusa Sanyog AAUC-1, AAUC-2 PCUC-F1, Pant Sankar Khura-1 DCH-1, DCH-2 NDBGH-4, NDBGH-7 Pusa Manjari, Pusa Hybrid-2, Pusa Meghdoot PBOG-1, PBOG-2 Pusa Hybrid-1 Pusa Alankar Pusa Hybrid-1 Arka Kirthiman Arka Lalima

Source IARI Katrain IIHR Rahuri Ludhiana Durgapura IARI Katrain Jorhat Pantnagar IARI Faizabad IARI Pantnagar IARI Katrain IARI IIHR IIHR Hybrid-1 Katrain

Table 13. Heterosis breeding - Private sector hybrids of vegetables Crop Tomato Name of hybrid Karnatak, Vaishali, Rupali, Mangala, Naveen, Rashmi, Sheetal JTH-9 TC-161, TC-159 XLE-006, Sun-230 Gotya, NS-386, NS-815, Summerset Cross B MTH-1, MTH-2, MTH-3, MTH-4, Cross B, S-16, Gulmohar, MTH-15, MTH-16,S-28, Sonali, Samridhi,S-15 Madhuri, Meenakshi, Manisha, Megha Ratna, Larica, Avinash-2 Arjuna, Krishna, Karna, Bhim, Nakul SG-9, SG-12, SG-18, SG Prolific, SG Wonder NA-601, NA-501, NA-701 Swarna, Maitri, Century-12, Rishi ARTH-3, ARTH-4, ARTH-13, ARTH-15, ARTH-16 NH-25, NH-15, NH-38 HOE-303, HOE-606, HOE-909, HOE-616 LHB-230 Source Indo-American Hybrid Seeds Zuari Agro Hindustan Lever Sun Seeds Namdhari Mahyco

Beejo Sheetal Novartis Sungro Suttons Nath Seeds Century Seeds Ankur Seeds Nijjar Seeds HOECHEST Pioneer

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Crop Brinjal

Name of hybrid Sungrow Mukta, Sungrow Pragati, Kanhaya, Navkiran Suphal Hybrid Seeds AHB-2, AHB-4, ARBH-201, ARBH-527, ARBH-258. Shyamal MHB-1, MHB-2, MH-10 (Kalpataru), MHB-39, MHB-10, MH-39 (Ravalya) HOE-404, HOE-414 Neembakar-01 PHB-10 Nisha, Vardan, Shiva Delhi Hot, Hot Green, Skyline Tejaswini Agni ARCH-236 BSS-141,Gayatri Champion HOE-808, HOE-888 HOE-80 Bharat Indira, Lario Early Bounty, Gem Giant Hira, NAFCR-101 Green Gold

Source Sungrow Indo-American Ankur Seeds Mahyco Hoechest Neembakar Pandey Beej Century Seeds Hung Nong Mahyco Novartis Ankur Seeds Bejo Sheetal Seoul Hoechest Hoechest Indo-American Hybrid Seeds Novartis Suttons Nath Seeds Mahyco Indo-American Ankur Seeds Century Seeds Pioneer Seeds Mahyco Nath Seeds Sungrow Sakata Century Nath Seeds Novartis Namdhari Indo-American Hybrid Seeds

Chilli

Sweet Pepper

Okra

Varsha, Vijay Hybrid Seeds AROH-8, AROH-9 Panchali, Adhunik NIHB-090, HIHC-083, Supriya No.7, No.8 Nath Shobha Sungrow-35 Candid Charm, White Flesh, Cashmere Early Himlata, Early Himangine Nath Ujwala, nath Shweta Serrano Namdhari-84 Himani

Cauliflower

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Crop Cabbage

Name of hybrid Nath-401, Nath-501 Questo Sri Ganesh Gol, Hari Rani, Cabbage No.8 Vishesh, Uttam, Uttara Green Express Bajrang, Suvarna, Sudha, BSS-44, BSS-32 Gloria, Runa, Rotan Rare Ball Green Boy, Green Express, Stone Head, Herculis Regalia KK Cross, OS Cross, Resistalke, Green Cornet, Green Challenger

Source Nath Seeds Novartis Mahyco Hidnsutan Lever Suttons Beejo Sheetal Daehanfeldt Kaneko Sakata Takli Hung Nong

Watermelon

Madhur, Milan, Mohini Hybrid Seeds MHW-4, MHW-5, MHW-6, MHW-11 Charlie Seeds Nath-101, Nath-102, Nath-202 NS-246, NS-295 Suruchi Century No.2

Indo-American Mahyco Sheetal Hybrid Nath Seeds Namdhari ProAgro Century

Muskmelon

MHC-5, MHC-6, MHC-2 Shweta Seeds Swarna, Sona Abhijit, NS-7455 Madhubala

Mahyco Sheetal Hybrid Indo-American Hybrid Seeds Namdhari Seeds Century Indo-American Seminis (Syngenta) Golden Seeds Namdhari Senp World Nunhems ProAgro

Cucumber

Priya Hybrid Seeds Malini Rajdhani NS-404 US-6125 Tripti Aman

Bottle gourd

Gutka,Harit Varad

Century Mahyco

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Crop Bitter gourd

Name of hybrid MBTH-101, MBTH-1202 No.49, No.711 Hybrid Seeds. Vivek Tijarti Surekha Rohini Gaurav Harita, MSGH-1 Utsav Hybrid-1

Source Mahyco Indo-American Sngrow Century Mahyco Sluisgroat Sungrow Mahyco Century Mahyco

Ridge gourd

Sponge gourd Carrot

Table 14. Future needs of introduction of vegetable materials with specific traits. Crop Tomato Cucumber Muskmelon Watermelon Onion Garlic Chillies Sweet Pepper Cole Crops Nature of germplasm to be introduced Bitoic and abiotic resistance, long shelf life and good paste type. Biotic resistance, gynoecious and breeding lines. Good storage capacity, multiple fruiting and early lines, male sterile lines. Yellow fleshed, good storage types and Fusarium wilt resistant. Lines with high TSS and resistant to storage diseases. Lines with large bulb and clove. Hot types (Mexican types) lines. Heat tolerant lines. Heat tolerant and lines to biotic stresses

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Annexure I Source
Katrain I.A.R.I. Ludhiana Solan Hisar Coimbatore Faizabad Kaliyanpur Pantnagar Periakulam Dapoli I.I.H.R. Bhubaneswar N.B.P.G.R. Almora West Bengal Anand Akola Kovilpetti Jabbalpur Rahuri Lam Madurai AADF Udaipur Nasik Sabour Ranchi Bangalore Godhra Durgapura Kurnool Vellanikara Palampur Parbhani Jobner Dholi Hyderabad Gwalior Jorhat Srinagar B.C.K.V. Ranchi (CHES) : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : IARI Regional Station, Katrain, Kullu Valley, Himachal Pradesh Indian Agricultural Research Institute, New Delhi. Punjab Agricultural University, Ludhiana. Agriculture College, Solan, Himachal Pradesh. Haryana Agricultural University, Hisar. Tamil Nadu Agricultural University, Coimbatore. Narendra Deva University of Agriculture and Technology, Faizabad, UP Vegetable Research Station, Kaliyanpur, C.S. Azad University of Agriculture & Technology, Kanpur, U.P. G.B.Pant University of Agriculture & Technology, Pantnagar, U.P. Horticulture Research Station, Periyakulam, TNAU, Coimbatore, T.N. Konkan Krishi Vidyapeeth, Dapoli, Maharashtra. Indian Institute of Horticultural Research, Bangalore. Orissa University of Agriculture & Technology, Bhubaneswar. National Bureau of Plant Genetic Resources, New Delhi. Vivekananda Krishi Anusandhanshala, Almora, U.P. Horticulture Research Station, Government of West Bengal, Krishnanagar. Gujarat Agricultural University, Anand Campus, Gujarat. Punjab Rao Krishi Vidyapeeth, Akola, Maharashtra. Regional Agricultural Research Station, Kovilpetti, Tamil Nadu. Jawaharalal Nehru Krishi Viswa Vidyalaya, Jabbalpur, M.P. Mahatma Phule Krishi Viswa Vidyalaya, Rahuri, Maharashtra. Regional Agricultural Research Station, Andhra Pradesh Agricultural University, Lam, Guntur. Department of Horticulture, Agriculture College, T.N.A.U. Madurai. Associated Agricultural Development Foundation, New Delhi. Rajasthan Agricultural University, Udaipur. Onion Research Station, Nasik, Maharashtra. Rajendra Agricultural University, Sabour, Bihar. Birsa Agricultural University, Ranchi, Bihar. University of Agricultural Sciences, Bangalore, Karnataka. Central Horticultural Research Station, Godhra, Gujarat (of IIHR). Agricultural Research Station, Durgapura, Department of Agriculture, Rajasthan. Kurnool Research Station, Kurnool, Andhra Pradesh. Kerala Agricultural University, Vellanikkara, Kerala. Y.S.Parmar University of Horticulture & Forestry, Himachal Pradesh. Marathawada Krishi Vidyapeeth, Parbhani, Maharashtra. Department of Horticulture, Rajasthan Agricultural University, Udaipur, Jobner Campus. College of Agriculture, Dholi, Rajendra Agricultural University, Bihar. Andhra Pradesh Agricultural University, Rajendra Nagar, Hyderabad. Regional Agricultural Research Institute, Gwalior, M.P. Assam Agricultural University, Jorhat. Sher-e-Kashmir University of Agriculture & Technology, Srinagar, Jammu & Kashmir. Bidhan Chandra Krishi Viswa Vidyalaya, West Bengal. Central Horticultural Experiment Station, Bihar, Ranchi (of IIHR).
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ADVANCES IN SPICES BREEDING


Peter, K.V.1 and K. Nirmal Babu2

Spices are defined as natural plant or vegetable products or mixtures thereof, which are used for imparting flavour, aroma, pungency and for seasoning the food. The International Standards Organization (ISO) listed about 112 plant species as spices but only 53 spices are included in spices Act, Govt. of India. Of these, only 12 are commercially important and are grown at large scale in one or the other states and play a major role in the economy. India is considered as the magic land of spices and is the native home of black pepper, cardamom, tamarind, curry leaf and to certain extent ginger, turmeric, garcinia and cinnamon where the good variability exists. From the Indian sub-continent, these spices spread over to most of the tropical part of the countries, around the world, and many of these countries eventually became competitors for India in production and trade of spices. Other seed spices like coriander, fennel, fenugreek, paprika and cumin were introduced from other countries. Spices are generally tropical, some especially herbal spices are of temperate and seed spices are sub tropical or arid in distribution. They are cultivated in many countries in wide variety of geographical regions. Each country has its own traditional cultivars/ races/ types of the different spices. India is blessed with varied agro-climatic and agro-ecological approaches that enable us to grow a large number of spices in one or the other. In fact, there is no state in India that does not grow spices and in turn play an important role for the lives of the people and for their own economic sustainability. The research and development programmes initiated by Indian Council of Agricultural Research and various State Agricultural Universities and Departments during last few decades led to the assemblage
1. Kerala Agricultural University, Thrissur, Kerala. 2. Indian Institute of Spices Research, Kozhikode.

of a large collection of germplasm and development of over 200 improved cultivars of various spices including the seed and tree spices. Conservation of genetic resources Conservation of genetic resources is extremely important in the context of rapid gene erosion that is taking place due to a variety of abiotic, biotic, social, political and economic factors. The loss of land races and traditional varieties is rapid in certain crops such as black pepper due to devastating diseases, spread of improved cultivars, deforestation etc. At the Indian Institute of Spices Research (IISR), National Conservatories have been established for all major spices. Germplasm collections are also being maintained at the All India Coordinated Research project on Spices (AICRPS) Centers (Table 1). The National Bureau of Plant Genetic Resources (NBPGR) also maintains germplasm collections of various spices at its regional stations. However, due to the specific agro-climatic requirements of most spices and their vegetatively propagated nature the conservation is mainly at Indian Institute of Spices Research (IISR). The germplasm of spices is conserved in clonal field repositories and also in in vitro gene banks in vegetatively propagated crop species and seed gene banks in paprika, seed and herbal spices as a safe additive (Krishnamoorthy and Rema, 1994, Madhusoodanan et al., 1994a, Mohanty and Panda 1994, Rao and Rao, 1994, Ravindran and Babu, 1994, Nirmal Babu et al. 1999, Ravindran et al., 2000, Sasikumar et al., 1992). Cultivars and land races Black pepper: Over 100 cultivars exist in

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black pepper. They might have had their origin from wild forms by domestication and selection (Ravindran et al., 2000). Considerable variability exists among cultivars with regard to morphology, yield and quality. Cultivar Karimunda is the most popular and it gives consistent yields under varying agro-climatic conditions. Others like Aimpirian, Kottanadan, Neelamundi, Balankotta, Chumala, Narayakodi, Kalluvally, Kuthiravally, Malligesara and Thommankodi are popular in certain locations. The hybrid Panniyur 1 is also as popular as Karimunda. Cultivar Kuching is most popular variety in Malaysia. Kottanadan, Kumbhakodi and Aimpirian are cultivars with high oleoresin and essential and hence give high quality pepper (Ravindran and Babu, 1994). There is very little variability in pepper germplasm for resistance to biotic and abiotic stresses. Recently a few tolerant lines were identified at IISR. Cardamom : Based on the adaptability, nature of the panicle, shape and size of fruits three types of cultivated cardamom -Malabar, Mysore and Vazhukka - have been identified. Good variability exists in cardamom with regard to quality characters such as essential oil content and the quantity of 1,8-cineole and alpha-terperyl acetate in essential oil (Zachariah et al., 1998). Variations have also been reported in important characters like branching of inflorescence, fruit (capsule) size, shape, leaf and plant pubescence, retention of green colour etc. (Madhusoodanan et al., 1994b). Ginger: There is no natural seed set in ginger which resulted in limited variability with regard to certain characters. This also hampers the conventional breeding programmes. However, many commercial cultivars of ginger are known. They are generally named after the localities from where they are cultivated or collected. Maran, Himachal, Nadia, Rio-de-Janeiro, Jamaica, China, Waynad local, Kuruppampady and Bhaise are some of the local popular cultivars (Mohanty and Panda, 1994). There is good variation for
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crude fibre contents and dry recovery with in the germplasm which determines the suitability of each cultivar for dry ginger making. Cultivars Assam and Thodupuzha have high dry recovery. Exotic cultivar Jamaica has very low fibre content making it highly suitable for making ginger powder. High variation was also observed for oleoresin and essential oil contents which contribute to the quality of the spice. Indian ginger is known for its quality and flavour. The variability in ginger germplasm against the dreaded rhizome rot and bacterial wilt is very narrow. No genotype is either tolerant or resistant to these diseases. Turmeric: There are many popular turmeric cultivars, which are specific to each region of cultivation. Duggirala, Armoor, Sugandham, Nandyal, Alleppey, Rajapuri, GLpuram, Bhavanisagar, Gorakhpur, Jobedi etc, are some of the popular local cultivars which are essentially named after the places where they are grown extensively. The cultivars are grouped into short duration kasturi types, medium duration kesari types and long duration types (Rama Rao and Rao, 1994). Cultivars Armor, Tekurpet, and Mydukur are long duration crops, Kothapeta is medium duration crop while Kasturi is short duration crop. Turmeric sets seed only in certain locations and IISR has developed over 100 seed generated lines. In India, over 22 high yielding varieties have been released for cultivation. There is reasonable variation with regard to reaction to pests and diseases. Cultivars Mannuthy local and Kuchipudi are tolerant to shoot borer. Cultivars Mannuthy local, Tekurpeta and Kodur are tolerant to leaf spot while Mannuthy local, Glpuram-2, Kasturi Tanuku and Armoor are tolerant to leaf blotch. Suguna and Sudarshana were reported to be field tolerant to rhizome rot. Dry recovery, curcumin and oleoresin contents determine the quality of turmeric and high variability was observed in turmeric germplasm with respect

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to these characters (Khader et al., 1994). Tree spices: Cinnamon (Cinnamomum verum Brecht. & Presl.), Nutmeg (Myristica fragrans Houtt.), Clove (Syzygium aromaticum (L.) Merr. et Perry) Tamarind (Tamarindus indica L.) and Curry leaf (Murraya koenigii (L.) Sprengel) are tree spices of importance. Cinnamon is the earliest known spice and is native to Sri Lanka. The quality of cinnamon depends on the appearance, content and aroma character of volatile oil for which there is significant variability in the cultivars (Krishnamoorthy and Rema, 1994). Nutmeg is a dioecious tree native to Moluccas and was introduced to India. Nutmeg produces two separate spices, the nutmeg and the mace. As it is an obligatory cross-pollinated tree (being dioecious), considerable variation is observed with respect to growth and vigour, sex expression, size and shape of nutmeg and quantity of mace. Myristicin is the most important component of nutmeg. High variability was observed in the chemical and aroma quality with in nutmeg populations. Seed fat ranged from 1048 per cent, oleoresin from 2-14 per cent and essential oil from 1.4-3.4 per cent (Gopalam and Sayed, 1987). Clove also is native to Moluccas and was introduced to India. In India the genetc variability for clove is very narrow because of its self pollinating nature. A few variants identified are Zanzibar clove with more anthocianin, king clove with extra bold flower bud and dwarf clove with short and spreading growth habit (Krishnamoorthy and Rema, 1994). Though India is the native home of tamarind not much work was done in this crop except a few dwarf and sweet types were selected from germplasm. A few selections from curry leaf were also identified and released as varieties with high oil and flavour. Seed and herbal spices: Coriander (Coriandrum sativum L.), Cumin (Cuminum
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cyminum L.), Fennel (Foeniculum vulgare Miller) and Fenugreek (Trigonella foenumgraecum L.) are the seed spices of relevance in India. None of these are native to India. Yet Coriander, Fennel and Fenugreek are cultivated over wide variety of agro climatic regions in the country. A reasonable amount of genetic diversity is available in India. Except fenugreek, all the seed spices are cross pollinated and hence the traditional varieties of these crops exist in the form of complex gene mixtures. Good range of variability exists for important characters such as days for flowering, plant height, branches per plant, yield per plant, days to maturity etc (Sarma, 1994). But variability for resistance to pests and diseases is limited. Breeding and development of varieties In the effort to raise production and productivity of spices, primary importance was given for evolving high yielding varieties with good quality attributes. Evaluation and selection within the germplasm has led to the isolation of many elite varieties. Most of these varieties were evolved by clonal selections from germplasm, while a few are from seedling selection and very few are due to recombination breeding (Edison et al., 1991, Ravindran and Johny, 2000). The varieties released so far in various spices, their pedigree, the centers responsible for developing the variety, areas of adoption and important agronomic characters are given in Table 2. Black pepper: Black pepper has good variability for various agronomic and quality attributes but variability is limited or resistance to biotic and abiotic stresses. Hence pepper breeding was essentially dependent on clonal selections, selections from germplasm and selections from open pollinated progenies of popular cultivars. But presently, most improvement programmes are based on inter cultivar hybridization and recombination breeding to develop varieties resistant to biotic and abiotic stresses. So far, 12 black pepper

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varieties were released for cultivation in India. Of these, only two are hybrids while others are of clonal selections from germplasm or from open pollinated progenies. PLD 2 is a high quality variety suitable for industrial extraction of oils and oleoresins while Pournami is tolerant to root knot nematode. Panniyur 1 has bold berries while Panniyur 5 is suitable for mixed cropping. Malaysia and Indonesia have research programmes on black pepper. Malaysia has developed two important varieties. The variety Semongok Perak was developed by clonal selection and Semongok Emas by hybridization followed by back crossing. The latter is highly tolerant to Phytophthora foot rot disease. In Indonesia, two selections Natar 1 and Natar 2 have been evolved. In Madagascar selections Sel IV.1, Sel IV.2 have been developed from cultivars introduced from Indonesia (Ravindran et al., 2000). Cardamom: Cardamom breeding depend on selections from germplasm and from open pollinated progenies of popular cultivars. Nine high yielding varieties of cardamom were released for cultivation while one more line NKE 12, a katte virus tolerant line is in the final process of release. RR1 is a variety tolerant to rhizome rot disease of cardamom while ICRI 4 is relatively field tolerant. PV 1 has long and bold capsules while CCS 1 was highly suitable for high density planting because of its compact plant type. Hybridization between NKE, RR, extra bold and Multibranch types are in progress to pyramid these characters into single varieties. Ginger and Turmeric: Five ginger and eighteen turmeric varieties were released so far for cultivation. In ginger variety IISR Varada has low fibre while Suruchi has bold and attractive rhizomes. Surabhi is an induced mutant suitable for both rainfed as well as irrigated conditions. Himgiri is suitable for green ginger and reported to have tolerance to rhizome rot. In turmeric most of the varieties are clonal selections from germplasm except Prabha and Prathibha which
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were the first ever varieties developed from seedling progenies. They are also rich in curcumin content. Varieties Suguna and Sudarshana are short duration varieties with field tolerance to rhizome rot. In turmeric, we have varieties suitable for every turmeric growing state. Mutant and polyploid lines were also developed and are in various stages of evaluation. Tree spices In cinnamon, priority is given to develop lines with high cinnamaldehyde. The varieties Navashree and Nithyashree have high cinnamaldehyde (Krishnamoorthy and Rema, 1994, Krishnamoorthy et al., 1996). So far, five high yielding varieties of cinnamon, two high quality and high yielding nutmegs selected from germplasm were recommended for release. In curry leaf, only one high yielding high essential oil variety with good flavour, named Suvasini was released for cultivation. Seed and herbal spices: Among seed spices, powdery mildew and Fusarium wilt in coriander, Fusarium wilt and Alternaria blight in cumin, powdery mildew and sugary disease in fennel and powdery mildew and wood rot in fenugreek are the major production constraints. So far, 18 coriander, 5 cumin, 6 fennel and 4 fenugreek varieties were released for cultivation. Though most of the released varieties are high yielders, only few of them have shown partial field tolerance to these diseases and resistant varieties are not available. Only Gujarat cumin 3 was reported to be resistant to wilt (Vedamuthu et al., 1994). Fennel variety PF35 is moderately tolerant to leaf spot, leaf blight and sugary disease. Fenugreek variety Lam selection-1 has field tolerance to major pests and diseases. Coriander varieties Co 2, Co 3 and Hissar Anand are dual purpose varieties while Sadhana and Swathi are tolerant to white fly. Most of the earlier work on spices

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improvement concentrated mainly on developing high yielding lines alone. Some of them incidentally have high quality and good adaptability. Lesser importance was given to other characters like high quality and diseases and pest resistance, though they were not lost from the programme. Only in seed spices, mass or pureline selection and in some case recurrent selection methods were adopted. Occasionally, mutation breeding was used in ginger, turmeric and cumin which resulted in development of new varieties. Recently, more emphasis is being given to convergent breeding programmes of various spice crops to develop high quality lines and resistant lines to biotic and abiotic stresses, in addition to higher yield. For example, high priority is now given to develop varieties tolerant/ resistant to Phytophthora foot rot. A large number of inter cultivar hybrids, open pollinated seedling progenies and accessions in germplasm are being evaluated for this purpose. A few intercultivar hybrids in black pepper, inter varietal hybrids and natural katte escapes in cardamom have been developed. Seedling progenies in turmeric are highly promising and are in advanced stages of evaluation. Promising and high yielding black pepper genotypes suitable for mixed cropping system in coffee and tea plantations which can give good yields at low shade and high elevations (3,000 ft MSL) are in advanced stages of evaluation (Madhusoodanan et al., 1994b, Ravindran and Babu, 1994, Ravindran et al., 2000). Biotechnological approaches for spices crop conservation and improvement The past few years have witnessed a dramatic increase in our ability to manipulate and study tissues and has resulted in commercial propagation of many crop species, development of new varieties and new breeding lines via somaclonal variation, anther culture and protoplast fusion. Production of secondary metabolites, flavour and colouring components through bioreactor technology, recombinant
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DNA technology and use of transgenics with increased production levels have great significance in spices (Nirmal Babu et al 2005). Micro rhizomes: Rhizome formation in vitro, was reported in long term cultures of ginger, turmeric and Kaempheria. In vitro formed rhizomes are important source of disease-free planting material ideally suited for germplasm exchange, transportation and conservation similar to that of microtubers of potato. In vitro conservation of germplasm: Storage of germplasm in seed banks is not ideal in many spices as most of them are vegetatively propagated and seeds are recalcitrant and heterozygous. Hence, storage of germplasm in vitro is a safe alternative. Conservation of pepper, cardamom, seed and herbal spices, vanilla and ginger germplasm in in vitro gene bank by slow growth and through cryopreservation was reported. Conservation of genetic resources in invitro gene banks is now an established convention and two gene banks for conservation of spices germplasm functions at IISR and National Bureau of Plant Genetic Resources. Somaclonal variation and in vitro selection for tolerance to diseases Somaclonal variation is an important source of variability in crops like ginger, turmeric and vanilla where the native variability is very low and seed set is either absent or difficult. Attempts on induction of variability on somaclones for important agronomic characters and tolerance to diseases through both in vitro and in vivo selection were reported in black pepper, cardamom, ginger and galangal. Variants with high curcumin content were isolated from tissue cultured plantlets. Genetic transformation Recent advances made in developing techniques for transfer of foreign DNA into plant cells have aroused much interest in the

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possibility of utilizing recombinant DNA technology in crop improvement. Reports are available on Agrobacterium mediated gene transfer system in black pepper, bell pepper and direct gene transfer by particle bombardment in ginger and cardamom. Production of secondary metabolites Biotechnology can be utilized to exploit the potential of spices for bioproduction of useful plant metabolites. Plant cells cultured in vitro produce wide range of primary and secondary metabolites of economic value. This technique is all the more relevant in recent years due to the ruthless exploitation of plants in the field leading to reduced availability. Trials are in progress for production of primary and secondary metabolites and flavour and colouring compounds like capsaicin and biotransformation of ferulic acid vanillamine to capsacin and vanillin in immobilised cell cultures of Capsicum frutescen and in vitro synthesis of crocin, picrocrocin and safranel from saffron stigma and colour components from cells derived from pistils. Production of essential oils from cell cultures and accumulation of essential oils by Agrobacterium tumefaciens transformed shoot cultures of Pimpinella anisum was also reported. Regulation of the shikimate pathway in suspension culture cells of parsley and production of anethole from cell cultures of Foeniculum vulgare, production of monoterpene by transformed shoot cultures of Mentha , biosynthesis of sesquturpenic phytoalexin capsidol in elicited root cultures of chilli, production of rosmarinic acid in suspension cultures of Salvia officinali, production of phenolic flavour compounds using cultured cells and tissues of vanilla, in vitro production of petroselinic acid from cell suspension cultures of coriander are also available. Though the feasibility of in vitro production of spice principles has been demonstrated, methodology for scaling up and reproducibility need to be developed before it can reach commercial levels.
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Genomics In recent times there is increased emphasis in molecular markers for characterization of the genotypes, genetic fingerprinting, in identification and cloning of important genes, marker assisted selection and in understanding of inter relationships at molecular level. Molecular markers were used for crop profiling, molecular taxonomy, identification of duplicates, hybrids, estimation of genetic fidelity of micropropagated and in vitro conserved plants in pepper, ginger, turmeric vanilla cardamom, tree spices etc. Mapping population was also developed for construction of molecular map and to tag important genes in black pepper (Nirmal Babu et al 2005).Studies are also in progress for tagging important genes for useful agronomic traits and QTLs for marker aided selection in black pepper and cardamom. Comparative genomics has already made much headway in US for solanaceous crops to which capsicum belongs (Tanksley et al 1988, Livingstone et al 1999). Similarly Global Musa Genome Consortium involving 27 institutions in 18 countries was in operation to elucidate musa genome architecture. The Musa Genome Resources Centre (MGRC) was established at the Laboratory of Molecular Cytogenetics and Cytometry of the Institute of Experimental Botany (IEB), Olomouc, Czech Republic in 2003. The information generated helps in better understanding of other related sub families like Zingiberaceae to which important spices like cardamom, ginger and turmeric belongs. REFERENCES Edison, S., Johny, A.K., Nirmal Babu, K. and Ramadasan, A. (1991) Spices Varieties. A Compendium of morphological and agronomic characters of improved varieties of spices in India. National Research Centre for Spices (ICAR), Kerala, 63 p. Gopalan, A. and Sayed A.A.M (1987)

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Evaluating chemical and aroma quality of nutmeg accessions, Myristica fragrans L, Indian Spices 14: 9-11. Khader, M.A., Vedamuthu, P. G. B. and Balashanmugam, P. V. (1994) Improvement of Turmeric. In Advances in Horticulture, Plantation Crops and Spices. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, Vol. 9. p. 315- 332. Krishnamoorthy, B. and Rema, J. (1994) Genetic Resourses of Tree Spices.In Advances in Horticulture, Plantation Crops and Spices. K L Chadha and PRethinam (eds.) Malhotra Publishing House, New Delhi, p. 169 -192. Krishnamoorthy, B., Rema, J., Zachariah, T.J., Abraham, J. and Gopalam, A. (1996) Navashree and Nithyashree two new high yielding and high quality cinnamon (Cinnamomum verum Bercht & Presl.) selections, J. Spices and Aromatic Crops, 5 : 28 33. Livingstone, K.D., Lackney, V.K., Blauth, J.R., van Wijk, R. and Jahn, M.K. 1999. Genoome mapping in Capsicum and the evolution of genome structure in the Solanaceae. Genetics. 152 : 1183-1202. Madhusoodanan, K. J., Kuruvilla, K.M. and Priyadarshan, P.M. (1994a) Genetic Resourses of Cardamom. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, p. 121 - 130. Madhusoodanan, K. J., Kuruvilla, K .M. and Priyadarshan, P. M. (1994b) Improvement of Cardamom. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, p. 307-314. Mohanty, D. C. and Panda, B. S. (1994) Genetic Resourses of Ginger. Advances in Horticulture, Vol. 9. In. K L Chadha and P Rethinam (eds.)Plantation Crops and Spices.
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Malhotra Publishing House, New Delhi, p. 150 -168. Nirmal Babu, K., Geetha, S. P., Minoo, D., Ravindran, P. N. and Peter, K. V. (1999) In vitro conservation of germplasm. pp :106-129, In. Biotechnology and its application in Horticulture. In S P Ghosh (ed) Narosa Publishing House, New Delhi. Nirmal Babu, K., Sasikumar, B., Ratnambal, M. J., Johnson George, K. and Ravindran, P. N. (1993) Genetic variability in turmeric (Curcuma longa L.) Indian J. Genetics. 53: 91-93. Nirmal Babu, K., Minoo, D., Geetha, S.P., Ravindran, P.N. and Peter, K.V. (2005) Advances in Biotechnology of Spices and Herbs. Ind. J.Bot.Res. 1: 155-214. Rao, M. R. and Rao, D. V .R. (1994) Genetic Resourses of Turmeric. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, p. 131 150. Rattan, R. S. (1994) Improvement of Ginger, Advances in Horticulture, Vol.9. Plantation Crops and Spices. In. KL Chadha and P Rethinam (eds.)Malhotra Publishing House, New Delhi, p.333 344. Ravindran, P.N. and Nirmal Babu, K. (1988) Black pepper cultivars suitable for various regions. Indian Cocoa, Arecanut & Spices J. 11 : 110-112 Ravindran, P. N. and Nirmal Babu, K. (1994) Genetic resources of Black pepper. In. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. K L Chadha and P Rethinam (eds.). Malhotra Publishing House, New Delhi, p. 99-120 Ravindran, P. N., Nirmal Babu, K., Sasikumar, B. and Krishnamoorthy, K. S. (2000) Botany and crop improvement of black pepper, pp. 23-142, In. Black pepper, Piper

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nigrum. P N Ravindran (ed.).Harwood Academic Publishers, Amsterdam, The Netherlands. Ravindran, P.N. and Johny, A.K. (2000) High yielding varieties in Spices, Indian Spices 37: 17-19. Ravindran, P.N., Sasikumar, B., Johnson George, K., Ratnambal, M. J., Nirmal Babu, K., Zachariah, T.J. and Ramakrishnan Nair, R. (1994). Genetic resources of ginger and its conservation in India. Plant Genetic resources News letter, (IPGRI) 98: 1-4. Sarma, Y.R., Ramana, K.V., Devasahayam, S. and Rema, J. (eds) (2001) The Saga of Spice Research A voyage through history of spice research at Indian Institute of Spices Research. Indian Institute of Spices Research, Calicut, Kerala. Sasikumar, B., Nirmal Babu, K., Jose Abraham. and Ravindran, P. N. (1992) Variability, correlation and path analysis of ginger germplasm. Indian J. Genetics, 52 : 428431. Sharma ,A. K. (1994) Genetic Resourses of Seed Spices. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, p. 193 - 208. Sukumara Pillay, V., Ibrahim. K. K. and Sasikumaran, S. (1994) Improvement of

Black pepper. Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.). Malhotra Publishing House, New Delhi, p. 293-206. Tanksley, S.D., Bernatzky, R., Lapitan, N.L. and Prince, J.P. 1988. Conservation of gene repertoire but not gene order in pepper and tomato. Proc. Natl. Sci. USA. 85 : 64196423. Vedamuthu , P. G. B., Khader, M. A .and Rajan, F. S. (1994) Improvement of Seed Spices Advances in Horticulture, Vol. 9. Plantation Crops and Spices. In. K L Chadha and P Rethinam (eds.) Malhotra Publishing House, New Delhi, p. 345 374. Zacharia, T. J., Mulge, R. and Venugopal, M. N. (1998) Quality of cardamom from different accessions. In. Developments in Plantation Crops Research, Mathew N M and Jacob C K (Eds.). Allied publishers, India. pp. 337-340

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Table 1. Germplasm collections of spices at major canters in India Crop Black pepper Cardaman Ginger Turmeric Clove Cinnamon Nutmeg Garcinia Vanilla Paprika Cumin Fennel Fenugreek Coriander IISR 2299 395 659 899 235 408 482 61 68 40 AICRPS centres 367 336 406 1136 42 41 42 420 944 1467 Maintenance centres IISR, Panniyur, Sirsi, Chinthapalli, Yercaud, Pundibari, Dapoli IISR, ICRI, Mudigere,Pampadumpara IISR, Solan, Pottangi, Kumarganj, Pundibari, Raigarh, Dholi IISR, NBPGR, Jagtial, Dholi, Pottangi, Raigarh, Pundibari, IISR, Yercaud, Dapoli, Pechiparai IISR, Yercaud, Dapoli, Pechiparai IISR, Yercaud, Dapoli, Pechiparai IISR, KAU IISR, ICRI, KAU IISR 495 Jobner, Jagudan Jobner, Jagudan, Dholi Coimbatore, Guntur, Jobner, Jagudan, Hisar , Dholi, Coimbatore, Jobner, Guntur, Hisar, Dholi, Raigarh, Kumarganj Table 2. Improved varieties of Spices
Crop Breeding strategies Released varieties Important characters high yield, high oleoresin, high oil, high piperine, suitable for high elevation and resistant to Phytophthora and M.incognita

Black pepper Selection from clonal and open Panniyur 1,2,3,4,5,6,7, PLD-2, pollinated seed progenies and Sreekara, Subhakara, Panchami, Hybridization Pournami, IISR Thevam, IISR Shakti, IISR Malabar excel, IISR Girimunda Cardamom

Selection from open pollinated Mudigere 1, Mudigere 2 PV 1, High yield, high quality bold and seed progenies and Hybridization PV 2 CCS 1, ICRI 1, ICRI 2, elongated fruits, resistance to ICRI 3, ICRI 4, RR-1, IISR Katte and rhizome rot Avinash, IISR Vijeta Selection and mutation breeding Suprabha, Suruchi, Surabhi, High yield, low fibre, extra bold Himgiri, IISR Varada, IISR rhizomes, Rejatha, IISR Mahima

Ginger

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Turmeric

Selection from germplasm, from open pollinated and seedling progenies

Co.1, Krishna, Sugandham, BSR.1, Roma, Suroma, Rajendra Sonia, Suguna, Suvarna, Sudharsana, Ranga, Rasmi, BSR.2. IISR Prabha, IISR Prathiba, Megha turmeric 1, Kanthi, Sobha, IISR Kedaram, Sona, Varna .Alleppey Supreme, Suranjana, Pant Peethabh Nithyasree, Navasree, YCD.1, Konkan Tej, RRL(B) C-6, Sugandhini, , PPI (C)-1 Konkan Sugandha, Vishwasree, Konkan Swad Guj. Cor.1 Co.1, Co.2, Co. 3 and Co.4 Guj.Cor.2, Rajendra Swathi, RCr.41, RCr 436, RCr 684,Sadhana, Swathi CS 287 CO.3 Sindhu Hisar Anand, Azad Dhania-1 RCr 20 RCr 435, Pant Haritima, Hisar Sugandh, Hisar Surabhi, CIMPO-33, CIMPO33 Mc.43, 5-404, Guj. Cumin 1,RZ-19 Guj Cumin 2, Guj. Cumin 3, Guj. Cumin 4, RZ-209, RZ-223 PF 35, Co.1, Guj Fennel 1 Guj fennel 2 RF 101, RF 125, Azad snauf-1, S-7-9, Pant Madhurika, Rajendra Saurabh Co.1 Rajendra kanti RMt.1 Lam sel.1 Hisar Sonali, Co 2 ,RMt 303 Guj Methi 1 , Rajendra Abha, Hisar Madhuri, Hisar Suvarna, Hisar Muktha, , Guj Methi 1, RMt 1, RMt 143, RMt 305, Rajendra Khushbu, Pant Ragni, Pusa early bunching

High yield, high curcumin, short duration, field resistance to rhizome rot, suitable for both rainfed and irrigated conditions

Cinnamon

Selections from elite lines and seed progenies

High yield, high quality

Nutmeg

Selections from elite lines and seed progenies Bulk, pure line and recurrent selections

High yield, high myristicin

Coriander

High yield, high quality

Cumin

Bulk, pure line and recurrent selections,Mutation breeding

High yield, high quality

Fennel

Bulk, pure line and recurrent selections

High yield, high quality

Fenugreek

Bulk, pure line and recurrent selections

High yield, high quality, duel purpose types, early maturing types, bold grains, short plant types,

Chilli

Bulk and pure line selection, Convergent breeding

About 56 vareties

High yield, good colour, bacterial wilt and virus resistance, short plant High oil Dwarf high yielding and sweet types

Curry leaf Tamarind

Clonal and seedling selection Clonal and seedling selection

DWA-1, DWA-2, PKM-1, DTS 1, Prathisthan, MH- 263

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ENHANCING UTILIZATION OF PLANT GENETIC RESOURCES IN CROP IMPROVEMENT


Upadhyaya, H.D1. and C.L.L. Gowda

ABSTRACT
Crop plant genetic resources (PGR) including landraces, old and new cultivars, mutant etc., are vital to crop improvement. These were used in research to develop improved cultivars that has resulted in increase of productivity and production considerably of various crops. The need for collecting and conserving germplasm was realized during 1960s, when there was threat of loss of landraces due to large adoption of improved varieties. Currently over six million-germplasm accessions are held in over 1300 genebanks across the world. This paper discusses assembly and management of genetic resources of sorghum, pearl millet, chickpea, pigeonpea, groundnut and six small millets at the Rajendra S Paroda Genebank at ICRISAT-Patancheru, India and means to further enhance their utilization for sustainable agriculture globally. Various institutes and organizations worldwide have donated germplasm to the ICRISAT genebank. In addition, two hundred and thirteen germplasm collection missions were organized in 62 countries securing 33,194 germplasm accessions. The entire holding is over 118,800 accessions of the above crops from 130 countries. The germplasm accessions receive high priority for regeneration, characterization, conservation and distribution. The focus of research is on diversity assessment and on developing representative core, mini-core and composite collections to enhance utilization by the breeders. Molecular characterization of diverse germplasm sets is pursued for value addition and to enhance their utilization. Most of the accessions have been characterized. Germplasm seeds are conserved under very precise (cool and dry) conditions. Adequate seed of each accession is conserved to meet the requests of researchers and for posterity. The ICRISAT genebank has been supplying over 21,000 germplasm samples annually to scientists across the countries. ICRISAT has restored crop germplasm to several countries including India. From the basic germplasm supplied from ICRISAT genebank, 66 varieties were released for cultivation in 44 countries.

Introduction The wealth of plant genetic resources that includes landraces, old and new cultivars, genetic stocks, mutants etc., has contributed enormously towards achieving the global objectives of food security, poverty alleviation, environment protection and sustainable development. The value of genetic resources in developing superior crop cultivars is well recognized. The utilization of Norin 10 gene in wheat and Dee Geo Woo Gen in rice (sources of reducing plant height) have revolutionized the production of these crops globally. Wheat productivity increased by 137%

and of rice by 93% in last 40 years due to the improved cultivars (Table 1), coupled with good agronomic management. Diverse genotypes were used in developing improved cultivars of soybean (resistance to diseases and insectpests, tolerance to pod shattering, promiscuous nodulation and high yield; cf Dashiell and Fatokun, 1997) and groundnut (broadening genetic base, adding disease resistance and high yield; cf Singh and Nigam, 1997) that resulted in 93.2% productivity increase in soybean and 69.6% in groundnut in the last 40 years. Similarly, diverse germplasm sources having traits of

1. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India

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short-duration, large seed size and disease resistance were used to develop new and high yielding cultivars of Chickpea (cf Singh et al., 1997) and pigeonpea (cf Remanandan and Singh, 1997). The concern of PGR exploration and ex-situ conservation was not serious until 1960s. The development and spread of high yielding varieties of wheat and other crops by 1960s started replacing the local cultivars very rapidly leading to erosion of plant diversity. This loss of native crop landraces and cultivars prompted the international organizations such as the Food and Agriculture Organization (FAO) and the World Bank to create new institutional structures for the collection and preservation of valuable plant genetic resources in ex-situ genebanks. Since the last four decades, this program has achieved spectacular success. Over six million germplasm accessions have been collected and/or assembled in 1308 genebanks world over (FAO, 1998). Created in 1971, the Consultative Group on International Agricultural Research (CGIAR) is an association of public and private members supporting a system of 15 Future Harvest Centers that work in more than 100 developing countries to achieve sustainable food security and reduce poverty through scientific research and development activities in the fields of agriculture, forestry, fisheries, policy and environment. The CGIAR germplasm collections are a unique resource, available to all researchers. Germplasm contributions have helped lay the foundations of recovery by jumpstarting agricultural growth in countries emerging from conflict such as Afghanistan, Angola, Mozambique and Somalia. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), one of the 15 CGIAR centers, is responsible for germplasm assembly, characterization, conservation and distribution of germplasm of five mandate crops
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(sorghum, pearl millet, chickpea, pigeonpea and groundnut) and six small millets (finger millet, foxtail millet, kodo millet, little millet, proso millet and barnyard millet) and their wild relatives. Germplasm Assembly in the ICRISAT Genebank When ICRISAT was established in 1972, efforts were begun to assemble the germplasm of the mandate crops that existed with various research institutes in India and other countries. The Rockefeller Foundation had assembled over 16,000 sorghum germplasm accessions from major sorghum areas, and ICRISAT acquired 11,961 accessions of this collection in 1974 that existed in India and USA, besides 2000 pearl millet accessions. ICRISAT also obtained 2000 accessions of pearl millet collected by the Institut Francais de Recherch Scientifique pour le Development en Cooperation (ORSTOM) in francophone West Africa. The germplasm material of chickpea and pigeonpea originally collected and assembled by the former Regional Pulse Improvement Project (RPIP), a joint project of the Indian Agricultural Research Institute (IARI), the United States Department of Agriculture (USDA) and Karaj Agricultural University in Iran, formed the initial collection. Sets of this germplasm, which were available in several agricultural research institutes in India and Iran, and at the USDA, were donated to ICRISAT in 1973. ICRISAT also acquired over 1,200 chickpea accessions from the Arid Lands Agricultural Development (ALAD) program in Lebanon. Similarly, much of the groundnut germplasm was received from the Indian groundnut research program, [now the National Research Center for Groundnut (NRCG), Junagadh], and USDA. Besides germplasm donations by the All India Coordinated Research Projects on various crops, considerable number of germplasm were received from agricultural universities at

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Pantnagar (Uttranchal), Rajendranagar (Andhra Pradesh), Ludhiana (Punjab), Coimbatore (Tamil Nadu), Jabalpur (Madhya Pradesh), Rahuri (Maharashtra) and IARI at New Delhi. Fifteen Indian organizations that donated highest number of germplasm are listed in Table 2. Recently, in 2004-05, we obtained chickpea germplasm samples from Washington State University, Pullman, USA (2083 cultivated, 68 wild) and ICARDA, Syria (682 cultivated, 21 wild). We also received 622 groundnut germplasm samples from the National Institute of Agrobiological Sciences, Japan. Over 400 accessions of sorghum collected in Niger were received from our regional genebank in Niamey. ICRISAT initiated activities to add new germplasm of its mandate crops from areas that were not adequately represented in the germplasm collection. Between 1975 and 2000, a total of 213 joint missions were launched in 62 countries, from which 33,194 accessions (sorghum 9011; pearl millet 10841; chickpea 4228, pigeonpea 3873, groundnut 2776; and small millets 2465) were collected. A large number of breeding lines or germplasm selections are developed and evaluated at important locations. The promising/improved germplasm lines were also registered in the genebank and conserved for future utilization. The genebank currently holds 118,833 accessions of which 73.8% have been conserved as base collection and 93.0% are designated with FAO (Table 3). Germplasm Management Phenotypic characterization and evaluation Agronomic and morphological characterization is necessary to facilitate the utilization of germplasm. To achieve this, germplasm accessions of all the crops were sown in batches over the years and characterized for morphological and agronomic traits. Germplasm screening against biotic and abiotic stresses were conducted in collaboration with various
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disciplinary scientists. Grains were tested for nutritional value. Germplasm sets were evaluated over locations jointly with scientists in India, Nepal, Thailand, Indonesia, Ethiopia, Kenya and more intensively with the National Bureau of Plant Genetic Resources (NBPGR), India. The results of joint evaluations have led to a better understanding of the germplasm material. Regeneration Regeneration was carried out to meet the seed increase of (1) accessions that had reached a critical low level of seed stock or viability; (2) accessions required for mediumterm storage (MTS; 5 oC, 25-30%RH) or longterm storage (LTS; -20 oC); and (3) germplasm repatriation, particularly to the NBPGR, India. Some of the germplasm accessions that do not produce seeds under ICRISAT-Patancheru climatic conditions (some wild Arachis species) are maintained vegetatively in the greenhouse. Some other accessions (wild Cicer species) need long day length and cool weather to grow and produce seeds. These species are also regenerated in greenhouse facilities. Conservation Germplasm conservation requires cleaning the seed material, drying to minimal seed moisture content, storing in cool and dry conditions and regular monitoring of seed health during storage. In the ICRISAT genebank, the seeds are stored in medium-term storage (MTS) in aluminium cans. A recent monitoring of the health of seed conserved for 1025 years (MTS) indicated greater than 75% seed viability for majority of the accessions. Accessions with declining seed viability (less than 75% seed germination) are regenerated on priority and the old stock is replaced with fresh seeds. The germplasm accessions are also conserved in long-term storage (LTS) after packing in vacuum-sealed aluminium foil pouches. Before packing, the seeds are dried to about 5%

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moisture content in a walk-in drying room (100 m3 size; 15 oC and 15% RH) facility. At present, we have about 76% of the FAO designated germplasm in the LTS facility. Documentation and supply of information The vast germplasm data gathered on chickpea and pigeonpea germplasm has been summarized and presented to the users in the form of catalogs (Pundir et al., 1988; Remanandan et al., 1988). During the last 20 years, we had a very purposeful collaboration with NBPGR, India, on germplasm exploration, and evaluation at a number of locations, and results were published as Collaboration on Genetic Resources (ICRISAT 1989). The data on joint germplasm evaluations were analyzed and published two catalogs each on forage sorghum germplasm (Mathur et al., 1991, 1992), and pearl millet (Mathur et al., 1993b and 1993c), and one on chickpea (Mathur et al., 1993a). Core and minicore collections of ICRISAT mandate crops were established and the information was published for the benefit of fellow research workers. A Manual of Genebank Operations and Procedures was published (Rao and Bramel, 2000) documenting the procedures for germplasm acquisition, maintenance, documentation, conservation, and distribution. Existing procedures were reviewed and revised to maintain the collections according to international standards. A taxonomic key for the identification of wild species of the mandate crops has also been included in the manual. Global germplasm supply to scientists and institutions The ICRISAT genebank is holding germplasm that was donated by various institutes, organizations and farm communities and is ever willing to supply the same for research. From the beginning of our work (1973) until 2005, we have supplied 674,108 germplasm samples to scientists in 142 countries (Table 4).

Repatriation of germplasm to national programs The global collections held at ICRISAT serve the purpose of restoration germplasm to the source countries when national collections are lost due to natural calamities, civil strife, etc. We supplied 362 sorghum accessions to Botswana; 1827 sorghum and 922 pearl millet to Cameroon; 1723 sorghum and 931 chickpea to Ethiopia; 838 sorghum and 332 pigeonpea to Kenya; 1436 and 445 sorghum accessions respectively to Nigeria and Somalia; and 71 pigeonpea accessions to Sri Lanka. The germplasm collection maintained in the ICRISAT genebank includes 44,822 accessions received from or jointly collected with the Indian National Programs. The National Bureau of Plant Genetic Resources (NBPGR), India requested ICRISAT for restoration of this germplasm. As part of ICAR/ICRISAT Partnership Projects, the genebank has repatriated almost full set of this germplasm by July 2004 (Table 5). Thus the NARS of several countries have regained their precious heritage which could have been lost if this was not conserved in the ICRISAT genebank. Impact of germplasm supplied to NARS worldwide Besides the utilization of germplasm in ongoing research at other institutes, 66 germplasm accessions (sorghum 30, pigeonpea 7, chickpea 19, groundnut 6, finger millet 2, and 1 each of pearl millet and barnyard millet) supplied from the ICRISAT genebank have been directly released as cultivars in 44 countries (Figure 1). Pigeonpea germplasm accession ICP 8863 collected from farmers field in India was found very promising against fusarium wilt and was purified for the trait. The purified line was found high yielding and it was released for cultivation in 1986 as Maruthi in Karnataka state, India. This variety is also

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grown on large hectarage in adjacent states, namely, Maharashtra and Andhra Pradesh (Bantilan and Joshi 1996). A sorghum variety, Parbhani Moti was released in Maharashtra, India, in 2002. This variety is an excellent Maldandi-type [predominant postrainy (Rabi) sorghum landrace in Maharashtra and Karnataka states of India) with large lustrous grains and high yield. This was selected from a germplasm collection from Ghane Gaon, Sholapur, Maharashtra, made by ICRISAT genebank staff during 1989. Another example is the release of barnyard variety (PRJ 1) in Uttranchal state during 2003. This variety yielded 45.4% higher grain yield compared to the check variety VL 29. It provides substantial fodder yield as well. This variety is a selection from ICRISAT germplasm collection IEC 542 that originated in Japan. Present scenario of PGR utilization Much progress has been in developing stable and high-yielding cultivars using diverse germplasm resources. This has resulted in area increase under some crops. During the last 40 years, area under soybean increased by 250.9%; pigeonpea: 60.7%; groundnut: 47.9% and rice: 22.4%. For other crops such as wheat and chickpea, area remained nearly unchanged. Productivity has improved considerably in most of the crops (Table 1). However, in future, there is much to be done to further improve productivity of the crops to meet the food requirement of ever increasing population. A glance of ICRISAT genebank service to researchers revealed that on an average, 21,065 germplasm samples are supplied annually to users outside the ICRISAT (mean from 1974 to 2005). According to Marshall (1989), this figure indicates satisfactory germplasm distribution service of the genebank. However, the use of basic germplasm in breeding programs is scanty. For example, the summary of parental lines used in the ICRISAT groundnut-breeding
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program at ICRISAT (1986-2002) revealed that 986 unique parents were used in developing 8279 breeding lines, but this included only 132 unique germplasm accessions of groundnut and 10 of wild Arachis species. The two most often used cultivars were Robut 33-1 (3096 times) and Chico (1180 times). In the ICRISAT chickpea-breeding program (1978-2004), 12,887 parents (586 unique parents) were used in developing 3548 breeding lines, which included only 91 unique germplasm accessions of chickpea and five of wild Cicer species (Upadhyaya et al., 2006). The two most frequently used cultivars were L 550 (903 times) and K 850 (851 times). The data analysis from the Indian chickpea research program revealed that during 1967 - 2003, a total of 86 varieties was developed through hybridization that traced back to 95 unique parents. The top 10 parents contributed more than 35% to the genetic base of the released varieties. Most frequently used parents were Pb 7, IP 58, F 8, Rabat and S 26. About 41% varieties developed have Pb 7 as one of the parents in their pedigree (Kumar et al., 2004). There are similar reports from China (Jiang and Duan, 1998), and the USA (Knauft and Gorbet, 1989) in groundnut. Strategies to enhance germplasm utilization Assessment of diversity in the germplasm collection The germplasm characterization and assessment of diversity is important to plant breeders for crop improvement and to genebank curators for efficient and effective management of their collection. The chickpea germplasm collection (16,820 accessions) was characterized for seven morphological and 13 agronomic traits and reaction to fusarium wilt to determine phenotypic variation in different geographical regions. The means for different agronomic traits differed significantly between regions.

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The variances for all the traits among regions were heterogeneous. South Asia region contained the largest range of variation for all the traits. The Shannon-Weaver (Shannon and Weaver, 1949) diversity index (H) was variable in different regions for different traits. Analysis revealed the need to secure more germplasm collections from Mediterranean countries and Ethiopia. Cluster analysis delineated two regional clusters consisting Africa and South and Southeast Asia in the first cluster; and the Americas, Europe, West Asia, Mediterranean and East Asia in the second cluster (Upadhyaya, 2003) (Figure 2). An earlier study of chickpea germplasm data at ICRISAT (Pundir et al., 1988) revealed that in general, Indian accessions were highest yielding and the accessions from Chile had higher plant height and greater seed mass. The accessions from Spain and Syria had longer flowering duration and the accessions from Greece and Russia had erect growth habit. Resistance to fusarium wilt was more common in accessions from Bangladesh than from other countries. The groundnut germplasm collection (13,342 accessions) was characterized for 16 morphological and 10 agronomic traits in two seasons to determine the phenotypic variation in different geographical regions. The means for different agronomic traits differed significantly among regions. The variances for all the traits among regions were heterogeneous. South America, which showed 100% range variation for 12 of the 16 morphological traits, also revealed highest range variation. From South America among regions, primary seed color among morphological traits and leaflet length among agronomic traits showed highest pooled H. Three of the six botanical varieties, aequatoriana, hirsuta, and peruviana were poorly represented indicating the need to be collected. PCA using 38 traits and clustering on first seven PC scores delineated three regional clusters; consisting North America, Middle East, and East Asia in
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the first Cluster, South America in the second cluster, and West Africa, Europe, Central Africa, South Asia, Oceania, Southern Africa, Eastern Africa in second cluster and Southeast and Central Asia and the Caribbean in the third cluster (Upadhyaya et al., 2002b) (Figure 3). The pigeonpea germplasm collection (11,402 accessions from 54 countries grouped into 11 regions) was analyzed for patterns of variation for 14 qualitative and 12 quantitative traits. Semi-spreading growth habit, green stem color, indeterminate flowering pattern, and yellow flower color were predominant among qualitative traits. Primary seed color had maximum variability and orange color, followed by cream were the two most frequent seed colors in the collection. Variances for all the traits were heterogeneous among regions. The germplasm accessions from Oceania were conspicuous by short growth duration, short height, fewer branches, pods with fewer seeds, smaller seed size, and lower seed yields. The accessions from Africa were of longer duration, taller, with multiseeded pods, and larger seeds. The germplasm diversity, indicated by H pooled over all traits, was highest for Africa and lowest for Oceania. The cluster analysis delineated three clusters: cluster 1 includes accessions from Oceania; cluster 2 from India and adjacent countries, and cluster 3 from Indonesia, Thailand, The Philippines, Europe, Africa, America and the Caribbean countries. Pigeonpea-rich countries such as Myanmar, Uganda, and others like Bahamas, Burundi, Comoros, Haiti, and Panama are not adequately represented in the collection, and need priority attention for germplasm exploration (Upadhyaya et al., 2005c). Developing core collections One of the reasons that plant breeders are using less basic germplasm in research is the lack of information on traits of economic importance, which often shows high genotype x environment interactions and requires

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replicated multilocational evaluations. Evaluation is very costly and resource-demanding task owing to the large size of the germplasm collections. To overcome this, our research now focuses on studying the diversity of germplasm collection and developing core collections, which are about 10% of the entire collection, but represent almost full diversity of the species. From the germplasm collection in the ICRISAT genebank, we have already developed core collection of sorghum (2,247 accessions, Grenier et al., 2001); pearl millet (1,600 accessions, Bhattacharjee, 2000); chickpea (1,956 accessions, Upadhyaya et al., 2001a); groundnut (1,704 accessions, Upadhyaya et al., 2003); groundnut Asia core (504 accessions, Upadhyaya et al. 2001c); pigeonpea (1,290 accessions, Reddy et al., 2005); finger millet (622 accessions, Upadhyaya et al., 2005a) and foxtail millet (155 accessions, Upadhyaya unpublished data) (Table 6). Developing mini-core collection When the size of the entire collection is very large, even a core collection size becomes unwieldy for evaluation by breeders. To overcome this, ICRISAT scientists developed a seminal two-stage strategy to develop a minicore collection, which consists of 10% accessions in the core collection (and hence only 1% of the entire collection) (Upadhyaya and Ortiz, 2001). This mini-core collection still represents the diversity of the entire core collection. The first stage involves developing a representative core collection (about 10%) from the entire collection using all the available information on origin, geographical distribution, and characterization and evaluation data of accessions. The second stage involves evaluation of the core collection for various morphological, agronomic, and quality traits, and selecting a further subset of about 10% accessions from the core collection. At both stages standard clustering procedures should be used to form groups (clusters) of similar accessions and then

select desired number of accessions from each cluster. At ICRISAT, we have already developed mini-core collections of chickpea consisting of 211 accessions (Upadhyaya and Ortiz, 2001), groundnut (184 accessions) (Upadhyaya et al., 2002a), pigeonpea (146 accessions), and finger millet (65 accessions) (Upadhyaya unpublished data) (Table 6). Developing composite collection The revolution in molecular biology, bioinformatics, and information technology has provided the scientific community with tremendous opportunities for solving some of the worlds most serious agricultural and food security issues, and has led to the formation of Generation Challenge Program (GCP) entitled Unlocking Genetic Diversity in Crops for the Resource-Poor ( www.generationcp.org). The GCP is designed to utilize molecular tools and comparative biology to explore and exploit the valuable genetic diversity existing in germplasm collections held at the CGIAR and NARS genebanks, with particular focus on drought tolerance. In recent years, several studies conducted on plants have detected DNA markers associated with ecology, geography, disease resistance, and quantitative traits (Thornsberry et al., 2001; Turpeinen et al., 2001; Ivandic et al., 2002, 2003; Russel et al., 2003; Sun et al., 2001, 2003; Gebhardt et al., 2004; Sabharwal et al., 2004; and Amirul Islam et al. 2004) demonstrating that it is a viable alternative to classical QTL analyses, which were time taking and costly measurements. ICRISAT and collaborating institutes have constituted composite collections of chickpea (Upadhyaya et al., 2006a) and sorghum (3000 accessions each) and groundnut, pigeonpea, finger millet (1000 accessions each) (Table 7) that contain maximum diversity known in the species, accessions with economic traits and some representation of the related wild species. The composite collections will be genotyped using SSR markers. The data generated will
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be used to define the genetic structure of the collection for functional and comparative genomics. The analysis of genetic diversity will help to elucidate population structures that influence the analysis of the associations between molecular markers and the morphological or reaction traits. Using all available information, about 10% accessions will be selected containing maximum diversity and those could be used in the breeding programs. Identification of new sources for traits of economic importance for use in crop improvement program Due to the reduced size, the core collection can be evaluated extensively to identify the useful parents for crop improvement. By evaluating core collection of chickpea, we identified new sources of important traits, namely, early maturity (28 accessions), large seeded kabuli (16 accessions) and high-yielding (39 accessions) types. The clustering of 28 early maturing accessions along with four controls revealed three clusters. Cluster-1 was formed of five entries including three controls (ICCVs 2, 96029 and Harigantars). Cluster-2 was formed of 14 entries including control Annigeri. Thirteen entries constituted cluster-3 and no control among them. It can be presumed that these 13 accessions are more distant from controls than other accessions (Figure 4). The phenotypic diversity index was highest between ICC 14648 and ICCV 96029, compared to the other entry pairs. Such information has high value to chickpea breeders. The evaluation of groundnut core collection resulted in identification of 21 accessions with early maturity (Upadhyaya et al., 2005c). The cluster analysis done on these 21 accessions and three controls revealed three clusters. Cluster-1 comprised of four entries including two controls (Gangapuri and Chico). Cluster-2 contained 13 entries including one control (JL-24). Seven test accessions formed cluster-3 and these accessions are more distinct from the three
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controls used in this study (Figure 5). In the groundnut core, 158 accessions had low temperature tolerance at germination (Upadhyaya et al., 2001b). Also found were 15 Valencia, 20 Spanish, and 25 Virginia type germplasm lines in groundnut with high yield, good shelling percentage and 100-seed weight through multilocational evaluation of the Asia region core collection (Upadhyaya et al., 2005b). These new sources performed better than or similar to the best control cultivars for particular trait (s), but were diverse from them. Holbrook et al. (1997) achieved similarly through examining all accessions in the groundnut core collection (Holbrook et al., 1993) for resistance to the groundnut root-knot nematode (Meloidogyne arenaria (Neal) race 1) and resistance to pre-harvest aflatoxin contamination (PAC) (Holbrook, 1998) while Franke et al. (1999) later did similarly for resistance to Rhizoctonia limb rot (Rhizoctonia solani Kuhn AG-4). The mini-core collections of chickpea and groundnut have been evaluated and diverse sources of useful traits were identified. From the chickpea mini-core, 18 accessions having traits related to drought tolerance (Kashiwagi et al., 2005) and 29 accessions tolerant to soil salinity (Serraj et al., 2004) have been identified. Similarly, Pande et al. (2006) screened the minicore collection for resistance to various diseases and identified 67 accessions resistant/highly resistant to fusarium wilt, moderate resistance to ascochyta blight in 3 accessions, botrytis grey mold in 55 accessions, and to dry root rot in 6 accessions. Some accessions also with multiple resistances were identified. The evaluation of groundnut mini-core resulted in identification of 18 diverse accessions with high water use efficiency (Upadhyaya, 2005). The evaluation of chickpea mini-core at the Indian Institute of Pulses Research (IIPR), Kanpur, India during 2002 to 2004 seasons revealed 12 very promising accessions. Of these six accessions

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were involved in hybridization to develop large seeded kabuli cultivars. The evaluation of groundnut mini-core in Thailand (2004-05) indicated ten accessions high-yielding. The groundnut mini-core evaluation in China during 2005 resulted in identification of 14 accessions highly resistant to bacterial wilt, six with high oil content and four with high Oleic and low Linoleic acid. Three accessions had highest Oleic: Linoleic acid ratio. Molecular characterization of germplasm Characterization of germplasm with molecular markers can help improve their utilization. It can form the basis for mining and cloning of genes of agronomically important traits. Genotyping chickpea accessions A total of 288 chickpea accessions including 211 mini-core subset accessions consisting of 75% desi type (Upadhyaya and Ortiz, 2001), 57 accessions of kabuli chickpea, and 20 accessions of wild Cicer species from ICARDA were genotyped using 40 SSR markers. The results indicated that the chickpea mini-core developed at ICRISAT was allelically more diverse than the germplasm from ICARDA. The accessions from ICARDA consisted of more heterozygous individuals compared with mini-core accessions. The dendogram constructed based on shared allele distance using unweighted pair group mean average (UPGMA) method indicated two main groups: one consisting mainly of accessions from the Indian subcontinent and the other group of accessions from Mediterranean, Middle-East and Ethiopia. The accessions of wild species (C. reticulatum and C. echinospermum) formed two groups of their own flanking two ends of the chickpea accessions (Upadhyaya et al., 2006b). Validating the chickpea mini core collection: Discriminant function analysis was used to determine the level of congruence between the genotypic data set and the 28 phenotypic clusters
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of the chickpea mini-core (Upadhyaya and Ortiz, 2001) based on morphological and agronomic traits. For DFA analysis, genotypic data from 210 accessions screened with 40 SSR markers was used. Overall most individuals were assigned with a high degree of confidence to the original (phenotypic) clusters from which accessions constituting mini core collection were selected. Only 27% of the individuals were reassigned into new clusters according to genotypic data, which were mainly identified within clusters 4, 6, and 7 of the mini-core (ICRISAT, 2004). This confirmed that the chickpea mini core was well selected. Genotyping chickpea accessions of varying maturity duration Sixty-two chickpea germplasm accessions (50 early-, 6 medium- and 6 late-maturing) were analyzed with 37 SSR markers. A total of 673 alleles were found. The number of alleles per marker varied from 4 to 28 with an average of 18. The polymorphic information content (PIC) values ranged from 0.53 to 0.94 with an average of 0.85. Mean heterozygosity was low (0.0276). The principal component analysis (PCA) plot of Rogerss distance indicated three distinct clusters (ICRISAT, 2004). Genotyping groundnut accessions In groundnut, 26-accessions were analyzed with random amplified polymorphic DNA (RAPD) assays. The genetic similarity (Sij) ranged from 59.0 to 98.8% with an average of 86.2%. Both multidimensional scaling and unweighted pair-group method with arithmetic averages (UPGMA) dendograms revealed the existence of five distinct clusters. Some accessions with diverse DNA profiles (ICGs 1448, 7101, and 1471, and ICGVs 99006 and 99014) were identified for mapping and genetic enhancement in groundnut (Dwivedi et al., 2001). Molecular marker based diversity estimates are useful to select diverse lines for developing populations that may be used for

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mapping studies to identify DNA markers linked with resistance to rosette disease in groundnut. Nine amplified fragment length polymorphism (AFLP) using primer pairs were performed on nine rosette resistant and one susceptible accessions. Across the 10 accessions, the nine primer pairs identified 94 unique markers, with an average of 10.4 markers per primer pair. The genetic dissimilarity (Dij) values ranged from 3.92 to 50.53% with an average of 19.56%. Groundnut accessions, namely, ICG 11044 with ICGs 3436, 9558 and 11968 showed greater genetic diversity (36.59 to 50.53%) amongst the nine rosette resistant accessions used. These accessions possess high levels of resistance to rosette, average d2% compared to e90% infection in susceptible control ICG 7827 across four seasons evaluation at Lilongwe, Malawi. These accessions therefore could be intercrossed among themselves to produce diversified rosette resistant breeding populations (Dwivedi et al., 2003). Conclusion Crop genetic resources have contributed enormously towards sustainability of agriculture and alleviation of poverty. These are being assembled and conserved at several genebanks for future use. Using raw germplasm resources, a large number of crop varieties and hybrids have been developed and released for cultivation. New strategies on core and mini-core collections were developed to enhance the precision of germplasm characterization and reducing cost on germplasm regeneration and conservation. Composite sets of ICRISAT mandate crops are being developed under the Generation Challenge Program. Phenotypic and genotypic characterization of these sets will provide vast scope of identifying useful and unique germplasm resources for utilization in crop improvement. Molecular characterization of the germplasm of agronomic importance has been pursued for value addition and to enhance their utilization.
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REFERENCES Amirul Islam, F.M., Beebe, S., Munoz, M., Tohme, J., Redden, R.J. and Basford, K.E. 2004. Using molecular markers to assess the effect of introgression on quantitative attributes of common bean in the Andean gene pool. Theoretical and Applied Genetics 108:243-252. Bantilan, M.C.S. and Joshi, P.K. 1996. Adoption and impact pigeonpea ICP 8863. Pages 3639. in Partners in impact assessment: summary proceedings of an ICRISAT/ NARS workshop on methods and joint impact targets in Western and Central Africa, 3-5 May 1995, Sadore, Niger. ICRISAT, Patancheru, 502 324, India. 116 pp. Bhattacharjee, R. 2000. Studies on the establishment of a core collection of pearl millet (Pennisetum glaucum). Ph. D. Thesis, CCS Haryana Agricultural University, Hisar 125 004, India. 162 pp. Dashiell, K. and Fatokun, C. 1997. Soybean. Pages 181-190 in Fuccillo D, Sears L and Stapleton P (eds.). Biodiversity in Trust. Cambridge University Press, Cambidge, UK Dwivedi, S.L., Gurtu, S., Chandra, S., Upadhyaya, H.D. and Nigam, S.N. 2003. AFLP Diversity among selected rosette resistant groundnut germplasm. International Arachis Newsletter 23: 2123. Dwivedi, S.L., Gurtu, S., Chandra, S., Yuejin, W. and Nigam, S.N. 2001. Assessment of genetic diversity among selected groundnut germplasm. I: RAPD analysis. Plant Breeding 120: 345-349. FAO. 1998. The state of ex-situ conservation. Page 90 in The state of the worlds plant genetic resources for food and agriculture. Rome, Italy: FAO. Franke, M.D., Brenneman, T.B. and Holbrook,

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C.C. 1999. Identification of resistance to Rhizoctonia limb rot in a core collection of peanut germplasm. Plant Dis. 83: 944-948. Gebhardt, C., Ballvora, A., Walkemeier, B., Oberhagemann, P. and Schuler, K. 2004. Assessing genetic potential in germplasm collections of crop plants by marker-trait association: a case study for potatoes with quantitative variation of resistance to late blight and maturity type. Molecular Breeding 13: 93-102. Grenier, C., Bramel, P.J. and Hamon, P. 2001. Core collection of the genetic resources of sorghum: 1. Stratification based on ecogeographical data. Crop Science 41: 234 240. Holbrook, C.C., Anderson, W.F. and Pittman, R.N. 1993. Selection of a core collection from the U.S. germplasm collection of peanut. Crop Science 33: 859-861. Holbrook, C.C., Stephenson, M.G. and Johnson, A.W. 1997. Level and geographical distribution of resistance to Meloidogyne arenaria in the germplasm collection of peanut. Agron. Abstr.:157. Holbrook, C.C., Wilson, D.W. and Matheron, M.E. 1998. Sources of resistance to preharvest aflatoxin contamination in peanut. Proceedings of the American Peanut Research & Education Society 30:21. ICRISAT (International Crops Research Institute for the Semi-Arid Tropics). 1989. Collaboration on Genetic Resources: summary proceedings of a joint ICRISAT/ NBPGR (ICAR) workshop on germplasm exploration and evaluation in India, 1415 Nov 1988, ICRISAT, Patancheru, India. Patancheru 502 324, Andhra Pradesh, India: International Crops Research Institute for the Semi-Arid Tropics. ICRISAT (International Crops Research Institute for the Semi-Arid Tropics). 2004. Harnessing Biotechnology for the Poor
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Archival Report 2004. Patancheru, India: ICRISAT. Ivandic, V., Hackett, C.A., Nevo, E., Keith, R., Thomas, W.T.B. and Forster, B.P. 2002. Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology, geography and flowering time. Plant Molecular Biology 48:511-527. Ivandic, V., Thomas, W.T.B., Nevo, E., Zhang, Z. and Forster BP. 2003. Association of simple sequence repeats with quantitative trait variation including biotic and abiotic stress tolerance in Hordeum spontaneum. Plant Breeding 122:300-304. Jiang, H.F. and Duan, N.X. 1998. Utilization of groundnut germplasm resources in breeding programme. Crop Genetic Resources 2:24-25. Kashiwagi, J., Krishnamurthy, L., Upadhyaya, H,D,, Krishna, H., Chandra, S., Vincent Vadez. and Serraj, R. 2005. Genetic variability of drought-avoidance root traits in the mini-core germplasm collection of chickpea (Cicer arietinum L.). Euphytica 146: 213-222. Knauft, D.A. and Gorbet, D.W. 1989. Genetic diversity among peanut cultivars. Crop Science 29:1417-1422. Kumar, S., Gupta, S., Chandra, S. and Singh, B.B. 2004. How wide is the genetic base of pulse crops? In: Ali M, Singh BB, Kumar S and Dhar V (eds.). Pulses in new perspective. Indian Society of Pulses Research and Development, IIPR, Kanpur, India. pp. 211-221. Marshall, D.R. 1989. Limitations to the use of germplasm collections. In: Brown AHD, Frankel OH, Marshall DR and Williams JT (eds.). The use of plant genetic resources Cambridge Univ. Press, New York, pp. 105-120.

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Mathur, P.N., Prasada Rao, K.E., Singh, I.P., Agrawal, R.C., Mengesha, M.H., and Rana, R.S. 1992. Evaluation of Forage Sorghum Germplasm, Part-2: NBPGR-ICRISAT Collaborative Programme. NBPGR, New Delhi, India. 296 pp. Mathur, P.N., Prasada Rao, K.E., Thomas, T.A., Mengesha, M.H., Sapra, R.L. and Rana, R.S. 1991. Evaluation of Forage Sorghum Germplasm, Part-1: NBPGR-ICRISAT Collaborative Programme. NBPGR, New Delhi, India. 269 pp. Mathur, P.N., Pundir, R.P.S., Patel, D.P., Rana, R.S. and Mengesha, M.H. 1993a. Evaluation of Chickpea Germplasm, Part-1: NBPGRICRISAT Collaborative Programme. NBPGR, New Delhi, India. 194 pp. Mathur, P.N., Rao, S.A., Agrawal, R.C., Mengesha, M.H. and Rana, R.S. 1993b. Evaluation of Pearl Millet Germplasm, Part1: NBPGR-ICRISAT Collaborative Programme. NBPGR, New Delhi, India. 200 pp. Mathur, P.N., Rao, S.A., Sapra, R.L., Mengesha, M.H., and Rana, R.S. 1993c. Evaluation of Pearl millet Germplasm, Part2: NBPGR-ICRISAT Collaborative Programme. NBPGR, New Delhi, India. 215 pp. Pande, S., Kishore, G.K., Upadhyaya, H.D. and Raom, J.N. 2006. Identification of sources of multiple fungal diseases resistance using mini-core collection in chickpea. Plant Disease (in press). Pundir, R.P.S., Reddy, K.N. and Mengesha, M.H. 1988. ICRISAT Chickpea Germplasm Catalog: Evaluation and Analysis. Patancheru, A. P. 503 324, India: International Crops Research Institute for the Semi-Arid Tropics. 94 pp. Rao, N.K. and Bramel, P.J. 2000. Manual of Genebank Operations and Procedures. Technical Manual no. 6. Patancheru, A. P.
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502 324, India: International Crops Research Institute for the Semi-Arid Tropics. 190 pp. Reddy, L.J., Upadhyaya, H.D., Gowda, C.L.L. and Sube Singh. 2005. Development of core collection in pigeonpea (Cajanus cajan (L) Millsp. Genetic Resources and Crop Evolution. 52:1049-1056. Remanandan, P., Sastry, D.V.S.S.R. and Mengesha, M.H. 1988. ICRISAT Pigeonpea Germplasm Catalog: Evaluation and Analysis. Patancheru, A P 502 324, India: International Crops Research Institute for the Semi-Arid Tropics. 89 pp. Remanandan, P. and Singh, L. 1997. Pigeonpea. Pages 156-167. in Fuccillo D, Sears L and Stapleton P (eds.). Biodiversity in Trust. Cambridge University Press, Cambidge, UK. Russel, J.R., Booth, A., Fuller, J.D., Baum ,M., Ceccarelli, S., Grando, S. and Powel, W. 2003. Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan. Theoretical and Applied Genetics 107: 413-421. Sabharwal, V., Negi, M.S., Banga, S.S. and Lakshmikumaran, M. 2004. Mapping of AFLP markers linked to seed coat color loci in Brassica juncea (L) Czern. Theoretical and Applied Genetics 109: 160-166. Serraj, R., Krishnamurthy, L. and Upadhyaya, H.D. 2004. Screening of chickpea minicore germplasm for tolerance to soil salinity. International Chickpea and Pigeonpea Newsletter Shannon, C.E. and Weaver, W.1949. The mathematical theory of Communication. Univ. Illinois Press, Urbana. Singh, A.K. and Nigam, S.N. 1997. Groundnut. Pages 114-127. in Fuccillo D, Sears L and

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Stapleton P (eds.). Biodiversity in Trust. Cambridge University Press, Cambidge, UK. Singh, K.B., Pundir, R.P.S., Robertson, L.D., van Rheenen, H.A., Singh, U., Kelley, T.J., Parthasarthy Rao, P., Johansen, C. and Saxena, N.P. 1997. Chickpea. Pages 100113. in Fuccillo D, Sears L and Stapleton P (eds.). Biodiversity in Trust. Cambridge University Press, Cambidge, UK. Sun, G.L., William ,M., Liu, J., Kasha, K.J. and Pauls. K.P. 2001. Microsatellites and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings. Molecular Breeding 7:1324. Sun, G., Bong, M., Nass, H., Martin, R. and Dong, Z. 2003. RAPD polymorphism in spring wheat cultivars and lines with different level of Fusarium resistance. Theoretical and Applied Genetics 106:1059-1067. Thornsberry, J.M., Goodman, M.M., Doebley, J., Kresovitch, S., Neilsen, D. and Buckler, I.V. E.S. 2001. Dwarf 8 polymorphism associate with variation in flowering time. Nature Genetics 28:286-289. Turpenien, T., Tenhola, T., Manninen, O., Nevo, E. and Nissila, E. 2001. Microsatellite diversity associated with ecological factors in Hordeum spontaneum populations in Israel. Molecular Ecology 10:1577-1591. Upadhyaya, H.D. and Ortiz, R. 2001. A mini core subset for capturing diversity and promoting utilization of chickpea genetic resources. Theoretical and Applied Genetics 102: 12921298. Upadhyaya, H.D., Bramel ,P.J. and Sube Singh. 2001a. Development of a chickpea core subset using geographic distribution and quantitative traits. Crop Science 41:206 210. Upadhyaya, H.D., Nigam, S.N. and Sube Singh. 2001b. Evaluation of groundnut core
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collections to identify sources of tolerance to low temperature at germination. Indian J. Plant Genet. Resources 14:165-167. Upadhyaya, H.D., Ortiz, R., Bramel, P.J., and Sube Singh. 2001c. Development of groundnut core collection from Asia region. Hundered years of post Mendelian genetics and plant breeding retrospect and prospects, 6-9 November 2001, IARI, New Delhi Upadhyaya, H.D., Bramel, P.J., Ortiz, R. and Sube Singh. 2002a. Developing a mini core of peanut for utilization of genetic resources. Crop Science 42:21502156. Upadhyaya, H.D., Bramel, P.J., Ortiz, R. and Sube Singh. 2002b. Geographical patterns of diversity for morphological and agronomic traits in the groundnut germplasm collection. Euphytica 128:191204. Upadhyaya, H.D. 2003. Geographical patterns of variation for morphological and agronomic characteristics in the chickpea germplasm collection. Euphytica 132:343352. Upadhayaya ,H.D., Ortiz, R., Bramel, P.J. and Sube Singh, 2003. Development of a groundnut core collection using taxonomical, geographical and morphological descriptors. Genetic Resources and Crop Evaluation 50:139148. Upadhyaya, H.D. 2005. Variability for drought resistance related traits in the mini-core collection of peanut. Crop Science 45: 1432-1440. Upadhyaya, H.D., Gowda, C.L.L. , Pundir, R.P.S., Gopal Reddy, V., and Sube Singh. 2005a. Development of core subset of finger millet germplasm using geographic origin and data on 14 morpho-agronomic traits. Genetic Resources and Crop Evolution (in press).

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Upadhyaya, H.D., Mallikarjuna Swamy, B.P., Kenchana Goudar, P.V., Kullaiswamy, B.Y. and Sube Singh. 2005b. Identification of diverse groundnut germplasm through multienvironment evaluation of a core collection for Asia. Field Crops Research 93:293-299. Upadhyaya, H.D., Pundir, R.P.S., Gowda, C.L.L, Reddy,K.N. and Sube Singh. 2005c. Geographical patterns of diversity for qualitative and quantitative traits in the pigeonpea germplasm collection. Plant Genetic Resources: Characterization & Utilization 3(3): 331-352. Upadhyaya, H.D., Furman, B.J., Dwivedi, S.L., Udupa, S.M., Gowda, C.L.L., Baum, M.,

Crouch, J.H., Buhariwalla, H.K. and Sube Singh. 2006a. Development of composite collection for mining germplasm possessing allelic variation for beneficial traits in chickpea. Plant genetic Resources: Characterization and Utilization (in press). Upadhyaya, H.D., Gowda, C.L.L., Buhariwalla, H.K. and Crouch, J.H. 2006b. Efficient use of crop germplasm resources: identifying useful germplasm for crop improvement through core and mini-core collections and molecular marker approaches. Plant genetic Resources: Characterization and Utilization (in press).

Table 1. Area under cultivation and productivity of the selected crops during last four decades1

Crop Wheat Rice (Paddy) Soybean Sorghum Chickpea Groundnut in shell Pigeonpea Wheat Rice (Paddy) Soybean Sorghum Chickpea Groundnut in shell Pigeonpea

1963-65 213.2 123.5 25.3 47.3 11.7 16.9 2.8 1196 2062 1172 970 577 853 632

1983-85 Area: m ha 230.4 143.8 51.7 47.7 9.8 18.4 3.5 Grain yield kg ha-1 2173 3201 1747 1466 682 1089 750

2003-05 213.1 151.2 88.8 43.8 10.6 25.0 4.5 2841 3976 2265 1328 780 1447 708

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Table 2. Institutions in India that donated a large number of germplasm to ICRISAT, 19732003. Institution AICSIP, Hyderabad AICRPO, Hyderabad ANGRAU, Hyderabad ARS, Niphad, Maharashtra GAU, Junagadh GBPUAT, Pantnagar HAU, Hisar IARI, New Delhi JNKVV, Jabalpur MPKV, Rahuri NBPGR, New Delhi PAU, Ludhiana RAU, Samastipur, Bihar TNAU, Coimbatore Total Sorg hum 175 115 33 90 13 11,796 Pearl millet 66 155 164 234 170 106 45 2,022 2,962 Chick pea 345 96 211 3,022 127 173 149 1,029 63 5,215 Pigeo npea 3,035 174 479 191 40 3,919 Groun dnut 529 1,366 1,167 267 161 496 197 590 4,773 Small millets 285 469 531 1,246 2,531 To t a l 175 529 4,801 345 1,233 251 211 3,229 770 865 1,039 1,631 197 1,282 14,638 31,196

Rockefeller Foundation (India) 11,370

Table 3. Germplasm holdings in the Rajendra S Paroda Genebank, ICRISAT, Patancheru, December 2004.

Crop Sorghum Pearl millet Chickpea Pigeonpea Groundnut Finger millet Foxtail millet Proso millet Little millet Kodo millet Barnyard millet Total

Active collection1 37,257 21,594 20,116 13,632 16,041 5,949 1,535 842 466 658 743 118, 883

Base collection 2 31,669 15,150 15,984 10,266 6,820 4,620 1,054 576 384 630 487 87,640

Accessions held in-trust 3 35,836 21,329 16,970 12,712 14,419 4,979 1,535 835 462 656 743 110,476

1. Active collection: germplasm seeds stored in medium-term storage facility and available for current utilization. 2. Base collection: germplasm seeds stored in long-term storage facility for utilization in posterity. 3. Accessions held in-trust: FAO designated germplasm freely available for use to the researchers.
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Table 4. Global distribution of germplasm samples to scientists, 1974 - 2005 Crop Sorghum Pearl millet Chickpea Pigeonpea Groundnut Small millets Total 1974-83 58,627 15,302 52,015 19,546 20,908 20,067 186,465 1984-1993 158,762 62,769 45,413 30,593 44,034 17,352 358,923 1994-2005 31,382 11,536 24,893 16,278 29,182 15,449 128,720 Total 248,771 89,607 122,321 66,417 94,124 52,868 674,108

Table 5. Restoration of basic germplasm from ICRISAT genebank to different countries Number of accessions Country Botswana Cameroon Ethiopia Kenya Nigeria Somalia Sri Lanka India 14,637 7,189 7,488 Sorghum Pearl millet Chickpea 362 1,827 1,723 838 1,436 445 71 5,988 6,060 3,460 922 931 332 Pigeonpea Groundnut Small millets Total 362 2,749 2,654 1,170 1,436 445 71 44,822

Table 6. Core and mini -core collections of ICRISAT mandate crops. Crop Number of accessions used 22,473 16,063 16,991 12,153 14,310 5,940 1,474 4,738 1,704 1,956 1,290 622 155 Number of traits involved Core Sorghum Pearl millet Chickpea Pigeonpea Groundnut Finger millet Foxtail millet Groundnut Groundnut Chickpea Pigeonpea Finger millet Foxtail millet 20 11 13 14 14 14 13 Asian core 15 Mini-core 31 22 16 14 13
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Number of accessions 2,247 1,600 1,956 1,290 1,704 622 155 504 184 211 146 65 -

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Table 7. Composite collections of seected crops Crop Size of the composite collection (accessions) Genetic markers used Institutes collaborating with ICRISAT

Chickpea Sorghum Groundnut Pigeonpea Finger millet

3000 3000 1000 1000 1000

50 SSR markers 50 SSR markers 20 SSR markers 20 SSR markers 20 SSR markers

ICARDA, Syria CIRAD, FranceCAAS, China EMBRAPA, Brazil Only ICRISAT AICSMIP, India

32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0

30

19

No. of varieties

6 2

1 Sorghum Pearl millet Chickpea Pigeonpea Groundnut

1 Barnyard millet

Finger millet

66 varieties released in 44 countries

Fig 1. Number of cultivars released worldwide from the basic germplasm supplied from ICRISAT genebank 1976-2003
12

10

8 Linkage Distance

0 Arica Southeast Asia South Asia Americas Europe Mediterranean West Asia East asia

Fig 2. Dendogram of eight regions for the entire chickpea germplasm based on first three principal components.
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20 18 16 14 12 Linkage Distance 10 8 6 4 2 Middle East West Africa Oceania North America Central Africa Eastern Africa Southern Africa Southeast Asia South America Central Asia South Asia Caribbean East Asia Europe 0

Fig 3. Dendogram of 14 regions in entire groundnut germplasm based on sores of first seven principal components.
40 35 30 25 Linkage Distance 20 15 10 5 0 ICCV 96029 Harigantars ICCV 2 ICC16644 ICC16641 ICC14648 Annigeri ICC14595 ICC10822 ICC8931 ICC10232 ICC2171 ICC16947 ICC12424 ICC11059 ICC10996 ICC10981 ICC8618 ICC2023 ICC11180 ICC11021 ICC11039 ICC11160 ICC10976 ICC11040 ICC10926 ICC2859 ICC10629 ICC1398 ICC6919 ICC8378 ICC1097

Cluster 1

Cluster 2

Cluster 3

Fig 4. Dendogram based on first three principal components of 16 quantitative traits of 28 earlymaturing germplasm lines and four control cultivars capturing (74.3%) variation.

Cluster3

Cluster2

Cluster1

Fig 5. Dendogram of 21 early maturing groundnut landraces with three control varieties based on the first 10 principal components
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RICE BIODIVERSITY AND ITS UTILIZATION


Subramanian, M1. and S.Tirumeni2

ABSTRACT
Rice (Oryza sativa L.) the most essential food crop of the world, is popularly known as Global Grain. Unlike other crop plants, rice is endowed with enormous biodiversity, spread in many countries of the globe. The land races, indigenous cultivars, modern varieties, genetic stocks, breeding lines and wild species form the major components of the rice biodiversity found abundantly in South East Asia, Africa, Australia and Southern Central America. This germplasm with wide variability is the wealth of the country because of its valuable gene system. These genetic differences are very much useful to breed high yielding rice varieties resistant to biotic and abiotic stresses and quality traits improvement. Therefore, exploration and conservation of these valuable rice genotypes had been initiated already during 60s with a view to investigate their origin, variability and to evaluate their relationship for utilization. The responsibility of collection, conservation and regeneration of those germplasm is vested with international and national research institutes and stations of all rice growing countries. In spite of countless problems and constraints, these efforts have already been in vogue to collect and conserve the variability found in the globe and to utilize them in rice improvement work.

Introduction Biological diversity or biodiversity is the variability among living organisms from all sources. Agricultural biodiversity focuses a portion of the biodiversity that has undergone selection and modification over millennia by human civilization to better serve their needs. Genetic diversity, one of the components of biodiversity, refers to the variety of genetic information contained in all the individual plants, animals and microorganism. The plant diversity is not only distributed over the globe and India is also recognized as one of he 17 mega biodiversity areas of the world with enormous diversity in many flora and fauna. 1. Former Director of Research TNAU, Plot No.9, VOC Street, Chokkanathapuram, Madurai - 625 014, Tamil Nadu, India. 2.Associate Professor (Plant Breeding) AJANCOA & RI, Karaikal - 609 603. Rice, the worlds most important staple food crop needs continuous improvement to feed the

ever-growing population of the world. This crop warrants resistance against abiotic and biotic stresses besides other quality characters to be improved. The components of agro biodiversity used in the development of new plant varieties or hybrids are called genetic material. Rice genetic resources comprising land races, modern and obsolete varieties, genetic stocks, breeding lines and wild races are the basis of food security. Currently, the land races and varieties under cultivation are declining. The wild species are threatened with extinction through changes in land use, extension of agriculture into marshal areas and deforestation. Besides, the future progress in the improvement of rice crop largely depends on exploration. In view of the above, the conservation of rice biodiversity is taken up with great care and importance in South East Asia, South Asia, Africa, Australia and South Central America. Collection and conservation are in progress in many places of the aforesaid regions and the details are discussed in this paper.

1. Former Director of Research TNAU, Plot No.9, VOC Street, Chokkanathapuram, Madurai - 625 014, Tamil Nadu, India. 2. Associate Professor (Plant Breeding) PAJANCOA & RI, Karaikal - 609 603.

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Rice belongs to the family Poaceae (Gramineae) and the tribe Oryzeae. The tribe Oryzeae consists of 12 genera (Table 1) including the genus Oryza, with specific differences among their traits. Examples of potentially useful traits in genera related to Oryza are plants adapted to cold water (Zizania), salt water (Porteresia an eco type of Leersai oryzoides) and plants with unisexual spikelets (Zizania, Luziola zizanopsis). Most of the species in genera related to Oryza have not been studied in detail. However, two species in the genus Zizaniza are well - known in parts of America and Asian cuisine. Z.palustris L. is the wild rice of North America commonly served during the United State. Thanks giving Day meal and Z.latifolia is eaten as a vegetable, particularly in Chinese dishes.
Table 1. Genera, number of species, chromosome number and spikelet structure in the tribe Oryzeae (Duistermaat 1987, Pyrah 1969, Second 1985)
Genus Chromosome number (2n) Spikelet structure

bamboos. Rice and its relatives are quite unrelated to other major cereal seeds and maize, wheat and sorghum (Watson, et al 1985). A comprehensive numerical taxonomy analysis of the grass family, which probably reflects evolutionary relationship, shows the association between rice and bamboos and the divergence of rice from other cereals. The genus Oryza consists of species adapted to a broad range of habitats. Several species grow in shady forest and others in vast stands in deep water swamps. Wild rices can be found, for example, in the Himalayan foothills, Asian river deltas, tropical Caribbean islands, Amazon basin, and the inland swamp lands of southern and western Africa as well as in temporary pools of the arid savannas of the tropics. The wild species of Oryza are found almost exclusively within the boundaries of the tropics. Cultivated rice, however, is grown as far as 50 S in Argentina.
Table 2. Chromosome number, genomic composition, and geographical distribution of Oryza species (Khush and Brar, 2001)
Species O.sativa complex O.sativa L. 2n 24 Genome AA AA Distribution World Wide Tropical and Subtropical Asia Tropical and Subtropical Asia, tropical Australia Africa West Africa Africa Tropical Australia

Oryza Leersia Chikusichloa Hygroryza Porteresia Zizania Luziola Zizaniopsis Rhynchoryza Maltebrunia Prosphytochloa Potamophila

24,28 24,48,60,96 24 24 48 30,34 24 24 24 Unknown Unknown 24

Bisexual Bisexual Bisexual Bisexual Bisexual Unisexual Unisexual Unisexual Bisexual Bisexual Bisexual Unisexual and bisexual

O.nivara Sharma et 24 Shastry O.rufipogon Griff 24

AA

O.breviligulata A. Chev, et Roehr.

24

AA AA AA AA

O.glaberrima Steud. 24 O.longistaminata A. 24 Chev. et Roehr. O.meridionalis Ng 24

The genus Oryza to which cultivated rice belongs, has 22 wild species and two cultivated species viz., O.sativa and O.glaberrima (Table 2). Oryza is closely related to the bamboos and some of the forest wild rices look like miniature
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Rice is believed to have originated in the southeast and South Asia, which included North

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O.glumaepatula Steud O.officinalis complex

24

AA

South and Central America Africa Philippines and Papua New Guinea Tropical and subtropical Asia tropical Australia Sri Lanka South Asia and East Africa South and Central South and Central America South and Central America Tropical Australia

0..punctata Kotschy 24,48 BB,BBCC ex Steud. O.minuta J.S. Presl. 48 ex C.B. Presl. 0.officinalis Wall ex Watt O.rhizomatis Vaughan O.eichingeri A. Peter O.latifolia Desv. America O.alta Swallen O.grandiglumis (Doell) Prod. O.australiensis Domin. O.meyeriana complex O.granulata Nees et Arn. Ex Watt 24 GG GG 24 BBCC

CC

24 24 48 48 48 24

CC CC CCDD CCDD CCDD EE

South and Southeast Asia Southeast Asia

O.meyeriana (Zoll. 24 et Mor. ex Steud.) Baill O.ridleyi complex O.longiglumis Jansen 48

HHJJ

Irian Jaya (Indonesia) and Papua New Guinea South Asia Africa Papua New Guinea -

and cultivated annual (O. saliva L) (Sharma and Sastri, 1965). Corresponding members of African rice are O.longistaminata Chav. and Roehr, O.barthi A Chev. and O.glaberrima. He also asserted that O.sativa L. could have evolved in a broad area extending over the foothills of Himalayas in South Asia and Southwest China. The Asian cultivated rices have formed three eco-geographic races (indica, japonica and javanica ) and three distinct cultural types in monsoon area (upland, lowland and deep water). Based on local needs and traditions, many such groups have been recognized. Chinese have traditionally recognized Hsien and Keng types. Indonesian has grouped them into Bulu, Gundil and Tjereh and Bengal rice varieties are grouped into Aus, Bow and Aman types. Based on isolation and selection, O.sativa was divided into two geographic races viz., O.sativa var indica and O.stiva var japonica (Kato et al 1930). The differentiation also involved morphological and serological characters as well as inter varietal fertility. The former is grown all over the tropics and latter confined to temperate and subtropical regions. One more geographical race javanica, has also been recognized originating in Indonesia which is somewhat intermediate between indica and japonica, resembling the former morphologically and the latter physiologically. Genetic erosion In many areas, high yielding modern varieties were adopted by the farmers and the cultivation of land race varieties declined as high as 85100% (Saxena et al, 2003) which also resulted in the loss of genetic diversity and increased the genetic erosion. The wild species are threatened with extinction through changes in land race, changes in land use, extension of agriculture in the marginal areas, deforestation and natural disorders. They contributed for abundant habit fragmentation of destruction of wild as well as land races (OECD, 1996). Unless these losses are checked, genetic
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O.ridleyi Hook F. Unclassified O.brachyantha A. Chev. et Roehr

48 24

HHJJ FF HHKK -

O.schlechteri Pilger 48 O.schweinfurthiana -

Eastern zone of India. Therefore, a large number of indigenous varieties of cultivated rice and forms of wild speices are found prominent in these regions. The Asian cultivated rice (O.sativa L) has evolved from wild rices following the sequence of wild perennials (O.rufipogon Griff.), wild annual (O.nivara)

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erosion will invariably increase and replacement of such biodiversity will cost more. This can be reduced by strategic and timely conservation action. Therefore, exploration and conservation of biodiversity are given importance. Germplasm collection The collecting activities are closely linked to conservation and use. Most samples in the collection are land race varieties of O.sativa. Farmers throughout Asia usually maintain the identity of each rice variety and help to identity different varieties for effective collection of germplasm. Using this method more than 2000 samples of O.sativa were collected during the second half of 1995 from Southern provinces of the Lao Peoples Democratic Republic (PDR). It is estimated that about 60% of these samples are unique varieties. IRRI received almost 700 samples of Oryza sativa and 84 samples of different wild species form the Lao PDR, Tanzania, Philippines and Costa Rica during 2000. More than 24,700 samples of cultivated rice and 2400 samples of wild rice were collected in 165 missions from 22 countries (Anon 2000). India, the primary centre of origin of cultivated rice (O.sativa) obviously conserves a very high genetic diversity of rice with its diverse eco geographic conditions. Collecting the variability observed in indigenous rice cultivars began in India around the turn of this century. The work received special attention following establishment of the attention following establishment of the Agricultural Research Station at Dacca (Eastern India) in 1961 and Paddy Breeding Station, Coimbatore (Southern India) in 1912. Setting up the Indian Council of Agricultural Research Institute (ICAR) at New Delhi in 1929 and the Central Rice Research Institute (CRRI) at Cuttack in 1946 further strengthened these efforts. The Jaipur Botanical survey explored south Orissa and adjoining areas of Madhya Pradesh during 1955-60 and collected 1745 cultivars. During 1965 - 67, 900 traditional cultivars of Manipur in Eastern India were
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collected. The Plant Introduction of IARI, New Delhi was converted as National Bureau of Plant Genetic Resources (NBPGR) in 1976 and acted as a nodal agency for exploration collection, conservation, characterization, evaluation and documentation of germplasm. A large number of collections were made by Sharma and his team from 1968 to 1983, with the help of IARI (Sharma 1982). Those collections were known as Assam Rice Collection (ARC). The Raipur collections of 19,116, rice cultivars grown locally in Madhya Pradesh region were made from 1971 - 76. Additional collections of 1938 cultivars were made through a special drive for upland varieties in Andhra Pradesh, Karnataka, Madhya Pradesh, Orissa and West Bengal. Collaborative explorations by NBPGR and State Agricultural Universities added 7000 cultivars during 1978 - 80. The Vigyan Parishad Kendra Agricultural Station at Almorah collected 1247 cultivars from hilly regions of Uttarpradesh. NBPGR and CRRI jointly explored Sikkim, South Bihar and parts of Orissa in 1985 and collected 447 local types. Exploration by NBPGR during 1983 - 89 led to further addition of 4862 cultivars to the National Germplasm Bank. ICAR and the 82 research stations established at various agroclimatic regions of the country collected more than 80,000 accessions of rice (Table 3). The International Rice gene bank (IRG) of IRRI Philippines represents the largest and most diverse collection of rice in any gene bank. The collection comprises about 2% of all germplasm samples conserved world wide donated from more than 110 countries. The collection currently holds 1,02,700 samples of Asia rice Oryza sativa (95%) and West African Rice O.glaberrima (15%). The accessions were mostly collected from land races varieties nurtured by farmers for generations, modern

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and obsolete rice varieties, some breeding lines and all the 22 wild species in the genus Oryza (8.5%).
Table 3. Rice germplasm maintenance at major rice research stations in India
Name of S.No station/ centre 1 2 3 4 National centre Andhra Pradesh Assam Bihar Location(s) No. of accessions maintained 19718 1076 3000,150

CRRI, Cuttack DRR, Rajendranagar Titabar, Katimganj Pusa, Patna, Ranchi, Sabour and Hazaribagh Nawagaon Karnal Palampur

800,1252 30 960 100 426 1850 600 20758 1119 1038 552

5 6 7 8 9 10 11 12 13 14

Gujarat Haryana Himachal Pradesh

Jammu and Khudwani, R.S. Kashmir Pura Karnataka Kerala Madhya Pradesh Manipur Orissa Mandya Pattambi I.G.K.V.V., Raipur Wangbal Bhubaneswar, Jeypore and Ranital (OUAT) Kapurthala (PAU) Banswara, Kota Almora (VPKAS), Pantnagar, Faizabad and Kanpur,

Wild rice collection in India In addition to spectacular variability in its traditional cultivars, India is also rich in wild rice, particularly O.nivara, O.rufipogan, O.officinalis and O.granulata. These species were collected by the pioneer research workers. Subsequently, S.V.S. Shastry and his coworkers at Indian Agricultural Research Institute (IARI) made extensive collections of wild species of Oryza from Northern, Western, Central and Eastern India and assembled striking variability in O.nivara and 0.officinalis. Variability in Portersia coarctata has also been collected form coastal areas. Besides Indian Scientists, the foreign scientists like H. Kihara in early 1960s, Watanabe in the late 1960s and 1970s, French Scientists in 1986 came to India and in collaboration with Indian Council of Agricultural Research (ICAR) and IRRI undertook more intensive exploration all over the country and collected the wild species. Conservation Conservation is the management of resources to derive sustainable benefits and to meet the needs of future. It preserves the genetic resources for a longer time without loss of viability of frequent rejuvenation and to distribute the required accessions to needy countries with collaborative approach. The two major approaches conserve the rice for diversity are in situ conservation and ex situ conservation. In situ conservation In situ conservation means the conservation of ecosystem and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings and, in the case of domesticated or cultivated species in the surroundings where they have developed their distinctive properties (UNEP 1995). It preserves the evolutionary processes of generating new germplasm under natural selection and the maintenance of important field
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Maharastra Karjat

15 16 17 18

Punjab Rajasthan Uttar Pradesh

1178 2370 2306 1577,1003 1098

Tamil Nadu Coimbatore

19 20

West Bengal Chinsura and Kalimpong NBPGR Cuttack, Shillong, Thrissur Total

1013,1037 2248, 2600, 2873 81018

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laboratories for crop biology and biogeography. It serves as a continuous source of germplasm for ex situ conservation, and also to conserve potentially useful alleles, protects specially adapted species. It allows to natural evolution to continue, preserve pest and disease resistant species which can coevolve with their parasites, serves several sectors at one place. (Crop breeding and forestry maintained with in the same protected area facilitate research on species in their natural habitats). On farm conservation It is also an in situ conservation of the rice genetic resources under continued cultivation and management of a diverse set of rice population by the farmers in the agro ecosystem where rice has evolved. It is a dynamic complex process of crop evolution involving origin, domestication, spread, diversification and evolution. Four components of farmers management of diversity are seed flows, variety selection, variety adaptation and seed selection and storage. There is general consensus that farmers are not conservationists in nature but are conservationists through use. Therefore, farmers have to be provided with the right technical and economical options, so that they would be benefited by sowing the varieties targeted for conservationists. Nature and objectives of on farm conservations Maintain and enhances allelic diversity Access to and control over the diversity at the local level Promotion of genetic diversity conservation a house hold security. The competition between traditional and modern varieties is increasing, and adaptation of modern high quality varieties affects the cultivation of traditional varieties. Besides, the higher market price for traditional varieties does not compensate their lower yield and longer duration. Therefore, farmers will cultivate the
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traditional varieties only if their cultivation does not penalize them. The seed stores generally carry only modern varieties and certified seed growers, a part of the Department of Agriculture system of seed procurement strategy, grow only modern varieties. Even under a scheme Plant now Pay later scheme, the farmers are given seeds at no cost, but upon harvest are expected to pay for them. The seeds given in the scheme are from the certified seed growers, and are always modern varieties and sometimes only the recommended varieties. Traditional varieties are not planted by certified seed growers and were not included in the scheme. The varieties that were planted in irrigated plots were obviously less affected by the drought than the varieties planted on rainfed plots. Therefore, irrigation sustained the use of modern varieties, as farmers plant only modern varieties in irrigated plots (Morin et al 1998). Poor storage condition is a cause of genetic erosion. Therefore, a simple and cheap seed drying and storage device that farmers could use to store the seeds for several years need to be supplied. Changing land use patterns may have an influence on diversity. It was suggested that land fragmentation may permit farmers to grow landraces on one plot of land, and commercial varieties on others. Since the development of onfarm conservation approaches beyond current practices have hardly been started, and the mechanism poorly understood, need to increase awareness of the potential of onfarm conservation through dialogues, training and education at all levels was felt must. Interaction with farmers to enhance their understanding of the broader issues of plant genetic conservation would be one approach. It was further agreed that since on farm conservation is important for many groups, this is one area in which government; non-government organizations, the science community and

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farming groups should have a common interest. Ex situ conservation Ex situ conservation refers to the conservation of germplasm away from its natural habitat. It is now being practiced to some extent in almost all countries as a means to conserve crop species diversity for posterity. This strategy is particularly important for crop gene pools and can be derived by propagating and maintaining the plants in genetic resource centres, botanical gardens, tissue culture respositories or in seed gene bank (OECD, 1996). Periodical regeneration and rejuvenation of collections kept in the short, medium and long term storages are either done in the field in suitable conditions or in special situations such as green houses, grow houses, screen houses etc. At IRRI about 500 rice accessions are grown every season in such a way as to characterize them and rejuvenate them. NBPGR now keeps its field collection numbering about 5000 in three centres in the field as well as in the store. A long-term seed store also caters to the needs of safer storage of collections immediately after field characterization and evaluation. Conservation of wild species * The IRGC of IRRI shall preserve a complete set of genotypes. Other national and inter national centres help IRRI on rejuvenation. * IRRI shall preserve, rejuvenate and distribute Indica, Japonica cultivars of O.sativa and Oryza species except those from Africa. * The National Institute of Agrobiological Resources in Tsukuba Japan shall preserve, rejuvenate and distribute Japonica varieties of East Asia. * The National Plant Germplasm System (NPGS) of USA shall preserve the accessions from USA, South America and Mediterranean area. The USA also shall continue to store duplicate samples of
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conserved IRRI stock. * IITA (International Institute of Tropical Agriculture, Ibadan Nigeria) will preserve,rejuvenate and distribute cultivars of O.glaberrima and wild species of Africa. * Institute de Recharche Scientistique et Technique out line - Mer FranceThe international network for rice germplasm conservation has the following components (ORSTOM) and West African Rice Development Association, Bouake, Ivory Coast (WARDA) plan to collaborate with IITA.The above centres shall exchange and carefully compare the accession lists to minimize the maintenance of obviously duplicate accessions. Now, the need of multilevel (National/ State/ Lesser entities) public and private collaboration in various conservation activities is felt. In vitro conservation This technology is used to ensure the survival of seed lots with low viability. Seeds may have low viability when they are sent to gene banks for long term conservation. Anther culture, embryo cultures and cold treatment of flower buds or panicles (at 9-1lc for 14 days) induced more number of plantlets. Further, anther float culture or cell suspension culture were also utilized. Meanwhile culture of isolated pollen was also carried out to induce plantlets. The development of isozyme classification provides an unequivocal biological framework for the use and analysis of diversity patterns of germplasm based on other molecular markers. DNA markers (RFLP, AFLP, RAPD and SSR) are routinely used for the management and evaluation of crop germplasm collections (Westman and Kresovich, 1997). Molecular biology, by generating new technologies and methods of analysis that proved new approaches of supplement classical

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methods of analysis, has contributed significantly to increased understanding of many aspects of plant biology. Promising areas of biotechnology that may serve plant genetic resources activities and research are shown in Table 4.
Table 4. Biotechnological tools and their potential applications in plant genetic resources activities.
Activities of Research Collection or acquisition Helpful new technologies In vitro technology, recombinant DNA technology (gene / DNA library and cloned genes) RFLP technology, protein / isozyme electrophoresis

of their national collections are duplicated at IRRI. Nevertheless, the IRG has provided an important safety net for national conservation efforts. Maintenance of Germplasm The maintenance of germplasm bank is to conserve it in state in which it can be indefinitely propagated. Storage of seeds for long term in the case of orthodox species is done based on the Harrington rules of thumb which define the relative influence of temperature and seed moisture content on seed longevity or viability. The first rule says that for every reduction in seed moisture content, the longevity of seed viability is double. Similarly, second rule says that for reduction of every 10f or 5.66 - 5.56c temperature seed longevity is doubled. The main task of a germplasm bank is to conserve germplasm in a state in which it can be indefinitely propagated. The term base collection is applied to collections stored under long-term conditions (-10 to -20c at 4% moisture), whereas the term active collection is used for collections stored under mediumterm collections (10c at 4% moisture) and working collection refers to breeders collections usually stored under short-term conditions (10 to 20c at 4% moisture). For safety reasons, duplicates of the base collections should be conserved in other germplasm banks. At IRRI long term conservation of this strategically important germplasm collection has been achieved by exploiting the seed production environment in Los Banos to achieve maximum seed longevity in storage for all the diverse rice accessions (Kamaeswara Rao and Jackson, 1996). Utilization of Rice Germplasm The land races have an inherent genetic value because of their adaptation to different farming conditions and resistance to pests and diseases. Knowledge of these traits, their genetic and
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Characterization, Biosystematics, Genetic diversity, Identifying duplicates, Genetic stability Maintenance and preservation

In vitro technology, cryopreservation, recombinant DNA technology (gene / DNA library) In vitro technology, recombinant DNA technology (disease indexing, gene / DNA library and cloned genes)

Dissemination and exchange

The International Rice Genebank (IRG) The long-term preservation of rice genetic resources is the principal aim of the IRG. Formerly known as the International Rice Germplasm Centre, the gene bank has operated since 1977, although genetic conservation activities started in the early 1960s, just after the Institute was founded. It meets all the approved or preferred international genebank standards adopted in 1994 by the FAO Commission on Genetic Resources for Food and Agriculture. For several countries, including Sri Lanka, Cambodia, Lao PDR and the Philippines, the germplam conserved in the IRG represents a more or less complete duplicate of their national collections. For other countries, such as India and the Peoples Republic of China, only parts

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molecular control and stability under different conditions enhances the value of the conserved germplasm. The use of germplasm in crop improvement could be facilitated by systematic evaluation and documentation of the acquired data. Rice breeders of India have made effective use of the indigenous gene pools which provides resistance to pests or tolerance to eco-edaphic stresses. The drought resistant N22 was used in breeding Bala. TKM 6, which has multiple resistance to insects and disease, became a parent of Ratna, Saket 4, Parjat, CR 44-1, W 1256 and W 1263; the latter lines were widely used inside India as well as in Sri Lanka and Thailand. The tungro virus-resistant PTB 10 has been bred into improved varieties such as Aswini, Bharathi, Jyothi, Rohini, Sabari and Triveni. Similarly, PTB 18 possessing multiple resistance has been widely used in India. For tolerance to submergence by floodwaters, FR13 A is an outstanding source. Indian breeders were also developing saline-tolerant varieties from indigenous sources such as Pokkali, Getu and
Table 5. Donors utilized and varieties bred for abiotic stress (Sharma et.al., 1988)
Stress situation Drought / Upland Donor utilized N22 Varieties developed Akashi, Bala, Kanchan, Kiran, Prasanna Abha, Cauvery, CR 128-928, Kalinga 3, Madhu, Neela, Parijat, Poorva, Pusa 2-21, Pusa 33, RatnamRudra, Saket 4, Sankar, Sarasa, Tripti. Birsadhan 201, C 7306, Cauvery, Himdhan, HM 95, Kanchi, Kusuma, Madhu, MR 118, Padma, Pennai, Parijat, Radhanagari 185-2, Rajendra, Ratnagiri 24, Rasi, Sarjoo 49, Sarjoo 50, Sarjoo 52, Sattari, Subhadra, Suma Suphala, Tella-Hamsa MR 118, Narshing, Vishnu 60

Donor Stress situation utilized Deegeowoogen Salt IR8 tolerance BR 4-10 Dasal SR26B

Varieties developed Pathara, ADT 31, ADT 33, ADT 34, Govind, Karuna, Manhar, MDU1, Narendral, Narendra2, Palman 579, Paramkudi 1, PR 103 Pusa 4-1, Thirupathisaram 1, TKM 9 Vytilla 2 CO 43 PVR1

Dasal. R 575, a local variety of H.P. state was used to breed Himdhan, which is adapted to altitudes above 1,000 meters (Table 5). Similarly the germplasm collections were
Table 6. Germplasm utilized and varieties bred for resistance to pests and diseases
Pest Insect pests TKM6 Stem Borer ARC 6650 CR 138-928, Madhu, Parijat, Radh, Ratna, Saket 4, Sayasree Pratibha, vajram Donor utilized Varieties developed

Manoharsali Sonasali Brown plant hopper PTB10 Bharti, Jyoti

TKM 6

Karivennel Pavizham PTB21 Daya, Neela, Pratap, Sarasa, Udaya Bharatidasan

PTB33 Disease Tadukan Blast

T(N)1

Archana, Asha, Deepa, Himalayan, IR 579, Samridshi, Usha MR 118, Narsing Rasi, Vaigai, Vishnu Asha, Bhagawathy, Govind, Hema, IGP1-37, Jaya, Jayanti, Kakatiya,

CO 29

CO 29 CO 18

IR8

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Karjat 14-17, Lakshmi, Narandra 2, Rajarajan, Ratnagiri 1, Ratnagiri 68-1, Ratnagiri 78-1, Samalei, Samridhi, Usha, Vani, Vijaya CO 25 Bacterial leaf blight TKM6 Bhagawathy, Rajarajan Govind, IR20, IR 36, Karjat 1, Radha, Ramakrishna Bharatidasan, CR 138-928, IR20, IR 50, Narendra 2, Pusa 2-21, Radha, Ratna, Saket 4 Vikramarya Annapurna, Triveni CR 94, IR 36, Neela, Sarasa, Shakti CR 57, CR 94, Daya, IR 36, Neela, Pratap, Sarasa, Shakti, Udaya CO 44

TKM6

PTB2 Rice Tungro virus PTB10 PTB18 PTB21

Kheer, Polao and Chokuwa and soft rice, and rices used for preparation of flaked rice, puffed rice and bar boiled rice were also found (Ahmed et al., 2000 a,b). Over the decades, the germplasm collections at IRRI have been systematically characterized for a range of morphological and agronomic traits that facilitate conservation, as well as selection of suitable phenotypes by breeders (Table 7). Thousands of individual rice accessions have been evaluated for their resistance to or tolerance of a wide range of pests, diseases and abiotic stresses, such as brown plant hopper (BPH), rice blast and bacterial leaf blight (BLB) and adaptation to cold temperature or saline soils (Jackson et al., 1996). The Genetic Evaluation and Utilization (GEU) Programme has made successful use of the following gene pools viz., Chinese semidwarfing source, vertical resistance to several diseases and insects, early maturity and photoperiod insensitivity, drought and resistance and recovery and tolerance to certain adverse soil factors. For instance, the recent IR varieties are highly resistant to bacterial leaf blight, the tungro virus, grassy stunt virus, biotypes 1 and 2 of the brown plant hopper, leafhopper and tolerance to one or more adverse soil factors. The genes for grassy stunt resistance were derived from the wild relative, Oryza nivara. Nearly all of the national centres have made profitable use of the semi dwarfing gene (sdi) contributed by Dee-geo-woo-gen and a varying number of the pest resistance genes derived for IRRI lines or IR varieties. Moreover, through local screening and selection, several national centres have incorporated additional resistance or tolerance genes from other sources into their improved varieties.

ASD5

evaluated and screened for pests and diseases, used as donors and many varieties were bred in rice (Table 6). Assam Rice Collections (ARC) had many valuable genes for various pests and diseases (Sastry et al., 1971) tolerance to cold, drought (Hakkim and Sharma, 1974) flood, high protein (Srivastava and Nanda, 1977) amylase content and also for the genes for dwarf stature. Seetharaman et at (1974) found that whole assemblage of japonica characters such as their culm, shy tillering, short panicle etc., in ARC and they have pointed out that racial differentiation in O.sativa might have taken place in this region. Rao and Srinivasan (1978) found that ARC also have high field potential under low P in the soil. The ARC have also possessed diversity for glutinous or waxy traits in rice. This is special class of rice for preparation of confectioneries (Pithus, Kurum), rice beer (Apong, Haj) as break fast food (Salpan) and industrial use. Besides, aromatic rices (Jolia) for the preparation of

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Table 7. Number of O.sativa accessions in the international Rice Genebank collection evaluated at IRRI for their reaction to insect pests and diseases (Jackson, 1997)

Stress

O.sativa accessions Number Resistant (%)

Insects pests Brown plant 44335 15.4 hopper biotype Brown plant 10053 1.9 hopper biotype Brown plant 13021 1.8 hopper biotype Green leaf hopper 50137 2.8 Rice whorl 22949 3.0 White backed 52042 1.7 plant hopper Zigzag leaf 2756 10.1 hopper Rice leaf folder 8115 0.6 Yellow stem 15656 3.8 borer More than 1,80,000 rice accessions were screened at IRRI for soil related stresses and tolerant lines were identified (Table 8).
Table 8. Summary of screening tests for adverse soil tolerance in rice 1969-1986 (Neue, 1994)
Stress No. tested No. found Tolerant tolerant (%) (score 1-3) 19,873 3,848 1,525 1,109 390 233 151 84 27,214 19.6 8.7 7.3 13.6 7.3 9.4 12.9 9 14.8

programme for various stress situations and hybrid rice development has been described by Siddiq (1991) Table (9). Under IRRI- genetic evaluation and utilization programme; Villegas (1991) has enlisted certain wild species used to enhance the value of agronomical traits in cultivars by way of transferring the insect resistant genes (Table 10).
Table 9 . Distribution of useful gene among wild rice (Siddiq 1991)
Character A) Biological stress Diseases Grassy stunt virus Rice Tungro virus Species

Bacterial leaf blight

O.nivara O.grandiglumis O.latifolia O. malampuzhensis O.minuta O .officinalis O.longistaminata O .officinalis O.officinalis O.nivara O.minuta O .officinalis O.minuta O.eichengeri O.brachyantha O.ridleyii O.resseranti O.perieri O brachyantha O.coaractata O.eichengeri O.granulata Portersia caractata O.perennis

Insects pests Brown Plant hopper (all three biotypes) Striped borer

Yellow borer

Gall midge

Wet land rice Salinity Alkalinity Zinc deficiency Phosphorus deficiency Iron toxicity Peat soil Upland rice Al / Mn toxicity Iron deficiency Total 1,01,293 44,052 20,784 8,139 5,376 2,485 1,169 891 1,84,189

Physical stress Salinity Drought

Physiological features High photosynthetic efficiently Under low light conditions Hybrid rice Cytoplasmic sterility O.granulata O.malampuzhensis

Floral characteristics

Utilization of wild species The use of wild rice species in breeding


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O.f.spontanea O.rufipogon O.nivara O .officinalis, O.perennis

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Table 10. Use of wild rice to transfer useful traits

Useful traits Resistance to BPH Resistance to WBPH Resistance to GLH O.australiensis Resistance to BPH Tolerance to drought O.minuta Resistance to BPH Resistance to WBPH O.officinalis Resistance to BPH Resistance to WBPH O.punctata Resistance to BPH Resistance to GLH Resistance to Bacterial Leaf Streak Resistance to BLB O.latifola Increased biomass

Species O.eichengeri

conserved at IRRI and in other genebanks, the use of conserved germplasm for breeding is really rather limited. What has had real significance is the contributions to rice science through the many studies of land race varieties and wild species concerning their reaction to pests and diseases, the nature of biochemical pathways and molecular basis of resistance, which guide more strategically the utilization of germplasm accessions in rice breeding. Genebank management. In theory and in practice at many locations, the production or collecting of high viability seed lots of Oryza sativa and O.glaberrima is less a problem than is the case for many other crops. There are many potential causes of poor viability, especially under hot and humid tropical environments. Seed processing problems (particularly inadequate seed drying procedures) and delays in receiving accessions at national centres are two of the more likely causes. The single most important factor in the successful maintenance of rice seed stocks in genebanks is the control of seed moisture content. Accordingly, it is necessary to improve seed drying procedures and the capability of genebenks to approach this target. A moisture content of 6-8% is acceptable, however, for centers that can provide subzero storage conditions (typically - 10 c). Rice seed viability monitoring is the second most widespread concern. In collections held under poor storage conditions, it is necessary to monitor and regenerate accessions frequently, a heavy workload in addition to the risks inherent in frequent generation. Conclusion Many rice growing and consuming countries continued to explore rice biodiversity and conserve them ever. It is to be strengthened at regional level and mutually benefited with exchange of germplasm at international level. Conservation of germplasm, the important
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The use of landraces and wild species in rice breeding had an enormous impact of rice productivity in many countries. For example, one accession of the wild species O.nivara (RGC 101508) was used to introduce resistance to grassy stunt virus into cultivated rice, which led to the release of IR 36. This variety also had 15 land races varieties in its pedigree (Plucknett et al 1987) and at one time, it was planted on more than 11 million ha, making it the worlds most widely cultivated cereal crop variety (Swaminathan, 1982). Now, hybrid between O.sativa and many wild species have been achieved through the use of various biotechnological tools (Khush et al., 1993). In Tamil Nadu CO 31 (O.perennis / GEB 24 (O.sativa)) and MDU 5 (O.glaberrima / Pokkali (O.sativa)) were the two rice varieties released by inter specific hybridization by utilizing the wild species of rice (Subramanian and Manual, 1998). The economic value of the rice germplasm collection for rice improvement has also been assessed. It is clear that over the past 15 years there has been a significant increase in the use of landraces in rice breeding. Nevertheless, relative to the large number of rice accessions

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and challenging task needs more attention and concentration. Though research institutes all over the world grow and regenerate the germplasm, in situ conservation of land races and indigenous rices through on farm cultivation in farmers field are effective, because it maintains more allelic diversity, very accessible and provides high security. It should be made as viable option to farmers and encouraged more intensively by supporting the farmers. Quality seed supply in affordable seed cost, procurement of seed at reasonable price, and providing simple effective seed storage facilities are very important needs for successful seed conservation of local races. The farmers should also be trained in seed production and conservation. They should be often discussed to solve the problems and constraints encountered in preserving seeds then and there. This is a novel way not only to conserve more gene pools and also to prohibit the genetic erosion of valuable germplasm. In situ conservation is also a method of conserving the wild species and related genera of the genus Oryza. These germplasm need specific location and environment to grow well and attain maturity to produce quality seeds. These regions need to be brought under the control of plant biodiversity authority to prevent the loss of the valuable species and genera and the seeds collected from them should be spared to needy countries freely on mutual understanding. Use of biotechnological tools such as in vitro techniques have to be further strengthened and practiced. Very large number of rice accessions are being maintained in many research institutions for very long time, their regeneration is very much essential to prevent the loss of viability. Growing all these germplasm every year for the aforesaid purposes is very difficult and expensive. Therefore biotechnological approach through anther culture, cell suspension culture, pollen culture etc., may ease the regeneration of large number of germplasm.
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Research on seed technology is yet another attempt to study the quality, viability, dormancy and storability of rice seeds to raise healthy plants ever as germplasm. Documentation on the details of biodiversity of the rice germplasm and their characteristics is the most useful approach for the researchers choice of useful germplasm to achieve their goal in rice improvement, besides it serves as a basic compendium for the plant science students and scientists. Policy on intellectual property right (IPR) should be well documented and implemented to protect the property right of the rice germplasm from every country and also to exchange genetic materials freely on mutual under standing to breed desirable rice plants by the rice growing countries in the world. REFERENCES Ahmed, T., Sharma, K.K. and Pathak, 2000 a. Export potential of bora rice of Assam (Abstract) 4 th Agricultural Sciences Congress, Jaipur. Ahmed, T., Sharma, K.K. and Pathak, 2000 b. Market potential of Jolia rice of Assam (Abstract) 4 th Agricultural Sciences Congress, Jaipur. Anonymous, 2000. Rice genetic resources: Conservation, safe delivery and use. Bettencourt, E. and Konopka, J. 1990. Directory of Germplasm Collections. Cereals: Avena, Hordeum, Millets, Oryza, Secale, Sorghum, Triticum, IRRI program report for Rice 2000. pp. 102 - 108. 3. Chang, T.T. 1995. Rice: O.sativa, and O.glaberrima. Evolution of Crop Plants (eds.) Zea and Pseudocereals. Internation Board for Plant Genetic Resources. Rome. Chattarji S.M. Prasad, K.Mishra B.C and Rajamani, P. 1977. Identification of gall midge (Orseolia oyzae wood - Masan) resistant germplasm and their utilization in

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breeding J. Entomological Research; 1: 111 -113. Duistermatt, H. 1987. A revision of Oryza (Graminae) in Malaysia and Australia, Blumea, 32 :157 -193. Evenson R.E. and Gollin 1994. Genetic resources, international organizations, and rice varietal improvement. Centre Discussion paper 7123, Economic Growth Centre, Yale Univ. New Heven, CT. FAO. 1994. Gene bank standards, FAO, Rome. Glaszmann, J.C. 1987. Isozymes and classification of Asian rice varieties. Theor. Appl. Genet., 74 : 21-30. Hakkim, K.K. and Sharma, S.D. 1974. Localized distribution of certain characters of rice in North East India. Indian, J. Genet. Plant Breeding. 34 :16-21. Jackson, M.T. 1997. Conservation of rice genetic resources. The role of the International rice gene bank at IRRI. Plant. Mol. Biol, 35 : 61-67. Jackson, M.T., Loresto, G.C., Appa Rao, S., Jones, M., Guimzarases, E.P. and Nga, N.Q. 1996. Rice Biodiversity in Trust. Chapter 20. SCRP / CGIAR. Cambridge University Press. Jackson M. 2001. Managing Genetic Resources and Biotechnology at IRRIs Rice Genbank, 1999. Managing Agricultural Biotechnology. Addressing Research Programme and Needs and Policy Implications (Ed. J.I. Cohen). pp 102 -109. Kameswara Rao, N. and Jackson, M.T. 1996. Effect of planting date and harvest tie on longevity of rice seeds. Seeds. Sci. Res. (inpress). Kato, S., Kosaka, H. and Hara, S. 1930. On the affinity of rice varieties as shown by the fertility or rice plants. Centre Agricultural Institute Kyushu Imp. Univ 2: 241 - 276. Khush, G.S., Brar, D.S., 2001. Rice: In: Evolution and adaptation of crops. I. Cereals (V.L.
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Chopraed.), Oxford and IBH, New Delhi. Khush, G.S. and Brar, D.S., Zapata, F.J., Nelson, R. Nmecough, S. and Bottrell, D.G. 1993. Biotechnology for rice improvement. Proceedings of the Tenth Australian Plant Breeding Conference, April 18-23, 1993, Vo. Focused Plant Improvement: Towards Responsible and Sustainable Agriculture. Morin, S.R, Parm, J.L. Sebastian, L.S., Bellon, M.R., Calibo, M. and Jackson, M.T. 1998. Integrating indigenous technical knowledge and in situ conservation: Collaborative research in Cagayen valley, Philippines. Indigenous knowledge for conservation and management of biodiversity. Ceby city, Philippines, 4-6, March 1998. Neue, H.U. 1994. Variability in rice to chemical stresses of problem soils and their method of identification. In Rice and Problem Soils in South and Southern Asia. (Edited by D. Senadhira). IRRI, 115 - 144. Plucknett, D.L., Smith, N.J.H., Williams, J.T. and Murthi Anishetty, N. 1987. Gene Banks and the Worlds Food. Princeton University Press, Princeton. Pyrah, G.L. 1969. Taxonomic and distributional studies in Leersia (Graminae). Iowa State J.Sci, 44 : 215 - 270. Rao, U.P .and Srinivasan, T.E. 1978. Evaluation of Assam Dwarfs-suitability under low P and N conditions. Madras. Agric. J. 65 : 626 - 62. Saxena, S.K. Chandak, S.B., Ghosh, R, Sinha, N. Jain, and Anil, K., Gupta, 2003. Costs of conservation of agrobiodiversity in India. In : Efficient conservation of Crops Genetic Diversity. Theoretical Approaches and Emprical Studies Detlef Virchow (Ed.). Springer Verleg, Berlin. 137 - 174. OEDC 1996. Saving Biological Diversity economic Incentives, France. OECD, 1999.

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Handbook of Incentives Measures for Biodiversity. Design and Implementation. Second, G. 1985. Relation evolutives chezlegenere O. precessus der domestication des riz. Orstom etudes and theses: 1-189. Paris. Seetharaman R and Ghorai D. P. 1976. Occurrence of types with characters of glaberrima. In Assam Rice Colelction Curr. 50: 62-69. Seetharaman, R., Srivastava, D.P. and Ghorai, D.P. 1974. Preliminary studies in rice cultivars from North East India. Indian J. Genet. Plant Breeding., 34 : 3-149. Sharma, S.D. 1982. Collection and evaluation of rice germplasm form North East India. IBPGR Plant Genetic Resources Newsletter, 50 : 62 - 69. Sharma, S.D. and Shastry, S.V.S. 1965. Taxonomic studies in the genus Oryza O.rufipogon Griff. Sensustricks and O.nivara Sharma et Shastry nom. Nov. Indian Genet., 25 :157 - 165. Sharma, S.D., Krishnanusti, A. and S.R. Dhua 1988. Genetic diversity in rice and its utilization in India. Plant Genetic Resources (Indian Perspectives) pp 108 -120. Shastry, S.V.S., Sharma, S.D., John, V.T. and Krishnaiah, 1971. New sources of resistance to pests and disease in the Assame Rice collections. Intern. Rice Comm. Newsletter, 22 :1-16. Siddiq, E.A. 1991. Genes and rice improvement. Oryza, 28 :1-17. Srivastava, D.P. and Nanda, B.B. 1977. variation

in grain protein in some groups of rice varieties from the collection of North East India. Oryza, 14 : 45-46. Subramanian, M. and Manual, W.W. 1998. Varietal description of rice, Aduthurai, Platinum Jubliee Publication, pp. 1-80. Swaminathan, M.S. 1982. Beyond IR 36: Rice research strategies for the 80s. Paper presented at the International Centers; Week, World Bank, November 20.1982. Washington, D.C. Tzvelev, N.N. 1989. The system of grasses (Poaceae) and their evolution. Bot. Rev., 55 :141 - 204. UNEP, 1992. Guidelines for country studies on Biological Diversity UNEP, Nairobi. UNEP, 1995. Global Diversity Assessment. Cambridge University Press. Cambridge. Villegas, V.N. 1991. Rice germplasm collecting, preservation in USA. Proc. of the third international workshop. International Rice Research Institute, Manila. Philippines, 10-12, March 1991. p.120. Watson, L., Cliford, H.T. and Dallwitz., M.J. 1985. The classification of Poaceae: Subfamilies and supertribes. Aust. J. Bot., 33 : 433 - 484. Westman, A.L .and Kresorich, S. 1997. Use of molecular marker techniques for description of Plant Genetic variation. In : Biotechnology and Plant Genetic Resources. Conservation and use; edited by J.A. Callow, B.R. Ford - Lloyd and H.J. Newbury. Walling Ford.

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GENETIC DIVERSITY OF ROBUSTA - ARABICA HYBRIDS OF COFFEE AND UTILIZATION IN BREEDING


Santa Ram, A1., D. Ganesh, N. Sandhyarani, S.R. Mythrasree, C. Murugan, R.K. Sabir, K.P. Dinesh, A. Manoharan, M.K. Mishra and Jayarama

ABSTRACT
Coffee is an important commodity of international trade and India is one of the important exporting countries. Leaf rust caused by Hemileia vastatrix is a devastating disease of coffee. Coffea arabica is susceptible to this disease. Through selection over years, a number of resistant selections were developed. Sln.5A, Sln.5B, Sln6 and Sln.8 were tested against susceptible check Sln.3 in three locations. All the new resistant selections showed a high degree of resistance ranging from 81.25 to 95.0 per cent. In the check Sln.3 an average of 92.0 per cent plants were susceptible. Preliminary observations on RAPD markers in Sln.6 and Sln.8 indicated distinctions between resistant and susceptible plants in these selections. A general description of the genetic architecture, diversity, inheritance of rust resistance, quality and possible use of RAPD markers in selecting resistant plants in advanced generations is presented.

Introduction Coffee is an internationally important commodity in trade volume and money value. It is not an exaggeration to state that the economies of most of the coffee producing developing countries depend on the earnings from this crop (Marshal, 1985). Leaf rust is a devastating disease of great economic significance on this crop (Kushalappa and Eskes, 1989). This disease is caused by the Basidiomycete fungus Hemileia vastatrix B. et Br. Of the two commercially important species of Coffea, C. arabica L. (Arabica) is more susceptible than C.canephora Pierre ex Froehner (Robusta). Incidentally, Arabica is the species, which produces quality coffee with fine aroma and taste attributes. C. arabica is also the species susceptible to pests and diseases. Leaf rust disease has wiped out Arabica in Sri Lanka and Indonesia where only Robusta is grown now. The susceptibility of Arabica is possibly due to the narrow genetic base of the commercial populations which are known to have been derived from very few plants (Smith, 1985). Another possible reason for the susceptibility of

Arabica coffee is the perennial nature of the crop plant and quick adaptation of the rust fungus to the resistance offered by the host. Added to these, is the autogamous reproductive behaviour of C. arabica, which tends to fix the traits and reduce the variability in adaptive genotypes or land races. Another important point is the tetraploidy of C. arabica versus the diploidy of all other species of Coffea preventing ready flow of genes between other species and Arabica. Thus, improving Arabica coffee with the specific objective of rust resistance without compromising on yield and quality is a task of considerable dimensions. Resistance to leaf rust in Arabica coffee is known to be conditioned by 9 genes symbolised SH1 SH9 (Rodrigues et al., 1975; Eskes, 1989). Old cultivars of C. arabica such as Bourbon and Typica are highly susceptible to this devastating disease. Four genes of resistance viz. SH1, SH2, SH4 and SH5 were identified in the cultivated/wild gene pool of C. arabica (Rodrigues et al., 1975,2000). However, the resistance of coffee plants carrying these genes in different combinations

1. Division of Botany, Central Coffee Research Institute, Coffee Research Station 577117, Chikmagalur District, Karnataka, India

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was defeated by the virulent races of the rust fungus (Rodrigues et al., 1975; Eskes, 1989). Current coffee breeding programmes utilize the resistance genes resident in some spontaneous interspecific hybrids. Thus, the early Indian coffee selections are known to carry the SH3 resistance gene putatively derived from C. liberica (Rodrigues et al., 1975). Hibrido de Timor (HDT) spotted in an Arabica field in Timor (Bettencourt, 1973) is the extensively used source of rust resistance genes. This is commercially exploited in our country as Selection-8 (Sln.8). HDT manifests resistance to all known races of the rust fungus and was shown to be SH6, SH7, SH8, SH9 in genotype (Bettencourt et al., 1992). Resistance of the commercially exploited hybrids of HDT ancestry was, at least, partially defeated by the new races of the rust fungus while HDT has been maintaining its high resistance (Rodrigues et al., 1993; Sreenivasan et al., 1994). This situation prompts that breeders should generate or identify alternative sources of resistance to this important disease. In the coffeebreeding programme of India, certain unique Robusta Arabica hybrids carrying a high degree of resistance to leaf rust were created. Sln.5A and Sln.5B were derived by crossing Devamachy, a natural Arabica Robusta hybrid with S.881 (Rume Sudan Arabica) and S.333 (a natural hybrid of Arabica and Liberica from Doobla, India) respectively. Sln.6 (Robarbica) is an artificial hybrid developed by crossing Robusta (S.274) and Arabica (Kents) and backcrossing the hybrid to Kents. However, the allelic composition of these hybrids is not yet elucidated. This led to gaps in the knowledge of behaviour of genes and resulted in a set back in understanding the stability of resistance in these important sources of resistance. Present study attempted a comparison of the manifested diversity of these interspecific hybrids in a bid to assess their utility as sources of resistance genes. Materials and Methods Observations on various morphological
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characters were recorded from a sample of 10 plants of each of the selections Sln.5A, Sln.5B, Sln.6 and Sln.8. Sln.3 (S.795) was included as control. Summary of observations is presented in table 1. Incidence of leaf rust was recorded from all individuals of three populations of each of these selections in three different locations and summary of observations is presented in table2. Beverage quality of the samples of all selections was assessed by the quality lab at Coffee Board Head Office, Bangalore. Results of three consecutive tests are presented in Table 3. RAPD markers were generated from the DNA of resistant and susceptible plants of Sln.6 and Sln.8 by the method described earlier (Ram and Sreenath, 1999, Williams et al., 1990). Results and Discussion The Genetic System of Coffea arabica A brief consideration of hereditary dynamics of Arabica coffee is important to explain the observed durable resistance in the genus Coffea and propose a model breeding strategy for imparting durable resistance to C. arabica without compromising on quality. Arabica is the lone tetraploid in the genus and has many biological distinctions apart from its chromosome number. This species was considered a segmental allotetraploid on the basis of its diploid cytological behaviour with occasional quadrivalent formation at meiosis (Carvalho, 1952; Grassias and Kammacher, 1975). Most of the allopolyploid species manifest variations that are illustrative of the processes of natural selection (Darlington, 1946) and C. arabica is an example. An important point to be noted is that the evolutionary processes which led to the isolated populations of Coffea (with their morphological peculiarities) from a possible common ancestor were unable to proceed as far as creating biological species showing absolute isolation or a marked chromosomal restructuring. Thus, the diploid groups that are

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mostly homosequential in chromosome structure form a vast genetic continuum (Kammacher, 1977). The wild populations of Coffea adapted balanced heterotic breeding as the basic strategy of evolution that facilitates relatively easy lateral transfer of genes across sympatric populations even as the so-called species maintain their relative identities (Stebbins, 1971). The singular major distinction of C. arabica is its tetraploidy, even while it carries a considerable degree of genetic homology with several diploids. The large genetic variability of this species in its center of origin and diversity and its ability to assimilate the genes of several diploid species indicate that it could be a compilospecies (Ram, 2004). This has tremendous implications for breeding and potential materials that can be used in exploiting this feature of Arabica are already available for breeding purposes (Ram et al., 2004). In this context, the observed resistance of Arabicoid descendants from interspecific hybrids can be effectively exploited to evolve gene pyramids (Ram, 2001). Another important point is that an allotetraploid is a permanent hybrid whose recessive gene mutations cannot segregate when it is self-fertilized. The self-sterility system of its parents need not necessarily work in the allopolyploid species rendering it effectively endogamous. The only possible mode of enlarging its variation is by secondary segregation of ancestral differences (Darlington, 1946; Dawson, 1962) or induced mutations. The large diversity of C. arabica in the land of its origin (Lashermes et al., 1995,1996; Anthony et al., 2001) is not exactly reflective of this situation and renders credibility to the possibility of its being a compilospecies. Dual modes of inheritance in the tetraploid Arabicoid interspecific hybrids (Lashermes et al., 2000; Teixeira-Cabral et al., 2004) and gene conversion in diploid interspecific hybrids (Ky et al., 2000) were reported and can lead to inconsistencies in realizing expected results in resistance breeding programmes. The heredity and durability of disease resistance
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should be understood by superimposing it on this basic genetic system that is responsible for the observed inconsistencies. Genetic Diversity and Inheritance in Robusta Arabica Hybrids Four selections of the present study were all derived from the natural or artificial hybridization of the commercially important species C. arabica and C. canephora. These are morphologically very similar to C. arabica and are distinct from each other (Table 1). These distinctions indicate that the genetic architecture of each of these selections is unique even though they are derived from similar parents. Distinctions in leaf shape, size and petiole length appear to have been a contribution of the ancestral Arabica parent. Morphological homogeneity of each of these selections is a result of continuous selection for characteristic features. Relative uniformity of plants in each of the selections is a reflection of this. Selection for young leaf colour, angle of insertion of primary branches, fruit colour, and frequency of A-grade beans contribute largely to this morphological and genetic homogeneity of the selections. Heredity of Leaf Rust Resistance All these selections also manifest a high degree of resistance to the leaf rust disease ranging from 81.25 to 95.00 percent of the population being resistant (Table 2). In the plots of Sln.3 (S.795), the control variety, a high frequency of plants averaging 92.00 percent were observed to be susceptible. An important point to be considered is that these commercial populations are derived from selected plants and hence segregations do not necessarily reflect genetic laws. However, the observed ratios in Robusta Arabica hybrids approach 15:1 of resistant and susceptible plants respectively, indicating that two pairs of genes are involved in conditioning resistance of these materials. In Sln.3 (S.795) also a similar ratio is observed

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with susceptible plants in higher frequency. This pattern is in conformity with tetrasomic heredity as elucidated in other studies (Ram, 1995, Lashermes et al., 2000; Teixeira-Cabral et al., 2004). Plants in the commercial populations are third generation descendants from the parents in the case of Sln.5A and Sln.5B. It is the third generation from second backcross in the case of Sln.6 and second generation in the case of Sln.8. Sln.3 (S.795) included as control is the sixth and seventh generation. Rust resistance genes of Sln.5A, Sln.5B and Sln.8 are derived from C. canephora while that of Sln.3 is derived from C. liberica (Rodrigues et al., 1975; Eskes, 1989; Sreenivasan et al., 1993,1994). From the observed resistance patterns of various selections it is evident that the resistance genes are gradually getting eliminated with advancing generations, a possible manifestation of negative natural selection (Sreenivasan et al., 1994). Thus, imposing artificial selection for leaf rust resistance in the seed plots and isolating them can maintain resistance of a high order for a long time in the commercial populations. Complementary action of the vertical and horizontal resistance genes in these selections was elucidated (Ram, 2006) and can be superimposed on the cytogenetics of interspecific hybrids to explain the longevity of rust resistance of Robusta Arabica hybrids. A simplified picture of the dynamics of cytogenetic phenomena involved in gene transfer is as follows. The genotype of an Arabicoid derived from interspecific hybridization of Robusta and Arabica and stabilized through backcrossing to Arabica is shown in the figure 1. This genotype is derived from the homologous recombination between the chromosomes of Arabica and Robusta. Hereditary dynamics of this chromosomal genotype is shown in the checkerboard 1.
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In the progeny shown in checkerboard 1, normal homozygotes (AA) carrying a pair of Arabica chromosomes form 1/16 of the progeny (~6%). Structural homozygotes A/RA/R, R/A-R/A and the substitution line RR breed true for the character of interest (such as disease or pest resistance) and comprise a proportion of 3/16 of the progeny (~18%). Structural heterozygotes carrying a Robusta chromosome or an Arabica chromosome and the structurally aberrant Arabica chromosomes carrying a segment of Robusta chromosome or the structurally aberrant Robusta chromosome carrying a segment of Arabica chromosome form the entire remaining progeny of 12/16 (~ 76%). Structural heterozygosity maintains the manifested characters of these plants by suppressing chromosomal recombination in a large frequency of spore mother cells. In essence, this leads us to realize about 94% of the progeny manifesting the character of interest, leading to an apparent fixation of heterosis (Brewbaker, 1964). This situation is described as balanced polymorphism or functional homozygosity of a heterozygote. Dynamic reproductive selection processes (selective fitness of structural heterozygotes, genetic drift etc.) lead to a stabilized population over three to four generations. This is the existing situation in all the Robusta Arabica hybrid selections of the present study. If natural selection does not favour structural heterozygotes, the population tends to revert to pure Arabica type and substitution types with a few structural homozygotes. Among them, only the latter two categories manifest the trait of interest, as often observed in the case of rust resistance in advanced generations of Arabica coffee hybrids. Sixth and seventh generation Sln.3 populations represent this situation. This explains the importance of a genomic imprint in the context of evolutionary fitness. This cytogenetic model explains the process of introgression of genes from diploid species into

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C. arabica and holds good for the genes conditioning VR as well as HR as introgression of these two types of genes can be parallel. Transmission of Quality Traits There is a belief that coffee quality is compromised in interspecific hybrids. Quality of coffee is assessed on the basis of bean size and organoleptic quality of beverage. Bean sizes above 6.6mm (A- or AA-grade) are considered important in trade and achieving higher frequency of this grade in the produce is an important breeding objective (Walyaro, 1983,1997). However, beverage quality was known not to be dependent on the bean size (Roche, 1995). Thus, bean grades assumed importance because uniform size gives uniform roasting that is important to realize good beverage quality (Ram, 2003). Fair Average Quality (FAQ) that is generally accepted in international markets is realized in the four Robusta Arabica hybrid selections. From the data in table 3, the quality of various selections derived from diverse parents combining the genes of C. arabica, C. canephora and C. liberica appears not to be differing significantly. This manifestation has powerful implications for breeding to improve the quality of beverage in Arabica as Robusta and Liberica produce a very inferior beverage (Charrier and Berthaud, 1988; Barre et al., 1998). Thus, C. liberica genes introgressed into the Arabica variety Sln.3 (S.795) are confined to the resistance factors (SH genes) and all others appear to have been eliminated or neutralized in the course of evolution of this selection. Similarly, Sln.5A, Sln.5B, Sln.6 and Sln.8 that incorporate the genes of C. canephora are also not expressing them in the context of quality. It is possible that some of the genes of C. canephora and C. librica that contribute to quality are suppressed in their expression in C. arabica genomic background. As Robusta and Liberica are diploid species (carrying only two sets of chromosomes and thereby two sets of genes) the alternative states of good versus bad
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quality character come to expression, as they exist in the genetic set-up. Arabica, on the other hand, is a tetraploid (carrying four sets of chromosomes) in which several other mechanisms of gene expression are likely to be operational. One such mechanism is homology dependent or repeat induced gene silencing in which, the expression of a character is driven from a single gene when more than two copies of the gene conditioning that character are present in the same genome (Jorgensen, 1995). In Arabica, apparently the genes introgressed from other species and influencing quality appear to be unable to find expression as not only the quality of Sln.3 carrying the genes of C. liberica but also the quality of Sln.5A, Sln.5B, Sln.6 and Sln.8 carrying the genes of C. canephora is appreciated well in the various cup tests. In these cases, it is possible that co-suppression is operating to prevent the expression of genes coming from the diploid species. Another genetic mechanism that can possibly cause the observed quality in selections is gene conversion. In the interspecific hybrids generated by crossing closely related species, the reproductive process eliminates most of the non-homologous chromosomes received from the male parent and only those carrying the maximum homology with those of the female parent are retained in the first backcross progeny. This gets further reduced with each advancing cycle of reproduction of the backcross progeny and within three cycles only a few blocks of genes of the introgressed species will remain in the genome. This retention of genes is usually because they are favourably selected in each reproductive cycle. Such conscious selection was practiced in our country with reference to the rust resistance genes. The selection for genes conditioning quality of coffee was also evidently very successful as reflected in the data of Table 3 even though it was not intended. How does this

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happen? Available evidence indicates that inheritance of genes in Arabica does not always conform to the simple Mendelian order. Thus, for the characters native to C. arabica Mendelian inheritance was recorded (Krug and Carvalho, 1951). Conversion of genes was reported in the interspecific hybrids of Coffea (Ky et al., 2000). Considering the basic interspecific hybrid nature of C. arabica and the dual modes of inheritance for single loci observed in the tetraploid interspecific hybrids of C. arabica x C. canephora, it is plausible to infer that natural selection favours the genes of C. arabica conditioning the various traits of quality (which are well conserved in all the above hybrids) (Narasimhaswamy, 1960, 1971; Ganesh et al., 2002) through conversion of diploid genes. Thus, natural selection played a key role in the quality improvement of Indian coffee selections. This inference gains support from reports on the beverage quality of Icatu hybrids of Brazil (Fazuoli et al., 1977; Petracco, 2000), Hibrido de Timor and derived lines such as Catimor, Sarchimor and Colombia (Bertrand et al., 2003) and a recently developed hybrid of Catimor x (Congensis x Robusta) (Srinivasan et al., 2004) as all of them are reported to possess good beverage quality. RAPD Markers in Advanced Generations It is hypothesized that in advanced generations all characters and the genes conferring them are well stabilized and hence, unique markers found to be associated with a complex of characters breed true. Preliminary results obtained from the amplification experiments on the DNA of Sln.8 and Sln.6 (Figs. 2 and 3) indicate distinctions between resistant and susceptible plants in these selections. These observations appear to confirm the hypothesis. Some of the unique RAPD fragments inherited from specific parent were also identified in Sln.6 (Fig. 4). Further work in this aspect is expected to lead to the identification of additional unique markers in the resistant plants
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producing good beverage quality. These markers have a diagnostic utility in identifying plants possessing these traits at an early developmental stage like nursery for the establishment of seed gardens and constitute the initial approach to a marker assisted selection programme. Foregoing discussion provides a deep insight into the genetic and cytogenetic phenomena underlying the evolution of Robusta Arabica hybrids of coffee. The insight gained is of great practical utility in identifying the particular generation that can be exploited commercially. The four selections of Robusta Arabica hybrids are derived from diverse parents but possess similar quality. They also manifest high resistance to leaf rust. The diversity of parents implies that these traits are conferred by a different complex of genes in each of the selections. Thus, a combination of these selections yields produce of relatively uniform quality and forms a resistance gene pyramid that stands highly resistant to the leaf rust disease for a very long time. These insights also help in identifying the mother plants that can be excellent seed bearers that maintain the resistance genes without compromising productivity and quality. This analysis also provides a clear understanding of the direction and method of selection and backcrossing to be undertaken when loss of resistance is experienced in advanced generations. Thus, a new strategy combining the cytogenetic results and molecular markers is expected to result in greater efficiency of the coffee breeding programme. This study also revealed that Indian Robusta Arabica hybrids could be potential sources of new rust resistance genes. REFERENCES Anthony, F., Bertrand, B., Quiros, O., Wilches, A., Lashermes, P., Berthaud, J., Charrier, A. 2001. Genetic diversity of wild coffee (Coffea arabica L.) using molecular

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markers. Euphytica. 118: 53-65. Barre, P., Akaffou, S., Louarn, J., Charrier, A., Hamon, S., Noirot, M. 1998. Inheritance of caffeine and heteroside in an interspecific cross between a cultivated coffee species Coffea liberica var. dewevrei and a wild species caffeine free C. pseudoz anguebariae. Theor. Appl. Genet. 96: 306-311. Bertrand, B., Guyot, B., Anthony, F., Lashermes, P. 2003. Impact of the Coffea canephora gene introgression on beverage quality of C. arabica. Theor. Appl. Genet. 107: 387394. Bettencourt, A.J. 1973. Consideracoes gerais sobre o Hibrido de Timor. Circular # 23. Sao Paulo (Brazil): Instituto Agronomico de Campinas. 20 p. Bettencourt, A.J., Lopes, J., Palma, S. 1992. Factores geneticos que condicionam a resistencia as racas de Hemileia vastatrix Berk. et Br. dos clones tipo dos grupos 1, 2 e 3 de derivados de Hibrido de Timor. Broteria Genetica. XIII (LXXX): 185-194. Brewbaker, J.L. 1964. Agricultural Genetics. Englewood Cliffs, New Jersey (USA): Prentice-Hall. 156 p. Carvalho, A. 1952. Taxonomia de Coffea arabica L., caracters morfologicos dos haploides. Bragantia 12: 201-212. Charrier, A., Berthaud J. 1988. Principles and Methods in Coffee Plant Breeding: Coffea canephora Pierre. In: Clarke RJ, Macrae R, editors. Coffee (Vol.IV) Agronomy. London (UK) and New York (USA): Elsevier Applied Science. p.167-197. Darlington CD. 1946. The Evolution of Genetic Systems. London (UK): Cambridge University Press. 151 p. Dawson, G.W.P. 1962. An Introduction to the Cytogenetics of Polyploids. Oxford (UK): Blackwell Scientific. 95 p.
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Eskes, A.B. 1989. Resistance. In: Kushalappa AC, Eskes AB, editors. Coffee Rust: Epidemiology, Resistance and Management. Boca Raton (Florida, USA): CRC Press. p.171-292. Fazuoli, L.C., Carvalh, A., Monaco, L.C., Teixeira, A.A. 1977. Qualidade de bebida do caf Icatu. Bragantia. 36: 165-172. Ganesh, D., Ram, A.S., Prakash, N.S., Ahmed, J., Mishra, M.K., Jagadeesan, M., Reddy, A.G.S., Srinivasan, C.S. 2002. Evaluation of Coffea liberica x Coffea eugenioides and its progenies for yield, leaf rust tolerance and quality. In: Sreedharan K, Vinod Kumar PK, Jayarama, Chulaki, BM, editors. Proceedings of PLACROSYM XV, 10-13, December 2002. Mysore (India): Indian Society for Plantation Crops. p. 72-77. Grassias, M., Kammacher, P. 1975. Observations sur la conjugaison chromosomique de Coffea arabica L. Caf Cacao The 19: 177-190. Jorgensen, R.A. 1995. Cosuppression, Flower color patterns and Metastable gene expression states. Science 268: 686-691. Kammacher, P. 1977. Utilisation des ressources genetiques du genre Coffea pour lamelioration des cafeiers cultives. In: Proceedings of VIII International Scientific Colloquium on Coffee. 28 November 3 December 1977. Abidjan (Ivory Coast). Association Scientifique Internationale du Caf. p. 335-358. Krug, C.A, Carvalho, A. 1951. The Genetics of Coffea. Adv. Genet. 4: 128-158. Kushalappa, A.C., Eskes, A.B. 1989. Coffee Rust: Epidemiology, Resistance and Management. Boca Raton (Florida, USA), CRC Press. 345 p. Ky CL, Barre, P., Lorieux, M., Trouslot, P., Akaffou, S., Luarn, J., Charrier, A., Hamon, S., Noirot, M. 2000. Interspecific genetic

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linkage map, segregation distortion and genetic conversion in coffee (Coffea sp.). Theor. Appl. Genet. 101: 669-676. Lashermes, P., Combes, M.C., Trouslot, P., Charrier, A. 1996. Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica. Euphytica. 87: 59-64. Lashermes, P., Paczek, V., Trouslot, P., Combes, M.C., Couturon,E., Charrier, A. 2000. Single locus inheritance in the allotetraploid Coffea arabica L. and interspecific hybrid C. arabica x C. canephora. J. Heredity. 91: 81-85. Lashermes, P., Combes, M.C., Cros, J., Trouslot, P., Anthony, F., Charrier, A.1995. Origin and genetic diversity of Coffea arabica L. based on DNA molecular markers. In : Proceedings of XVI International Scientific Colloquim on Coffee, 9 14 April 1995. Kyoto City (Japan): Association Scientifique Internationale du Caf. p.528-536. Marshall, C.F. 1985. World Coffee Trade. In: Clifford MN, Willson KC, editors. Coffee: Botany, Biochemistry and Production of Beans and Beverage Westport (Connecticut, USA): Avi Publishing Co. Inc. p.251-283. Narasimhaswamy, R.L. 1960. Arabica selection S.795: Its origin and performance A study. Indian Coffee 24:197-204. Narasimhaswamy, R.L. 1971. S.795 Arabica and Quality. Indian Coffee 35: 371-372. Petracco, M. 2000. Organoleptic properties of espresso coffee as influenced by coffee botanical variety. In: Sera T, Soccol, CR, Pandey A, Roussos S, editors. Coffee Biotechnology and Quality. Dordrecht (The Netherlands). Kluwer Academic Publishers. p. 347-353. Ram ,A.S. 1995. New dimensions in understanding inheritance of coffee rust resistance: A Mendelian perspective. In : Proceedings of XVI International Scientific
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Colloquim on Coffee, 9 14 April 1995. Kyoto City (Japan): Association Scientifique Internationale du Caf. p.548556. Ram, A.S. 2001. Breeding for rust resistance in coffee: The gene pyramid model. J. Plantn. Crops. 29: 10-15. Ram, A.S. 2003. Coffee primary processing in Kenya and Tanzania. Indian Coffee 67: (9):9-11. Ram, A.S. 2004. Coffea arabica L- A Compilospecies: Implications for Breeding. In: Proceedings of XX International Scientific Colloquium on Coffee. Bangalore (India): 10-14 October 2004. Association Scientifique Internationale du Caf. (Electronic Publication). Ram, A.S. 2006. Genetic basis of rust resistance in Arabica coffee. (in preparation). Ram, A.S, Ganesh, D., Srinivasan, C.S., Reddy, A.G.S. 2004. Ligenioides A source of new genes for Arabica coffee breeding. J. Plantn. Crops. 32 (Suppl.): 5-11. Ram, A.S, Sreenath, H.L. 1999. A method for the isolation and amplification of coffee DNA with random octamer and decamer primers. J. Plantn. Crops. 27:125-130. Roche, D. 1995. Coffee genetics and quality. In : Proceedings of XVI International Scientific Colloquim on Coffee, 9 14 April 1995. Kyoto City (Japan): Association Scientifique Internationale du Caf. p.584588. Rodrigues, J.r. C.J., Bettencourt, A.J., Rijo, L. 1975. Races of the pathogen and resistance to coffee rust. Annu. Rev. Phyto pathology 13: 49-70. Rodrigues, Jr. C,.J., Varzea, V.M.P., Godinho, I.L., Palma, S., Rato, R.C. 1993. New physiologic races of Hemileia vastatrix. In: Proceedings of XV International Scientific Colloquium on Coffee,

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Montpellier (France). Association Scientifique Internationale du Caf. p. 318321. Rodrigues, Jr. C.J., Varzea ,V., Silva, M.C., Guerra-Guimares, L., Rocheta, M., Marques, D.V. 2000. Recent advances on coffee leaf rust. In: Prakash NS, Raghuramulu Y, Devasia J, editors. Proceedings of International Symposium on Coffee. Central Coffee Research Institute. p. 179-193. Smith, R.F. 1985. A history of coffee. In: Clifford MN, Willson KC, editors. Coffee: Botany, Biochemistry and Production of Beans and Beverage. Westport (Connecticut, USA): Avi Publishing Co. Inc. p. 1-12. Sreenivasan, M.S., Ram, A.S., Prakash, N.S. 1993. Tetraploid interspecific hybrids in coffee breeding in India. In: Proceedings of XV International Scientific Colloquium on Coffee. Montpellier (France) 6-11 June 1993. Association Scientifique Internationale du Caf. p. 226-233. Sreenivasan, M.S., Ram, A.S., Prakash, N.S. 1994. Search for new sources of resistance to coffee leaf rust. Report on the International Collaborative Project Pathology and Improvement of Coffee (Coffea arabica) for the Main Diseases, Central Coffee Research Institute (India). 24 p. Srinivasan, C.S., Kumar, A., Amaravenmathy, V.S., Ram, A.S. 2004. Robusta like coffee plants with arabica like coffee quality. Myth or possibility. In: Proceedings of XX International Scientific Colloquium on Coffee. Bangalore (India). 10-14 October 2004. Association Scientifique Internationale

du Caf. (Electronic Publication). Stebbins, G.L. 1971. Processes of Organic Evolution (2nd Ed.). Englewood Cliffs (New Jersey, USA): Prentice-Hall, 193 p. Teixeira-Cabral, T.A, Sakiyama, N.S., Zambolim, L., Pereira, A.A., Schuster, I. 2004. Single locus inheritance and partial linkage map of Coffea arabica L. Crop Breeding and Applied Biotechnology 4: 416-421. Walyaro, D.J. 1983. Considerations in breeding for improved yield and quality in arabica coffee (Coffea arabica L.). Ph.D. Thesis. Wageningen (The Netherlands): Agricultural University of Wageningen. 119 p. Walyaro, D.J. 1997. Breeding for disease and pest resistance and improved quality in coffee. In: Proceedings of XVII International Scientific Colloquium on Coffee. Nairobi (Kenya). 20-25 July 1997. Association Scientifique Internationale du Caf. p.391-404. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535.

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Table 1. Dinstinctive Characters of Robusta-Arabica Hybrids of Coffee

Character Angle of Branch insertion Leaf shape Leaf apex Leaf length Leaf width Young leaf colour Petiole length Stipule shape Fruit colour Fruit ripening Yield (kg/ha) A-grade beans (%)

Sln. 5A Sln. 5B Semi-erect to Horizontal to drooping drooping Linear Lanceolate Acuminate 110-180mm 55-75mm Green 7-10mm Traingular Red 260 days 1020 30.00 Broad Lanceolate Acuminate 126-190mm 63-85mm Light Bronze 5-8mm Ovate

Sln. 6 Semi-erect

Sln. 8 Sln. 3 (S.795) Horizontal to Semi-erect drooping Broad Lanceolate Acuminate 150-200mm 68-90mm Bronze 7-10mm Triangular Deep Red 240 days 900 60.00 Lanceolate Acuminate 135-200mm 62-78mm Bronze 7-12mm Triangular Orange Red 240 days 975 75.00

Broad Lanceolate Acuminate 125-220mm 67-79mm Light Bronze

7-13mm Triangular Deltate Orange Red - Orange Red Red Red 240 days 240 days 1100 960 65.00 69.00

Table 2. Rust Resistance Patterns in Robusta Arabica Hybrids

Selection Sln.5A Sln.5B Sln.6 Sln.8 Sln.3 (S.795)

Resistant (%) 81.25 87.50 90.00 95.00 8.00

Susceptible (%) 18.75 12.50 10.00 5.00 92.00

Table 3. Quality of Robusta Arabica Hybrids

Selections Sln.3 (S.795) Sln.5A Sln.5B Sln.6 Sln.8 1999 FAQ FAQ + FAQ-FAQ+ Good

Years of Quality Testing 2000 2001 FAQ Sl.Below FAQ-FAQ FAQ+-Good FAQ+ as Special Coffee + FAQ-FAQ FAQ+-Good FAQ FAQ+ FAQ-Good Sl.Below FAQ

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Checkerboard 1.Segregation of Chromosome genotype in RobustaArabica Hybrids

A A/R R/A R

A AA A/R-A R/A-A RA

A/R A-A/R A/R-A/R R/A-A/R R-A/R

R/A A-R/A A/R-R/A R/A-R/A R-R/A

R AR A/R-R R/A-R RR

1 2 3 4

6 7 8 9 M

Fig.2. RAPD profiles of Hibrido de Timor (Sln.8) A (Resistant) and R (Susceptible) types generated by the primers OPF-15 (5 CCAGTACTCC 3) and OPF-04 (5 GGTGATCAGG 3). Lanes 1& 2: HDT A (OPF-15) Lanes 3 & 4: HDT R (OPF-15) Lanes 6 & 7: HDT R (OPF-04) Lanes 8 & 9: HDT A (OPF-04) Lane 5 : PCR products of HDT (Open pollinated plant) (OPF-15) Lane M - Marker Hind III-Eco R1 double digested Lambda DNA

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EVALUATION AND UTILIZATION OF BIODIVERSITY IN CASSAVA (MANIHOT ESCULENTA CRANTZ)


Santha V. Pillai1, R.R. Nair, M.S. Palaniswami, C.S. Ravindran, S.N. Moorthy, V. Ravi and S. Sree Lekha

ABSTRACT
Collection, evaluation and utilization of the biodiversity available in the crop is the basic requirement in any plant breeding program. As such, more than 1600 accessions of Cassava, consisting of both indigenous and exotic accessions, were assembled at Central Tuber crops Research Institute, Trivandrum. These accessions were evaluated for economic characters like tuber yield and quality parameters like starch content, cooking quality, cyanogen content, keeping quality and tolerance to Cassava Mosaic Disease (CMD), white fly, drought etc and genetic stocks were identified for each character. Some of the promising accessions, combining high yield, good quality and tolerance were evaluated in replicated trials and the selections are undergoing on-farm trials in Kerala and Tamil Nadu. CMD tolerant accessions are being utilized in the hybridization program. They were also evaluated for unconventional characters like leaf yield and also quality of starch. Cassava leaf is increasingly being used in cattle feed, pet animal feed and also silviculture. The quality of starch namely, Amylose content, Amylopectin and AP/Am ratio which determine the suitability of starch for specific industrial use, was analysed. The germplasm was also screened for morphological characters, biochemical markers (Isozyme) as well as molecular markers (DNA-RAPD) in tune with international standards for identification of varieties and isolation of duplicates. This information, along with that on economic characters and passport data were utilized to arrive at a core collection of cassava germplasm. At present, the land races of cassava are being analysed for microsatellite markers to study the molecular variability and diversity available in the population and also for DNA fingerprinting of farmers varieties. Microsatellite markers are also being utilized to identify varieties resistant to CMD and white fly. Details are presented in the paper.

Introduction Cassava (Manihot esculenta Crantz) is a popular tuber crop grown in the tropical belt of Asia, Africa and South America. In India, it is mostly grown in the Southern region, especially, Kerala, Tamil Nadu and parts of Andhra Pradesh. In Kerala, it is grown as a subsidiary food crop, whereas in Tamil Nadu and Andhra Pradesh, it forms the raw material for starch and sago industry. Research on cassava started in Kerala about 50 years ago, under the University, by introduction of varieties from other cassava growing areas and improving them by intercrossing, mutation breeding, polyploidy
1. Central Tuber Crops Research Institute, Trivandrum 695017

breeding etc. The Central Tuber Crops Research Institure, Trivandrum was established in 1963 and it is the main centre for tuber crops research.Research on cassava is going on in a few Universties as well including TNAU, Coimbatore. India can boast of the highest productivity in the world; 26t/ha, against the global average of 10 t/ha. This high yield in India is mostly a contribution of Tamil Nadu, where improved varieties developed at research centres are grown under irrigated conditions. Still, development of improved varieties, suited to different needs, at different times is a major

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item of research. Germplasm is the raw material for the purpose. The Central Tuber Crops Research Institute is maintaining more than 1600 accessions of cassava collected from different countries. Over the years, a number of improved varieties, suited for both edible purpose as well as for stach extraction were developed. The varieties H-226 and H-165 developed at the Institute occupy sizeable area in the starch factory areas. At present the germplasm is also evaluated for non-conventional characters and for varying purposes and more sophisticated tools are employed for evaluation and utilization of germplasm. Some of the items of work going on at CTCRI, Trivandrum in this direction are presented in the paper. Materials and Methods The germplasm collection of cassava, numbering about 1600, available at the Institute formed the material for the study. They were screened for presence or absence of Cassava Mosaic Disease (CMD) symptom and a subset of about 75 symptom free accessions were evaluated for yield, quality and other special characters. The accessions showing any of the yield / quality component in high level were selected as genetic stocks for that character, for further use in the breeding program. The best promising accessions were evaluated on replicated trial for 3 consecutive years and selected ones are undergoing on-farm trials both in Kerala and Tamil Nadu. The subset was screened for special characters like tolerance to drought , white fly, keeping quality etc and the best performers were identified. The subset was also screened for nonconventional characters like leaf yield and quality of starch. The germplasm, was also characterized based on different markers namely, morphological, biochemical (Isozyme) and molecular (DNARAPD), to identify and isolate duplicates and eliminate them from the field in due course. This
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information, along with that on genetic stocks and geographical representation, was used to identify a tentative core collection of the germplasm. Results and Discussion Seventy five out of the 1300 accessions, screened for the incidence of CMD were found to the symptom free. The symptom free accessions (75) were evaluated for yield and quality and subjected to genetic analysis. Phenotypic coefficient of variation was the highest for weight of shoot (69.74), followed by cyanogen content (66.74) and tuber yield per plant (54.85) (Table 1). Weight of shoot per plant was found to have the highest correlation with yield (0.80), followed by number of tubers per plant (0.60). Genetic stocks for yield, starch and low cyanogen were identified for use in recombination breeding [Table 2]. Tuber yield above 2.55 kg/plant, starch above 30% and cyanogen below 20 ppm were kept as yardsticks. Six promising lines identified from the subset were evaluated in replicated plot trial. Two of the selected accessions are undergoing on-farm trials in 6 districts of Kerala and the performance is good. Some of the selections are undergoing on-farm trial in Tamil Nadu. These selections were found to be better suited to the hilly regions of the state. Sixty three CMD free accessions were screened for drought tolerance under rain fed condition in upland. Four months drought period existed during the season. Nineteen accessions showed very high drought tolerance based on one or other criteria namely, high Leaf Area Index (LAI), high tuber yield or high starch content (Table 3). Now cassava cultivation is spreading to dry areas as well, especially in Andhra Pradesh and there is a need for drought tolerant varieties. At present H-165, a short duration variety, which escape drought, is cultivated in these areas.

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Twenty one CMD free accessions were evaluated for the incidence of white fly. Two accessions were found to have very low incidence (Table 4). CMD is a serious disease in cassava and it is spread through white fly. Varieties having resistance to white fly infestation may be able to evade CMD and hence this approach. About 50 CMD free accessions were evaluated for keeping quality of tuber. Tubers were cut into 2 pieces and kept under net. Two accessions could be kept up to 5 days without black spot. Fast perishability is a very big limitation in cassava and we had observed that variability for this character exists. But the number of varieties with keeping quality is very low. Six CMD free accessions with branching character were screened for leaf yield. Accession no E-34 gave the highest leaf yield of 1.68 kg per plant. Cassava leaf is found to be suited for pet animal feed, in addition to cattle feed and is in great demand (Metha Wanapat, 2002). And hence, this work was initiated to create a database. Twelve accessions were screened for quality of starch namely Amylose content, Amylopectin content and Ap/Am ratio. The accession no E108 gave the highest Amylose content of 30 %, and accession no E-109 showed the highest Ap/ Am ratio of 4 (Table 5). The proportion of these components of starch determines the suitability of starch for specific purposes. Satrch with high Amylose content is best suited for textiles whereas that with high Ap/Am ratio is best suited for fish feed, by virtue of its binding property. The germplasm was also characterized based on morphological markers as well as biochemical (Iszoyme) and molecular markers (DNA / RAPD). This information was used to identify duplicate accessions in the germplasm as per international standards (Ocampo et al. 1995).
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About 90 accessions were found to be duplicate. DNA/RAPD analysis was also used for DNA fingerprinting of released varieties as well as that of elite breeding lines. This is very important to safe guard the Plant Breeders Right as well as the Farmers Right in the new IPR (Intellectual Property Right) regime. The data on genetic stocks, duplicates and geographical representation were utilized to arrive at a core collection of the germplasm consisting of 15% of the accessions (Table 6). Identification of a smaller subset, representing the variability is essential when the number of collection become very large (Santha and Nair, 2002). Researches are going on to use more powerful molecular markers like SSR and ISSR to screen the varieties for resistance to CMD and white fly,and also to study the variability and diversity available in the local collection. REFERENCES Metha Wanapat. 2002. The role of cassava hay as animal feed. Paper presented in Seventh Asian Cassava Research Workshop, October 28-November 1, 2002. CIAT, Bangkok. P.21. Ocampo, C., Angel, F., Jimenez, A., Jaramillo, G., Hershey, C., Granados, E., Iglesias, C. 1995. DNA fingerprinting to confirm possible genetic duplicates in cassava germplasm .Paper presented in the Second International Scientific Meeting of the Cassava Biotechnology Network held at Bogor, Indonesia: August 22-26, 1994, CIAT, Cali, Columbia. P.145-151. Santha, V., Pillai Nair, R. R. 2002. Germplasm management in cassava with special emphasis on core collection Paper presented in the National Vegetable Conference held at Bangalore, Nov. 1014, 2002.

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Table 1. Variability and correlation of 10 characters

Sl. No. 1 2 3 4 5 6 7 8 9 10

Characters Tuber yield /plant (kg) No of Tubers One Tuber (kg) Length of Tuber Girth of Tuber Starch percent Cyanogen (ppm) Height of plant (cm) No of branches Weight of shoot (kg)

Mean 1.8 4.8 0.3 25.7 15.6 28.7 42.4 110.1 5.7 2.7

PCV% 54.8 45.9 49.4 35.1 19.0 18.8 66.8 28.5 37.3 69.7

Correlation with yield 1.00 0.60** 0.05 0.08 0.04 0.03 (-) 0.01 0.08 0.22 0.80**

** Significance at 1% level of probability

Table2 . List of elite genotypes selected

Sl.No 1 2 3 4 5 6 7

Acc.no E 393 E 329 E 88 E 111 E 480 E 127 E 135

Desirable characters Y (2.66), S (31) , C(34) Y (1.80) , S (29), C (10) Y(1.15), S (35), C (33) Y (1.75), S (34) , C(40) Y (2.60), S(33), C(15) Y(2.25), S(35),C(42) Y (2.66), S(36) ,C(5.4)

Y-Yield/plant(kg),SStarch percent,C-Cynogen content ppm

Table 3. Accessions having tolerance to drought, based on LAI, Tuber Yield and Starch content

High LAI (>7.20) 1 2 3 4 5 6 7 8 9 E -165 E -282 E-328 I -192 I -82

High Tuber yield (>7.0 kg/plant) E-272 E-273 E-274 E-354 I-82

High starch content (>25%) E-33 E-39 E-430 E-440 E-459 E-500 E-534 I-82 I-120

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Table 4. White fly incidence in CMD symptom free accessions

Sl. No. 1 2 3 4 5 6 7 8 9 10

Variety E-144 E-152 E-97 E138 I-775 E-347 E-39 E-96 E-301 E-142

Nymph 8 5 32 68 61 22 24 13 42 35

pupae 0 0 27 3 7 17 0 6 3 0

Female 0 1 2 3 6 3 1 0 0 3

Male 0 0 0 0 1 0 0 0 0 0

Whitefly 8 6 61 74 75 42 25 19 45 38

Table 5. Starch quality in promising accessions

Sl.No 1 2 3 4 5 6 7 8 9 10 11 12

Genotype I-101 I-102 I-103 I-104 I-105 I-107 I-108 I-109 I-110 I-111 I-112 I-113

Starch Extractability 30.1 32.2 18.4 19.8 29.7 24.5 27.4 31.8 26.2 24.9 24.4 21.0

Amylose (%) Amylopectin(%) 23.7 23.1 27.9 26.9 26.0 28.1 30.0 19.7 22.3 29.8 26.5 27.0 76.3 76.9 72.1 73.1 74.0 71.9 70.0 80.3 77.7 70.2 73.4 73.0

AP/Am Ratio 3.21 3.30 2.80 2.70 2.80 2.50 2.30 4.07 3.40 2.30 2.76 2.70

Table 6. Tentative core collection

Sl. No. 1 2 3 4 5 6 7 8 9

Criteria High yield (>5kg/plant ) High starch(>33%) Low cyanogens(<10ppm) High carotene (>450ppm) CMD Symptoms free Released varieties Local popular varieties Geographic representatives Wild relatives Total
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No. of accessions 30 25 25 40 75 15 20 2 8 240

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AGRO - MORPHOLOGICAL CHARACTERIZATION AND EVALUATION OF RICE GERMPLASM FOR MAJOR BIOTIC STRESS TOLERANCE
Subba Rao, L.V., T. Ram, N. Shobha Rani, V. Ravindra Babu, I. C. Pasalu, C. S. Reddy, A. S Rama Prasad, B. C Viraktmath and S. V. Subbaiah

ABSTRACT
One thousand and fifty six rice accessions were characterized for 21 agro-morphological characters at directorate of rice research and also screened for major biotic stresses at 20 hot-spot locations across the country. Agro-morphological characterization of 1056 accessions revealed that 54 percent of them showed very good early plant vigour, 41 percent exhibited intermediate vigour while 4.5 percent accessions were found to exhibit poor plant vigour. The study revealed that 57 percent accessions exhibited green basal leaf sheath colour, 18 percent possessed purple colour and 12 percent showed light purple colour while another 12 percent accessions exhibited purple lines. 57 percent of accessions recorded high number of effective tillers per plant (upto15), 29 percent accessions showed effective tillers upto10, while the remaining 14 percent of accessions exhibited less than 10 effective tillers. Days to 50 percent flowering ranged from 74 days to 112 days and based on the flowering duration total germplasm accessions can be grouped into early (9 percent), mid early (45 percent),medium(34 percent) and late(12 percent). Seed weight of 100 grains ranged from a minimum of 1.00 g to a maximum of 3.48 g. Less than 2 g of 100-grain weight was recorded by 5.7 per cent of accessions, while 86 per cent of the accessions showed 2-3 g and the remaining 8 per cent accessions recorded more than 3 g. Single plant yield of less than 15 g was recorded by 35 per cent of accessions Almost 50 per cent of accessions registered a single plant yield of 20-25 g. Of the 1056 accessions screened, 14.7 per cent germplasm showed tolerance / resistance for major biotic stresses, which includes blast (2 per cent), BLB (1.4 per cent), RTD (1.0 per cent), plant hoppers (3.3 per cent), GM (2.9 per cent) and stem borer (4.1 per cent). Some of the promising accessions with resistance / tolerance to major biotic stresses are IC 115330 and IC 115481 for BPH; IC 113990, IC 113999, IC 114322 and IC 115924 for gall midge; IC 115957 for stem borer; I.C 14335 and I.C 114507 to leaf blast and bacterial blight ; I.C 114653 and IC 114787 to bacterial blight and tungro. Promising germplasm with more than 25 g of single plant yield coupled with resistance to BPH are IC 114419 and IC 114430; IC 115905 and IC 114847 to GM; IC 114725 to blast and to BLB IC 114335 and IC 115738. The present results indicated that ample genetic variability exists for improving the yield potential as well as resistance to major diseases and insect pests in modern high yielding varieties.

Directorate of Rice Research, Rajendranagar, Hyderabad 500030

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CHARACTERIZATION OF COTTON (GOSSYPIUM HIRSUTUM L.) GENOTYPES AND EVALUATION OF GENETIC DIVERGENCE
Preetha, S1. and T.S.Raveendran2

ABSTRACT
An investigation was taken up to compare the genetic variability of 150 cotton (Gossypium hirsutum) genotypes after grouping them visually into three different growth habits. The evaluation led to the grouping of accessions into 67 robust, 66 semicompact and 17 compact genotypes. The genotypes in the above three groups came under 13, 20 and 5 clusters respectively, when Mahalanobis D2 technique was applied. The grouping of genotypes supported that the visual evaluation was in good agreement with the character evaluation of robust and compact types but not in the case of the intermediate semicompact types. Relatively higher contribution towards genetic divergence was noticed from quality characters, leaf area index and earliness characters.

Introduction Cotton, known as the King of fibres, continues to be the predominant fibre in the Indian textile scene, despite stiff competition from the man-made synthetic fibres. It assumes a place of pride in Indian economy, as cotton production, processing and trade in cotton goods provide employment to about 60 million people in our country. Further, the export of raw cotton, yarn, textile, garments, cotton seed cake, oil and other byproducts earn valuable foreign exchange. In India, cotton is grown in three agro climatic zones - northern zone where cotton is raised entirely under irrigation, central and south zones where it is predominantly a rainfed crop. Under rainfed cultivation a compact plant type with short internodes, low leaf area and high harvest index is preferred to get the best yield besides withstanding the drought in different phases of crop growth. However, under irrigated conditions, the crop attains a luxuriant growth with large leaves, open plant type, big bolls and longer duration. A specific plant type has acclimatized in a particular tract and is able to interact well with the weather parameters and perform well in respect of yield. The studies on suitability of particular ideotype to a particular environment have not been taken up by breeders
1. 2.

either in tetraploid or diploid species. Such studies will be useful to pinpoint and fix the most efficient genotype for a particular location. Further the characterization of the robust, semicompact and compact genotypes in terms of crop growth, physiological efficiency, agronomic characters and quality parameters will be useful not only to increase the yield level in this important fibre crop but also helps to classify and select the most desirable ones for each of the target environments. Therefore, the present study was attempted to define the robust and compact plant types and a group intermediate between them using the agronomic, physiological and yield parameters for attaining the highest biological efficiency and fibre yield. Material and Methods One fifty genetic accessions of Gossypium hirsutum were raised in an experimental layout in Randomized Block Design (RBD) with two replications during kharif 2002-03. The genotypes were sown in six meter long ridges spaced 75 cm apart and with an interplant distance of 30cm so as to accommodate 20 plants in each row. Five randomly selected plants were tag-labelled for recording

Ph.D. scholar, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore Director, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore

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observations. Average of data recorded on each character from these five plants represented the mean of that replication. For determining the physiological traits fourth leaf from the top was used. For analyzing the biochemical constituents youngest, fully unfold, disease free leaves were collected from the sample plants and pooled to form the composite sample. Sampling was done at flowering stage. Observations were recorded on morphological, yield and quality traits viz.,plant height (PH), number of sympodia per plant (NOS), number of monopodia per plant (NOM), length of sympodia (LOS), number of flower bearing nodes in sympodia (NFBN), days to first flowering (DFF), internode length (IL), petiole length (PL), number of flowers per plant (NOF), number of bolls per plant (NOB), boll weight (BW), number of locules (NOL), number of seeds per locule (NOSL),days to first boll bursting (DFBB), days to fifty percent boll bursting (DFFBB), seed cotton yield (SCY), seed index (SI), lint Index (LI), ginning outturn (GOT), 2.5 per cent span length (2.5%SL),bundle strength (BS),uniformity ratio (UR),micronaire (MIC),elongation per cent (EL). Apart from this physiological parameters namely leaf area per plant (LA), specific leaf area (SLA),specific leaf weight (SLW),photosynthetically active radiations (PAR),canopy temperature (CT),diffusive resistance (DR) and transpiration rate (TR),root length (RL) and biochemical traits like chlorophyll content (CC),soluble protein (SP),total phenols (TP),nitrate reductase activity (NRA) were also recorded. By visual evaluation the accessions were grouped into robust, semicompact and compact plant types. Based on all the above characters robust, semicompact and compact plant types were characterized and they were analysed for their genetic divergence. Results and Disscussion The genotypes were visually evaluated based
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on their stature, branching habit, leaf size, internode length and grouped into three distinct morphological groups viz. robust, semicompact and compact. In order to characterize the three groups in terms of agronomic, physiological and yield traits a grade index was formulated for the three plant types which would be highly useful to visualize robust, semicompact and compact types. For each of the characters, low, intermediate and high range was fixed based on the expression (minimum and maximum values) and they were assigned with scores 1, 2 and 3. Then the grade index was calculated as follows: grade1 x number of accessions in grade 1 (A1) + grade2 x number of accessions in grade 2 (A2) + grade3 x number of accessions in grade 3 (A3) Grade index = Total number of accessions (A1 + A2 + A3) The grade indexes for different characters are presented in the Table 1. Based on this, the robust plant types can be characterized as tall, with longer petioles, more number of sympodia, longer fruiting branches, late flowering, more number of bolls and high yield. They also had high lint index, high span length and medium bundle strength. Moreover, it occupies more ground area with more number of leaves and consequently high total leaf area but relatively low specific leaf area, specific leaf weight, canopy temperature and transpiration rate. A compact plant type can be characterized by short plant with intermediate petiole length, less number of sympodias, short fruiting branches, early flowering, low number of bolls and low seed cotton yield. Compact genotypes had superior fiber quality like high bundle strength, high lint index, medium span length

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

and seed index. Compact types occupy less ground area with low total leaf area but they had relatively high specific leaf area, high leaf temperature and low transpiration rate. Compact genotypes had high leaf soluble proteins and chlorophyll contents. The semicompact types were intermediate for all the characters. The percentage of genotypes of each group under different range for different characters was calculated. In robust group, high frequency of genotypes were in the minimum range of expression for the characters number of flower bearing nodes, days to first boll bursting, days to fifty per cent boll bursting, specific leaf area, canopy temperature, diffusive resistance, chlorophyll a and oil content. For the characters petiole length, internode length, number of sympodia, length of sympodia, number of bolls, seed cotton yield, ginning outturn, 2.5 per cent span length, uniformity ratio, micronaire, bundle strength, elongation length, root length, photosynthetically active radiations, transpiration rate, phenol content, high frequency of genotypes fell in the intermediate range. More than fifty per cent of the robust genotypes had high total leaf area, soluble proteins and nitrate reductase activity. Compact group had majority of genotypes under low expression for the characters plant height, internode length, number of flower bearing nodes, number of bolls, seed cotton yield, specific leaf weight, diffusive resistance and chlorophyll a. All the accessions had registered low range for length of sympodia. Intermediate range was predominant for the character petiole length, number of sympodia, boll weight, lint index, ginning outturn, 2.5 per cent span length, uniformity ratio, micronaire, elongation percentage, canopy temperature and phenol content. High frequency of plants fell under the high range for bundle strength and nitrate reductase activity. Semicompact genotypes fell under the
86

intermediate range for most of the characters. The distribution of genotypes under the different levels of expression indicated that in general, robust genotypes can serve as donors for earliness, leaf area, soluble protein and nitrate reductase activity while compact genotypes can be considered for improving bundle strength, photosynthetically active radiations, and nitrate reductase activity. The genetic divergence in the genotypes was estimated by subjecting them to distance analysis, using Mahalanobis D2 statistics. A groupwise analysis of genetic divergence indicated that the sixty seven robust genotypes could be grouped into 13 clusters. It was observed that cluster I was the largest including 54 genotypes followed by cluster XIII comprising of two genotypes. All the other clusters had only one genotype. In a similar way, the 66 semicompact genotypes came under twenty clusters. Cluster I comprised the maximum number of 14 genotypes followed by cluster II (13 genotypes) cluster III (11 genotypes), cluster VII (4 genotypes), cluster IX and XII (3 genotypes), cluster XIII, XV and XX (2 genotypes). All the other clusters had only one genotype. The 17 compact genotypes which were subjected to diversity analysis using 12 characters after stepwise elimination of less important characters were grouped into five clusters. Cluster I comprised the maximum number of nine genotypes followed by cluster II (5 genotypes). Cluster III, IV and V had only one genotype each. The above grouping supported that visual evaluation was in good agreement with the character evaluation in respect of robust and compact types as most of the genotypes came under a single cluster. However, the agreement in respect of semicompact types was not as much as in the other two groups because for some characters it is towards robust type and for others it is towards compact type and so further detailed study is needed. The clustering pattern of the

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genotypes from various geographical regions into different clusters was random indicating the absence of parallelism between genetic grouping and diversity. Earlier studies of Kowsalya and Raveendran (1996) and Gururajan and Manickam (2002) also indicated more are less similar observations. This may be due to frequent exchange of breeding material between the breeders and common objectives of selection in different locations. Murthy and Arunachalam (1966) also suggested that the forces of genetic drift and natural selection under diverse environmental conditions within a country cause considerable diversity than geographic isolation. So, selection of parents for hybridization programmes should be based on genetic rather than the geographical diversity. However, a comparison between the two methods of parental selection based on geographical and genetic diversity, and study of segregating progenies of the hybrids synthesized within each group will give a better result on further use of parents. Inter cluster distances were greater than intra cluster distances, revealing considerable amount of genetic diversity among genotypes studied. Use of genetically distant genotypes as parents to get most promising hybrids or segregants have been suggested by Kowsalya and Raveendran (1996), Manimaran and Raveendran (2001) and Gururajan and Manickam (2002). In case of robust genotypes (Table 2) the minimum inter cluster distance was recorded between the genotypes S-1622 and 560 whereas highest distance was noticed between cluster II(Able - 51(P)) and cluster XIII (Gregg and 5143) followed by cluster XII (920) and cluster XIII. Cluster XIII recorded highest mean value for the characters ginning outturn, specific leaf area, micronaire and elongation percentage (Table 3). Cluster II showed low mean values for all the characters. Cluster XII recorded high mean values for number of bolls, specific leaf weight and 2.5 per cent span length. It would be
87

a good effort to hybridize the genotype 920 with genotypes of cluster XIII to get better segregants showing good performance for yield components, earliness and fibre quality. Cluster IX (Empire-16 WR) also can be involved in hybridization programme to improve the seed cotton yield. In semicompact group, the lowest inter cluster distance recorded was between clusters IV and XI and highest distance was recorded between clusters XIII and XIV followed by clusters XIII and XVI and cluster VI and XVIII(table 4). Cluster XIII (Stoneville and Acala-1577-D) recorded the highest mean value for specific leaf area (table 5). Cluster XIV (47-2) showed high expression for plant height, number of sympodia and number of bolls. These results indicate that Stoneville and Acale-1577-D can be crossed with 47-2 to get desirable recombinants. Further cluster XVI (Nectariless) which had recorded second highest distance with cluster XIII showed desirable expression for quality traits viz. uniformity ratio, micronaire value and elongation percentage. Thus, to combine high physiological efficiency and good fibre quality characters, cluster XIII and cluster XVI can be used in crossing programme while, cluster VIII (Buri147) will serve as a good source for yield improvement. To produce hybrids with wide genetic base and with pronounced hybrid vigour this genotype can be crossed with any other highly divergent cluster having desirable genotypes. The compact genotypes (table 6) registered highest inter cluster divergence between cluster II (Kapland, BP-52NC-62, Stardel, Brazos and Deltapine) and cluster III (72/1). Cluster II showed high expression for bundle strength whereas cluster III registered high sympodial number and specific leaf weight (table 7). The cluster IV (199F) recording high mean values for seed cotton yield, number of bolls, length of sympodia, plant height, internode and petiole

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Plant Breeding in Post Genomics Era

length showed high divergence with cluster II. So 199F can be hybridized with the genotypes of cluster II to improve the seed cotton yield. The data pertaining to robust and semicompact genotypes (table 8) have also shown that quality characters were found to be good indices for selection of genotypes in the present study. As the yield and yield components failed to exhibit high degree of influence on genetic divergence, care should be taken to identify segregants for good yield performance from the limited variability available in the material under study. Similar reports have been given by Amudha et al. (1997).

REFERENCES Amudha, K., Raveendran, T.S, Krishnadoss, D. 1997. Genetic diversity in coloured linted cotton varieties.Madras Agric. J.,84:334337 Gururajan, K.N., Manickam, S. 2002. Genetic divergence in Egyptian cotton (Gossypium barbadenseL.).J. Indian Soc. Cotton Improv., 27: 77-83. Kowsalya, R., Raveendran, T.S. 1996. Genetic variability and D2 analysis in upland cotton. Crop Res. 12: 36-42. Manimaran, R., Raveendran, T.S. 2001. Relationship between genetic diversity and heterosis in cotton. Crop Res. 22 : 72-77. Murthy, B.R, Arunachalam, V. 1966. The nature of genetic divergence in relation to breeding systemin crop plants. IndianJ.Genet., 26(A):188-198

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Plant Breeding in Post Genomics Era

Table1. Grade index for the three plant types

Characters Plant height Petiole length Internode length Number of sympodia Length of sympodium Number of flowering bearing nodes Days to first flowering Days to first boll bursting Number of bolls Boll weight Seed cotton yield Seed index Lint index Ginning outturn 2.5% Span length Uniformity ratio Micronaire Bundle strength Elongation percentage Total leaf area Specific leaf area Specific leaf weight Leaf area index Root length Canopy temperature Photosynthetically active radiations Transpiration rate Diffusive resistance Phenol content Soluble proteins Chlorophyll a Chlorophyll b Nitrate reductase activity Oil content

Grade index for plant type Robust 2.37 2.16 1.87 2.24 2.43 1.31 1.97 2.34 2.07 1.94 2.04 1.42 2.25 2.39 2.10 2.27 2.25 1.85 2.00 2.36 2.70 1.72 2.36 1.78 1.91 2.30 2.10 1.20 2.10 2.20 1.40 2.40 2.20 1.60 Semi compact 1.91 1.82 1.50 1.97 1.55 1.29 2.20 2.30 1.79 2.09 1.79 1.94 1.65 2.24 1.94 2.33 2.03 1.83 1.95 1.88 2.70 1.67 1.88 1.62 2.20 2.60 1.80 1.20 2.40 2.00 1.90 1.90 2.10 1.80 Compact 1.47 1.88 1.35 1.65 1.00 1.24 2.12 2.12 1.47 1.88 1.47 1.94 2.24 2.35 1.88 2.29 2.12 2.59 2.00 1.59 2.41 1.41 1.59 1.79 2.24 2.50 2.30 1.30 2.10 2.80 1.80 2.30 2.30 1.90

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Table 2. Inter and intra cluster distances (D) (D2 values in brackets) in robust genotypes

Clusters I I 9.09 (82.68) II

II 10.01 (100.11) 0.00 (0.00)

III

III 10.01 (100.28) 11.35 (128.91) 0.00 (0.00)

IV

IV 10.00 (99.91) 11.09 (122.97) 10.24 (104.83) 0.00 (0.00)

Second National Plant Breeding Congress 2006

V 10.20 (104.03) 10.93 (119.39) 10.53 (110.79) 8.19 (67.05) 0.00 (0.00)

VI

VI 10.16 (103.17) 11.81 (139.58) 12.16 (147.75) 9.39 (88.26) 9.51 (90.35) 0.00 (0.00)

90

VII

VII 10.66 (113.67) 13.07 (170.86) 10.76 (115.76) 9.93 (98.54) 11.14 (124.04) 9.91 (98.20) 0.00 (0.00)

VIII

VIII 10.44 (109.09) 12.08 (146.04) 9.42 (88.71) 11.85 (140.46) 11.00 (121.04) 11.58 (134.01) 11.81 (139.46) 0.00 (0.00)

IX

IX 10.23 (104.60) 9.91 (98.29) 9.62 (92.53) 11.22 (125.83) 12.16 (147.87) 12.75 (162.59) 11.60 (134.64) 12.21 (149.17) 0.00 (0.00)

X 10.01 (100.26) 10.78 (116.27) 9.53 (90.83) 10.97 (120.37) 12.29 (150.94) 12.12 (146.86) 11.54 (133.18) 10.99 (120.73) 9.22 (85.04) 0.00 (0.00)

XI

XI 10.11 (102.21) 10.33 (106.65) 11.42 (130.42) 11.79 (139.05) 12.65 (160.08) 11.47 (131.56) 12.04 (144.93) 11.24 (126.23) 10.64 (113.23) 8.68 (75.36) 0.00 (0.00)

Plant Breeding in Post Genomics Era

XII

XII 11.17 (124.80) 11.93 (142.24) 9.65 (93.18) 10.70 (114.40) 8.27 (68.43) 11.92 (142.07) 12.06 (145.54) 10.24 (104.89) 12.37 (152.90) 12.84 (164.77) 13.44 (180.60) 0.00 (0.00)

XIII

XIII 12.34 (152.20) 14.43 (208.23) 11.71 (137.02) 11.26 (126.85) 13.18 (173.73) 12.15 (147.70) 9.94 (98.72) 12.79 (163.58) 12.79 (163.48) 11.03 (121.76) 12.45 (155.08) 13.96 (194.98) 0.00 (0.00)

Table 3. Mean values of 13 clusters for different characters in robust genotypes


DFF NOB DFBB DFFBB BW (g) SI SCY (g/ plant) 51.98 48.64 38.79 53.96 47.50 53.26 44.52 32.57 55.73 38.32 48.37 51.02 48.43 8.77 7.24 6.94 5.24 5.18 5.50 7.05 4.07 35.64 38.62 32.86 43.14 7.57 3.58 37.58 8.92 7.48 9.09 10.6 8.50 8.55 5.54 39.36 8.92 116.26 111.72 135.85 120.30 106.55 136.02 8.15 5.06 38.34 8.32 124.00 7.04 4.15 37.40 10.0 93.53 3.50 4.50 1.47 4.25 1.76 1.94 2.47 1.28 8.99 5.36 38.39 8.90 108.60 2.72 26.45 31.70 30.40 16.45 20.75 15.95 27.00 18.10 30.85 8.61 5.15 36.23 7.89 133.33 2.15 33.60 27.20 30.10 24.30 25.20 27.90 25.70 25.00 23.40 31.40 23.30 8.17 5.30 39.37 8.30 121.74 2.09 37.05 27.10 8.95 5.15 36.49 8.21 123.27 3.41 22.70 26.51 47.00 2.80 43.00 3.20 47.00 4.10 46.00 4.10 47.00 4.70 48.00 4.70 46.00 4.00 47.00 2.80 50.00 3.60 52.00 3.70 43.00 3.60 49.00 4.90 7.98 4.68 37.03 9.06 114.71 3.34 24.87 26.21 47.78 3.79 19.85 18.80 20.20 17.70 20.00 17.70 19.10 23.00 19.10 19.80 19.80 21.70 18.00 LI UR GOT (%) SLW LA LAI (mg/cm) (cm/g) RL (cm) 2.5% SL MIC BS (g/tex) EL

PH (cm)

IL (cm)

PL (cm)

NOS

LOS NFB (cm) N

I 59.50 60.00 61.00 61.00 62.00 53.50 64.50 60.50 58.50 55.50 62.00 58.50 12.50 167.00 172.00 3.81 18.00 125.50 130.00 2.90 13.84 131.50 138.00 3.60 13.50 165.00 169.00 2.84 17.33 140.00 149.00 3.13 9.84 140.00 149.50 3.37 15.67 130.50 137.00 3.32 10.34 137.00 142.00 5.00 12.83 137.50 143.00 4.75 12.50 140.00 149.50 4.21 15.33 141.50 149.50 2.59 11.00 136.50 141.50 4.96

120.75

5.20

11.28 18.78

40.44

1.52

58.66

14.58

141.43

147.21

3.73

6.00 5.40 6.20 6.10 4.70 5.10 7.40 5.00 7.70 7.30 7.00 4.51 8.20

II

136.30

5.08

9.35

16.92

38.17

2.50

III

141.95

4.50

11.22 19.50

39.50

2.42

IV

121.07

6.05

11.55 19.50

43.50

1.33

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124.82

5.50

9.50

21.50

37.83

1.00

VI

102.50

4.70

12.25 19.33

30.92

2.00

VII

126.92

5.28

12.14 19.50

41.50

1.83

91

VIII

98.59

6.27

12.65 14.50

33.00

1.00

IX

120.85

5.03

10.78 21.00

52.34

1.00

130.84

6.45

12.54 13.17

33.67

2.58

XI

116.56

4.59

10.45 16.00

32.33

1.50

XII

111.48

3.78

8.67

16.50

41.34

1.42

Plant Breeding in Post Genomics Era

XIII

89.38

4.44

12.59 10.09

35.34

1.50

Table 4. Inter and intra cluster distances (D) (D2 values in brackets) in semi compact genotype III 7.95 (63.21) 8.33 (69.34) 6.45 (41.62) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 6.59 (43.45) (49.10) 7.01 9.70 (94.18) 10.05 (100.99) 0.00 (0.00) (76.49) (102.44) (116.46) 8.75 10.12 10.79 (66.61) (42.53) (76.66) (77.64) 8.16 6.52 8.76 8.81 7.71 (59.39) 9.54 (90.99) 9.16 (83.92) 9.97 (99.40) 7.42 (55.10) 6.57 (43.19) (58.09) (50.05) (51.83) (71.69) (101.40) (86.44) 7.62 7.07 7.20 8.47 10.07 9.30 (55.35) (50.20) (81.64) (110.37) (101.46) (70.54) (53.71) 10.07 (101.38) 8.38 (70.27) 9.18 (84.30) 10.34 (107.00) 11.66 (136.06) 9.69 (93.87) 8.04 (64.60) 0.00 (0.00) 7.44 7.09 9.04 10.51 10.07 8.40 7.33 (54.43) (81.51) (57.57) (69.70) (60.63) (61.25) (93.37) 7.38 9.03 7.59 8.35 7.79 7.83 9.66 7.51 (56.47) 7.21 (51.95) 7.60 (57.74) 5.95 (35.36) 7.61 (57.92) 7.44 (55.38) 9.46 (89.53) 9.05 (81.84) 7.99 (63.78) 8.07 (65.17) 0.00 (0.00) IV V VI VII VIII IX X XI

Clusters

II

6.61

8.91

(43.74)

(79.47)

II

6.35

(40.28)

III

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IV

92

VI

VII

VIII

IX

Plant Breeding in Post Genomics Era

XI

Table 4. contd...

Clusters I

II

III

IV

VI

Second National Plant Breeding Congress 2006

VII

VIII

IX

93

XI

XII

XII 9.37 (87.83) 9.34 (87.24) 10.71 (114.64) 8.75 (76.59) 10.70 (114.54) 10.44 (108.97) 11.51 (132.55) 8.30 (68.92) 8.06 (64.90) 7.73 (59.80) 8.58 (73.65) 6.67 (44.44)

XIII

XIII 10.32 (106.51) 7.62 (58.10) 8.98 (80.62) 8.93 (79.70) 7.44 (55.33) 9.87 (97.36) 11.06 (122.39) 11.67 (136.13) 9.99 (99.76) 9.16 (83.85) 8.98 (80.62) 11.12 (123.58) 5.99 (35.88)

XIV

XIV 8.86 (78.55) 10.80 (116.56) 10.91 (118.99) 9.59 (91.88) 11.53 (132.94) 10.34 (106.98) 10.87 (118.26) 6.23 (38.78) 8.78 (77.05) 10.12 (102.41) 9.62 (92.53) 8.42 (70.86) 12.52 (156.70) 0.00 (0.00)

XV

XV 9.18 (84.33) 10.23 (104.65) 8.89 (78.99) 8.71 (75.92) 10.08 (101.55) 7.64 (58.40) 7.36 (54.10) 10.66 (113.67) 9.93 (98.70) 11.40 (129.96) 9.80 (96.06) 11.68 (136.31) 10.44 (108.95) 11.54 (133.22) 6.24 (38.89)

XVI

XVI 7.97 (63.47) 11.13 (123.78) 9.72 (94.47) 9.58 (91.72) 11.06 (122.22) 9.01 (81.26) 8.78 (77.10) 8.10 (65.69) 9.75 (95.12) 11.61 (134.85) 9.59 (91.92) 10.57 (111.77) 12.46 (155.14) 8.38 (70.19) 10.33 (106.68) 0.00 (0.00)

XVII

XVII 8.95 (80.13) 10.29 (105.87) 10.89 (118.68) 8.95 (80.15) 11.36 (129.08) 10.25 (105.08) 10.97 (120.39) 6.33 (40.12) 7.94 (63.12) 9.28 (86.14) 9.27 (85.86) 7.10 (50.48) 11.97 (143.32) 6.26 (39.17) 11.31 (127.82) 9.43 (88.85) 0.00 (0.00)

Plant Breeding in Post Genomics Era

XVIII

XVIII 9.74 (94.78) 8.81 (77.70) 10.71 (114.62) 9.29 (86.29) 10.24 (104.92) 10.86 (117.95) 11.99 (143.87) 9.00 (81.03) 8.26 (68.23) 6.41 (41.10) 8.90 (79.18) 7.36 (54.24) 10.78 (116.15) 8.96 (80.27) 12.04 (145.01) 11.20 (125.51) 8.51 (72.39) 0.00 (0.00)

XIX

XIX 8.32 (69.18) 9.42 (88.65) 8.13 (66.02) 8.11 (65.81) 8.94 (80.01) 7.94 (63.07) 8.37 (70.00) 9.93 (98.55) 9.54 (91.03) 10.47 (109.57) 8.47 (71.67) 10.68 (114.03) 10.22 (104.45) 10.51 (110.49) 8.64 (74.60) 9.27 (86.02) 10.51 (110.37) 11.08 (122.86) 0.00 (0.00)

XX

XX 7.88 (62.05) 9.83 (96.63) 9.50 (90.22) 8.45 (71.45) 10.39 (108.05) 8.67 (75.14) 9.28 (86.06) 7.59 (57.61) 7.83 (61.29) 10.02 (100.36) 8.76 (76.80) 9.54 (91.07) 11.06 (122.39) 8.81 (77.61) 9.59 (92.01) 8.93 (79.69) 8.74 (76.36) 9.83 (96.70) 10.33 (106.63) 0.00 (0.00)

Table 5. Mean values of 20 clusters for different characters in semi compact genotypes
LOS (cm) DFF SI SCY (g/ plant) EL 45.48 7.70 43.25 8.62 47.43 8.03 45.04 8.46 54.71 9.62 22.72 8.44 32.65 7.81 67.08 9.62 39.97 7.87 44.04 9.70 43.70 6.89 56.86 7.96 42.92 6.81 52.12 7.70 36.78 8.67 59.95 8.15 60.38 8.56 27.18 9.27 71.39 8.14 40.76 9.24 3.93 4.97 5.43 5.09 4.36 5.02 3.65 34.06 31.10 37.15 37.03 34.80 35.97 36.00 3.92 35.11 4.52 36.13 3.95 40.99 12.6192.11 9.93 101.66 6.84 155.17 10.6695.27 10.59104.77 9.03 122.67 9.44 103.52 8.81 113.57 8.30 121.57 9.05 122.07 4.65 33.05 7.85 122.72 4.07 33.69 9.96 115.93 3.11 3.27 2.92 4.26 1.41 4.48 1.47 3.46 4.47 3.86 2.29 2.98 5.07 33.96 8.86 109.87 3.90 32.75 25.58 26.70 26.85 23.88 20.80 30.10 19.68 21.35 29.25 18.50 13.60 23.95 4.24 35.69 9.35 112.61 2.00 24.65 4.96 37.53 8.93 111.48 2.30 27.75 24.90 23.60 25.10 26.97 30.00 26.80 26.30 26.75 26.60 25.55 24.70 25.60 29.20 26.10 28.30 4.66 39.43 8.03 122.75 1.68 33.00 26.80 5.68 40.16 7.39 128.69 2.76 31.65 26.00 4.55 37.04 8.38 121.53 1.89 23.38 24.41 48.83 4.24 49.00 3.91 47.00 4.30 48.00 4.20 50.50 3.83 47.00 3.00 46.67 3.33 46.00 3.80 47.00 4.60 47.75 3.90 47.50 3.40 45.00 3.51 47.50 3.40 51.00 4.60 46.00 3.20 44.00 3.80 47.00 3.80 45.00 3.40 4.65 35.92 8.05 127.64 2.35 24.08 27.12 47.28 4.10 20.51 18.53 19.20 18.51 18.20 20.20 18.80 21.60 21.10 19.10 20.05 18.65 18.60 23.55 20.00 19.20 20.30 19.90 21.00 4.60 37.08 8.76 116.58 2.85 24.33 25.37 49.23 3.90 19.51 6.55 4.93 6.23 6.20 6.00 7.10 6.83 6.40 5.27 5.10 6.30 5.08 5.25 7.00 5.55 7.60 5.90 5.10 6.90 6.70 LI UR 27.48 25.34 28.73 25.00 29.50 24.59 26.15 27.34 26.20 25.00 28.00 30.56 24.83 22.00 26.50 28.00 26.08 29.00 27.08 24.17 1.33 68.50 16.50 166.50 140.50 1.00 68.50 15.09 130.50 167.00 4.73 2.49 1.33 68.50 12.00 134.50 139.00 2.27 2.00 65.50 14.00 161.00 166.50 4.26 1.00 69.00 17.67 140.00 149.00 3.43 1.87 68.75 13.84 154.50 160.75 2.64 1.00 69.00 18.67 150.50 156.00 2.92 1.54 68.00 10.54 136.00 143.50 4.20 2.00 67.63 13.56 153.88 160.50 4.21 1.33 66.00 10.17 156.00 159.00 4.29 1.50 68.00 13.00 141.50 149.00 3.41 2.25 67.17 17.50 134.50 140.67 2.31 2.17 69.00 17.34 140.50 143.50 3.95 1.46 64.38 9.65 148.88 156.88 3.56 1.42 63.50 7.17 167.00 170.00 3.09 1.83 62.50 15.67 130.00 137.00 3.49 1.33 69.00 11.33 164.50 169.00 3.96 1.66 66.08 11.51 137.17 142.83 4.15 1.37 67.42 12.36 136.42 142.96 3.56 1.43 66.03 12.86 137.63 144.20 3.90 NFB N NOB DFBB DFFBB BW (g) GOT SLW (%) (mg/ cm) LA LAI (cm/g) RL (cm) 2.5% SL MIC BS (g/tex)

PH (cm)

IL (cm)

PL (cm)

NOS

103.22

4.52

10.97

17.89

II

98.84

4.52

10.28

15.00

III

104.20

4.38

9.84

16.75

IV

101.04

5.17

11.15

14.17

101.73

4.21

11.26

15.00

VI

104.43

4.50

11.27

13.00

VII

102.15

3.74

10.74

18.37

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VIII

126.51

5.59

9.67

14.25

IX

97.42

5.87

11.39

13.39

107.86

3.82

8.97

16.25

94

XI

77.40

3.67

8.75

14.09

XII

95.61

4.97

11.05

17.35

XIII

63.83

3.54

8.54

11.83

XIV

160.52

5.55

11.92

24.83

XV

122.92

4.89

11.92

18.29

XVI

124.10

5.15

11.42

21.83

XVII

118.75

8.10

12.00

16.38

XVIII

151.42

4.58

9.67

19.00

XIX

111.54

3.50

8.92

23.92

Plant Breeding in Post Genomics Era

XX

121.09

4.40

11.59

15.67

Table 6. Intra and Inter cluster distances (D) (D2 values in brackets) in compact genotypes

Clusters 5.77 (33.33) 8.41 (70.70) 7.49 (56.07) 6.49 (42.16) 7.84 (61.45)

II

III

IV

Second National Plant Breeding Congress 2006

II

5.91 (34.95)

33.39 (111.03)

9.63 (92.67)

9.88 (97.65)

III

95

0.00 (0.00)

6.73 (45.30)

7.91 (62.60)

IV

0.00 (0.00)

8.93 (79.67)

Plant Breeding in Post Genomics Era

0.00 (0.00)

Table 7. Mean values of five clusters for different characters in compact genotypes

Character IL (cm) NOB 11.15 10.37 11.16 15.66 7.66 21.29 7.71 117.03 68.53 8.04 129.20 3.05 0.87 32.16 9.05 110.29 3.01 25.30 24.90 28.20 30.83 9.00 120.85 1.59 25.38 21.36 16.90 18.40 19.50 40.67 7.61 137.38 2.46 25.46 19.19 LAI 3.95 4.89 3.49 8.08 4.31 11.58 12.00 14.83 11.91 15.00 20.49 9.21 20.83 17.83 11.34 12.61 19.50 10.56 14.71 18.64 PL NOS (cm) LOS (cm) 2.5% SL BS(g/ tex)

Cluster

PH (cm)

SCY (g/ plant)

SLW SLA (mg/ (cm/g) cm)

87.14

Second National Plant Breeding Congress 2006

II

87.30

III

85.57

IV

97.50

96

83.15

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Table 8. Percentage contribution of different characters to total genetic divergence in robust, semicompact and compact genotypes

Character

Contribution (percent) Robust Semicompact 0.09 2.08 1.96 0.70 5.83 3.08 2.33 44.76 38.69 100 2.21 72.79 18.38 4.41 0.74 100 Compact 1.47 -

Plant height Days to first boll bursting Days to fifty percent boll bursting Seed cotton yield Bundle strength Micronaire Uniformity ratio Elongation length Leaf area index Specific leaf area Specific leaf weight Total

1.04 0.09 0.14 17.37 22.57 22.52 28.99 7.28 100

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INTERFAMILY VARIATION AND FAMILY SELECTION IN INTERVARIETAL CROSSES IN SUGARCANE UNDER EXCESS WATER STRESS CONDITION
Govindaraj, P

ABSTRACT
In sugarcane breeding programmes two methods of selection viz., family selection and individual progeny selection are followed. Family selection even though laborious gives much dividend compared to individual selection. Excess water stress is one of major limiting factor of productivity in North Central Zone. Even though targeted breeding programes have not been initiated so far, sugarcane breeders have always look for clones with water logging tolerance as an ancillary character in addition to cane yield, sucrose content in juice, red rot resistance and tolerant to top borer in the regular breeding programme. The main effect of excess water stress is not only yield but also sugar recovery due to accumulations of low sucrose in juice at harvest. In order to study the family variation under excess water stress conditions, two hundred progenies developed from 8 families of sugarcane intervarietal crosses were planted clonally in single row plot of 6m and were evaluated for their performance to 4 quality parameters recorded in 8th and 11th months and 3 yield contributing traits. High variation was observed among the families for all the characters. Family mean values for CCS % at 11th month ranged from 11.52 (UP 22 X Co 775) to 10.30 (CoSe 92423 X CoS 510) with overall mean value of 11.09 and two families exceeded this mean value. Sucrose % in juice at 8th month ranged from 12.33 (CoS 88216 X Co 87272) to 14.89 (CoG 93076 X Co 93009) with overall family mean value of 14.22. For single cane weight, the range was from 1.41kg (UP 22 X Co 775) to 0.82 kg (CoS 932 X BO 91) with the family mean value of 1.11 kg. Range for each characters also varied among the families. While the range for the family CoG 93076 X Co 93009 was the highest (13.30 to 7.05), the family CoS 88216 X Co 87272 had the narrow range (11.74 to 10.37) for sucrose % at 11th month. For single cane weight, the highest range was recorded by UP 22 X Co 775 (2.30 0.70 kg) and the lowest range was observed with CoS 90269 X CoS 510 (1.15 0.75 kg). Variance estimate also differed among the crosses. The highest variance component for sucrose was exhibited by CoG 93076 X Co 93009 (24.35) and the lowest variance was recorded by Co 1158 X CoJ 64 (7.92). Results clearly indicated that variation was observed for both quality and yield traits among the families. The families with the highest variance resulted in the progenies with maximum per se values for CCS % at 11th month and single cane weight the important quality and yield contributing trait respectively. Hence it is concluded that in the early generation of selection family selection followed by individual selection will improve the efficiency of selection and such families with wider variation should be repeated as proven crosses. Interfamily differences for economic traits and the importance of family selection is discussed.

Introduction Modern sugarcane varieties are the complex hybrids involving different species of Saccharum. A wide range of variability among seedlings ranging from wild cane (Saccharum spontaneum) to noble cane (S. officinarum) is observed among the intervarietal progenies. The
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frequency of seedlings having desirable agronomical traits depend upon the parental combination used, genetic control of the trait and selection efficiency. Very limited studies on inheritance of agronomically important traits have been made in sugarcane due to its complex genetic architecture and non-

Senior Scientist (Plant Breeding), Sugarcane Breeding Institute, Coimbatore 7

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fulfillment of certain assumption or design. Sugarcane breeders worldwide differ in their opinion on selection in the early segregating generation ie individual performance or family per se. In the individual selection the assumption of gene x environmental variance is considered as negligible hence the genotype selected in the first generation is fixed in the later clonal generations. However another school of thought argues that in the absence of any statistical procedure adapted, the families with high mean performance is selected and further individual selection is within the selected families only. In addition, the families with high mean performance is repeated (proven crosses) to produce larger families to recover more elite segregants. Increasing population, improved standard of living and demand for sugar necessitated to expand the cultivation to the sub optimal areas like water logging. In North central zone of India, the main constraints are the early drought and late water logging. Water logging is mainly due to excess rain during Aug-Sep and poor drainage in many parts of Bihar, Eastern Uttar Pradesh and certain pockets of Orissa. Under severe waterlogging, growth of the crop is reduced and sugar recovery also affected. Since it is very difficult to manage waterlogging stress through either agronomical or physiological manipulations, development of water logging tolerant varieties is the most appropriate solution and incorporation of water logging tolerance is an integral part of varietal development for these areas. Hence, it is essential to breed varieties suitable to waterlogging condition and the breeders should know appropriate breeding procedures to be adapted for this purpose. In the present study variability for economic traits and their relationship to selection efficiency is discussed. Materials and method Eight different parental combinations were constituted with seven pistil parents viz., Co
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1158, CoS 932, UP 22, CoG 93076, CoS 92423, CoS 88216, CoS 90269 and seven pollen parents viz., CoJ 64, BO 91, Co 775, Co 93009, Co 62198, Co 87272 and CoS 510 among them UP 22, CoS 92423, BO 91 and Co 87272 were tolerant to water logging. Crosses were effected in the lantern method and fluff were sown to raise segregating progenies. Two hundred progenies developed from these 8 families of sugarcane intervarietal crosses were planted clonally in single row plot of 6m and were evaluated. Data on juice brix% and sucrose% were estimated at 8th and 11th month and CCS % and Purity % were worked out. CCS % = (1.022 x Sucrose %) (0.292 x Brix %); Purity % = Sucrose % x 100/ Brix % Three important yield-contributing traits viz., single cane weight (SCW), cane length (CL) and cane girth (CG) were recorded in all the progenies and mean, variance and range were estimated. Results and discussion Genetic potential of sugarcane families to produce superior seedlings (elite genotypes) can be estimated through several methods which include factors for superior performance (FSP) by Arceneaux et al. (1986), the probability of exceeding target value (PROB) (Milligan and Legendre, 1991) and a univariate cross prediction method (Chang and Milligan, 1992). The factors for superior performance (FSP) method is easy to use, but a FSP value can only be obtained after the original seedlings have been carried through all stages of selections. The univariate cross prediction method described by Chang and Milligan (1992) requires extensive data collection. Sugarcane breeder needs a method, which is very simple to estimate but reliable and repeatable. Simple estimate of co efficient of variation, mean and range can also bring out required information to understand the genetic potential of the segregating families.

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The selection percentage is a measure of the overall merit of the cross, which represents all the aspects of desirability considered in these stages and the weight given to each component character by the selector (Walker, 1963). A high selection percentage indicates that the population had a high mean and/or variance for some or all desirable characters. Tai and Miller (1989) reported that selection rate between early stages of selection was highly correlated. A progeny test with small number of individuals is routinely used to estimate the selection rate or the evaluation of proven crosses in sugarcane breeding programmes in Australia (Hogarth, 1987). The progeny assessment trials also have been routinely used to identify the best families and select superior clones from these families (Cox et al. 2000). Stalk height and stalk weight were strongly correlated with cane yield both in dry and wet zone and can be considered as ideal trait for selection in these stress conditions (Bissessur et al., 2001 and Brown et al., 1968). Under water and salt stress conditions, stalk diameter has the highest heritability and is the most reliable character for selection (Bakshi Ram et al., 2001). Hence all these three easily measurable traits were recorded as yield contributing traits (Malavia and Ramani, 1992) in the progenies. In the present study, variance estimate differed among the crosses for yield and quality characters (Table 1). The highest variance component for sucrose at 11th month was exhibited by CoG 93076 X Co 93009 (24.35) and the lowest variance was recorded by Co 1158 X CoJ 64 (7.92) and the same trend was observed for CCS %. Many families recorded lower variance (<10) and the existence of low variability for quality characters indicates the difficulty in improving quality traits by selection. The large amount of variability in single cane weight is in agreement with the observation of Wright (1956) on the existence of large amount of variability in hybrid population of asexually
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propagated out breeding species. Variability was observed among the families for mean values for all the characters except for cane length, cane girth and purity %. Family mean values for CCS % at 11th month ranged from 11.52 (UP 22 X Co 775) to 10.30 (CoSe 92423 X CoS 510) with over all mean value of 11.09 and two families exceeded this mean value. Sucrose % in juice at 8th month ranged from 12.33 (CoS 88216 X Co 87272) to 14.89 (CoG 93076 X Co 93009) with over all family mean value of 14.22. For single cane weight, the range was from 1.41kg (UP 22 X Co 775) to 0.82 kg (CoS 932 X BO 91) with the family mean value of 1.11 kg. Range for all characters also varied among the families. While the range for the family CoG 93076 X Co 93009 was the highest (13.30 to 7.05), the family CoS 88216 X Co 87272 had the narrow range (11.74 to 10.37) for sucrose % at 11th month. For single cane weight, the highest range was recorded by UP 22 X Co 775 (2.30 to 0.70 kg) and the lowest range was observed with CoS 90269 X CoS 510 (1.15 to 0.75 kg). In accordance with these results, in a random population Balasundaram and Bhagyalakshmi (1978) reported the range for stalk thickness (1.28 to 3.21 cm), stalk length ( 112.6 to 190.1 cm) single stalk weight (0.28 to 1.52 kg) and sucrose % (11.14 to 16.90). Number of selection for various traits in relation to mean and variance are given in table 2. Selections were made for each character based on selection cutoff value (single cane weight :1.0 kg, cane length : 200 cm, cane girth : 2.5 cm and sucrose % 16.0). For single cane weight, high variance (10.12) and high mean value (1.14 kg) resulted in high selection (12) in UP 22 x Co 775 and the same trend reflected in CoSe 92423 x CoS 510. The estimates are less in either variance or mean number of selections are also less as evidenced from other two crosses. Sucrose % at 11th month, more number of selections were obtained in UP 22 x

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Co 775 and CoSe 92423 x CoS 510 due to high mean and variance respectively. For cane girth CoG 93076 x Co 93009 and UP 22 x Co 775 produced more number of selectable sergeants due to variance and high mean respectively. The families UP 22 x Co 775 and CoSe 92423 x CoS 510 recorded more family mean values whereas CoG 93076 x Co 93009 registered more variance to produce more selections. Hence it is evident that selection based on family per se followed by variance will yield more selections than selection based on individual performance to harness additive genetic variance. Simultaneous selection for different economical traits is possible only when the components have positive association. A number of studies have examined relationships among components of cane and sugar yield with genetically different populations and varying sample size. Relationships among component traits have been found to be affected by different genetic background of populations (Bakshi Ram and Hemaprabha, 1991), water stress (Bakshi Ram et al., 2001) and selection ( Bakshi Ram et al., 2000). Under excess water stress condition, the highest significant positive correlation was observed between sucrose % and brix % at 8th (0.9726) and 11th (0.9475) months indicating that brix % which can be estimated easily can be used as indirect selection for sucrose % in larger segregating population (Table 3). Like wise, Brix at 8th month had significant positive association with brix % (0.3575) and sucrose % (0.3508) at 11th month signifying the possibility of early election for quality traits in early generation. All the three yield contributing traits had positive significant inter correlations among themselves. In the earlier studies also the association between stalks weight and stalk diameter (Madhavi et al., 2002, Kang et al., 1983, Verma et al., 1988) and stalk weight and stalk length (Madavi, et al., 2002) were found to be positive and significant. Earlier studies indicated that stalk
101

weight had low positive correlation with sucrose percent (Balasundaram and Bhagyalakshmi, 1978; Hogarth, 1971). Hence it is concluded that stalk diameter can be regarded as most stable character under different environmental conditions and can be considered for selection in various stress environment (Bakshi Ram et al., 2001; Tai and Miller, 1989; Ortiz and Caballero, 1989; Bakshi Ram and Hemaprabha 1991 and Bakshi Ram et al., 1996) which can be also measured in larger population in short time. Summary Improvement of sugarcane seedling population by eliminating inferior progenies should increase the frequency of elite clones and increase the selection efficiency. The objective of this study is to evaluate the different families for their efficiency in giving higher selectable segregants and suggest that the families with higher frequency of selectable segregant would be designated as proven cross and the same can be repeated for raising larger families and effect selections. Instead of selecting the best progeny from the populations raised, selecting the best individual from the best families would be a stable performer. Results clearly indicated that variation was observed for both quality and yield traits among the families. The families with the highest variance resulted in the progenies with maximum per se values for CCS % at 11th month and single cane weight the important quality and yield contributing trait respectively. Hence it is concluded that in the early generation of selection family selection followed by individual selection will improve the efficiency of selection and such families with wider variation should be repeated as proven crosses. The families with high mean value threw more number of selections for yield components. Single cane weight had significant positive association with cane length and cane girth. Hence these two easily observable

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characters cane be used for selection in early generation with the greater emphasis on stalk diameter. For quality traits Brix % had significant positive association with sucrose per cent both in 8th and 11th month hence spindle brix % can be used for selection in early generation. REFERENCES Arceneaux, G.J.F., Van Breemen. and Despradel, J.O. 1986. A new approach in sugarcane breeding: Comparative study of progenies for incidence of superior seedlings. Sugar Cane 0 7-10 Bakshi Ram. and Hemaprabha, G. 1991. Character relationship in cultivar x species progenies in sugarcane. Indian J. Genet. 51: 89-95 Bakshi Ram Sahi, B.K. and Chaudhary, B.S. 2000. Effect of selection stages on relationships between attributes in sugarcane. Sugarcane International 8: 511 Bakshi Ram, Chaudhary, B.S. and Singh, S. 1996. Repeatability of important traits among seedling, ratoon of seedlings and settling stages in three population of sugarcane (Saccharum spp. Hybrids) Indian J. Agric Sci. 66: 546-548 Bakshi Ram, Kumar, S., Sahi, B.K. and Tripathi, B.K. (2001). Traits for selecting elite sugarcane clones under water and salt stress conditions. Proc. IISCT 431-438. Balasundaram, N. and Bhagyalakshmi, K.V. 1978. Variability, heritability and association among yield and yield components in sugarcane. Indian J. Agric. Sci. 48:291295. Bissessur, D., Tiney-Bassett, R.A.E and Lim Shin Chong Lim. 2001. Genetic potential of sugarcane progenies grown in extremely wet and dry environments in Mauritius. Sugarcane International Nov: 5-10 Brown, A.H.D., Daniel, J. and Latter, B.D.H.
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1968. Quantitative genetics of sugarcane II. Correlation analysis of continuous characters in relation to hybrid sugarcane breeding. Theoretical and Applied Genetics. 38: 1-10 Chang, Y.S. and Milligan, S.B. 1992. Estimating the potential of sugarcane families to produce elite genotypes using univariate cross prediction methods. Theoretical and Applied Genetics. 84: 662-671 Cox, M.C., Hogarth, D.M. and Smith, G.R. 2000. Cane breeding and improvement. In. D.M. Hogarth and Allsopp P.G. (Eds.) Manual of cane growing. Bureau of Sugar Experiment Station, Brisbane, Queensland, Australia pp 91-108 Hogarth, D.M. 1971. Quantitative inheritance studies. II. Correlation and predicted response to selection. Aust. J. Agric. Res. 22: 103-109 Hogarth, D.M. 1987. Genetics of sugarcane. In. Heinz (Editor), Sugarcane improvement through breeding. Elsvier, New York. Pp 255-272 Kang, M.S., Miller, J.D. and Pai, P.Y.P. (1983). Genetic and phenotypic path analysis and heritability in sugarcane breeding. Crop Science, 23: 643-647 Madhavi, D., Reddy, C.R., Reddy, P.M., Reddy G.L.K., Reddy, K.R. and Reddy, K.H.P. 2002. Correlation Studies in sugarcane. Co-operative Sugars. 22: 379-381 Malavia, D. D. and Ramani, V.V 1992. Correlation path analysis of cane yield in sugarcane. Indian Sugars. 4: 19-22 Milligan, S.B. and Legendre, B.L. 1991. Development of a practical method for sugarcane cross appraisal. J. Am. Soc. Sugarcane Tech. 11: 59-68 Ortiz, R. and Caballero A. (1989). Feasibility of using family selection at the sugarcane seedling stage. Cultivos Tropicales, 11:

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27-33 Tai, P.Y.P. and Miller, J.D. 1989. Family performance in early stages of selection and frequency of superior clones from crosses among canal Point cultivars of sugarcane. J. Am. Soc. Sug. Tech, 9:62-70 Verma, P.S., Dhaka, R.P.S. and Singh, H.N. 1998. Genetic variability and correlation

studies in sugarcane. Indian J. Genet. 48: 2132-217 Walker, D.I.T. 1963. Family performance at early selection stages as a guide to the breeding programme. Proc. IISCT 11:469483 Wright, S. 1956. Modes of selection. Am.Nat, 90:5-24

Table 1. Estimates of different parameters for families under water logging conditions at 8th and 11th months
Families Para meter Brix % Brix Suc. Suc. % CCS CCS Purity Purity Single Cane CaneGirth % 11th % 8th 11th % 8th % 11th % 8th % 11th Cane length cm 8th month month month month month month month month Weight cm kg 19.02 20.24 16.68 18.80 11.49 13.30 90.07 95.14 2.30 3.20 3.30 13.62 15.46 10.86 13.27 7.12 9.05 79.74 85.83 0.70 1.55 2.00 16.45 18.15 13.99 16.46 9.49 11.52 84.89 90.67 1.41 2.55 2.74 9.94 7.46 14.07 8.45 12.17 6.80 8.71 4.38 10.12 4.91 3.62 20.95 20.75 17.50 18.94 11.78 13.30 91.64 95.25 1.55 3.35 3.70 13.16 13.16 10.66 10.66 7.05 7.05 81.00 81.00 0.30 1.30 1.90 17.27 17.14 14.89 15.60 10.17 10.94 86.09 90.90 1.06 2.41 2.56 19.11 22.02 21.22 24.35 16.82 19.25 9.37 13.11 12.91 10.39 5.93 19.05 20.96 16.44 19.18 11.78 13.48 89.45 93.08 1.20 3.10 2.80 13.16 14.96 10.36 12.78 6.59 8.69 75.62 82.83 0.25 1.20 1.50 16.50 18.15 14.07 15.90 9.71 10.95 85.06 87.54 0.88 2.20 2.27 12.19 11.12 17.30 13.47 14.61 11.23 11.26 8.05 5.17 11.40 4.74 17.88 19.96 15.54 17.58 10.66 12.14 88.83 89.86 1.60 3.00 2.80 14.10 14.28 10.86 12.58 6.98 8.69 77.02 85.20 0.65 2.10 1.80 16.41 17.02 13.81 14.94 9.32 10.30 83.98 87.75 1.19 2.59 2.48 8.16 23.22 14.60 22.82 13.53 16.71 12.96 1.99 11.06 3.21 3.81 16.10 19.66 12.97 17.10 8.55 11.74 81.02 88.05 1.75 2.50 3.30 14.70 17.16 11.30 15.11 7.26 10.37 76.87 86.98 1.05 2.10 2.50 15.50 18.06 12.33 15.77 8.08 10.85 79.48 87.36 1.40 2.27 2.83 3.35 10.69 6.58 8.37 6.29 5.47 6.51 0.42 8.75 1.91 6.12 19.08 20.06 16.44 18.30 11.25 12.85 86.16 91.23 1.15 3.00 2.70 14.70 17.76 11.80 15.54 7.77 10.70 80.27 85.39 0.75 2.10 1.90 17.00 18.94 14.25 16.62 9.99 11.45 83.63 87.71 0.94 2.49 2.33 18.90 4.66 25.34 8.44 27.23 7.98 7.18 7.14 5.04 5.79 5.27 18.88 19.96 16.68 18.06 11.53 12.63 88.35 90.48 1.40 2.80 2.70 14.30 16.36 11.85 14.19 7.94 9.73 82.87 86.06 0.50 1.80 1.90 17.06 18.28 14.52 16.01 9.86 11.03 85.06 87.55 0.82 2.20 2.26 11.47 8.57 13.03 9.46 10.21 7.37 2.58 2.02 13.09 6.48 3.12 19.18 19.06 16.68 17.23 11.45 12.04 86.97 91.43 1.25 2.65 2.80 13.40 15.68 10.41 13.70 6.73 9.42 77.69 86.25 0.70 1.80 2.00 17.39 17.73 14.65 15.73 9.90 10.90 84.00 88.73 0.89 2.27 2.30 20.14 8.74 27.70 7.92 23.17 5.98 10.81 5.10 5.87 3.34 2.48 20.95 20.96 17.50 19.18 11.78 13.48 91.64 95.25 2.30 3.35 3.70 13.16 13.16 10.36 10.66 6.59 7.05 75.62 81.00 0.25 1.20 1.50 16.72 17.87 14.22 15.96 9.70 11.09 84.88 89.24 1.11 2.39 2.50 103

Max Min Mean CV Max CoG Min 93076 X Co 93009 Mean CV CoS Max 92423 X Min Co 62198 Mean CV Max CoSe 92423 X Min CoS 510 Mean CV Max CoS 88216 X Min Co 87272 Mean CV Max CoS 90269 X Min CoS 510 Mean CV Max CoS Min 932 X Mean BO 91 CV Max Co 1158 X Min CoJ 64 Mean CV Over all l Max Min Mean UP 22 X Co 775

Table 2. Number of selections, mean and variance in different families at 11th month

Traits 6 5 0 5 2.58 2.47 22.82 1.19 11.06 3.21 3.81 3.40

UP 22 X Co 775 Select Mean Variance ions Single cane 12 1.14 10.12 weight Cane length 5 2.55 4.92 Cane girth 9 2.74 3.62 Sucrose % 6 16.4 8.45 4 2 5 2.41 2.56 15.6 10.39 5.93 24.35 3 0 5 2.20 2.27 15.6 11.4 4.74 13.47

CoG 93076 X Co 93009 Select Mean Variance ions 2 1.06 12.91

CoS 92423 X Co 62198 Selections Mean Variance ions 0 0.88 5.17

CoSe 92423 X CoS 510 Select Mean Variance

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Table 3. hips among yield and quality characters under water logging conditions at 11th month

Brix % 11 Sucrose % Sucrose % Purity % 11 Single cane month 8 month 11 month month weight 0.3290* 0.9475** 0.1678 -0.0630 0.0970 -0.0456 0.3391* 0.3290* -0.1620 -0.0091 -0.0644 0.4726** 0.0159 0.1534 0.0412

Cane length

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Brix % 11 month Sucrose % 8 month Sucrose % 11 month Purity % 11 month Single cane weight Cane length Cane girth

Brix %8 month 0.3575* 0.9726** 0.3508* 0.3575* -0.1668 0.0069 -0.0557 0.2202* 0.2000 0.2538*

Plant Breeding in Post Genomics Era

0.6951** 0.7761**

0.4213**

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Fig 1. Frequency distribution for Sucrose % at 11 months


25 20 Sucrose % 15 10 5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 Genotypes Family I Family II Family III

Family I: UP 22 x Co 775 Family II: CoG 93076 x Co 93009 Family III: CoS 92423 x Co 62198

Fig 2. Frequency distribution for SCW at 11 months


2.5 2 SCW in kg 1.5 1 0.5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 Genotypes Family I Family II Family III

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DEVELOPING HIGH YIELDING RICE VARIETIES FOR KERALA-A NEW APPROACH


Chandrasekharan, P

ABSTRACT
Crosses were done between promising rice accessions from different parts of Kerala during 1996-97, hybrids were identified and subsequent selections continued based on height (80 -120 cm), higher productive tillers, longer panicles and high grain numbers and matta (bold grain and red rice). Those selected were given PC numbers. PC 1 was identified in F6, PC 2 in F7 and both were released in February 2003. Four more short duration varieties have been identified: PC 5 in F9, PC 3, PC 4 and PC 6 in F10. These were evaluated in yield trials in the second crop of 2004-05 and in the first crop 2005-06 and the superiority of their performance was confirmed. PC 3 Sivam and PC 6 Sundaram are two varieties where genes responsible for high grain number exceeding 400 grains from TKTM have been transferred in full since the latter was the donor for this trait. PC 1 and PC 2 have now spread to about 600 and 500 acres respectively during the last 4 seasons in almost all districts of Kerala.

Introduction Thavalakkannan is a tall indica variety and phenotypically very similar to Chenkazhama and both were very popular among Palakkad rice farmers before the introduction of high yielding dwarfs by IRRI, Philippines. Extreme palatability of their rice, the ability of cooked rice to preserve in cold water for 12 hours without losing hardness and taste and their high recovery from paddy after milling (above 60 %) were the welcome traits. Main difference between these two varieties is that Chenkazhama is a week earlier than Thavalakkannan (TK) of 135 days .To ensure maximum plant diversity, TK was collected from a farmer in Thiruvilvamala (TKTM) and Chenkazhama from another in Ottappalam (OTP), in 1995. In the first crop of 1996-97, when TKTM population was studied, it was surprising to observe one plant of height 182 cm, main panicle length of 31 cm and had 430 spikelets (potential grains), two plants had 344 and 327 and others between 188 and 282 spikelets. This population of TKTM represented grain production of a very high order when compared with the grain numbers of IR 36, IR 8, Athira and Matta Thriveni, the dwarfs then in cultivation, their maximum -grain number being
Tamil Nadu Agricultural University, Coimbatore 641003

214. Other varieties, or TK (PTB 8, PTB 9) and Chenkazhama (PTB 26) and the one collected from Ottappalam (OTP) and included in the present study had the maximum of 250 grains on the main panicle. Thus TKTM is a distinctly superior variety from the point of view of the plant breeder. Since all characters are governed by genes, it should be possible to transfer this high grain number to the currently cultivated semi - tall and dwarf varieties. Crosses were done from 1996 - 97, hybrids were identified and subsequent selection continued based on height (80-120 cm) higher productive tillers, longer panicle, high grain number and Matta (bold grain; red rice) which farmers of Kerala prefer. Rice farmer of Palakkad gets Rs. 300 more for 500 kg paddy if it belongs to the category, Matta. Materials and methods Rice varieties, PTB 8, 9 (TK Matta and TK White rice), IR 8 and IR 36 were obtained from Pattambi Rice Research Station as also PTB 26 (Chenkazhama). Other varieties, Athira and Matta Thriveni (dwarfs of Kerala Agricultural University) were obtained from local farmers. Since the first crop commencing May is the most suitable for TK and Chenkazhama,
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crossing work was invariably done in first crop. Farmers do not grow TK and Chenkazhama in second (II) crop since their yield performance is poor. Seeds collected from crossing, had to be sown and therefore, in the majority of cases, sowings had to be done in the second crop for the identification of hybrids. Dwarf varieties are grown in both crops (I and II) and therefore, selections in succeeding generations have been done in both the crops and better looking plants carried forward. Selection work up to II crop was done at Mannapra. From the year 2000, the work was done at Alampallam, Vandazhi and Vadakkencherry, all in Palakkad district. Since the aim of the study was to transfer the high grain-number of the tall Indica variety of Thavalakkannan from Thiruvilvamala (TKTM), four important characters, (I) height of the plant in cm, (2) number of productive tillers, (3) length of panicle and (4) number of spikelets on the main panicle were recorded for all plants selected. Only in the final stages was the duration of the crop recorded. Those selected were initially given culture numbers and yield trials were conducted following standard procedures. Those finally selected, based on grain yield, were given PC (my initials) numbers and also named, to distinguish them from other varieties. PC 1 was identified in F6, PC 2 in F7 (in the farmers fields at Alampallam and Vandazhi respectively) and named second coming of Thavalkkannan and Chenkazhama respectively by Karshaka Sree, a monthly Publication of Malayala Manorama for Kerala farmers in its February 2003 issue. Four more short duration varieties were identified: PC 5 Santham in F9 and PC 3 Sivam, PC 4 Sathyam, PC 6 Sundaramin F10. These short duration varieties were all evaluated in the second crop of 2004-05 and their yield performance confirmed in first crop of 2005-06 before naming them. Details of these varieties were brought to the notice of the Palakkad farmers by the Malayalam daily, Mathrubhoomi on February 6th
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2006 followed by Karshaka Sree, in March 2006. The Scientific aspects of the study are presented and discussed in this paper. Results and Discussion Table 1 gives details of these characters for I and II crops. Data for the second (II) crop clearly show reduction in height, length of panicle and number of spikeiets while the tiller number gets increased in the II crop in some (e.g., TKTM;Chenkazhama (OTP), IR 8). Excepting PC 6, Sundaram, all others showed the ability of producing more tillers, especially in the II crop (Table, 4). Table 2 gives details of number of inter varietal hybrids between tall Indica (TKTM) and Chenkazhama (OTP) and dwarf rice varieties IR 8, IR 72 (IRRI) matta Thriveni, Athira (Kerala Agr. Univ.varieties). Spikelet fertility or seed - fertility percentage provides an index of relationship (close or otherwise) of varieties involved in the cross. Usually, it is the maximum fertility that should be compared and for the study to be completed a number of F1 hybrids should be available. Seed fertility of IR 8 x PTB 8 and Chenkazhama (OTP) X IR 8 are similar indicating that Thavalkkannan Matta of Pattambi research .station (PTB 8) is similar in genetic relationship to Chenkazhama of Ottappalam (OTP). Seed fertility of TKTM X IR 8, TKTM X Matta Thriveni and its reciprocal (about 30 %) showed that IR 8 and Matta Thriveni are the most diverged from TKTM. Seed fertility of IR 36, Athira with TKTM (maximum 69 %) compared with those of Chenkazhama (maximum 99 %) do indicate great divergence of TKTM and Chenkazhama (OTP). If fertility indicates close relationship, seed sterility (100seed fertility per cent) percentage shows how far are the two varieties distant. An attempt has been made to pictorially represent these varieties, TKTM and Chenkazhama (OTP) based on sterility per cent. While Chenkazhama

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(OTP) is comparatively recent in evolutionary scale, TKTM is ancient. Table 3 gives details of the maximum grain number recorded and the number of selections made in F2 to F10 as well as details of range of number of spikelets in F1 generation. However, it is the responsibility of the plant Breader to select a variety if it is likely to be superior to the current variety under cultivation by the farmer. This happened in the case of the cross, IR 36 X TKTM. In this cross although the range of number of spikelets in the 3 F1 hybrids suggested maximum of 322, maximum of 272 was reached in F6 in 135 days of duration; further increase was difficult to be achieved. The figures underlined indicate those studied in II crop and the number of 272 was achieved in the II crop and since this variety had grains which are bold and 1000 grains weighed 28 g (more than the weight of TKTM) the plants of this line was harvested and seeds mixed to form a culture. After yield trials, this was named PC 1. In the case of the cross, Athira X Chenkazhama, there were eight F1 hybrids raised in the second crop. Subsequently in each generation there was rise in the maximum of spikelets if the crop was in I crop, a temporary decrease if it was in II crop and a dramatic increase in the subsequent I crop (216,388 in I crops in F2, F3 289 in second crop of F4and 379 in I crop of F5). Maximum of 496 was reached in F8. By that time the panicles started showing symptoms of disease and selection work had to be restricted to F7 (maximum of 417 spikelets). As can be seen in table 4 this was a semi - tall (about 120 cm). One thousand grains weighed 25.0 g. Yield trial of this culture was done at Vandazhy Village of Palakkad District. PC 1 is dwarf and capable of yielding 6.0 mt/ha in a duration of 135 days while P6 2 gives 5.6 mt/ha in 120 days (Table 5). Subsequently PC 1 and PC2 have now spread to 600 and 500 acres respectively in almost all the districts of Kerala, during the last four Seasons.
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TKTM X Athira This cross needs special mention because it has yielded two short duration varieties, one of 100 and the other of 110 days duration. This cross succeeded only when Thavalkkannan was used as mother. Three F1 hybrids were identified in the second crop (Table 3) and their maximum spikelets ranged from 127 to 184. Thereafter it rose to 369 in F3 and the maximum number of 413 was reached in F10 in the I crop of 2004 - 05. One particular plant needed special mention. It was semi-tall (122 cm), had 6 tillers: main tiller carried 413 spikelets and 370, 345, 337, 174 and 158 in second to sixth respectively, of spikelets totaling 1797 grains per plant in the first crop. However, the progeny of the same plant showed maximum of 216 in the II crop (Table, 4). The culture emanating from the progeny of this line recorded maximum of 6.375 mt/ha in the second crop of 2004-2005, the maximum under the short-duration category (Table 5), with 110 days duration. Another distinct group of plants in this cross was shorter (107 cm), with 5 productive tillers and 435 spikelets in I crop of 2004-05; in the II crop, the height was reduced to 80 cm and spikelets to 181. However, the culture that emanated from this plant - progeny proved to be shortest in duration of 100 days and the grain yield recorded from the yield trial was 5.579 mt/ha. Higher yielding 110 days duration, PC 3 , was named Sivam and the shortest duration of 100 days, PC 6, Sundaram. TKTM X matta Thriveni In this cross, both the cross and their reciprocals were successful; seven F1s were identified in the former and two in the latter (Table 2). Subsequently, the aim was to select all progenies showing higher fertility, more productive tillers and number of grains, reduction in height of the plant and in the process the progenies of Matta Thriveni X TKTM got eliminated. Here also plant

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parameters got reduced in the II crop compared to the I; however, F3 plant 24 proved to be the better in the I and II crop, the difference being the minimum. A number of progenies of this plant figured in subsequent generations and in the tenth (F10) it was decided to make a culture of seeds of all the plants and conduct yield trial in the II crop of 2004-05. The best one, recorded 5.963 mt/ha. This was numbered PC 4 named Sathyam. It is a dwarf capable of producing more tillers (upto 17) in II crop and maximum of 266 spikelets (Table 4), of 110 days duration. TKTM X IR 8 Since this cross with 14 F1 hybrids with maximum spikelet fertility of 26.3 %, selection from second generation onwards was based on higher fertility, more productive tillers and spikelet number (Tables 2,3). Maximum spikelet number of 299 was achieved in F9. In the first crop, the plant had height of 105 cm, 6 tillers, panicle length of 32 cm. This culture recorded the lowest yield of 4.083 mt/ha in II crop (Table 5). This culture had largest sized seeds and highest weight of 30.5 g for 1000 grains (Table 6). The variety is PC 5 and named Santham. In the yield trial conducted in the first crop of 2005-06, with more rains received, it yielded 6.114 mt/ha. The larger sized grain of this variety is preferred for consumption in Thrissur, Kollam and Kottayam in addition to making avil (beaten rice), a value added product fetching a premium price for the farmer. Currently, it is Jyothi, another existing variety, derived from crossing IR 8, that is used for making avil. In the trial in the I crop of 200506, it was found that Santham produced grain yield of 0.85 mt/ha more than Jyothi, with a 7 days more duration. It is interesting to note that a medium duration variety like TKTM (Tavalakkannan from Thiruvilvamala) and a near medium duration variety, Athira had given rise to two short duration varieties, one with 110 days and another of 100 days in the second crop, where Athira takes 125 days for maturity. This is the only cross where
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genes responsible for high grain member exceeding 400 grains from TKTM have been transferred in full. This can only be explained by gene -transfer as a result of chiasma formation in meiosis between homologous regions of genes (chromatids) responsible for determining duration of these two varieties. Since linked genes near the centromere are difficult to be broken, chromatid ends of chromosomes generally take part in chiasma formation (Elliott, 1958; Darlington and Mather, 1950). If the above assumption is correct, it would mean that duration determining loci constitute a complex unit of a few genes which can give rise to shorter duration varieties under certain conditions. A Palakkad Rice Farmer at present gets maximum of 4 mt/ha and these varieties would be a welcome addition to him. That Palakkad district, enjoying 35 % of its land under irrigation from river valley projects, produces only 2 mt/ha and Kerala is a deficit State producing only one fifth of its requirement, calls for more short duration very high yielding rices suited to varying agroclimatic Zones and it is this need that varieties like the above (PC 3 to PC 6) would fulfil. As far as I am aware, this is the first report of transfer of genes responsible for 430 grains from a tall Indica like TKTM to two shorter duration (100 to 110 days) varieties in F10, while both parents had duration of 125 to 135 days. PC 3, Sivam had 413 and PC 6 Sundaram 435 grains; the increased grain yield in these two varieties is not solely due to high grain number but also due to higher productive tillers. Higher tillers which are productive were also observed in all these varieties, excepting PC 6, Sundaram. PC 2, Chenkazhama (secondcoming) and PC 3 Sivam are two semi-tall varieties selected not only for grain-yield, but also due to need for straw for cattle that a small farmer will maintain in his farm. The highest grain yielder that ushered in the green revolution in Rice, IR 8 of International

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Rice Research Institute, Philippines produces, on an average, 150 grains per main panicle and as part of upgrading rice production, IRRI is trying to bring 200 grains per panicle and in this project IR 72 has been made use of. The climatic conditions of Palakkad are different from those of Philippines and would account for low yields especially the water deficit. This situation can be changed only when more short duration, very high grain yielders like the PC 3 Sivam, PC 4 Sathyam, PC 5 Santham and PC 6 Sundaram with 100 to 115 days in duration are introduced. Grain size and weight of 1000 grains of these varieties are given in Table 6. Palakkad farmers prefer grains like Thavalkkannan and Chenkazhama while Kottayam and Kollam farmers prefer a little more longer and wider grains that Jyothi or Santham (PC 5) provide. Gradual introduction of Varieties like these short duration varieties depending on their suitability, in different climatic zones in the State would bring about self sufficiency in rice production in the State. By 2020 it is feared that the growth of population would outstrip grain production in India and Kerala can face the future, with confidence. Significance of Thavalakkannan from Thiruvilvamala, tall Indica rice Variety Vavilov (1926 and 1949 - 1950 In Elliott, 1958) designated eight main ancient and independent world centres of diversity for .our major crop plants. Of these, South east Asia was considered the second main centre of origin involving Hindustan (including Burma and Siam), the Malay Archipelago, Java, Borneo, Sumatra, the Philippines and Indo-china. This area was the centre of origin for rice, sugarcane, numerous legumes and many tropical fruits. This region includes India and Palakkad district is considered the Rice-Bowl of Kerala. Almost all varieties cultivated here are grown through out Kerala. Krishnaswamy and Chandrasekharan (1957) reported a naturally occurring tetraploid species of Oryza, O.mlampuzhaensis from
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Malampuzha of Palakkad. Although the diploid of the species O.officinalis occurs naturally in Assam, its tetraploid species occurred here. The fact that a variety like Thavalakkannan has many varied known forms (matta TK - PTB 8, white rice TK-PTB 9, the present TKTM studied now) and its close phenotypic similarity to Chenkazhama (PTB 26) and currently studied Chenkazhama (OTP) which are all genetically different show great diversity. Both Thavalakkannan and Chenkazhama have violet (reddish-purple) coloured leaf sheath, leaf margin and leaf - junction, tip of glumes and when exposed to continued sunshine the upper surface of the flag-leaf also assumed violet colour. Sterile glumes of PTB 8 as well as the tip of glumes show extended violet colour. Excepting that Chenkazhama is a week earlier in duration to TK, there is no difference between the two. The maximum grain number in PTB 8, PTB 9, PTB 26 (Chenkazhama) and Chenkazhama (OTP) does not exceed 250. Therefore the variety, TKTM which possessed 430 grains is an exceptionally superior variety. Another important point to note is that while IRRI varieties show panicle length of about 24 cm, more frequently, varieties like Thavalakkannan and Chenkazhama show panicles of 28 - 30 cm indicating that our forefathers, as practical breeders, had preferred longer panicled rice plants with more grains per panicle for improvement in grain yield. All these would suggest that the superiority of TKTM among rice genotypes of the region and a secondary centre of origin for the cultivated varieties of rice. It is a reasonable hypothesis to suggest that both Thavalakkannan and Chenkazhama have evolved from a common ancestor with high grain number or that the varieties now known as land races, TK and Chenkazhma have originated from TKTM. Another important aspect is the tendency of TKTM to form more productive tillers in the II crop which it passes on to its progenies (genetic).

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PC 1 to PC 5 clearly showed this trait (Table 4). More Productive tillers would certainly increase the grain yield as indeed the experience of other farmers of Kollam, Kottayam and Thrissur where PC 1 and PC 2 have been tried and tested for grain yield, getting pure seeds from progressive farmers associated with this work. Some details are given below. PC 1 and PC 2 were released for general cultivation in February 2003 and has grown in cultivation through the effort of individual farmers and Seed procuring Agencies to the extent of 600 and 500 acres respectively. The observation of different farmers of Kerala in different parts of the state are: Palakkad region Many farmers have reported that 6.0 mt/ha can be obtained from PC 1 for both I and II crop. Shri. Rajakrishnan of Alampallam, Palakkad who supplied pure seeds of the variety to others have shown that upto 7.5 mt/ha can be obtained by varying cultural practices. PC2, semi-tall variety yields 5.6 mt/ha. Kole cultivation, Thrissur Lvla Ravindranath of Kanattukara found that PC 1 can multiply 47 times by volume and PC 2, 40 times. Her neighbouring farmers who inspected the crop in the I crop season said that even though PC 2 yielded grains less, they wanted straw for their cattle and liked PC 2 also. The farmer then tried PC2 for puncha third crop and obtained a bumper yield of 8.4 mt/ha. Cool region, Wvanad Mr. L.G.Robinson and A.M. Joseph tested PC 1 and found they could obtain 6.72 mt/ha. Kottayam, Kumarakom region Here too much water is their problem. Since Kuttanad is a region below the sea level, they frequently get water level rising rapidly remaining for some time and draining. Alexander Chako, a farmer of the region, Kumarakom
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obtained 28 quintals per acre despite the crop drowning in water twice (7 mt/ha). Mr. C.K.Das, Managing Director, Rajiv Gandhi Paddy Procurement and Marketing Society has obtained 10.8 metric tones of pure seeds for sowing and planting large area, has informed me that the performance of the crop is excellent in the area. George Joseph of Alappuzha who has sown both PC 1 and PC 2 feel that a very good crop of paddy, 6.75 mt/ ha, is now grown where his farm of 3 acres was running at a loss of Rs.4000 every season. All the above facts clearly bring about the fact that TKTM is an ancient and most successful variety of Palakkad, being cultivated by our fore-fathers for hundreds of years in all the varied conditions of Kerala and should have accumulated necessary genetic changes in its genome and developed built - in tolerance for varied adverse conditions which occur in the State. I have discovered it accidentally and transferred its yielding ability to the currently grown dwarfs resulting in very high yielding dwarfs (Table 5) which are also Matta (Table 6) preferred in the State. I believe these short duration varieties would aiso perform similarly and help increase the average yield of rice which remains static around 2 mt/ ha in Kerala State. Highest grain number found in the variety is proof that it can tolerate higher temperature prevalent in the district. On the other hand, studies done by the IRRI scientists (Peng et al 2004) have shown that rise of one degree Celsius in night temperature decreases rice grain yields by 10 per cent, of IRRI varieties, which form the dwarf parents in crosses. REFERENCES Darlington, CD., and K. Mather. 1950. The elements of Genetics. The Macillan Company, New York. Elliott Fred, C. 1958. Plant Breeding and Cytogenetics. McGraw Hill Book

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Company, London. Krishnaswamy, N. and P.Chandrasekharan. (1957) Note on a naturally occurring tetraploid species of Oryza. Sci. and Cult. 23: 307-310. Peng Shaobing, Jianliang Huang, John, E., Sheehy, Rebecca, C., Laza, Romeo, M., Visperas, Xuhuazhong, Grace, S,. Centeno, Grudev, S., Khush. and Kenneth, G., Cassman. 2004. Rice yields decline with higher night temperature from global warming. PNAS 101, 27 : 9971 - 9975.

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Table 1. Some important parameters of varieties used in crosses Chenkazhama (OTP) I Crop 193 81 7 20.5 70 105 100 184 107 214 130 27.5 23 24.5 5 5 5 4 23 135 125 90 73 3 28.4 213 128 109 76 II Crop I Crop II Crop I Crop Matta Thriveni IR 8 Athira IR 36

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Thavalakkannan Thiruvilvamala (TKTM) II Crop 161 12 24.2 160

I Crop

II Crop I Crop II Crop I Crop II Crop 96 5 26.7 169 125 71 13 22.5 159 120 97 5 24.5 211 125 70 5 23 134 120
Plant Breeding in Post Genomics Era

Height (cm)

182

113

Tillers

Length of Panicle cm)

31

No. of Spikelets

430

Duration (Days)

135

Table 2. Seed fertility among F1 hybrids of TKTM, Chenkzhama (OTP) with dwarf varieties of Rice SI.No Female parent Male parent Number Percent seed fertility in of hybrids F1 hybrids 1 29.6

SI. Female parent No


fertility in F1 hybrids

Male parent Number of Percent seed hybrids

1.

10.6-26.3

IR8

Chenkazhama (OTP)

2.

1 3 11 7 2 3 13 26.1-27.6 62-68 17.8-30 12 matta Thriveni Chenkazhama(OTP) Athira 62-69 49-69 10 11

41.8

IR8 IR72 Chenkazhama (OTP)

2 10 10 7

42.1-50.3 66-95 70-95 90-99

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3. 4.

ThavalakkannanIR8 14 Thiruvilvamala (TKTM) IR8 PTB8 (TK matta) IR36 TKTM TKTM IR36 Chenkazhama (OTP) -doIR36

5. TKTM Athira

TKTM

matta Thriveni

6. 7.

Matta Thriveni TKTM

Chenkazhama (OTP)

55-95

Table 3. Maximum grain number (Spikelets) in F1 and subsequent generations of varieties evolved

Variety No. Name of Variety Second coming Thavalakkannan (3) 226-322 (23) 180 (2) 246 (17) 252 (7) 272

Varieties inCross

F1(Range) N
F3 F4 F5 F6 F7 F8 F9

F 10

PC1

IR36xTKTM

PC2

PC3

AthiraxChenkazhama (OTP) TKTMxAthira

Second coming Chekazhama Sivam

(8) 113-139 (3) 127-184

(13) 216 (18) 241

(8) 388 (4) 369

(59) 289 (10) 208

(4) 211 (16) 203 (62) 379 (2) 313 (74) 266 (7) 172 (66) 496 * (8) 273

(5) 294

(2) 413

115

PC4 266

TKTM x matta Thriveni

Sathyam

(7) 61-206

(20) 295

(25) 240

(9) 217

(10) 233

(5)

PC5 Sundaram (18) 241

TKTMxlR8

Santham

PC6

TKTM x Athira

(14) 73-190 (3) 127-184

(33) 335 (4) 369

(4) 200 (10) 208

(6) 256 (2) 313

(5) 202 (4) 157 (0) 239 (7) 172

(88) 417 (13) 346 (7) 223 (8) 210 (8) 265 (2) 175 (13) 346

(2) 211 (8) 273 (7) 223

(4) 301 (2) 270 (2) 299 (5) 294

(2) 435

Figures with in ( ) indicate number of selection involved Figures underlined indicate study in second crop * Symptoms of disease affecting panicle with maximum spikelets

Table 4. Some parameters of varieties now evolved P C 3 Sivam TKTM x Athira P C4 Sathyam TKTM x matta Thiriveni I Crop 88 7 25.5 237 115 105 6 32 299 115 84 17 25.5 266 110 82 15 23 230 110 107 5 27.5 435 107 II Crop I Crop II Crop I Crop II Crop 80.4 22 18.1 100
PC5 Santham TKTM x IR 8

PC 1 IR36xTKTM PC2 Ahira x Chenkazhama P C6 Sundaram TKTM x Athira II Crop 90 17 28 272 128 120 8 29 401 120 22 6 30.5 413 117 98 17 26.5 30.5 11.5 75 10 23 216 110 I Crop II Crop I Crop II Crop

I Crop

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Height (cm) Tillers Length of Panicle cm) No. of Spikelets Duration (Days)

101 6 25 211 135

Table 5. Grain yield of varieties evolved


Duration (days) Parentage Metric Tonnes per

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No. of variety Name of the variety hect-are

PC1

Thavalakkannan

135

IR36xTKTM

6000 -7000

(Seond coming) 120 zhama OTP 110 110 Thriveni 115 100 TKTMxlR8 TKTMxAthira 4083 - 6114 5579 TKTM x matta TKTMxAthira Athirax Chenka5600 - 8440 6375 5963

PC2

Chenkazhama

(Seond coming)

PC3

Sivam

Plant Breeding in Post Genomics Era

PC4

Sathyam

PC5

Santham

PC6

Sundaram

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Table 6. Gain size and weight of short duration varieties

GRAIN SIZE Thavalakkannan Thirvilvamala (TKTM) Chenkazhama (OTP) Aiswarya Jyothi P C3 Sivam P C4 Sathyam PC5Santham P C6 Sundaram Length (mm) Mean 7.77 Range 7.0-8.5 Width (mm) Mean 3.4 Range 3.0-3.5

Weight of 1000 grains (g)

27.0

7.70 8.80 9.10 8.30 8.08 9.10 7.70

7.5-8.0 8.5-9.0 8.0-10.0 8.0-9.0 8.0-8.5 8.5-9.5 7.0-8.0

3.4 3.2 3.1 3.02 3.40 3.55 3.15

3.0-3.5 3.0-3.5 3.0-3.5 3.0-3.5 3.0-3.5 3.04.0 3.0-3.5

27.2 26.0 25.7 27.0 25.0 30.5 24.0

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TECHNICAL SESSION II QUANTITATIVE GENETICS AND ANALYSIS OF GENOTYPE X ENVIRONMENT INTERACTION

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QUANTITATIVE GENETICS - WHERE ARE WE TODAY?


Arunachalam, V

ABSTRACT
The evolution of Quantitative Genetics concurrently with Population Genetics, from the time Charles Darwin discovered variation and provided a logical frame, has been documented extensively. The monumental work of the greats - Sir Ronald Fisher, Sewall Green Wright and John B.S. Haldane that shaped Quantitative Genetics, has been taken to greater heights by a number of well-known geneticists including Kenneth Mather, John L. Jinks, Oscar Kempthome, Ralph .E. Comstock, Harold F. Robinson, to name a few.

There are two avenues of utilizing a vast subject like Quantitative Genetics - one as an appetizing theory and its development that need a theoretical bent of mind including good knowledge of Statistics and elements of Mathematics, and the other as a beacon for targeted plant breeding. In the context of the former, in other countries good foundation of Mathematics and Statistics is laid at the school and undergraduate level; but diversity of test crops and of growing environments is not always available for practical testing of biological and quantitative genetic concepts. In India, the reverse is true with weak or absence of strong foundation of Mathematics and Statistics but unlimited diversity of crops and growing environments. This imbalance defies any innovative correction in India, leading, at times, to misplaced doubts of the practical relevance of the subject of Quantitative Genetics. However, any theory initiates under restricted and often indefensible assumptions and developed relaxing the assumptions in stages. For instance, the popular model, P = G + E or its extensions have untenable built-in assumptions. Good conceptual strides have been made in targeted plant breeding, with designs of field experiments, whose strong foundation were laid by Fisher, playing an important role. But the gaps between practical plant breeding and the prompts to it through quantitative genetics theory continue to remain wide.

In general, there are accepted lines of practices to breed for improvement of quantitative traits (QTs), like diversity evaluation, parental choice for hybridization designs and selective breeding. How far Quantitative Genetics guides a breeder for targeted improvement is an open question. At this point, strides made in molecular biology from around 1980s, were set to provide the new avenue. Capacities to identify markers of QTs using molecular tools started increasing; with a good funding back-up, research priorities got shifted to the new avenue at the cost of time-tested Mendeiian breeding. What pathways are explored to integrate the best of Mendelian and Molecular breeding (a relatively new subject)? What is the role of Quantitative Genetics here? Such questions are intriguing and annoying. Until we address them, sooner than later, the scope for realizing more than additive benefits from integrating Mendeiian and Molecular approaches would be elusive. This paper addresses issues central and peripheral to them. Charles Darwin first discovered variation after his voyage on H.M.S. Beagle. The genetic basis of variation became clear after the first Classical experiments on Plant hybridisation by Gregor Johann Mendel that helped in the formulation of the famous Laws of Heredity. Further work by various researchers on variation and evolution, including Francis Galton
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M.S. Swaminathan Research Foundation, 3rd Cross Road, Taramani Institutional Area, Chennai 600113

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led to two differing schools of thought - (1) Darwins school of continuous evolution (with those professing this school being called Biometricians) and (2) Galtons school of discontinuous evolution (with those professing this school being called Mendelians). But it was a lingering question whether the two schools were consistent with Darwins theory of evolution by natural selection. It was R.A. Fisher who synthesized the two schools and established the principle of natural selection, more rigorously than Darwin, as the cause of evolutionary change using a mathematical theory developed on the basis of extant genetic research (cf. Fisher, 1930). Further work by another peer geneticist, J.B.S. Haldane reestablished that natural selection was the premier mechanism of evolution. He explained it in terms of mathematical consequences of Mendeiian genetics. His book, The Causes of Evolution (1932), came to be known as the modern evolutionary synthesis. Sewall Wright, another great geneticist and contemporary of Fisher and Haldane, founded the theory of genetic drift (that later came to be known as the Sewall Wright effect), a random effect caused by random sampling of genotypes. Through this theory, he could explain how evolution occurred. This was hugely influential in the evolution of evolution theory itself. Wright further drew a pathway to visualise the relationship between genotype and phenotype. Thus Fisher, Haldane and Wright whose monumental work shaped Quantitative Genetics are known as the founders of Quantitative Genetics. Their work was taken to greater heights by a number of well-known geneticists including Kenneth Mather, John L. Jinks, Oscar Kempthorne, Ralph .E. Comstock, Harold F. Robinson, to name a few. The subject of Quantitative Genetics has two important utilities: 1. Analysis of genetic phenomena underlying QT expression, variation, and evolution using advances in theory [has specific congruence
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with Population Genetics]. To deal with this area without adequate knowledge of statistics and probability theory would be profoundly difficult. 2. Optimization of methods of plant breeding using the leads given by Quantitative Genetics concepts that would provide firm (with a probability) responses leading to more hits than misses. This are a is within the reach of plant breeders and applied geneticists and we shall deal with this area therefore in this paper. Any basic conceptualization initiates under restricted and often indefensible assumptions. A number of theoretical results obtained this way have to be understood in their proper perspective and great caution is needed before applying them to a practical problem. An example is given here to illustrate this point. It is known that a genotype needs an environment to express and manifests itself as phenotype; in this process genotype interacts with environment in a very complex way and in general, it is not possible to model phenotype (P) in terms of genotype (G) and environment (E). However, the theory starts with the simplest additive model, P = G + E Known Unknown

As indicated, in the above equation only phenotypic value (dependent variable) can be measured and the independent variables, G and E are unknown. Fisher circumvented this problem using the strategy of growing the phenotypes in the field using an appropriate design and estimating environmental variance (= error mean square). Again by simplifying assumptions that G and E are independent which is not so anyway, he showed that variance of G can be estimated as Variance of G = Variance of P - Variance

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of E, and symbolically,

genotypes, TT and tt. GY: genotype; f: frequency; A: additive effect; D: dominance effect; GV: Genetic value Hence in utilizing various test expressions, as for example, heterosis, heritability (both narrow and broad sense) and the like, it is crucial to be aware of the limitations before extrapolating decisions for further breeding in practical situations. Table 1. Genetic values partitioned into additive (A) and dominance (D) components
GY TT Tt tt f P2 2pq Att A D GV Remark A TT -2q2h G T T where h is the ATt +2pqh GTt phenotypic Gtt value of the -2p2h heterozygote, Tt, measured as the deviation

F2G = F2F - F2E


Likewise, the genetic value G in P = G + E is partitioned into two orthogonal components, A (Additive Effect) and D (Dominance Effect). It can be shown that A is also equivalent to Breeding Value and gca effect (Arunachalam, 1993). Without going to details, we recognize that a fit of a best possible straight line to the 3 phenotypic values of a population governed by a single diallelic gene leads to the value of A (fuller details in Li, 1955; Arunachalam and Owen, 1971) and the deviations from the fitted straight line leads to D (Table 1). What often times is not recognized is the fact that the estimate of A involves the frequency of genotypes in the population and in fact, A is a function of not only the value (phenotypic) but also the gene frequency of T (equivalently the frequencies of genotypes, TT, Tt and tt, see Table 1) in general. In the particular case of a population where the frequencies of TT, Tt and tt are V*, Vz and % (implying p, the gene frequency of T = V2), the expression for A seemingly does not contain p (but actually it contains p with the numerical value, p= 1/4 !). In the context of treatises on Quantitative Genetics being naive and inexplicit on this point, the above explanation becomes crucial (see also Arunachalam, 1976). We thus note that in partitioning G into A and D, the following assumptions are implicit: 1. Population governed by a single diallelic gene 2. Fit of a straight line or additive model to the 3 phenotypic values 3. At times, the added simplification by confining oneself to a single diallelicpopulation with frequency of dominant gene = Vz ;for this, it is assumed that an F2 population is generated from the cross, TT x tt , noting that it is hard to ensure selection of parental phenotypes to be completely homozygous to be of
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However the theory is further developed after relaxing the assumptions in stages, though it is difficult to develop it to exactly match any practical situation. From the days of Mendel, breeders differentiate between qualitative (with defined discrete classes) and quantitative (continuous variation preventing to define discrete classes) traits. However, with scientific advances, it is now possible to measure traits in continuous scale and therefore argue that only QTs exist and qualitative trait is not defensible. For example, leaf colour that is defined using discrete scores, like 1- dark green; 2- green; 3pale green can now be quantitatively measured as colour intensity in a continuous scale. The same argument holds for other traits defined by colour, for example flower colour. Other traits that are common are seedling vigour (that can now be measured as dry weight of young seedlings) and disease incidence (that can be measured as leaf area affected and similar other measurements).

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Mendel provided the foundation for associating control of a gene with a QT, based on its phenotypic expression, more specifically, the number and frequency of various phenotypes. But he dealt elaborately with single gene diallelic phenotypes, though he extended his laws to QTs under two gene control also. However, with the present knowledge, it is known that QTs are under polygenic and possibly multiallelic control. As we observed earlier, it is essential in this context to understand the limitations of generalizing results based on single diallelic gene to QTs under multigenic control. Fisher provided the base to explain the variation between the 3 genotypes of a single gene diallelic population. The total genetic variation with 2 degrees of freedom (d.f.) between the 3 genotypes can be partitioned as Additive genetic variance with 1 d.f. and dominance variance with 1 d.f. A natural extension to the two gene - diallelic case will partition the 8 d.f. between the 9 genotypes (noting that we do not consider linked genes here) as follows: Component symbol d.f. Additive(gene 1) Additive(gene 2) Ai A2 1 1 1 1

area etc. each of which is measurable. In turn, the component QTs are also be controlled by genes some or most of which could be linked (Fig 1). Expression of QTs is also subject to genotype x environment interaction. Plant breeding aims to use QTs in this backdrop; in other words, it is developed based on phenotype and environment. Quantitative Genetics develops appropriate theories and ways of efficient breeding. In contrast, molecular breeding, that is of recent origin, aims to modify/ incorporate genes (targeting specific ones being an arduous and comparatively expensive process) and thereby QTs, as would be clear later.

Dominance (gene 1)D^ Dominance (gene 2)D2

Fig 1. QT - Genetic and phenotypic perception Breeding for improved performance gets highly complicated as it demands improvement of QTs defined by various areas like Biochemistry, Physiology, Pathology, Entomology, Agronomy, Soil Science, Microbiology, Seed Science and the like. Pyramiding those QTs to high performance demands co-adapted function of various genes governing QTs. This would involve multiple stages of hybridization, number of generations and selection cycles. Naturally this process would take time. Advances in molecular biology have
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Additive x Additive Ai x A2 1 Additive DominanceAi x D2 1 Dominance x Additive Dominance x Dominance D2 x A^ 1 Di x D2 1

But, in reality, QTs are governed by a number of genes, often with linkage between them. Further QTs also admit of component QTs. For example, days to maturity has imbedded component QTs like date of sowing, seedling vigour, days to flower, photosynthetic

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Fig 2. Projected and Unprojected information on Mendelian and molecular principles of breeding

provided ways of finding molecular markers (now, a large variety) that can be closely linked, in principle, to QTs of interest, though the process involved could be cost and time-extensive. The differences between Mendelian gene and molecular marker have earlier been made clear (Arunachalam, 2005). Let us consider the simple breeding process in which QTs are controlled by single diallelic genes (Fig 2). The segregation pattern of a QT like flower colour is clear from Mendelian and Molecular standpoint. While dominance does not help to identify heterozygotes inFi or F2 based on colour phenotypes, molecular marker, in principle, can identify them, the mandatory condition being that the marker should be tightly linked to the QT, flower colour. In that case, F2 segregation would always be in the ratio 1:2:1 (codomiance) compared to Mendelian segregation (dominance, 3:1). This is a special advantage of using molecular marker to identify genotypes, though, in practice, such situations are infrequent. If we extend the above logic to a QT like plant height, F1 itself can display variation in height measurements that could be
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pronounced in F2 (Fig 2). As is evident then, plant height shows continuous variation and hence grouping them in discrete genetic classes is impossible. One may argue that breeders use inbred lines as parents and therefore the above contingency usually do not arise. But it is known that few generations of inbreeding cannot bring homozygosis, particularly when several genes control plant height; quantitative variation can then be found in F-i that would manifest into greater variation in F2 generation. Molecular methods use one or more single gene markers (that are always independent, contrasting reality) to group individuals (genotyping) using pnenotypic (expressed QT) values. In that case, it is possible that a parental segregant with a value deviating from its original value could be classified as a recombinant and likewise, a real recombinant could be classified as a parent. In contrast, classical breeding characterizes and selects individuals on a set of key traits that are dependent and controlled by many genes (not ruling out linkage); misidentification of

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parents and recombinants is evaluated in the next generation. Such a process of iterative decisions, though time consuming enhances the probability of correct selection and success. The current status of Quantitative Genetics can be scanned in relation to various facets of improvement. We choose, as an example, a specific but popular use of Quantitative Genetics both in Mendelian and Molecular breeding. Hybridization is basic to crop improvement. Parental choice is crucial in hybridization. This needs assessing parental divergence and grouping together those parents that show comparatively low divergence or high similarity. Several methods of such grouping have been developed by various workers. One of them uses multivariate distance (D ) as a measure of genetic divergence and the process of estimating genetic divergence between individuals uses phenotypic variation corrected for environmental variation. The method uses a simple logic an optimal grouping is one where intra-group divergence is far smaller than the inter-group divergence. A simple but efficient method is that of Tocher described in Rao (1952). Grouping using similarity indices based essentially on band homology of two individuals based on a molecular marker is done following the logic set up by Sokal and Sneath (1953) initially. Further work on this area has now made available a number of computer software. The Unweighted Pairwise Group Method on Arithmetic Averages (UPGMA) is the mostpopular for grouping. A practical example illustrates the differences of grouping based on Tochers method and UPGMA (Fig 3).

Fig 3. Two contrasting methods of grouping based on genetic divergence A.based on multivariate distance B. based on similarity index Genetic divergence measured by D Unweighted Pair wise Group Method Tochers method (cf. Rao, 1952) on Arithmetic Averages [UPGMA] Note the groups obtained by Methods A and B are quite different. Nowadays it has become common to classify germplasm using a variety of molecular markers. The basic requirement that the markers chosen have to be completely associated with the trait of interest does not receive the attention it deserves. When a researcher is interested in a number of traits defining an individual, grouping on similarity index has to be on several markers, in which case one could be confronted with conflicting group configurations given by various markers.In sharp contrast, morphometric grouping uses a multivariate divergence measure taking into account several traits simultaneously avoiding such conflicts. There are several planes of variance between the molecular and Mendelian methods of grouping that is beyond the purview of this paper. One major aim of grouping on divergence is to select divergent parents to make a cross for initiating the process of breeding. Rarely this is an aim of molecular grouping; therefore there is no provision, however weak it may be, to verify the effectiveness of such grouping. More importantly, one is left with several grouping configuration, based, for example, on various types of markers, clouding clarity in decision making defeating the very purpose of the exercise. Further molecular grouping done essentially in a laboratory has obfuscated the essential need of examining the material in the field. The example of little millet (Panicum
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sumantrense L.) where 6 different groups from seven landraces were rightly detected by morphometries while no variation was found using RAPD or AFLP markers (Arunachalam et al., 2005) substantiates this observation. The basics behind Mendelian and Molecular perception of genetic variation and its characterization differ in many ways (Table 2). In general, evidence is not strong yet to observe that gene-articulated marker-based grouping holds an advantage over time-tested morphometric (multivariate) grouping. In this context, it seems that the basics of and developments in Quantitative Genetics are yet to make a visible impact on elementary but critical steps of breeding process. Quantitative Genetics does set in right perspective what we could target using genetic variation; however, in reality, the targets are not given the same due importance as routine methodologies (Table 3). There should be conscientious efforts on the part of researchers to mend the trend sooner than later. A close look at the trend in some areas relevant to plant breeding where Quantitative Genetics had made rich contributions (Table 4) highlights overdependence on turnkey software with insufficient understanding of principles and in many cases, inability to comprehend issues as knowledge and training continue to remain far from adequate. Molecular biology research tempted / driven by turnkey instrument packages that include hardware and software; Rare attempts to define very specific needs and engineer original solution (Peccoud, 1995). Little proof exists to counter this statement even now. Molecular biology research tempted / driven by turnkey instrument packages that include solution (Peccoud, 1995). Little proof exists to counter this statement even now. What then can we do to resurrect the
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Table 2. Differences between Mendelian and Molecular approaches to genetic variation


Basics Mendelian Molecular
Sophisticated means of identifying markers Variation deemed to be genetic based on single gene theory Additive model invoked for more genes Epistasis hence ruled out Relative distinctness of two individuals (marker genotypes) measured by similarity index based on RM (band homology) Simple distance measures (additive over loci?) Estimate genetic QTs variation governed by interacting multiple genes Quantitative models enabling estimation

Genetic characterisation

Defining an individual (genotype) on QTs Environment, G X E, multiple gene base of QT taken into account

Genetic divergence and grouping

Measured by multivariate distance; accounts for expressed variation including G and G X E

Table 3. Genetic variation - The Target and Reality


Target
Morphometric variation Diagnose the nature and magnitude of variation Comprehend pattern of genetic grouping across crops Genetic grouping on morpho- QTs (mostly D2?) Repeats of divergence analysis across many crops [QTs/markers]

Reality

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Target
Molecular variation Set paths of [QT] improvement based on the diagnosis Measure realised genetic advance Correct course or set new avenues of improvement

Reality

2. Software is the key triggering experiments and not necessarily the experimental objective or need Initial days around
1960s software dependence was low, but not now Linkage estimation etc used to be in the curriculum in schools like IARI; hence there was scope for understanding problems Fully softwaredependent

Large data base on genetic clustering? But restricted by commercial software (and perception?) now only UPGMA, and a few variants

Curricula do not focus on such topics They are referred to the realm of Mathematical Statistics. Hence poor understanding of linkage estimation between markers, Interval mapping, LOD score etc. and over-dependence on routine results from software, unaware of the logical bugs

Table 4. Some areas relevant to plant breeding


Mendelian Molecular

1. Genetic Divergence D2 software dependent NT-Sys software dependent Replete with Univariate distance numerous studies using multivariate distance Based on expressed single-gene specific, phenotypic traits independent markers on similarity index (based on Relative Mobility) Further used in Not easily visible practical breeding Grouping on easy UPGMA logic (Tochers method) Complicated pedigree Can use only to pyramid a number backcross, F2 or RILs of traits - Concrete] Pyramiding has examples available problems, for eg., relevant for ransfer of coadapted gene complexes Environment-specific, Environment has no hence relevant for relevance select environments or zones

situation? I suggest that, * Basic Statistics, Probability and Elements of Mathematics must at least, be introduced at the level of undergraduate classes, * Elements of Quantitative Genetics be taught at B.Sc level along with Mendelian laws and it should be elevated considerably at M.Sc level, * There should be full terminal question papers in all the above subjects with a high level of practical examinations, and * Those subjects must be made core courses and no exemption should be given even to students from pure Agricultural stream. * Molecular applications especially Markerassisted selection and Molecular Breeding require high level of knowledge on linkage and its estimation by various methods including maximum likelihood method,
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principles of Analysis of Variance etc. There should then be a separate compulsory course that should lead, through practical examples, to just-sufficient theory to avoid overload. Unfortunately, good teachers of Quantitative Genetics have become a vanishing breed. Until new good teachers become available, we could enlist known teachers [junior/senior] in a virtual knowledge forum (as suggested by President of India) to teach and interact with students across institutions. This will compensate for existing intra-institutional deficiency. Purposive funding is needed for this activity. To sustain efficiency, it is prudent to introduce evaluation of teachers by students on open discussion. Such an open evaluation would remove bad practices like high score cards given to students without justification in return for high rating of teachers. It would be innovative to introduce open valuation system of students performance so that scores do not become blocks in proper learning. It is equally important to encourage good teachers to move across institutions to teach and learn. Confidence need to be nurtured to encourage free and frank discussion among students and, between students and teachers. An indirect force imposing quality is to stop routine field experiments, analysis by routine software and unappetizing thesis containing routine results/discussion. Finally, we should Identify bright students and encourage them to remain in research and teaching, through innovative placements within the Institution. Honourable Presidents suggestion of adopting Singapore Biopolis model (Hindu, 23 Feb 2006) in which Organisations/Universities engaged in agricultural research could get their promising human resources trained in a known global Institution, meeting their expenses fully. On return, such human resources could be utilized on a covenant of 6-7 years. We must be strong to utilize the existing ICAR, CSIR, UGC etc. schemes for such human resource upgradation
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and should ask for more, effectively and decisively with reason. We need to help ourselves before looking for lucrative help from elsewhere!! REFERENCES Arunachalam, V. 1976. Evaluation of diailel crosses by graphical and combining ability methods. Indian Journal of Genetics and Plant Breeding, 36: 358-366. Arunachalam ,V. 1993. A genetic basis behind combining ability and breeding values in monogenic and digenic systems. Biometrical Journal, 2: 217-228. Arunachalam, V. 2005. Plant Breeding: Translation, Transgression or Transformation? Chapter 13, pp. 182-196. In: Perspectives of Agricultural Research and Developments). C. Ramasamy, S. Ramanathan and M. Dhakshinamoorthy, Tamilnadu Agricultural Univesity, Coimbatore, India, 675 pp. Arunachalam, V. and Owen, A.R.G. 1971 Polymorphisms with Linked Loci. Chapman & Hall, London. Arunachalam, V., Rangalakshmi, R. and Kubera Raj, M.S. 2005. Ecological stability of genetic diversity among landraces of little millet (Panicum sumatrense) in South India. Genetic Resources and Crop Evolution, 52: 15-19. Fisher, R.A., 1930. The Genetical Theory of Natural Selection. Oxford University Press, Oxford. Haldane, J.B.S. 1932. The causes of Evolution. London: Longmans, Green & Co., and New York: Harper Brothers. Li, C.C. 1955. Population Genetics. The University of Chicago Press, Chicago, U.S.A. Peccoud, J. 1995. Automating molecular

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biology: A question of communication. Biotechnology, 13: 741-745. Rao, C.R. 1952. Advanced Statistical Methods in Biometric Research. John Wiley, New York, USA. Sokal, R.R. and Sneath, P.H.A (1963) Principles of Numerical Taxonomy. San Francisco: Freeman.

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VARIABILITY AND ASSOCIATION ANALYSIS FOR FLORAL TRAITS OF COCONUT GENOTYPES


Augustine Jerard, B., V. Niral, V. Arunachalam and P. M. Kumaran

ABSTRACT
Coconut (Cocos nucifera L.) is an important plantation crop of India. The production and distribution of quality planting material has gained importance for high production and productivity as the crop is highly heterozygous and the yield vary greatly if suitable mother palms are not selected for seed production. Since, hybrids have been released involving selected cultivars as parents, the selection of mother palms gained more importance in the hybridization programmes for the production of quality planting material. The variability in the pollen yield greatly affects the hybrid nut production and may affect the yield in the subsequent progeny also. The present investigation involving seven distinct coconut cultivars showed that there is significant difference in the floral traits and the pollen yield among the cultivars. The tall genotypes Andaman Ordinary (ADOT) and Benaulim tall (BENT) recorded high pollen yield whereas the dwarf cultivars Gangabondam (GBGD) and Chowghat Orange Dwarf (COD) recorded low pollen yields. The association among the inflorescence traits and pollen yields is also discussed. The number of spikelets, length of spikelets and male flowers have positive and significant correlation with the pollen yield. The pollen yield correlated positively with the number of female flowers. Selection of mother palms based on the desirable inflorescence traits would help to increase the hybrid recovery and reduce the cost of hybrid seed production besides quality hybrid output.

Central Plantation Crops Research Institute, Kasaragod, Kerala

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BREEDING FOR IMPROVED YIELD AND YELLOW MOSAIC VIRUS DISEASE RESISTANCE IN BLACKGRAM (VIGNA MUNGO (L.) HEPPER)
Murugan. E1 and Nadarajan. N2

ABSTRACT
Five cross combinations considered in the study of the inheritance pattern of Mungbean Yellow Mosaic Virus (MYMV) disease revealed different gene actions. In the crosses Co5 X VBN (Bg) 4 and Co5 X VBG 66, the segregation was found to be governed by digenic complementary interaction. However, in Co5 X VBG 73 the MYMV inheritance was governed by digenic duplicate interaction. In contrast, in the crosses KBG 98005 X VBN (Bg) 4 and KBG 98005 X VBG 73, the incidence of MYMV was governed by trigenic inhibitory interaction. The putative gene symbols assigned for the five genotypes viz., Co5, KBG 98005, VBN (Bg) 4, VBG 66 and VBG 73 are r1r1r2r2r3r3r4r4, r1r1r2r2r3r3r4r4II, R1R1R2R2ii, R1R1R2R2 and R3R3R4R4ii respectively.

Introduction Mungbean Yellow Mosaic Virus is one of the most destructive diseases and is prevalent on mungbean, blackgram and soybean throughout India mainly in the Kharif season. The virus is transmitted by whitefly, Bemisia tabaci. The yield losses up to 100 per cent have been reported by Nair (1971) in blackgram. Khattak et al. (2000) reported 32.2 to 78.6 per cent decrease in grain yield under field condition in mungbean. Hence, the present investigation was taken up to study the inheritance pattern of MYMV in blackgram as the prerequisite to evolve high yielding blackgram varieties combined with resistance to MYMV. Materials and Methods To study the inheritance pattern of Yellow Mosaic Virus (MYMV), the following five cross combinations viz., Co5 X VBN (Bg) 4, Co5 X VBG 73, Co5 X VBG 66, KBG 98005 X VBN (Bg) 4 and KBG 98005 X VBG 73 were chosen. The lines (Co5 and KBG 98005) are highly susceptible and three testers (VBN (Bg) 4, VBG 73 and VBG 66) are highly resistant for MYMV. Six generations viz., P1, P2, F1, F2, B1 and B2 for each of the five crosses were generated

to understand the inheritance pattern of MYMV. During Kharif, 2004, six generations of the five selected cross combinations were raised at National Pulses Research Centre, Vamban, which is a hot spot area for Yellow Mosaic Virus disease (MYMV). The above said materials were raised on ridges of two meter length spaced at 30 cm between ridges and 10 cm between plants in two replications. For every 10th row and as border rows of the experimental plot the check Co5 (highly susceptible to MYMV) was raised as infector row so as to effectively spread the inoculum. The Yellow Mosaic Virus Disease (MYMV) incidence was recorded on all the plants based on the visual scores on 50th day. The classification was made into scales 1 9 as follows based on the scale adopted by Singh et al. (1988). The mean disease scale of parents and F1 was calculated as follows (Singh, 1980). The mean disease scale of parents and F1 was calculated as follows (Singh, 1980). Mean scale = (Infection rate x

1. Department of Plant Breeding and Genetics, Agricultural College & Research Institute, TNAU, Madurai 625104 2. Professor and Head, Department of Pulses, TNAU, Coimbatore 3

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Scales

Percentage of plant foliage affected Mottling of leaves covering 0.1 to 5.0 per cent of the leaf area. Mottling of leaves covering 5.1 to 10.0 per cent of the leaf area. Mottling and yellow discoloration of 10.1to 25.0 per cent of the leaf area. Mottling and yellow discoloration of 25.1to 50.0 per cent of the leaf area. Severe yellow mottling on more than 50.0 per cent and up to 100 per cent of the leaf area.

presented in Table 1.
Reaction Resistant

Moderately resistant

Moderately susceptible

Susceptible

Highly susceptible

The F1 of the crosses viz., Co5 X VBN (Bg) 4), (Co5 X VBG 73) and (Co5 X VBG 66) were resistant to MYMV, while it was susceptible in the crosses KBG 98005 X VBN (Bg) 4 and KBG 98005 X VBG 73. In the segregating generations, in crosses Co5 X VBN (Bg) 4 and Co5 X VBG 66 the chisquare test for the expected ratio of 9:7 (resistant: susceptible) in F2 and 1:3 (resistant: susceptible) in B1 was not significant. In B2 generation, all plants were resistant. In the case of Co5 X VBG 73 cross, the chi-square test revealed that the F2 generation showed a expected ratio of 15:1 for resistance: susceptible and B1 showed a ratio of 3:1 (resistant: susceptible). In B2, all plants were resistant. In the crosses KBG 98005 X VBN (Bg) 4 and KBG 98005 X VBG 73, the chisquare test was non-significant showing fitness of the expected ratios of 15:49 (resistant: susceptible) and 1:1 (resistant: susceptible) in F2 and B2 generations respectively. All the plants in B1 were susceptible to MYMV. Among these five crosses, three crosses viz., (Co5 X VBN (Bg) 4), (Co5 X VBG 73) and (Co5 X VBG 66) were found to be resistant to MYMV in F1, where the female parent was Co5. Therefore, resistance is dominant over susceptibility. In blackgram, Dahiya et al. (1977) and Verma and Singh (1980) and in greengram Reddy and Singh (1993) and Selvi (2002) reported that the resistance was dominant over susceptibility. The segregation of MYMV resistance in the present study appeared to be governed by digenic complementary interaction as seen from the ratio of 9:7 (resistant: susceptible) in F2 of Co5 X VBN (Bg) 4 and Co5 X VBG 66. The pattern of segregation in B1 (1 resistant: 3 susceptible) and B2 (all resistant) of these two crosses (Co5 X VBN (Bg) 4 and Co5 X VBG 66) also confirmed the result of complementary
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Frequency) / Total number of plants scored. The plants in the F 2 and back cross generations were classified as resistant (1-3) and susceptible (5-9) following Reddy and Singh (1990). The goodness of fit to Mendelian segregation ratio for MYMV (resistance: susceptible) in the segregating population was tested by Chi square test. Results and Discussion For the study of inheritance of MYMV disease resistance, the Chi-square test for the deviation from the expected genetic ratios of the segregating generations viz., F2, B1 and B2 of five crosses was made and the results are

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interaction (duplicate recessive genes) (Table 1). A similar type of duplicate recessive genes for MYMV resistance in blackgram was reported by Verma and Singh (1980), Sandhu et al. (1985) and Shukla and Pandiya (1985) and in greengram by Selvi (2002). However, in the cross (Co5 X VBG 73), the segregation of F 2 showed a ratio of 15:1 (resistant: susc eptible). The segregation ratio in B1 (3 resistance: 1 susceptible) and B2 (all resistant) also confirmed the ratio observed in F2. This revealed that, in the cross Co5 X VBG 73 the MYMV was governed by interaction of two duplicate genes (duplicate dominant genes). Shukla et al. (1978) and Singh (1980) reported the presence of duplicate dominant genes for MYMV in blackgram. From the above discussion it was found that, though the three crosses had same female parent (Co5), two different types of gene action of complimentary interaction in Co5 X VBN (Bg) 4 and Co5 X VBG 66 and duplicate interaction in Co5 X VBG 73 were noticed. The reason for the difference in gene action may be attributed to the presence of two different sets of genes in male parents of the crosses. The male parents of the crosses (Co5 X VBN (Bg) 4 and Co5 X VBG 66) namely VBN(Bg) 4 and VBG 66, may have same alleles (R1R1R2R2), which act in complementation. However, the male parent of the cross Co5 X VBG 73 namely VBG 73, may have another set of non-allelic genes (R3R3R4R4), which act in a duplicate manner. The common female parent of all these three crosses, namely Co5, may have recessive alleles for all these four loci (r1r1r2r2r3r3r4r4). In the crosses KBG 98005 X VBN (Bg) 4 and KBG 98005 X VBG 73, the F1 was found to be susceptible, where the female parent was KBG 98005. The segregation of F2 was observed to be 15:49 (resistant: susceptible). It showed that the reaction to MYMV was governed by trigenic inhibitory interaction. The segregation
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in B1 (all susceptible) and B2 (1 resistant:1 susceptible) also confirmed the trigenic inhibitory interaction. In blackgram, inhibitory gene action was reported by various authors like Solanki et al. (1982), Verma (1985), Verma and Singh (1986) and Reddy and Singh (1990) and in greengram by Mishra and Asthana (1996) and Khattak et al. (2000). From the above results, it could be concluded that the male parents of these two crosses namely VBN (Bg) 4 and VBG 73 respectively, may have recessive alleles for an inhibitory gene (ii) apart from two sets of alleles already indicated. The female parent KBG 98005 of these two crosses may have a set of dominant inhibitory alleles (II) at a locus apart from recessive alleles for susceptibility to MYMV at four loci. Therefore, the following putative gene symbols are proposed for the parents involved in the five crosses: However, the gene symbols are allotted subject to confirmation by allelic tests. The allelic tests may be conducted by intercrossing
Sl.No. Parent Reaction to Gene symbol for MYMV MYMV resistance

1. 2. 3. 4. 5.

Co5 KBG 98005 VBN(Bg) 4 VBG 66 VBG 73

Susceptible Susceptible Resistant Resistant Resistant

r 1r 1r 2 r 2 r 3 r 3 r 4 r 4 r1r1r2r2r3r3r4r4II R1R1R2R2ii R 1R 1 R 2 R 2 R3R3R4R4ii

all the three male parents and studying the resistant pattern for MYMV resistance. From the present investigation it was concluded that the MYMV was controlled by two and three genes with various types of interaction. Hence, recombination breeding with two or three cycles of recurrent selection is required to obtain desirable segregants of high yielding ability coupled with MYMV resistance. REFERENCES Dahiya, B.S., Singh, Kuldip. and J.S. Brar. 1977.

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Incorporation of resistance to mungbean yellow mosaic virus in blackgram (Vigna mungo L.). Tropical grain legume bulletin., 9: 28-32. Khattak, G.S.S., M.A. Haq, Muhammad Ashraf, and T. Elahi. 2000. Genetics of Mungbean Yellow Mosaic Virus (MYMV) in mungbean (Vigna radiata (L.) Wilczek). J. Genet. Breed., 54: 237-243. Mishra, S.P. and A.N. Asthana. 1996. Inheritance of yellow mosaic virus resistance in mung bean (Vigna radiata (L.) Wilczek). Recent advances in mungbean research. p 214-219. Nair,N.G. 1971.Studies on the yellow mosaic of urdbean caused by mungbean yellow mosaic virus Ph.D thesis,U.P. Agric.Univ., Pantnagar, India. Reddy, K.R. and D.P. Singh. 1990. Inheritance of resistance to mungbean yellow mosaic virus in blackgram. New Botanist, 17 : 99102. Reddy, K.R. and D.P. Singh. 1993. Inheritance of resistance to mungbean yellow mosaic virus. Madras agric. J., 80: 199-201. Sandhu, T.S., J.S. Brar, S.S. Sandhu and M.M. Verma. 1985. Inheritance of resistance to mung bean yellow mosaic virus in greengram. J. Res. PAU, 22: 607-611. Selvi, R. 2002. Genetics and molecular studies on mungbean yellow mosaic virus resistance as related to economic attributes in greengram (Vigna radiata (L) Wilczek). Ph.D. Thesis, Tamil Nadu Agric. Univ., Coimbatore.

Singh, D.P. 1980. Inheritance of resistance to MYMV in blackgram. Theor.Appl. Genet., 57: 233-235. Shukla, G.P. and B.P. Pandya. 1985. Research to yellow mosaic in greengram. SABRAO J., 17: 165-171. Shukla, G.P., B.P. Pandya, and D.P. Singh. 1978. Inheritance of resistance to yellow mosaic in mungbean. Indian J. Genet. Plant Breed., 38: 357-360. Singh, G., S. Kapoor and K. Singh. 1988. Multiple disease resistance in mungbean with special emphasis on mungbean yellow mosaic virus. In: International Symposium on Mungbean, 2nd Nov 16-20, Bangkok, Thailand. p 290-296. Solanki, I.S., B.S. Dahiya and R.S. Waldia. 1982. Resistance to mungbean yellow mosaic virus in blackgram. Indian J. Genet. Plant Breed., 43: 240-242. Verma, R.P.S. 1985. Inheritance of resistance to mungbean yellow mosaic virus in the interspecific and intervarietal crosses of greengram and blackgram. Ph.D.Thesis. G.B.Pant. Univ. of Agric. & Tech ., Pantnagar, India.80 p. Verma, R.P.S. and D.P. Singh. 1980. Inheritance of yellow mosaic virus in blackgram (Vigna mungo(L.) Hepper). Theor. Appl. Genet., 55: 233-235. Verma, R.P.S. and D.P. Singh. 1986. The allelic relationship of genes giving resistance to mungbean yellow mosaic virus in blackgram. Theor. Appl. Genet., 72: 737 - 738.

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Table 1. Chi- square test for inheritance of Yellow Mosaic Virus disease resistance in Blackgram Observed values Generation Resistant Co5 X VBN(Bg) 4F1 Resistant 151 F2 31 B1 121 B2 Co5 X VBG 73 Resistant F1 254 F2 80 B1 120 B2 Co5 X VBG 66F1 F2 B1 B2 KBG 98005 X VBN(Bg) 4F1 F2 B1 B2 KBG 98005 X VBG 73 F1 F2 B1 B2 Resistant 172 35 115 Susceptible 111 71 20 30 146 80 Expected ratio F2 values Probability between

9:7 1:3

0.21 1.59

0.70 0.50 0.30 0.20

15:1 3:1

0.52 0.31

0.50 0.30 0.70 0.50

9:7 1:3

0.6 1.8

0.50 0.30 0.20 0.10

54 52

Susceptible 212 112 56

15:49 1:1

1.44 0.15

0.20 0.30 0.70

57 57

Susceptible 218 115 50 15:49 1:1 1.13 0.45 0.20 0.30 0.50

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COMPLEX INHERITANCE IN RICE VARIETY MR1523 OF RESISTANCE TO GALL MIDGE BIOTYPES


Suneetha, K1, J.S. Bentur1, K. Hima Bindu1, P. Vijaya Lakshmi1, C. Cheeralu2,

P.Ram Mohan Rao3

ABSTRACT
Inheritance of gall midge resistance in rice variety MR1523 was studied by generating F3 families from individual F2 plants of various crosses and evaluating them against four biotypes in three greenhouse and two field tests at three locations. Results suggested involvement of two genes conferring resistance against biotypes 1 and 3, one dominant gene against biotype 4 and none against the new biotype 4M. Interactions of the two genes were different against the two biotypes used in greenhouse tests. While reciprocal crosses did not confirm to the ratio observed with straight cross, allelic tests with varieties having known resistance genes were also inconclusive. We are attempting to identify these genes with closely linked known SSR markers.

Introduction The Asian rice gall midge Orseolia oryzae (Wood-Manson) is a serious pest of rice in certain regions of South, Central and East India causing significant yield loss mainly during kharif season. Breeding rice varieties with pest resistance has been a viable, ecologically acceptable approach for the management of the gall midge. However, rapid development of virulent biotypes capable of overcoming the host plant resistance has been posing problem in recent years. Systematic germplasm evaluation has led to the identification of important sources of resistance like Eswarakorra, Ptb18, Ptb21, Siam 29, Leuang 152 etc. (see Bentur et al., 2003) which have been extensively used in breeding resistant varieties. Genetic studies have identified, so far, 10 major genes conferring resistance (Kumar et al., 2005). Most of the 60 plus gall midge resistant rice varieties developed to date contain one of the three major genes viz., Gm1, Gm2 and unidentified gene (s) in Ptb21 conferring immune level of resistance. Seven distinct gall midge biotypes have been characterized so far (Vijaya Lakshmi et al.,

2005), some of them being selected as direct consequence of extensive cultivation of these varieties. In most of the studies, resistance to gall midge has been found to be conferred by a single dominant or (as in case of gm3) a recessive gene (Kumar et al., 2005). Exceptionally, sources deriving resistance from Ptb18 and Ptb21 have displayed complex pattern (Kalode and Bentur 1989). We studied the genetics of resistance in the rice variety MR1523, derivative from Ptb21, in order to understand this complexity. Material and Methods The rice variety CR94-MR1523 (Ptb18/ Ptb21//IR8), developed at CRRI, Cuttack involving Ptb 21 with unknown resistance gene(s) was crossed with the susceptible check TN1 as well as with the varieties with known resistance genes viz., W1263 (with Gm1), Phalguna (Gm2), RP2068 (gm3), Abhaya (Gm4), ARC 5984 (Gm5), and with other resistance sources with unknown gene(s) like Bhumansan, NHTA8, Banglei, Aganni and ARC 6605. These crosses were made in kharif 2000. F1 seeds (direct and reciprocal crosses)

1. Directorate of Rice Research, Rajendranagar, Hyderanad 500 030, AP 2. Agricultural Research Station, ANGRAU, Warangal 506 007, AP 3. Agricultural Research Station, ANGRAU, Ragolu 532 484, Srikakulam Dist., 134

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were grown in rabi 2000. Individual F2 plants were raised in kharif 2001. F3 families consisting of progeny from individual F2 plants were evaluated in field at Ragolu and Warangal and in greenhouse at DRR against gall midge biotypes 1, 3 and 4 during kharif 2002. Nil damaged families across the locations/tests were identified and these were advanced to F 4 and F 5 generations and then tested in All India Coordinated Rice Improvement Programme (AICRIP) for two years (2004 & 2005). Individual families were scored for resistance reaction. Progenies showing plant damage of 45 per cent and above were considered to be susceptible, 1-44 percent damage as heterozygous and zero damage was considered as resistant. Chi-square test was carried out to test the goodness of fit for various segregation ratios. Results and Discussion Results revealed a complex segregating pattern of resistance depending upon the biotype used for evaluation (Table 1). Families derived from the cross MR1523 x TN1 showed segregation ratio of 13 resistant: 3 susceptible families when evaluated against Biotype 1, 15:1 ratio against Biotype 3 and 3:1 ratio against Biotype 4 in greenhouse test at DRR, Hyderabad. Interestingly, reciprocal crosses between TN1 and MR1523 did not show similar ratios. Crosses between MR1523 and rice varieties with known and unknown gall midge resistance genes did not show any distinct segregation pattern suggesting higher order of interaction and possible involvement of cytoplasmic inheritance. Thus it is apparent that genes controlling gall midge resistance in MR1523 interact differently against different biotypes tested. While two genes contributed resistance against biotype 1 and 3, only one gene was effective against biotype 4. These two genes interacted differently against biotypes 1 and 3. For biotype 1 the interaction appeared as one dominant and one recessive gene while for
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biotype 3 two dominant gene interaction was evident. For biotype 4 single dominant gene segregation ratio was observed. However, variations noted in reciprocal crosses need further investigation for a better understanding. Since allelic tests through crosses involving MR1523 with known gene sources did not reveal identity of the possible 2-3 genes in MR 1523, we propose to investigate using gene linked markers. Preliminary reports showed non-allelic amplification when Gm1 linked SSR markers RM444 and RM219, Gm2 linked marker RM241 were used for amplifying genomic DNA from MR1523. Other gene specific markers are being studied. Four of the cultures, RP 4516-3-8 and three selections of RP4518, derived from crosses MR1523 x RP2068 and MR1523 x Abhaya, respectively, identified in the present studies were nominated for multi location testing under AICRIP trials and showed a wide spectrum of resistance against gall midge biotypes across the locations (DRR, 2005). Few of the cultures identified in the present studies are being tested under AICRIP trails. Of these, RP4510-175 and RP4510-177 recorded resistance against biotype 3 while being susceptible to biotype 1 while RP4510- 260 showed resistance against the biotypes 1,3 and 4 (DRR, 2005). This type of reaction showing resistant for biotype 3 and susceptibility for biotype 1 has not been observed earlier in any of the cultures. Thus, unknown genes that are suspected to be present in MR1523 have been segregated and fixed in different lines which would serve as prebreeding material for breeding and genetic studies. The single gene conferring resistance in MR1523 against biotype 4 has been overcome by the newly reported biotype 4M (Vijaya Lakshmi et al., 2005). REFERENCES

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Bentur, J. S., Pasalu, I. C., Sarma, N. P., Prasada Rao, U., Mishra, B. 2003. Gall midge resistance in rice. DRR Research paper Series 01/2003, 20pp. Directorate of Rice Research, Hyderabad. DRR (Directorate of Rice Research) 2005. Progress Report for 2004. Directorate of Rice Research, Hyderabad, Vol. 2, 2.14p. Kalode, M. B., Bentur, J.S.1989. Characterization of Indian biotypes of the rice gall midge, Orseolia oryzae (Wood-Manson) (Diptera: Cecidomyiidae).Insect Sci. Applic. 10 : 219-224.

Kumar, A., Jain, A., Sahu, R. K., Shrisvastava, M. N., Nair, S., Mohan, M.2005. Genetic analysis of resistance genes for the rice gall midge in two rice genotypes. Crop Sci. 45: 1631-1635. Vijaya Lakshmi, P., Amudhan, S., Bentur, J.S. 2005. A new gall midge biotype characterized from Warangal population in Andhra Pradesh, India. In: Proceedings of 1 st Congress on insect science 15-17 December, 2005.Indian society for the advancement of insect science, Punjab Agricultural University, Ludhiana. p-54-55.

Table 1. Reaction of F3 families derived from the cross between MR1523 and TN1 against rice gall midge biotypes 1,3 and 4 in greenhouse at DRR, Hyderabad and in field at Ragolu (Biotype 4) Cross Biotype Number of F3 families Tested Resistant Susceptible MR1523 x 1 197 TN1 MR1523 x 3 194 TN1 MR1523 x 4 187 TN1 F2 (tab) value: 3.84(0.05), 6.63(0.01) 158 188 139 39 6 48 13:3 15:1 3:1 0.14 3.30 0.04 Ratio(R:S) F 2 value (calculated)

R: resistant including heterozygous; S: susceptible

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LEAF TRICHOME DENSITY AN INDICATOR OF JASSID TOLERANCE IN COTTON


Kannan, S., R. Ravikesavan and M. Kumar

ABSTRACT
A study was taken up to evaluate the number of trichomes present in the leaf as related to jassid resistance. Three cultivars KC 2, MCU 5 and MCU 12, their F1 and segregating populations F2 and F3 and backcrosses BC1 F1 were screened. Among the parents KC 2 recorded highest trichome density of 26.02, when compared to 14.11 and 17.88 per microscopic field of observation in MCU 5 and MCU 12 respectively. The F1 KC 2 MCU 12 registered more trichome density of 21.48 than KC 2 MCU 5 (15.47) and MCU 5 MCU12 (14.33). In F2 population the mean trichome density ranged from 15.33 to 21.20. The lowest value was observed in MCU5 MCU 12 and highest value had been observed in KC 2 MCU 12. In F3, the trichome density ranged from 14.53 to 20.42. The lowest value was observed in MCU 5 MCU 12 and the highest value in KC 2 MCU 12. Among the BC1 (KC 2 MCU 12) KC 2 recorded highest mean trichome density of 24.15 when compared to 20.17 and 17.85 of (KC 2 MCU 5) KC 2 and (MCU 5 MCU 12) MCU 5 respectively. In BC2 the (KC 2 MCU 12) MCU 12 recorded highest trichome density of 21.16 when compared to 19.38 and 14.81 of (KC 2 MCU 5) MCU 5 and (MCU 5 MCU 12) MCU 12 respectively. The parent KC 2 when used as female has contributed more trichome density in the F1 as well as in the segregating populations. This positive relationship between trichome density and jassid tolerance was also confirmed through artificial screening.

INTRODUCTION Cotton, the White Gold is an important commercial and industrial crop of many countries. In India, it plays a prominent role in economy through foreign exchange earnings. This crop is affected by number of insect pests, among which jassids cause considerable yield reduction. For managing the jassids farmers spend lot of money through chemical sprays. For reducing the use of chemical insecticides and in turn the input cost evolving jassid resistant genotypes is the need of the hour. Among the traits related to jassid tolerance trichome density is the primary indicator. Parnell et al. (1949) indicated that the hair length is most important and if length is maintained increased hair density confers more resistance. The present investigation, therefore, aims in assessing the relationship between trichome density and the
1 2

jassid resistance in a group of genotypes with a view to develop basic breeding material for evolving jassid resistant varieties. MATERIALS AND METHODS The experimental materials comprised three parents (KC 2, MCU 5 and MCU 12), three F1s, F2' s and F3s (KC 2 x MCU 5, KC 2 x MCU 12 and MCU 15 x MCU 12), three B1s [(KC 2 x MCU 5) x KC2, (KC2 x MCU 12) x KC 2, and (MCU 5 x MCU 12), MCU 5] and three B2s [(KC 2 x MCU 5) x MCU 5, (KC 2 x MCU 12) x MCU 12, and (MCU 5 x MCU 12) x MCU 12]. The materials were grown in Randomized Block Design (RBD) with three replications at the Department of cotton, Tamil Nadu Agricultural University, Coimbatore. The materials were sown in two rows of each replication with 6m length and spacing 75 x 30 cm. Data on ten randomly

PG Scholar, CPBG, TNAU Associate Professor (Cotton), CPBG * Corresponding Author (chithuragul@gmail.com) 3 Associate Professor (PB&G), CPBG, TNAU, Coimbatore 3 137

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selected plants in each genotype/population were collected and trichome density (per microscopic field), stomatal counts (per mm2 leaf) and leaf thickness (mm)was assessed. The leaf thickness was measured using screw gauge. The hopper burn injury was assessed as per the Indian Central Cotton Committee (ICCC, 1960) methods and based on resultant symptoms of infestation. Jassid resistance index was also calculated as proposed by Nageswara Rao (1973). RESULTS AND DISCUSSION The observations on three morphological characters which are found to confer jassid tolerance were recorded and the mean and range presented in Table 1. Among the parents KC2 recorded highest trichome density of 26.02, when compared to 14.11 and 17.88 of MCU 5 and MCU 12 respectively. In F1, F2, B1 and B2 generations the cross of KC 2 x MCU 12 was found to have more trichome density than other crosses. Sivasubramanian et al. (1991) reported that the number and weight of hairs were higher in resistant than susceptible cul tivars.Ravikesavan et al. (2002) have studied the trichome density and reported that trichome density had a positive correlation with jassid resistance and on the other hand glabarous trait gives tolerance to the boll worms. Stomatal counts were recorded per mm2 leaf area under microscope. This character is found to be negatively correlated with jassid resistance. For this character also KC 2 and the combination of KC 2 x MCU 12 recorded the lower number of stomata. However Shima Bhaskaran (2004) reported that there is no relationship between stomatal count and jassid resistance. The leaf thickness was found to be positively related to jassid tolerance and the genotype KC 2 and the cross combination of KC 2 x MCU 12 recorded more leaf thickness compared to other combinations. Similarly Tidke and Sane (1962) reported that thicker leaves
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conferred resistance to leaf hopper. Artificial screening was done for parents and the segregating population and the jassid injury index of different genotypes is tabulated (Table 2). In artificial screening the lowest grade index was recorded by parent KC 2 (1.0). The parents MCU 5 and MCU 12 registered 2.48 and 1.84 grade index respectively. Among the F1 KC 2 x MCU 12 (1.9) had low grade index compared to KC 2 x MCU 5 (2.26) and MCU 5 x MCU 12 (2.63). In F2 population, the grade index of KC 2 x MCU 12 was 1.65, when compared to 2.16 and 2.7 of KC 2 x MCU 5 and MCU 5 x MCU 12 respectively. In F3 population, the grade index of KC 2 x MCU 12 was 1.78 whereas 2.52 and 2.55 of KC 2 x MCU 5 and MCU 5 x MCU 12 respectively. Joint Scaling Test and Genetic Effects Leaf thickness: Scale A was significant in KC 2 x MCU 5. The scales B and C were significant in all the crosses. The results of joint scaling test presented in table 3 also indicated the inadequacy of the data to fit simple additivedominance model for all crosses. This is conformed by chi-square test values, which is significant in all the crosses. The m component was significant in all the crosses and the values ranged from 0.04 mm in MCU 5 x MCU 12 and 0.05 mm in KC 2 x MCU 12 and KC 2 x MCU 5. The [d] and [h] components were significant in KC 2 x MCU 12. The [i] component was significant in KC 2 x MCU 5 and KC 2 x MCU 12 and the values ranged from 0.01 mm in MCU 5 x MCU 12 to 0.04 mm in KC 2 x MCU 12. The [j] component was significant in KC 2 x MCU 5 and MCU 5 x MCU 12 and the values ranged from -0.02 mm in KC 2 x MCU 5 to 0.01 mm in MCU 5 x MCU 12. The [l] component was non significant in all the crosses (Table 4). Stomatal number: The scales A and B

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were significant in all the crosses, while the scale C was significant in KC 2 x MCU 12. The results of joint scaling test presented in table 3 indicated the inadequacy of the data to fit simple additivedominance model for all crosses. This is conformed by Chi-square test values that were significant for all the crosses. The m component was significant in all the crosses and the values ranged from 66.64 in KC 2 x MCU 12 to 95.00 in MCU 5 x MCU 12. The [d] component was significant in KC 2 x MCU 12. The [h], [i], and [l] components were significant in all the crosses. The values ranged from 47.97 in MCU 5 x MCU 12 to 161.46 in KC 2 x MCU 12 in [h] component. The values of [i] component ranged from 34.80 in MCU 5 x MCU 12 to 172.22 in KC 2 x MCU 12. The values ranged in [l] component from -312.16 in KC 5 x MCU 12 to -84.73 in MCU 5 x MCU 12. The [j] component was positively significant in KC 5 x MCU 12 (Table 5). Trichome density: The scale A was significant in KC 2 x MCU 5 and KC 2 x MCU 12. The scale B was significant in KC 2 x MCU 12 where as scale C was significant in MCU 5 x MCU 12. The results of joint scaling test are presented in table 6 which indicated the inadequacy of the data to fit simple additivedominance model for all crosses. This is also conformed by chi-square test values that are significant in all the crosses. The m component was significant in all the crosses and the values ranged from 17.33 in MCU 5 x MCU 12 to 19.80 in KC 2 x MCU 12. The [d] component was significant in KC 2 x MCU 12. The [h] component was significant in all the crosses and the values ranged from -10.73 in MCU 5 x MCU 12 to 8.87 in KC 2 x MCU 12. The [i] component was significant in all the crosses and the values ranged from -9.07 in MCU 5 x MCU 12 to 11.47 in KC 2 x MCU 12. The [j] component was significant in KC 2 x MCU 5. The [l] component was significant in
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KC 2 x MCU 5 and KC 2 x MCU 12 and the values ranged from -20.13 in KC 2 x MCU 12 to 17.51 in KC 2 x MCU 5 (Table 7). The overall scenario indicated that parent KC 2 had high trichome density and leaf thickness which makes it tolerant to jassid. When KC 2 was used as female parent in the crosses, the trichome density was more when compared to the other crosses. Artificial screening also indicated similar results. In the present study both additive and dominance components were significant for the crosses and hence reciprocal recurrent selection or multiple crossing can be used for further improvement of the traits under study in the respective crosses. This study revealed that the trichome density is the primary indicator for jassid resistance. Among the genotypes, the parent KC 2 was resistant to jassid, MCU 12 moderately resistant and MCU 5 susceptible to jassids. The trichome density and leaf thickness were positively correlated with jassid resistance. Uthamasamy (1994) also reported that the morphological traits such as leaf thickness, leaf hairiness, toughness of leaf veins, thickness of leaf lamina, length of hair, and angle of leaf insertion are positively related to leaf hopper resistance. The stomatal number had negative correlation with jassid resistance. Screening of segregating generation will result in high yielding plants having better resistance to leaf hopper. Further, studies in isolating jassid resistant progenies are in progress. REFERENCES Nageswara Rao, P. 1973. An index for jassid resistance in cotton. Madras Agric. J. 60: 264-266. Parnell, F.R., H.E. King and D.F. Ruston. 1949. Jassid resistance and hairiness of the cotton plant. Bull. Ent. Res. 39: 539-575. Ravikesavan, R., T.S.Raveendran, C.Parame shwari, S.Suganthi, S.Mohan and

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C.Surendran. 2002. Morphological, physiological and biochemical futures associated were biotic and abiotic stresses in cotton cultured derived through introgressive breeding. Paper presented in the 2nd meeting of the Asian cotton research and development network New Genetical Approaches to Cotton Improvement held on November 14-16,2002. Tashkent, Uzbekistan. Shimna Bhaskaran. 2004. Genetic and anatomical studies on jassid resistance in cotton (Gossypium spp.). M.sc., (Ag.) Thesis, TNAU, Coimbatore, India.

Sivasubramaniyan, P., Uthamasamy, S. and Parvathy, K. 1991. Resistance in cotton, Gossypium spp. to the leafhopper Amrasca devastans (Dist.). Madras Agric. J. 78 (1-4): 80-81. Tidke, P.M. and Sane, P.V. 1962. Jassid resistance and morphology of cotton leaf. Indian Cott. Grow. Rev. XVI (6): 324-327. Uthamasamy, S. 1994. Host resistance to the leafhopper, Amrasca devastans (Dist.) in cotton, Gossypium spp. Proceedings of the World Cotton Research conference, Australia pp. 14-17.

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Table 1. Mean of Morphological characters related to jassid resistance in cotton


Characters Leaf Thickness (mm) Stomatal Counts mm2 Trichome Density / / leaf microscopic field

Genotypes

RANGE 0.07-0.09 0.04-0.05 0.05-0.06 0.04-0.05 0.05-0.06 0.03-0.05 0.04-0.05 0.04-0.05 0.03-0.04 0.04-0.05 0.04-0.06 0.04-0.05 0.05-0.06 0.06-0.07 0.04-0.05

MEAN 0.08 0.04 0.05 0.05 0.06 0.04 0.04 0.05 0.03 0.05 0.05 0.04 0.06 0.06 0.05

RANGE 82-91 106-117 98-112 106-114 114-127 116-126 121-135 90-105 135-144 104-112 88-96 135-143 92-105 114-132 104-116

MEAN RANGE MEAN 86.33 112.33 105 110.67 119.67 121.33 127.33 95.67 139 107.33 92.33 139.33 99.67 124 108.67 23-28 9-15 10-19 11-18 18-22 13-15 14-20 14-24 14-20 12-18 16-22 12-17 16-22 18-26 14-20 26.02 14.11 17.88 15.47 21.48 14.33 17.91 20.79 16.13 17.26 19.32 14.92 20.17 24.15 17.85

Parents KC 2 MCU 5 MCU 12 F1s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 F2s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 F3s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 B1s (KC 2 X MCU 5) X KC 2 (KC 2 X MCU 12) X KC 2 (MCU 5 X MCU 12) X MCU 5 B2s (KC 2 X MCU 5) X MCU 5 (KC 2 X MCU 12) X MCU 12 (MCU 5 X MCU 12) X MCU 12 Overall mean Standard Error (SE)

0.05-0.06 0.04-0.05 0.03-0.05

0.05 0.05 0.04 0.049 0.003

99-108 89-96 104-116

103 92.33 109.33 110.74 3.61

18-22 18-24 12-16

19.38 21.16 14.81 18.51 0.84

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Table 2. Jassid injury index Injury index Parents KC 2 MCU 5 MCU 12 F1s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 F2s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 F3s KC 2 X MCU 5 KC 2 X MCU 12 MCU 5 X MCU 12 1.78 2.55 1.65 2.70 2.52 1.90 2.63 2.16 1.00 2.48 1.84 2.26

Injury index: 0.1 1.0 : Resistant, 1.1 2.0 : Moderately resistant, 2.1 3.0 : Susceptible, 3.1 4.0 : Highly susceptible

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Table 3. Scaling and Joint scaling test for leaf thickness (mm) and stomatal counts (Leaf area / mm2)

Scaling test for leaf thickness (mm) B C Joint scaling test J2 value A

Crosses

Scaling test for stomatal counts (Leaf area / mm2) Joint scaling B C test J2 value

0.01** r 0.00 89.12** -0.01** r 0.00 -0.01* r 0.01 -0.02* r 0.01 -0.06** r 0.01

-0.03** r 0.00

13.78 r 7.86

89.44** 996.28** 51.63**

KC 2 x MCU 5 -0.02** r 0.00 KC 2 x MCU 12 -0.01 r 0.00 MCU 5 x MCU 0.01 r 0.00 12 -32.29** r 6.42 15.13 r 7.32 Table 4. Genetic effects for leaf thickness (mm) m 0.05** r 0.00 0.05** r 0.00 0.04** r 0.00 0.00 r 0.00 0.00 r 0.01 0.02** r 0.00 0.02* r 0.01 -0.00 r 0.00 0.01 r 0.01 0.03* r 0.01 0.04** r 0.01 0.01 r 0.01 [d] [h] [i] [j] -0.02** r 0.00 0.00 r 0.00 0.01* r 0.00 [l]

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29.33** r 5.74 141.36** 115.33** r 4.87 19.62** 24.33** r 6.40

37.47** r 4.56 24.60** r 3.67 25.60** r 3.96

Crosses

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KC 2 x MCU 5

-0.02 r 0.01 -0.02 r 0.01 -0.00 r 0.01

KC 2 x MCU 12

MCU 5 x MCU 12

Table 5. Genetic effects for stomatal counts (Leaf area / mm2) [d] -8.60 r 2.88 36.73** r 2.17 -4.73 r 3.22 [h] 61.29** r 8.44 161.46** r 6.32 47.97** r 8.85 [i] 53.02 **r 7.96 172.22** r 5.77 34.80* r 8.53 [j] -4.07 r 3.28 45.37** r 2.69 -0.63 r 3.53 [l] -119.82** r 13.94 -312.16** r 10.81 -84.73** r 14.79

Crosses KC 2 x MCU 5 KC 2 x MCU 12 MCU 5 x MCU 12

M 83.58** r 1.38 66.64** r 0.95 95.00** r 1.96

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* Significant at 5 % level ** Significant at 1 % level

Table 6. Scaling and Joint scaling test for trichome density / microscopic field

Crosses

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KC 2 x MCU 5 KC 2 x MCU 12 MCU 5 x MCU 12

Scaling test for trichome density / microscopic field Joint scaling A B C test J 2 value -8.80** r 1.24 -1.73 r 1.53 -3.56 r 1.99 51.07** 4.53** r 1.42 4.13* r 1.69 -2.80 r 1.94 25.13** 1.33 r 1.38 -2.00 r 1.34 8.40** r 1.91 30.13**

Table 7. Genetic effects for trichome density / microscopic field [d] 2.20 r 0.79 4.53* r 0.97 0.27 r 0.77 [h] -7.91* r 2.22 8.87* r 2.49 -10.73** r 2.11 [i] -6.98*r 2.11 11.47* r 2.39 -9.07* r 1.99 [j] -3.53* r 0.90 0.20 r 1.02 1.67 r 0.86 [l] 17.51** r 3.73 -20.13* r 4.35 9.73 r 3.63

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Crosses KC 2 x MCU 5 KC 2 x MCU 12 MCU 5 x MCU 12

m 19.04** r 0.35 19.80** r 0.35 17.33** r 0.31

* Significant at 5 % level ** Significant at 1 % level

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VARIABILITY FOR YIELD AND QUALITY ATTRIBUTES IN INTERSPECIFIC PROGENIES OF SACCHARUM SP


Nagarajan, R., S.Alarmelu and R.M.Shanthi

ABSTRACT
Evaluation of 781 (439 S.officinarum x S. spontaneum and 342 S.robustum x Commercials / S.officinarum ) hybrid progenies for cane yield and quality traits revealed differences between the groups for number of millable canes, cane height, cane weight and juice quality characters. Heritability was moderate (33.8%) for cane height to high (73.18%) for single cane weight in S.officinarum x S.spontaneum group. Heritability was 38.82% for cane diameter to 62.87% for brix in S.robustum x Commercials / S.officinarum group. Heritability was moderate for sucrose in both the groups.The higher estimates of heritability coupled with higher genetic advance for number of millable canes and moderate sucrose indicated that the heritability of the traits is mainly due to additive effects and selection would be effective for these traits.High heritability combined with low genetic advance for single cane weight indicates the role of non additive effect.S.robustum x commercials /S.officinarum group had high heritability coupled with moderate genetic advance for brix and sucrose. Mean performance of the traits under study revealed the superiority of the S.robustum x commercial /S.officinarum group over S.officinarum x S.spontaneum for cane thickness,cane weight, brix, sucrose and yield in F1 for further use. S.officinarum x S.spontaneum group can be exploited in producing elite hybrids after back crossing and nobilization.

Introduction Genetic variability is essential for any successful crop improvement and use of wild germplasm in broadening the gene base need no emphasis..Over the decades, sugarcane breeding have been a sort of closed one though not strictly inbreeding.Initial involvement of a few clones from the cultivated S.officinarum , S.barberi and S.sinense and wild S.spontaneum in the beginning of the century provided the genetic base and needed variation for the varietal improvement in sugarcane for many decades.The wider genetic base is essential to have a better genetic gain for longer term and in sugarcane this is all the more needed, as the genetic gain in recent years has declined for many yield and quality attributes.In this connection the statement of Hawkes ( 1977) that in most crops the need for a wider genetic base is strongly apparent and this can be generally provided from

wild species and primitive cultivars is quite relevant. Narrowing of genetic base with slow rate of genetic improvement warranted the need for widening the genetic base by exploiting unutilised clones of cultivated and wild species of Saccharum in breeding programmes [Arceneaux (1965) , Price (1965),Roach (1968), Dunckelman and Breaux (1971) and Roach (1977)]. Apart from base broadening, interspecific hybridisation programme is important considering the increase in sugarcane area under adverse environments and many diverse use sugarcane is put into besides conventional sugar production. Walker (1987) reported that a range of improved nobles is available for utilisation, which have been evolved from a small programme of generationwise polycrossing and selection for yield and quality within this species. He also indicated that these improved nobles must form

Sugarcane Breeding Institute, Coimbatore- 641 007, India nagarajan_sbi@rediffmail.com 145

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a better base for sugarcane improvement. In general, S.officinarum clones are known to impart sucrose genes,clones of S.spontaneum confer vigour ,high tillering, resistance to biotic and abiotic stresses and wider adaptability and S.robustum for biomass and fibre.The developed improved clones of the three species were utilised in crossing and the progenies from two different nobilized groups involving improved S.officinarum, S.spontaneum and S.robustum were assessed for genetic variability, heritability and genetic advance for further exploitation. Materials and methods Improved clones of S.officinarum and S.spontaneum utilised in the present investigation were developed by selecting clones of S. officinarum followed by intercrossing among themselves and the progenies were subjected to selection for economic attributes through seedling and clonal stages. This resulted in a number of improved clones of S. officinarum. Similarly selected clones of S.spontaneum and S.robustum were also used in a crossing and selection cycle to produce improved clones of S.spontaneum and S.robustum.The improved clones developed were utilised in crossing and evaluated to study the genetic parameters in F1 population and exploit them for sugarcane improvement. The experimental material comprised seven hundred and eighty one (439 S.officinarum x S. spontaneum and 342 S.robustum x Commercials /S.officinarum) hybrid progenies from 30 crosses involving 9 S.officinarum, 12 S.spontaneum, 7 S.robustum and 5 commercials clones.The progenies were selected at seedling stage based on economic traits and the hybridity of the progenies was confirmed by distinct features of the species.The selected progenies along with checks were evaluated in 1R with 3m plot size in randomized block design with two replications.The crop was given normal package of practices and monitored. At the age
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of 12 months, data on number of canes /plot (NMC), cane diameter,cane length,single cane weight and H. R Brix were recorded. Cane yield/ plot was estimated as a product of number of canes /plot and single cane weight.Data on ten quantitative charcaters were recorded using standard procedures. Observations on NMC, cane height, cane diameter, brix, sucrose,single cane weight and sucrose were recorded.The data were tabulated and statistically analysed to derive heritability, genetic advance,GCV and PCV(Singh and Chaudary,1985). Results and Discussion The mean, range, PCV, GCV and coefficient of variation for various cane yield attributes and juice quality parameters for the two groups under study are presented in Tables 1 and 2 .The results are discussed below. a) S.officinarum x S.spontaneum progenies Number of millable canes The mean value was high (99.28 nmc) combined with a wide range of 27-170. The other genetic parameters viz.,genotypic and phenotypic variance, heritability ,PCV, GCV and genetic advance was observed high for the trait. There were many individual clones in this group possessing high NMC. Cane diameter The mean values were low with a range of 1.47-2.82. The population had low genotypic variance, phenotypic variance,heritability and GA. As expected the canes were thin due to high stalk populationwhich is probably due to predominant influence of S.spontanuem, which need further improvement through backcrossing. Cane height
For cane height, the population had high mean (200.67 cm), wide range (135-260) and low genotypic and phenotypic variance. Heritability and genetic advance were low indicating the

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role of nonadditive genes for cane height.

Single Cane weight Low mean values (0.56 kg) with wide range (0.27-1.01), high genotypic and phenotypic variances, high heritability (0.7318) and GA as% mean was observed for single cane weight. The presence of high PCV and GCV indicates the dominance of additive gene effect for the expresssion of the trait. Cane yield High mean (53.47 kg/row) and range (27.75 to 103.29) with low heritability, moderate to high GA was observed indicating the role of nonadditive effects in control of the character. Heterosis for cane yield was observed by Roach, 1968,1977.The group recorded higher cane yield in comparision to commercial varieties and S. officinarum parental stocks indicating the higher potentiality of their interspecific hybrid population for cane yield. Quality parameters Brix The mean value was generally low for brix (13.12%) with a moderate range of 9.2-17.65. Moderate to high genotypic and phenotypic variances was observed for the trait. and sucrose (9.74%). Though the population mean in general was low from commercial point of view, a number of types with brix above 17.0 % were identified. Sucrose The mean value was generally low for sucrose (9.74%) with a moderate range of 5.40 - 14.58. Moderate to high genotypic and phenotypic variances was observed for the trait. GA % mean was high due to moderate heritability and high variability for sucrose. Variation for this character was found to be moderate.Similar results were also reported in F1 progenies of S.officinarum x S.spontaneum
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(Roach, 1968,1977). Walker (1971) also reported low ranges for total sugars in such hybrid populations of S.officinarum and S.spontaneum. The studies clearly showed the superiority of this group for cane population..Variation was also very substantial for this important attribute which is neccessary for effective selection. Though high cane population/plot was observed,average cane diameter showed considerable reduction . In general, canes were thin which is normally expected and certainly back crossing either to a different S.officinarum clone or a good commercial clone is required to improve cane thickness. Mean single cane weight was in general low, although individual clones from families recorded single cane weight of around 0.8 to 1.0 kg. Improvement in single cane weight is essential along with good cane population to boost the cane yield. This improvement is possible through improved cane diameter in backcross generation without reduction in F1 vigour. Selection of parents for backcross and subsequent selection in backcross generation are very important to harness the benefit of improved S.officinarum clones and wild S.spontaneum clones. Cane population in general was lower in crosses involving S.officinarum x S.spontaneum compared to commercial variety x S.spontaneum (Pers.comm). But in respect to cane diameter and single cane weight, S.officinarum x S.spontaneum crosses had slightly higher values when compared with hybrid x S.spontaneum. This might be attributed due to the 2n+n chromosometransmission in S.officinarum x S.spontaneum and n+n transmission in commercial clones x S.spontaneum. The presence of wide variation, coupled with high heritability and GA for two important components NMC and single cane weight can be exploited to improve cane yield in this group. The hybrid progenies involving

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S.officinarum x S.spontaneum as suggested by Walker (1971) could be utilised for identifying genetic stocks with increased sugar content and better adaptability. Heritability and genetic advance was maximum for nmc indicating the reliability of this trait in selection of parents for hybridization. Earlier studies (James, 1971) emphasized the importance of number of stalks as the most reliable character on which selection has to be based High heritability coupled with moderate genetic advance was observed for single cane weight and cane diameter which needs further improvement through nobilization. Juice quality parameters particularly sucrose showed moderate heritability and high GA as % mean indicating that selection will be effective for sucrose % and it will be moderate for brix %. b) S.officinarum x S.robustum The mean values for all the characters except number of millable canes were higher in S.officinarum x S.robustum progenies with a wide range of expression..
Number of millable canes

Cane diameter High mean (2.57) combined with narrow range (1.67-3.20) was observed in this group. Genotypic variance, phenotypic variance, heritability and GA was very low . Single cane weight Single cane weight had higher magnitude of GCV and PCV and moderately high genotypic variance and phenotypic variance. The group mean was high (0.963 kg) with a narrow range of 0.48-1.67. High heritability combined with low genetic advance and high GAas % mean for single cane weight indicate higher role of additive gene in governing the trait. Cane yield For cane yield the overall mean was low (30.46 kg / row) and ranged from 12.75-50.74. Higher magnitude of GCV and PCV with moderate heritability was observed in the population. Genetic advance as % of mean was high which may be due to high variation in the population and enough variability for the trait. Brix The group had a high mean of 19.00% with a wide range between 11.2-23.3 %. The trait had low magnitude for genotypic variance, phenotypic variance , GCV and PCV.Though the heritability was high (63.0%),GA % mean was low indiacting low variation for the trait in this population. Sucrose The GCV, PCV, genotypic variance and phenotypic variancewas moderate. High mean (16.32 %) with wide range (8.12 -21.33%) was observed for the trait.High heritability coupled with moderate GA as % of mean suggest that selection will be effective for sucrose. The superior mean performance for the quality and yield attributes may be due to better
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A wide range of 13.75 -57.5 was observed for the trait. The other genetic parameters viz.,genotypic variance , phenotypic variance , GCV and PCV were high. The higher estimates of heritability (65.0%) coupled with higher genetic advance for nmc indicated that heritability of the trait is mainly due to additive effects and selection is effective for such trait.
Cane height Cane height registered a high mean value of 216.42cm with a wide range (180.00 -262.5). High magnitude of phenotypic variance with moderate genotypic variance and low heritability (37.61%) was observed in the population. Moderate GCV and PCV with moderate GA though GA % as mean was low was observed for the triat.

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exploitation of both additive and non additive gene effects present in the population.The estimates of variability, h2 and genetic advance for most of the characters in this group indicated greater variance in the progenies. Ramana Rao (1972) also reported that a large amount of variability for most of the characters and emphasized the importance of both additive and non additive variances for the characters. GCV in progenies involving improved S.officinarum x S.robustum was higher for nmc and single cane weight. The characters with wider range viz., number of millable canes, single cane weight, sucrose, brix and cane yield had higher magnitude of GCV and PCV except cane height.This suggests that there is scope for selecting better segregants in this group based on NMC, single cane weight and sucrose. Single cane weight (60.5%), number of millable canes (65.06%) and quality parameters (62.8%) showed high genetic advance.This suggests that gain from selection based on nmc, single cane weight would be higher in S.officinarum x S.robustum combination than in S.officinarum x S.spontaneum progenies. Comparison Yield components S.spontaneum progenies had high NMC and cane yieldwhich can be further improved through nobilization. In S.robustum progenies the

improvement of yield components viz., cane height, diameter and single cane weight offers scope for realization of more genetic gains for yield. Quality parameters S.robustum progenies had substantial increase for juice quality parameters and compared well with progenies of crosses involving Co varieties.S.spontaneum progenies had moderate brix and low sucrose low which suggests the need of back crossing to improve the trait. The breeding material developed from nobilization for various component traits of yield and quality could be utilized as genetic stocks in further breeding programmes.The occurrence of superior recombinants among the progenies of S.officinarum /S.spontaneum / S.robustum indicates the possibility of realizing new types with desired character. In general while the F1 clones combining high NMC, moderate brix, good vigour and medium thin to thick canes identified from both the groups in this study needs further exploitation,the promising first generation hybrids of S.robustum progenies can be directly inducted into general breeding programme for further evaluation and commercial use.

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Table 1. Genetic parameters in F1 progenies from S.officinarum x S.spontaneum

Characters

Mean/

Range

V 2g h2 GCV PCV GA

V2p

GA as % of mean

row 27-170 135-260 1.47-2.82 0.27-1.01 9.12-17.65 5.40-14.58 51.53-85.23 27.75-103.29 97.74 262.78 37.62 84.75 0.44 0.37 4.46 7.99 0.56 3.48 6.26 0.56 14.69 21.69 8.40 18.48 0.03 0.04 0.73 28.80 0.08 0.12 0.68 3.07 3.72 33.66 19.70 29.03 12.61 30.31 172.56 740.19 0.33 6.54 13.56 459.68 718.65 0.64 21.50 26.88 325.32 13.06 0.48 0.28 2.86 3.25 8.41 12.42

Number of

99.28

35.42 6.51 23.12 50.75 21.87 33.39 11.53 23.22

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millable canes

Cane height (cm)

200.67

Cane diameter

2.08

150

(cm)

Single cane

0.56

weight (kg)

Brix

(%)

13.12

Sucrose (%)

9.74

Purity (%)

72.99

Cane yield

53.47

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Kg/row

Table 2. Genetic parameters in F1 progenies of S.officinarum x S.robustum


Mean/ 13.75-57.5 180.00-262.5 1.67 -3.20 0.48-1.67 11.22 -23.3 8.12 -21.33 66.64-91.54 12.75-50.74 12.73 198.37 22.21 395.33 0.57 0.50 4.58 7.31 0.62 13.12 4.18 23.11 0.05 3.69 0.07 5.87 0.60 0.63 22.30 10.11 0.04 0.11 0.38 8.30 13.32 28.64 12.75 16.57 5.52 32.62 238.62 634.40 0.37 7.13 11.64 237.39 364.86 0.65 23.67 29.34 25.60 19.51 0.27 0.34 3.13 3.49 5.56 20.54 Range V2g h2 GCV PCV GA V2p GA as % of mean 39.33 9.01 10.65 35.74 16.51 21.40 6.52 33.72

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Characters

row Number of millable canes 32.54 216.42 2.57 0.96 19.00 16.32 85.35 30.46

Cane height(cm)

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Cane diameter (cm)

Single cane weight (kg) Brix (%)

Sucrose (%)

Purity (%) Cane yieldKg/row

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REFERENCES Arceneaux, 1965. Cultivated sugarcanes of the world and their botanical derivation . Proc. ISSCT 12:844-854. Dunckelman,T. H. and R.D. Breaux ,1971. Breeding of sugarcane varieties for Louisiana with new germplasm. Proc. ISSCT.14: 233-239.3. Hawkes,J .G. 1977.The importance of wild germplasm in plant breeding. Euphytica 26: 615-621. James, N. I. 1971. Yield component in random and selected sugarcane population. Crop Sci . 11: 906-908. Price, S.1965. Interspecific hybridization in sugarcane breeding. Proc. ISSCT. 12:10211026. Ramaa Rao,T.C. 1972. Breeding value of sugarcane genetic stocks with special

reference to Saccharum robustum. Roach, B.T.1968. Quantitative effects of hybridization in S.officinarum x S.spontaneum crosses. Proc.ISSCT.13: 939-954. Roach, B.T. 1977. Utilization of S.spontaneum in sugarcane breeding. Proc.ISSCT.16: 4357. Singh, R.K and Chaudhary,B.D.1985. Biometrical Methods in quantitative genetic analysis. Walker, D. I.T.1971. Utilization of noble and S.spontaneum germplasm in the West Indies Proc.ISSCT.14: 224-232. Walker, D . I. T. 1987. Manipulating the genetic base of sugarcane. Copersucar International Sugarcane Breeding Workshop. Copersucar Technology Center, Brazil. pp. 321-334.

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GENETIC STUDIES ON PLANT, MATURITY AND PHYSIOLOGICAL CHARACTERS OF MAIZE (ZEA MAYS L.) UNDER RAINFED AND IRRIGATED CONDITIONS
Subba Rao, M. and R.D. Singh

ABSTRACT
In order to study the gene effects for various morpho-physiological characters related to drought tolerance in maize, generation mean analysis was performed in 3 different crosses and the results are summarized below. Results indicated that mean values (m) were highly significant for all characters studied under rainfed as well as irrigated conditions. Under irrigated conditions, the interaction additive x additive was only found significant for days to 50% tasseling in cross Ib.62 x Ib.37. The type of epistasis was found to be complementary in one cross and it was duplicate in two other crosses studied. Under rainfed conditions, additive effect (d), dominance effect (h) and epistatic effects additive x additive (i) and dominance x dominance effect (l) were significant for the cross Ib.63 x Ib. 128. The type epistasis was found to be complementary in one cross while it was duplicate in nature in other two crosses studied. Dominance effect (h) and epistatic effect additive x additive (i) and dominance x dominance (l) were found significant for days to 50% silking under rainfed conditions. The type of epistasis was both duplicate and complimentary in different crosses studied. Dominance effect (h) was found to be significant in the cross Ib. 62 x Ib.128 while additive effect (d) was found significant for cross Ib.128 x Ib.145. Both additive effect (d) and additive x dominant effect (j) were found significant for cross Ib.62 x Ib. 37. The type of epistasis was duplicate in all the three crosses under rainfed and irrigated conditions. Dominance effect (h) and dominance x dominance interaction (j) were significant in cross Ib.62 x Ib.37 for the character plant height. Epistasis was duplicate in nature. For ear height under rainfed conditions, all the types of gene effects were found to be significant in different crosses studied. The epistasis was of duplicate nature. Dominance effect (h) was found significant in cross Ib.128 x Ib.145 under irrigated condition for number of leaves per plant while under rainfed condition dominance effect (h) and additive x additive effect (i) were significant in cross Ib.62 x Ib.37. The type of epistasis was both complementary and duplicate. Additive effect (d) and dominance x dominance (l) were found for the cross Ib.62 x Ib.37. The type of epistasis was duplicate. For wilt ratings under irrigated conditions, all the gene effects (d,h,i,j and l) were highly significant in the cross Ib.128 x Ib. 145. Under rainfed condition the gene effects were non-significant for wilt ratings and canopy air temperature difference. A perusal of the gene effects revealed that the estimates vary for each cross in different degrees. Significant occurrence of additive, dominance and epistatic components necessitates a breeding methodology like reciprocal recurrent selection or biparental mating or diallele selective mating which would be more useful to improve morpho-physiological characters related to drought tolerance.

Introduction Maize is one of the most important cereal crops in India grown for grain as well as fodder
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purpose. Nearly 65 per cent of maize is grown under rainfed condition. Moisture stress is a major limiting factor for maize production under

Division of Genetics, Indian Agricultural Research Institute, New Delhi

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rainfed conditions. Global estimate indicates that there is a loss of about 15 per cent due to drought. Therefore, it is essential for enhancing the breeding effort for drought tolerance in maize. Drought is a complex phenomenon and depends on several characters like plants, maturity and physiological traits. Hence, it is essential to generate information on genetics of these traits. Keeping this in view, an experiment was conducted involving three crosses, respective inbred lines, F2 and two parental back crosses (BC1 and BC2). The results of these studies are described in this paper. Material and Methods The material for the study consisted of three crosses viz., Ib.62 x Ib.128, Ib.62 x Ib.37 and Ib.128 x Ib.145 along with respective parental lines and the two respective back crosses (BC1 and BC2) with male and female parents. The experimental material was grown in a randomized block design with 3 replications at Indian Agricultural Research Institute, New Delhi. The irrigated crop was grown with recommended number (5 irrigations during crop season) while rainfed crop was grown under purely rainfed conditions without any supplementary irrigation. Data were recorded on 5 plants for parents and hybrids and on 20 plants for F2 and back crosses. Observations were recorded on plant height, ear height, days to 50 per cent tasseling, days to 50 silking, anthesis silking interval (ASI),number of leaves/ plant, specific leaf weight, wilt ratings at seedling stage (1-5 scale) and canopy air temperature difference (CATD). Recommended cultural practices were followed during crop growth. Genetic analysis was performed as per the six-parameter model described by Hayman (1958). Additive (d), dominance (h) and non-allelic effects i,j and l were calculated as per the generation mean analysis described by Hayman (1958).The estimates of h and l along with their sign were
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utilized to understand the nature of epistasis. Results and Discussion The results obtained from generation mean analysis for different characters studied are presented in table 1. Estimates of gene effects under irrigated and rainfed conditions are discussed below. Maturity and plant characters Results indicated that mean values (m) were highly significant for the characters studied for all the three crosses under rainfed as well as irrigated conditions (Table 1). Days to 50 % per cent tasseling The interaction additive x additive (i) was found to be significant under irrigated condition in the cross Ib.62 x Ib.37. The type of epistasis was complimentary in this cross while this was duplicate in nature in other two crosses. Under rainfed condition, additive effect (d), dominance effect (h) and interaction dominance x dominance (l) were found significant in the cross Ib.62 x Ib.128. The type of epistasis was complimentary in Ib.62 x Ib.37 while it was duplicate in nature in other two crosses. Estimates of gene effects indicated predominance of non-additive gene effects for this trait. Additive gene effects were found important for days to 50 per cent tasseling by Gousenard et al. (1989) and Hemalatha and Sarkar (1990). Guo et al. (1986) reported predominance of dominance effects. Both additive and non additive effects were reported to be important by Ramesha (1988). Days to 50 % silking None of the gene effects were found significant under irrigated condition and type of epistasis was duplicate for all the crosses. Under rainfed condition, dominance effect (h) and epistatic effects additive x additive (i) and dominance x dominance effect (l) were found significant. The type of epistasis was duplicate

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in Ib.62 x Ib. 128, Ib.62 x Ib.37 and complimentary in Ib.128 x Ib.145. Dominance effects were found important for this trait by Singh et al, (1979), while epistatic effects were found important by Saha (1981) and Ahuja et al. (1983). Anthesis, silking interval Dominance effect was found to be significant for the cross Ib.128 x Ib.145 under irrigated conditions. Under rainfed condition, dominance effect (h) and epistatic effect (i) were found significant in cross Ib.62 x Ib.128. In cross Ib.128 x Ib.145 cross, additive effect (d) was found significant while both additive effect (d) and additive x dominant effect (j) were found significant for cross Ib.62 x Ib.37. The type of epistasis was found to be duplicate in nature for all the three crosses under irrigated and rainfed conditions. Bonaparte (1977) found the importance of additive and dominance gene effects for this character, the magnitude of dominance effects being more important. Plant height Under irrigated condition, none of the gene effects were found significant. Under rainfed condition, dominance effect (h) and dominance x dominance effect (l) were found significant in cross Ib.62 x Ib.128 while additive effect (d) and additive x dominance effect (j) were found significant in the cross Ib.62 x Ib.37. The type of epistasis was of duplicate nature. Singh et al (1975), Khalidi (1982) reported the importance of non-additive gene action for plant height while Hauller and Mirinda (1988) and Crossa et al (1990) reported the significance of additive gene action. Pal et al (1986), Debnath and Sarkar, (1987) reported that both additive and dominance gene effects were important. Marker and Joshi, (2005) indicated the importance of dominance variance for this trait though additive genetic variance was also found significant under rainfed condition.
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Ear height Under irrigated condition additive effect (d) was found significant in cross Ib.128 x Ib.145 while dominance x dominance epistatic effect (l) was found significant for cross Ib.62 x Ib.37. Under rainfed condition, dominance effect (d) and epistatic effects additive x additive (i)and dominance x dominance (l) effects were found significant for the cross Ib.128 x Ib.145 while only additive x dominance effect (j) were found significant in the cross Ib.62 x Ib.37. The type of epistasis was of duplicate nature in all the three crosses. The importance of both additive and non-additive gene effects were found important by Pal et al. (1986) and Debnath and Sarkar, (1987). Physiological Traits Mean effects (m) were highly significant for physiological characters for most of the crosses in both the moisture regimes. Estimates of gene effects for four physiological characters under two growing conditions are presented in table 2. Number of leaves per plant Dominance effect (h) was found significant in cross Ib.128 x Ib.145 under irrigated condition. The type of epistasis was of duplicate nature. Under rainfed condition, dominance effect (h) and additive x additive epistatic effect (i) were found significant in cross Ib.62 x Ib.37. The type of epistasis was duplicate in Ib.62 x Ib.37 and complementary in other two crosses. Sharma, (1987) and Brar and Labana (1991) reported the significance of additive gene effects for leaf number per plant. Specific leaf weight Dominance effect (h) and additive x additive epistatic effect (i) were found significant for cross Ib.62 x Ib.37under irrigated condition. The type of epistasis was duplicate in two crosses while it was complementary in

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Ib.128 x Ib.145. Under rainfed condition, additive effect (d) and dominance x dominance effect (l) were found to be highly significant in cross Ib.62 x Ib.37. The type of epistasis was duplicate in the three crosses. Wilt rating at seedling stage All the gene effects (d,h,i,j and l) were found significant in cross Ib.12 x Ib.145. In the cross Ib.62 x Ib.128 dominance x dominance interaction (l) was found significant while additive effect (d) additive x additive effect (i) were found significant in cross Ib.62 x Ib.137. The type of epistasis was duplicate. Under rainfed condition, the gene effects were found to be non significant for this trait. Hemalatha and Sarkar (1990) reported greater importance of dominance variance for wilt ratings due to water stress. Canopy air temperature difference (CATD) The gene effects were non significant for this character under rainfed and irrigated condition. Estimation of various gene effects for different plant, maturity and physiological characters indicated the presence of additive, dominant and all the three digenic epistatic effects in various crosses studied. Hence, breeding procedures like reciprocal recurrent selection or biparental crosses or diallele selective mating would be useful tools to improve the characters related to drought tolerance. Blum (1979), Martinello (1983) and Ashok Kumar and Sharma (2005) earlier reported such breeding procedures . REFERENCES Ahuja, V.P., Mukharjee, B.K and Agarwal, K.N. 1983. Epistasis and its contribution to the expression of yield and other metric traits in maize (Zea mays L.). Genetika 15: 1-8 Ashok Kumar and Sharma, S.C.2005 Gene action and heterosis for some quantitative
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characters in bread wheat (Triticum aestivum L). Indian J. Genet. 65: 281283 Blum, A. 1979. Principles and methodology of selecting for dourght resistance in sorghum. Monografic de Genetica Agrano. 4: 205215 Bonaparte, E.N.A. 1977. Diallel analysis of leaf number and duration of mid silk in maize. Can.J.Genet. Cytol. 19: 251-258. Brar, G.P.S and Labana, K.S. 1991. Partitioning of quantitative variability for grain yield and its components in maize. In: Abstracts of Golden Jubilee Symposium on Genetic Research and Education: Currents trends and Next Fifty Years. Feb. 12-15,1991, New Delhi. 2: 385 Crossa, J, Vasal, S.K. and Beck, D.L. 1990. Combining ability estimates of CIMMYTs tropical late yellow maize germplasm. Maydica, 35: 273-278. Debnath, S.C and Sarkar, K.R. 1987. Genetic analysis of grain yield and some of its attributes in maize (Zea mays L.). Thei. J. Agric. Sci. 20: 263-276 Gouesnard, B, Gallas, A and Lefort-Buson, M. 1989. Intra and inter population genetic variability in two forage maize populations. Agronomie 9: 867-876. Guo, P.Z., Gardner, C.O and Obaidi, M. 1986. Genetic variation and gene effects controlling prolificacy and other traits in maize (Zea mays L.). Acta. Genet. Sin. 13 : 35-42. Hallauer, A.R and Miranda, J.B.1988. Quantitative Genetics in Maize Breeding. Iowa State Univ. Press, Ames. P. 267294. Hayman, B.I. 1958. The separation of epistasis from additive and dominance variation in

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generation means. Heredity 12: 371-390. Hemalatha, G.V and Sarkar, K.R. 1990. Genetical studies on some parameters of drought tolerance in maize. Phytobreedon. Khalidi, G.A. 1982. Genetic analysis of yield and other quantitative characters in heterozygous populations of maize (Zea mays L.) M.Sc. Thesis. IARI., New Delhi. Martinello, P. 1983. Outlines of major components of maize breeding progrmmes for semi-arid regions (Capitanata plain). Genet. Agr. 37: 361-390. Marker, S and Joshi, V.N. 2005. Genetic analysis of maize (Zea mays L.) composite under stress and non-stress conditions. Indian J. Genet. 65: 211-212 Pal, S.S, Khera, A.S and Dhillon, B.S. 1986. Genetic analysis of and selection advance in maize populations. Maydica. 31: 153162. Ramesha, M.S. 1988. Genetic study of some rare quantitative characters in maize (Zea mays L.), M.Sc . Thesis; UAS., Dharwad.

Saha, B.C. 1981. Nature of inheritance and heterosis manifestation in physiological and agronomic attributes in hybrids of maize (Zea mays L.). Ph.D. thesis, IARI, New Delhi. Sharma, J.K. 1987. Study of Genetics of some morphological, physiological and biochemical characters associated with drought resistance in maize (Zea mays L.). Thesis abstracts. H.P. Krishi Viswavidyalaya, Palampur, P.107-108. Singh, R.D, Joshi, A.B, Dhawan, N.L and Mukharjee, B.K. 1975. Studies on elite Indian maize composites. I. Genetic analysis of grain yield and other agronomic traits. Genetika. 7 : 26-268. Singh, A.K, Dixit, R.K and Singh, H.G. 1979. Combining ability analysis for yield and its attributes in maize (Zea mays L.). Indian J. Agric. Res. 13: 27-30. Spangole, W.E. 1986. Moisture stress response in maize. Diss. Abst. Internat. B46: 2135B

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Table 1. Estimates of gene effects for maturity and plant characters in maize under irrigated and rainfed conditions.
Character Environment Cross

Gene Effects m d
-1.67 2.67 -2.33 -2.67* 3.33 -0.33 -2.33 0.33 -4.00 -1.67 -0.67 3.00 -0.67 -2.33 -1.67 1.00 -4.00* 3.33* 3.33 -0.67 13.67 5.33 -36.00* 3.33 2.67 -10.00 13.00* 10.33 -16.00 7.33

Type of epistasis

1.Days to 50% Tasseling

a) Irrigated

b) Rainfed

2. Days to 50 % silking

a) Irrigated

b) Rainfed

3. Anthesis silking Interval

a) Irrigated

b) Rainfed

4. Plant height (cm)

a) Irrigated

b) Rainfed a) Irrigated

5. Ear height (cm)

b) Rainfed

1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib 1. Ib 2. Ib 3. Ib

54.33** 55.33** 58.67** 65.00** 64.67** 65.33** 61.67** 61.33** 65.00** 72.67** 72.00** 74.67** 7.33** 6.00** 6.33** 7.67** 7.33** 9.33** 149.33** 160.33** 138.67 127.00** 137.33** 107.33** 56.67** 56.00** 45.33** 50.67** 49.67** 40.00**

-2.33 4.67 0.33 5.33 8.00* 2.00 -4.00 -3.33 -2.67 -19.83* -18.67* 0.5 -5.00 -2.67 4.67 12.00 11.33 -0.33 -8.83 -6.67 -1.67 2.67 3.33 1.00 -11.33 -8.00 -3.00 -28.97** -25.99** 2.67 -10.17 -10.17 -0.17 2.83 2.00 1.83 -6.5 -5.33 -0.50 -2.67 -4.67 -3.00 -7.33* -4.67 -0.33 -9.17** -7.33* 2.17 -5.17 -8.00 -4.83* -9.17 -9.33 2.17 78.67 44.00 -12.00 11.83 -19.99 -7.17 25.33 15.33 17.99 137.50* 120.00 -7.50 60.83 29.33 -47.83** 67.99 59.99 -6.99 24.99 9.33 -4.00 7.67 -20.00 -10.00 14.00 8.67 12.33 48.33 40.67 3.33 38.83 24.00 -20.50* 26.67* 21.33* 0.67

-2.00 Complementary -14.67 Duplicate 16.00 Duplicate 32.99** Duplicate -7.33 Complementary -12.00 Duplicate 0.33 Duplicate -5.33 Duplicate 24.00 Duplicate 44.67** Duplicate 8.33 Duplicate 1.00 Complementary 2.33 Duplicate 9.33 Duplicate 8.00 Duplicate 11.67 Duplicate 15.67 Duplicate 13.00 Duplicate -89.33 Duplicate 24.33 Complementary -82.67 Duplicate -189.67* Duplicate -17.67 Duplicate -102.67 Duplicate -40.67 Duplicate 71.33* Complementary -44.00 Duplicate -63.00 Duplicate -4.33 Duplicate -40.00* Duplicate

*, ** : Significant at 5% and 1% level, respectively

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Table 2. Estimates of gene effects for physiological characters in maize under irrigated and rainfed conditions. Gene Effects Type of Character Environment Cross m d h I j l epistasis
1. No. of leaves per plant a) Irrigated
1. Ib 62x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145 10.33** 1.20 5.33* 3.73 1.07 -2.93 Duplicate 12.20** -0.23 -0.83 -1.00 -3.99 3.27 Duplicate 13.07** 0.80 -2.73 -3.47 -1.00 2.93 Duplicate

b) Rainfed

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

11.27**

0.33

2.45

0.93

-0.58

0.69

Complementary

11.53**

-0.27

4.78**

3.47*

-1.05

-5.30

Duplicate

10.87

0.37

-1.75

-1.53

0.05

-1.17

Complementary

2. Specific a) Irrigated leaf weight (g)

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

6.39**

0.50

-2.00

-2.83

0.43

5.02

Duplicate

6.61**

0.51

-3.50*

-3.46*

0.68

4.66

Duplicate

5.84**

0.35

-0.74

-0.44

0.21

-0.67

Complementary

b) Rainfed

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

7.26**

1.83**

2.85

1.72

1.31

-6.49*

Duplicate

6.49**

2.08**

3.63

3.46

2.51**

-5.00

Duplicate

7.00**

0.10

0.42

-1.47

-0.26

-1.12

Duplicate

3. Wilt rating at seedling stage

a) Irrigated

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

1.33**

0.00

-2.00

-1.33

0.33

4.00**

Duplicate

1.00**

0.67*

0.67

1.33**

0.67

1.33

Duplicate

2.00**

-1.00**

-2.17**

-2.00** -0.83** 5.67**

Duplicate

b) Rainfed

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

2.67**

-1.00

-0.33

-0.67

1.00

3.33

Duplicate

3.33**

0.67

-0.50

0.00

0.83

-1.67

Complementary

3.00**

-0.33

3.83

3.33

-0.17

-5.00

Duplicate

4.Canopy air a) Irrigated temperature difference ( 0 C) b) Rainfed

1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145 1. Ib 62 x Ib 128 2. Ib 62 x Ib 37 3. Ib 128 x Ib 145

-2.06*

-0.97

-1.12

-3.27

-0.55

8.37

Duplicate

-1.60

-0.23

-10.85

-10.20

0.22

13.17

Duplicate

-2.53**

0.27

-2.30

-2.40

-0.37

3.53

Duplicate

-0.33

0.17

-8.00

-6.87

2.53

11.87

Duplicate

-1.70

2.00

-5.37

-6.00

2.03

13.80

Duplicate

-1.06

-0.23

1.73

2.47

-0.20

2.53

Complementary

*, ** : Significant at 5% and 1% level, respectively


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GENETIC ANALYSIS OF LEAF ANATOMICAL CHARACTERS ASSOCIATED WITH JASSID RESISTANCE IN COTTON (GOSSYPIUM SPP.)
Shimna Bhaskaran, R.Ravikesavan and T.S.Raveendran

ABSTRACT
In a 6 x 6 diallel analysis three Gossypium hirsutum genotypes and three introgressed lines obtained by crossing Gossypium hirsutum and Gossypium arboreum were assessed for their combining ability and nature of gene action for nine anatomical characters, which contribute resistance to jassids. Parents and crosses showed significant differences for all characters. The parents KC 2, IS 30/68 and 376/4/3 were good general combiners. The crosses MCU 5 x MCU 12, 376/4/3 x IS 14/21, KC 2 x MCU 5, MCU 12 x MCU 5 were identified as good specific combiners for resistance contributing characters.

INTRODUCTION Cotton (Gossypium spp.) is one of the most important commercial crops of India, which plays major role in Indias industrial and agrarian economy. In India, cotton is grown in 8.75M ha with a production of 15.30 Million bales. Biotic constraints particularly insect pests are known to affect the stability in production. Bollworms and sucking pests are the two important groups of pests in cotton which cause considerable damage to cotton crop in terms of yield and quality. Jassids are regular, serious and most important sucking pests of cotton. The nymphs and adults of jassids suck the sap from the leaves and cause phytotoxic symptoms known as hopper burn which result in complete desiccation of plants (Narayanan and Singh, 1994). Use of resistant variety is a vital tool of integrated pest management (IPM). The choice of an appropriate breeding procedure for the development of pest resistant varieties depends on the nature and magnitude of genetic variation for factors governing resistance present in base population. It is also important to examine the mechanism of resistance in any breeding programme focussed to exploit host plant

resistance. Therefore, the present investigation was undertaken to study combining ability of resistant genotypes. Materials and methods The experimental material consisted of six genotypes: three G hirsutum genotypes viz., KC-2, MCU-5, MCU-12 and three introgressed lines IS 14/21, IS 30/68 and 376/4/3 obtained by crossing G. hirsutum and G. arboreum species and inducing polyploidy by treating colchicine. These parents were crossed in a 6x6 diallel mating design during September 2004 at Cotton Breeding Station, Tamil Nadu Agricultural University, Coimbatore. Thirty hybrids along with their parents were raised in RBD with two replications. Histological studies of leaves were carried out following the method described by Johannsen (1940) and trichome density was estimated following the procedure of Maite et al., (1980). A total of nine characters viz., phloem distance , phloem thickness, number of palisade cells, palisade cell height, spongy parenchyma height, tissue ratio, trichome density, stomatal density and leaf thickness were studied. The mean values of these anatomical and morphological features were used for combining ability analysis.

Department of Cotton, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore 160

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Combining ability analysis was done by the procedure outlined by Griffing (1956) for method I in model I. Results and discussion The analysis of variance for anatomical and morphological characters revealed highly significant difference among genotypes qualifying them for further study (Table 1). The combining ability analysis provides useful information regarding selection of parents in terms of the performance of their hybrid. Further, the analysis elucidates the nature and magnitude of various types of gene action involved in expression of quantitative traits (Dhillion, 1975). The analysis of combining ability revealed that the variance due to GCA and SCA were highly significant for all the characters studied, indicating the presence of both additive and non additive gene action controlling these characters. The magnitude of GCA variance (Table 2) was higher than that of SCA for all characters except spongy parenchyma height indicating preponderance of additive gene action which could be exploited for improvement of these traits by following simple selection, mass selection and pedigree selection. The GCA/SCA variance ratio was narrow for all the characters studied revealing the importance of both additive and non-additive gene action controlling these characters. Singh and Gupta (1970) also reported results similar to the present findings. Information on the per se performance and nature of general combining ability of characters is necessary for selection of suitable parents for developing hybrids. The parent KC 2 recorded highest mean performance for phloem distance and leaf thickness. Parent 376/4/3 was the best performer for tissue ratio and trichome density. IS 30/68-recorded superior performance for number of palisade cells and palisade cell height. The parent KC 2 was considered as best general combiner for characters phloem
161

thickness and leaf thickness (plate 2). The parent MCU 5 was best combiner for phloem distance and stomatal density. MCU 12 had higher gca effect for spongy parenchyma height and tissue ratio. Identification of parents based on either per se performance or gca effect alone was misleading in selection programme (Arumugam Pillai and Amirthadevarathinam, 1998). In the present study, considering gca effects and per se performance together, parents KC 2, 376/4/3 and IS 14/21 were selected as best. None of the parents was found to be good general combiner for all the traits. Hence, it would be desirable to have multiple crosses involving the parents and subject them to selection in segregating generations to detect superior genotypes with resistant characters. For all the traits some of the parents possessed favorable genes and some did not. The gca effects depend on the relative value rather than absolute value. Simmonds (1979) stated that the gca values relatively depend on the mean of the chosen material. But if a parent possessed significant gca effects for as many traits as possible it is ideal to consider it for hybridization rather than parents with low gca effects or based on mean performance. The other parent chosen for hybridization should possess favorable gca effects for other complementary traits, so that favorable recombinants for all traits could be obtained. sca effects and hybrid vigour of the crosses are considered frequently in cases where nonadditive component of genetic variance predominates the inheritance. The superior hybrids were selected on the basis of high per se performance and sca effects (Table 3) for each of the trait in the present investigation. The hybrid MCU 12 KC-2 recorded high mean performance for phloem distance and leaf thickness (plate 4) and the hybrid IS 30/68 IS

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14/21 had high mean performance for trichome density and low mean performance for tissue ratio. Hybrid 376/4/3 x MCU 5 showed high per se performance for thickness of phloem. The hybrid MCU 5 MCU 12 recorded highest positive significant sca effects for, phloem distance and tissue ratio (plate 3). The hybrid MCU 12 x MCU 5 recorded positive significant sca effects for phloem thickness and spongy parenchyma height. Out of thirty hybrids seventeen recorded positive significant sca effect for phloem distance and sixteen hybrids recorded positive sca effect for palisade cell height. Hybrid, 376/4/3 X IS 14/21, a combination resistant to jassid, had significant positive sca effect for that trait. The hybrid MCU 5 MCU 12 recorded positive significant sca effect for all the resistant characters studied. It is concluded that the parents, KC 2, IS 30/ 68 and 376/4/3 are good general combiners for resistant characters. The crosses MCU 5 x MCU 12, 376/4/3 x IS 14/21, KC 2 x MCU 5, MCU 12 x MCU 5 were identified as good specific combiners. REFERENCE Arumugam Pillai, N. and Amirthadevarathinam, A. 1998. Combining ability for economic traits using CMS system in cotton. Agric Sci.Digest 18: 54-58

Dhillion, B.S.1975. Application of partial diallel crosses in plant breeding. A review. Crop Improvement., 2:1-7. Griffing, B. 1956. Concept of general and specific combining ability in relation to diallel crossing systems. Australian J. Biol. Sci., 9: 463-493. Johannsen, D.A., 1940. Plant micro techniques. McGrew Hill Bock co. tuc. New York. Pp. 121-154. Maite, R. K., Bidinger, F.R., Reddy, K.V.S. and Davies, J.C. 1980. Nature of occurence of trichomes in sorghum lines with resistance to sorghum shootfly. Joint Progress report, Sorghum Physiology-3, sorghum entomology-3, ICRISAT, Patancheru, A.P. India, pp. 20-23. Narayanan, S.S. and Phundan Singh. 1994. Resistance to Heliothis and other serious insect pest in Gossypium spp. - A Review. Indian Soc. Cott. Improv., 19: 10-24. Simmonds, N.W. 1979. Principles of crop improvement. Longman Group Ltd., Londan .408p Singh, R.B. and Gupta, M.P. 1970. Combining ability for yield characters in upland cotton. Indian J. Genet., 30(3): 608-618.

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Table 1. Analysis of variance for combining ability

Source

df

Phloem P thickness (P) Tissue ratio


0.197 xx 0.561 xx 0.114 xx 0.802 xx 29.37 x 37.06 xx 2844.10 xx 6505.64 xx 127.52 xx 1013.31 xx 49.76 xx 2687.18 xx 2474.29 xx 4134.27 xx 2191.72 xx 2369.03 xx 9.02 x 2059.41 xx 429.89 xx 10.17 xx 256.10 xx 375.88 xx 12.48 xx 400.90 xx 319.48 xx 10.46 x 328.31 xx 369.37 xx

Phloem P distance (P)

Palisade height (P) P

Number of Spongy palisade parenchyma P cells height (P)


0.00032 xx 0.00098 xx 0.00021 xx 0.00005 x

Trichome Stomatal Leaf thickness density / (P) microscopic density/ mm2 P field

Genotypes

35

558.24

Parents

343.31 xx

Hybrids

29

604.72 xx

Parents Vs

285.15 xx

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Hybrids 1831.99 xx 2705.76 xx 31.39 0.26 119.74 xx 0.033 46.99 xx 9.87 xx 279.55 xx 328.42 xx 0.160 xx 5.497 xx 7.38 11.18 xx 242.38 xx 446.83 xx 0.074 xx 21.27 xx 114858 xx 3676.33 xx 14930.08 xx 0.00025 xx 0.00017 xx 0.00019 xx

F1s

14

1035.66 xx

Reciprocals

14

174.94 xx

163
11.46
xx

F1s Vs

588.44 xx

reciprocals 17.67 1882.64 1226.37 xx 1032.75 xx 5.566 xx 79.541 xx 209.47 xx 2.87 xx 224.71 xx 182.15 xx
xx

Error 11.30
xx

35 236.35
xx

1.48 117.89
xx

9.20 0.238

7.65

0.003 xx
xx

6.93 70.09
xx

154.56 2807.25
xx

0.00001 0.00029 xx

GCA

347.25

SCA

15

184.48 xx

0.118 xx 0.033 xx

15.69 xx 19.00 xx

771.40 xx 1427.88 xx

0.00017 xx 0.00011 xx

RCA

15

351.05 xx

Plant Breeding in Post Genomics Era

*Significant at 5 per cent level, ** significant at 1 per cent level

Table 2. gca effects of parents for nine characters

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Parents
-7.497** -2.539** -9.539** 24.490** -5.535** 0.619 -1.667** -7.642** 2.278** -0.542* -3.026** -2.449** 0.292 1.543** 1.919** -0.031** 0.086** 0.245* 0.542** -3.704** 1.374* -0.073** 1.083** -2.964** -4.335** -0.113** 0.292 2.458** -3.685** -0.114** 02.076** -0.213 2.182** 0.975 -2.953** -2.697**

Phloem distance (P) P

Palisade cell Phloem Spongy Number of height thickness parenchyma palisade cells P heightv (P) (P) (P) P P

Trichome Stomatal leaf thickness density / Tissue ratio microscopic density/ mm2 (P) P field
-15.189** -4.328 -7.563** -8.999** 26.139** 9.939** -0.002** 0.001* -0.002** 0.010** -0.003* -0.002**

376/4/3

4.733**

164

IS14/21

-7.033**

IS30/68

-0.537

KC-2

-0.146

MCU5

7.325**

MCU12

-4.342**

Plant Breeding in Post Genomics Era

* Significant at 5 per cent level, ** significant at 1 per cent level

Table 3. sca effects of hybrids for nine characters

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S.No.

Hybrids

Phloem distance (P) P Number of palisade cells Palisade cell height (P) P

Phloem thickness (P) P

Trichome Spongy density / Stomatal leaf thickness parenchyma Tissue ratio (P) microscopic density/ mm2 P heightv (P) P field

165

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

376/4/3 x IS14/21 376/4/3 x IS30/68 376/4/3 x KC2 376/4/3 x MCU5 376/4/3 x MCU12 IS14/21 x 376/4/3 IS14/21 x IS30/68 IS14/21 x KC2 IS14/21 x MCU5 IS14/21 xMCU12 IS30/68 x 376/4/3 IS30/68 x IS14/21 IS30/68 x KC-2 IS30/68 x MCU5 IS30/68 xMCU12 KC2 x 376/4/3 KC2 x IS14/21 KC2 x IS30/68 KC2 x MCU5 KC2 x MCU12 MCU5 x 376/4/3 MCU5 x IS14/21 MCU5 x IS30/68 MCU5 x MCU5 MCU5 x MCU12 MCU12 x 376/4/3 MCU12 x S14/21 MCU12 xIS30/58 MCU12 x KC-2 MCU12 x MCU5

5.226** -10.024** 22.947** 3.472 4.068* -32.375** -5.032** 3.789* 17.139** -14.015** 1.375 28.575** -28.786** -8.911** -6.890** 8.375** -1.325 -21.650** -5.515** 13.581** 31.375** -11.250** 25.700** 5.625* 47.431** -34.375** 42.500** -18.625** 14.125** 1.050

-2.092** -2.338 -8.979** 18.300** 7.467** 7.750** 1.629 11.787** -15.058** 2.358 -1.500 -3.050 -0.008 -12.079** 4.612** -1.000 2.900 -10.300** 5.929 -4.879* 27.00** -3.125 -8.650** 14.00** 6.700** 18.00** -3.625** -18.625** -0.225 27.425**

0.750 -0.208 1.292 0.125 -1.250 0.750 -1.000 0.500 -1.667** -1.292 -1.750** 2.750** 0.042 -0.125 0.500 -1.500** 1.500* -1.375 -0.750 -1.000 0.500 0.500 1.500** 2.083** -2.000** 0.250 3.000** -0.500 -3.000**

5.328** -7.162** -11.251** 5.317** 11.809** 7.875** 18.482** 8.528** -4.064** -5.072** -1.125 -1.125 -5.247** 4.232** 12.199** 4.375** 0.890 -3.375* 4.232** 12.199 1.125 -12.50 -9.425** -8.125** 5.067** 10.500** 4.375** -2.125 5.225** 13.475**

6.564** -4.019** 6.060** 5.781** -3.549** -5.500** 10.056** 8.085** -11.944** 1.976 3.125** 14.850** 5.076** -0.553 -12.357** -7.500** 0.775** -16.175** -5.074** -9.603** -7.500** 12.125** -4.025** -8.750** 19.898** 3.000* -5.625** 0.750 10.300** 23.425**

-0.001 0.092** 0.342 -0.045* -0.331 -0.183** -0.167** -0.057** -0.099** -0.065** 0.065* 0.158** 0.166** -0.081** -0.102** -0.212** -0.005 -0.127** -0.149** -0.432* -0.108** 0.180** 0.128** 0.048 0.091** -0.140** -0.192** 0.600* 0.033 -0.058*

-2.988* -1.148 -4.481** 0.772 1.749 1.582 1.023 -4.075** 3.033* 1.060 -3.453* -2.935* 1.967 0.695 -1.710 -0.963 8.853* 1.607 -0.510 0.877 -0.128 -2.325 3.262 2.060 -0.548 -1.520 -3.157* 0.487 -0.808 2.95*

-9.493** 2.967 -2.972 31.140** 8.340 -4.000 -2.420 -5.008 0.928 5.978 -10.475 -16.600* -0.768 -13.437* 5.363 -8.850 6.775 9.470 26.100** -1.470 -32.550** -55.00** -3.950 -54.600** 18.518** -15.950* 51.500** -2.750 8.320 -4.908

0.002* 0.000 0.001 0.004** -0.004** -0.012 -0.002* 0.002* -0.000 -0.002* -0.004** 0.004** -0.010 -0.002* -0.004** 0.008** 0.000 -0.011 -0.010 -0.003* -0.004** -0.001 0.007** -0.004** 0.022** -0.005 0.008** -0.006** -0.013** 0.006**

Plant Breeding in Post Genomics Era

* Significant at 5 per cent level, ** significant at 1 per cent level

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Plate No. 1. Leaf anatomy of parent IS 14/21

Plate No. 2. Leaf anatomy of parent KC-2

LE Lower epidermis P Phloem elements UP Upper palisade


Plate No.3.Leaf anatomy of hybrid MCU 5 x MCU 12 Plate No. 4. Leaf anatomy of hybrid MCU 12 x KC-2

LE Lower epidermis P Phloem elements UP Upper palisade

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TECHNICAL SESSION III UTILIZATION OF PLOIDY BREEDING IN CROP IMPROVEMENT

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PRE-BREEDING THROUGH PLOIDY MANIPULATION TO EXPLOIT ALIEN GENETIC VARIABILITY


Amala J. Prabhakaran

ABSTRACT
Plant breeding efforts to develop varieties/hybrids with desired economic characteristics are constrained by the narrow genetic base of the cultivated species. Wild relatives of the cultivar species are rich sources of novel genetic variability in terms of resistance to biotic and abiotic factors, quality parameters, plant types and other desirable agronomic characteristics and continue to serve as sources of cytoplasm in case of hybrid crops. Hence, concerted efforts are required to incorporate additional variability from these reliable sources so as to develop varieties/hybrids with desired characteristics. Successful introgression of desirable genes from the distantly related wild into cultivated species requires a clear understanding about the genomes and many severe problems like incompatibility, genetic distance, increased or decreased number, structural heterozygosity need to be overcome. Extensive genetic, cytogenetic, and molecular investigations will immensely help to plan the strategies to over come many of the problems. A wide gap exists between making initial hybrids and releasing cultivars with good agronomic performance and yield. While alien genes from primary gene pool could be easily transferred through conventional breeding, the introgression of traits of interest from the species from secondary and tertiary gene pools requires several manipulations both at chromosome and genome levels using recent techniques since the interspecific/ intergeneric hybrids showed sterility caused by ploidy differences, genomic incompatibility, cytoplasmic imbalances or other factors. Altering the ploidy levels of either the parental species or the interspecific hybrid itself through colchiploidy or in vitro techniques is necessary. The various approaches to transfer alien genetic variability into cultivars combining conventional methods with recent advancements in cytogenetics and crop breeding are discussed with a few representative crops.

Indian Agricultural Research Institute, Regional Station, Wellington, The Nilgiris

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WHEAT POLYPLOIDS AS A MODEL SYSTEM FOR CROP IMPROVEMENT


Dalmir Singh and P.K.P. Meena

ABSTRACT
Evaluation of polyploidy is younger than evaluation of life. It evolved by nature to suit environment conditions. Obviously, it evolved from low chromosome organism (like bacteria and virus) to organism with high chromosomes (plant and animals). Range of chromosome number varied from 2n=4, n=2 (Haplopaphus gtracillic and 2n > 1200 in some petridophytes. In nature polyploids are very common. Plants serve majority of requirements of not only of human being but also animals which are used by human being directly or indirectly. All these plants complied of diploids and polyploids. The contribution of wheat, cotton, Oat, Tabacum, Sugarbeet, Sugarcane, lilium, turnip, brassica as poliploid can not ignored. Polyploids can be classified mainly into four different classes, autoplolyploids, alloploiploids, segmental allopolyploids and autoallopolyploids. In a diploid, one chromosome of each kind will form a set of chromosomes known as genome so that in a diploid two similar genomes will be present, i.e. AA. An autotriploid and an autotetraploid will have three doses and four doses of the same genome i.e. AAA and AAAA. If A is equal to 7 chromosomes, a monoploid will have 2n = 7 chromosomes, a diploid, 2n = 14 chromosomes, a triploid 2n = 21 chromosomes and a tetraploid 2n = 28 chromosomes. In an allopolyploid more than one genomes, each having same or different chromosome numbers may be involved. For example, a tetraploid wheat has 2n = 4x = AABB = 28 and hexaploid wheat has 2n = 6x = AABBDD = 42, where A, B and D each genome comprises 7 chromosomes. Today hexaploid wheat is the only polyploid system where a maximum number of aneuploids are available. Thanks to Dr. Sears for his monumental work, for developing and providing such a huge cytogenetical stocks to to the wheat scientists. Before we learn about the details of aneuploids, we must be aware of the detailed architect of wheats.

Evaluation of wheat from common ancestor There are several diploid species of wheat which have been classified into genomes like AA ( T.monococcum Linn., T. boeoticum Boiss.), BB (Ae. speltoides, T. searsii Feldman et Kisl.), CC ( Ae. caudate Linn.), DD (Ae. squarossa Linn.) MM (Ae. comosa Sibth et Sm), Nn (Ae. unicaritata Vis.), SS (Ae. sharonensis Eig.) and TT (Ae. mutica Boiss). It is suggested that first a wild diploid wheat (AA, 2n=14=7II) got crossed with Ae. speltoides (2n=14=7II) and F1 hybrid got amphidiploid (AABB= 2n=28). A mutation for ph gene took place in the F1 hybrid resulting the hybrid to have regular chromosome pairing (forming 14II at
Indian Agricultural Research Institute, New Delhi-110012

meiosis). Several tetraploid wheat species originated from the amphidiploid. Subsequently, one of the tetraploid got crossed with another diploid species with DD genome and gave rise to wild hexaploid wheat species with genome constitution of AABBDD (2n=42). Identification of chromosomes belonging to different genomes in T. aestivum. Crosses were made between different monos (2n-1) and T. dicoccum (AABB) Meiosis of all 21 F1s was studied. In situation when there were 14II+ 6I, the monosomic line was grouped into D genome. On the contrary, when there were 13II+ 8I, then the monosomic line was grouped into A genome and B genome. Monosomics belonging to A and B genomes
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were assigned numbers arbitrarily from 1 to 14 and from 15 to 21 for D genome. Monos for D genome were established and identified by Matsumura (1952). Separation of A and B genome chromosomes was done by crossing the amphidiploid of T. aegilopoides (AA) and Ae. squarrosa (DD) withy monotelos of A and B genomes i.e. T. aegilopoides (AA) x Ae. Squarrosa (DD)

Amphidiploid of F1 (AADD) Amphidiploid (AADD) x Monotelo (A & B genome) F1 Meiosis was studied. In case of presence of heteromorphic bivalent, then the mono was classified to A genome, when there were normal bivalents, univalents and one telocentric, the monos were classified into B genome category. Thus, a classification and designation of the chromosomes of T. aestivum was proposed. GENOME
A New Old B D Old

chromosome 5B had a maximum of 15II in a cell with a mean of 12II per cell. He proposed that chromosome 5B, induces asynapsis. True picture emerged after the work of Sears and Okamoto (1958).Monosomic 5 B x T. aegilopoides (AA), in the F1 hybrid having 5B gene, showed 3II to 7II a mean of 5II per cell, while F1 hybrid lacking 5B showed 5II to 13II average 10 II per cell. In one cell, 27 chromosomes were involved in pairing. Sears and Okamoto (1958) concluded failure of homoeologous chromosomes pairing appeared to be due to suppression of pairing by chromosome 5B. At the same time Riley and Chapman (1958) working with nullihaploid of Holdfast, n=20. They observed 4.2II, 0.8III per cell. Cells with 3III were common, 29% of cells had 5 II to 7 II . They postulated that the chromosome deficient in nullihaploid carried a gene which reduced pairing. Important conclusions drawn about Ph gene 1. Okamoto (1957) First to indicate that chromosome 5B involved in the regulation of chromosome pairing. 2. Sears and Okamoto (1958) Failure of homoeologous chromosome pairing appeared to be due to suppression of pairing by chromosome 5B. 3. Riley and Chapman (1958) Chromosome 5B carried a gene which reduced pairing. 4. Riley (1960), Riley and Chapman (1964) Reported that the gene suppress homoeologous pairing may be located on long arm of chromosome 5B. 5. Feldman (1966) Increased dosage of 5 BL (absence of 5BS) reduces Chiasma frequency. 6. Riley et al. (1966) In tetrasomiccondition, meiosis is normal. Therefore, it seems 5BS increase synapsis.

Homoeologous Groups 1 1A 2 3 4 5 6 7

New Old New 1 2B 1D XVII

XIV 1B 2A 3A 4A 5A 6A 7A II

XIII 2D III 3D

XX XVI XV XVIII XIX XXI

XIII 3B IV IX VI XI 4B 5B 6B 7B

VIII 4D V X VII 5D 6D 7D

A study carried out by Okamoto (1957) indicated the involvement of chromosome 5B in the regulation of chromosome pairing. It was a cross between T. aestivum x Amphidiploid (T. aegilopoides x T. squarrosa. DD). In the F1 he observed a maximum of 6 univalents per cell against the expected 14 II + 7 I. When monosomic 5B, T. aestivum was crossed with amphidiploid ( as above). The F1 hybrid lacking
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7.

Riley (1960) None of the putative progenitors possess this gene, it might have arose by mutation.

8. Feldman (1966). The gene is operative in somatic cells also. 9. Riley et al. (1966) It is possible to induce mutations which suppress the effect of 5 B. 10.Riley (1966) The homoeologous chromosomes 5A and 5D also excise a regulatory influence over meiotic pairing. 11. Wall et al. (1971) Reported the location of gene on the long arm of chromosome 5B. 12. Riley and Chapman (1958, 1963) Riley (1966, 1967) Sears (1967), Joshi and Singh (1979) and Singh (1992) Reported the practical applications of 5B system in wheat improvement. 13. Riley (1966) It is recessive in nature. In hexaploid wheat, after the development of monosomic nullisomuics by Sears (1954), a large number of aneuploid lines have been added which are listed below. Aneuploidy in polyploids 1. Monosomics (2n 1) 2. Double monosomics (2n 1 - 1) 3. Nullisomics (2n 2) 4. Tetrasomics (2n + 2) 5. Trisomics (2n + 1) 6. Double trisomics (2n + 1 + 1) 7. Telocentrics (2n 1 chromosome arm) Monotelosomics (2n 2 + 1 arm) Monotelo disomic (2n-1+1 arm) Double monotelosomics (2n 2 + 1 arm1 + 1 arm2) Ditelosomics (2n 2 + 1 arm1 + 1 arm1) Double ditelosomics (2n 2 + 2 arm1 + 2 arm2) 8. Addition lines (alien chromosome)
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Mono addition (2n + 1) Diaddition (2n + 2) 9. Substitution line (2n) varietal hromosome 10. Nullipolyhaploid (n 1) 11. Nullitetra [2n 2 (1A) + 2 (1B)]

Uses of aneuploids of wheat Location of dominant gene To locate dominant gene, identify all the monosomic plants from the (2n=41) from the monosomic lines possessing the trait in recessive form. Cross the identified monosomic plants (2n=41) as female with the variety in question (2n=42) as male. Raise the F1 hybrids. Identify all the monosomic F 1 hybrids cytologically. All the monosomic (2n=41) and disomic (2n=42) hybrids will show dominant traits like the parent in question. Self all the monosomic F1 hybrids and one or two disomic F1 hybrids. Grow the seed obtained from monosomic and disomic F1 hybrids in the field. Evaluate all the F2 progenies derived from monosomic and disomic F1 hybrids. All the F2 progenies will segregate in a expected ratio of 3 dominant: 1 recessive, except one monosomic F2, where all the F2 plants will show dominant trait. this particular line is referred to as a critical line. Therefore the in question is located on this particular chromosome. Location of recessive gene To locate recessive gene, it is better to take a monosomic series possessing the trait in dominant condition. Identified monosomic (2n=41) plants are crossed as female with the variety in question as male parent. From F1 hybrids, monosomic (2n=41) F1 hybrids are identified. In this case, all the monosomic and disomic F1 hybrids will show dominant trait except one monosomic F1 hybrid, which will show recessive trait. The critical monosomic line can be determined on the basis of monosomic F1 hybrids, but it has to confirmed

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through the F 2 data. The particular monosomic F1 hybrid will produced plants with recessive trait while rest of the monosomic and disomic F2s will segregate in a expected ratio of 3 dominant: 1 recessive. The gene in question is therefore, located on the chromosome of that monosomic line which did not show any segregation. Locating hemizygous in effective gene The hemizygous ineffective gene can not be located simply by observing F2 ratios, closer study of the F2 segregation is required. Since in wheat, the disomics appears at a frequency of
Cross mono telo (S) disomic (Recessive) (mts) a

about 25%, the critical F2 also segregates in 3 dominant : 1 recessive. The critical family can however, be identified by cytological examination of the recessive segregates, all of which would be disomic. In other families, only about 25% of the recessive would be disomic. The recessive gene s (sphaerococcum) was located in this manner by Sears (1947). Location of dominant gene on specific chromosome arm Once the dominant gene is located on a chromosome, it needs to be located on specific arm of the chromosome. Follow the schemewith dominant male A A

Identify monotelo disomic F1 hybrids cytologically (mts) selfed for F2

A A Discarded A Phenotype like male A Phenotype like female

Study all the F2 plants cytologically

The gene is located on the long arm (in monotelo long arm is missing) In case the cytologically identified F2 plants with disomic, monotelo disomic and ditelosomic nature show dominant feature, the gene in question is located on the short (S) arm of the chromosome. Utilizing, aneuploid lines provided by Sears, several monosomic lines in different wheat background have been developed. The aneuploid lines thus developed have made it possible to locate genes like, disease resistance, grain weight, grain number, protein content, plant height, spike colour, spike length, spikelet number, tiller number, root
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number, root length, drought tolerance and maturity etc., it is practically a huge list to review. Transfer of monosomic series into a known variety The variety in question should be free from any meiotic abnormality, in other words there should be normal or regular chromosome pairing at meiosis. Procedure Identify all the monosomic plants cytologically at first meiotic metaphase (20II+ 1I).

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Cross all the monos of Var. Chinese spring x Variety in question (2n= 42, 21II). 1. Identify 42 and 41 hybrids, backcross 41 hybrid x Variety. 2. Identify 42 and 41 hybrids, IInd backcross 41 hybrid x Variety 3. Identify 42 and 41 hybrids, 3rd backcross 41 hybrid x Variety. Repeat the procedure upto 6th backcross. After six backcrosses, identify the monosomic plants showing 20II+ 1I at first meiotic metaphase. Like this, monosomics of all the 21 chromosomes are developed. A last step test the monosomic lines for their trueness. For this cross all the monosomic lines with their respective monotelosomic lines. Correctness of the line can be determined on the basis of meiosis of F1 hybrids. If at meiosis, a heteromorphic bivalent is present along with 20II regulator bivalents, the line is correct. If this situation is not observed, line is not correct and univalent shift has taken place. The line has to be developed again. Method of substituting a specific chromosome Using monosomics in hexaploid wheats Methodology Identify all the monosomic lines cytologically at first meiotic metaphase (2n=41, 20II+ 1I). 1. Cross all the monosomics of Var. Chinese spring (2n=41) x Variety in question (2n=42). Identify monosomic plant from F1 and self it.
2. Monosomics 1A of Chinese spring x Pollen from disomic selfed hybridIdentify mono somic F1 hybrid and self it. 3. Monosomic of Chinese spring x Pollen from disomic selfed hybrid.

backcross identify a monosomic plant in which most of nuclear part will be of var. Chinese spring, with a exception that it will have one chromosome from the variety in question. This way all the 21 chromosomes of a variety of interest can be substituted in the genetic background of var. Chinese spring by crossing all the monosomic lines of var. Chinese spring with the variety of interest. Disomic substitution lines can be obtained by selfing the monosomic plants. Critical analysis of these substitution lines can revealed the location of gene (s) on individual chromosomes in the variety of interest. Substitution lines were produced earlier using varieties Kenya Farmer, Mida and Marquis utilizing monoteloentric method. Using monosomics of var. Chinese spring, Swaminathan et al. produced substitution lines of var. Pb.C591. Wheat varieties, such as Hope, Thatcher, Red Egyptian and Timstein, resistant to stem rust were used to produce substitution lines (Sears et al., 1957). The substitution lines were compared for stem rust resistance with Chinese spring and the donor resistant varieties. A total of 9 different substitution lines (Chromosome 4B, 1D from Hope, 2B, 3B and 6B from Thatcher, 6A, 2B and 2D from Red Egyptian and 6B from Timstein) were found to be resistant to stem rust. Transfer of individual whole chromosome The addition of whole genome did not show much improvement except in few cases, it was thought that addition or substitution of individual whole alien chromosome might build desired results. A. Alien addition lines Rye chromosome additions An amphidiploid (2n=56) involving
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Continue the backcross till BC6. After 6

th

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hexaploid wheat and diploid rye is produced, by crossing hexaploid wheat and diploid rye followed by doubling of the chromosome number in F1 hybrid. The amphiploid is backcrossed to hexaploid wheat giving rise to a heptabloid with 2n = 49, 21 + 7, in which bivalents belong to wheat and univalents are of rye genome. The plants are selfed and monosomic (21 + 1 rye) and disomic (21 + 1 rye) addition lines are isolated. Wheat-rye addition lines have been produced using different varieties of wheat and rye. Individual addition lines can be identified morphologically. The addition lines thus produced can be used as source of desirable traits and can be transferred into wheat background through homoeologous recombination or translocation. Addition lines from Aegilops, Agropyron and Haynaldia to Wheat Barley chromosome additions to Wheat The wheat-barley crosses were made by Kruse (1973) and Islam et al. (1975) utilizing several approaches, including heptabloids. Islam et al. (1978) were able to produce six of the seven possible disomic additions of barley chromosomes to wheat. Alien substitution lines Like, addition lines, useful alien substitution lines have also been produced. In N. tabacum, a variety Samsoun resistant to mosaic virus, was developed by backcrossing the amphiploid (2n=72) involving N. glutinosa (2n=24) and N. tabacum (2n=48) to N. tabacum. In hexaploid wheat, alien substitution lines have been developed from Secale cereale, Agropyron elongatum, Ae. comosa, Ae. umbellulata, Hordeum vulgare, H. Chinense etc. Interchanges (through irradiation or homoeologous recombination) In case, plant breeders are interested in transferring the minimum chromatin material
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carrying the desirable traits, then efforts to be been made to produce translocations, either by irradiation or by using homoeologous recombination. Interchanges using irradiation The first useful transfer by irradiation involved a segment from Ae. umbellulata chromosome 6U, carrying resistance to wheat leaf rust (Lr9), to long arm of chromosome 6B of wheat (Sears, 1956). In the process, an amphidiploid (T. dicoccoides x Ae. umbellulata) was crossed with T. aestivum and after two more backcrosses with variety Chinese spring and selecting for leaf rust resistance, lead to a plant with 2n=43. In this plant the extra chromosome was from Ae. umbellulata carried the gene for leaf rust resistance. Along with this resistance it was also carrying some undesirable characters. The plant was given a high dose of X-rays before meiosis. Pollen from this irradiated plant was used for pollinating var. Chinese spring. Among 6091 off spring, 132 were resistant of which 40 had translocations, only one of them was intercalary. Plant with intercalary translocation was designated as Transfer. Giving irradiation of seeds and plants of an alien substitution line carrying Agropyron elongatum chromosome, a segment carrying leaf rust resistance was transferred to bread wheat variety Thatcher. It was possible to transfer a portion of Agropyron elongatum chromosome 6 el carrying stem rust resistance gene Sr26 to chromosome arm 6A. Another example is transfer of rye segment to wheat. Driscoll and Jensen (1964) produced 4A-2R translocation carrying resistance to wheat leaf rust and powdery mildew, Recently, translocations were induced between chromosomes of var. C306 and rye at IARI, New Delhi, possessing rust resistance from rye. In Avena, it was possible to transfer mildew resistance from A. barbata to Avena sativa.

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Recombination through chromosome pairing Besides irradiation, transfers of genetic mutational can also be achieved through recombination. In case of wheat the process of chromosome pairing is regulated by Ph. Gene which is located on the long arm of chromosome 5B. There are three distinct ways to isolate homoeologous recombinations. A. removal of 5B chromosome (i.e.nullisomic condition). B. suppression of 5B effect by the genome of Ae. speltoides or Ae. mutica, C. utilizing a recessive mutant of the Ph1 locus on 5B. Nullisomy for 5B was used by Riley (1966) for transfer of a segment from Ae. bicornis. Sears (1972) used nulli-5B-tetra 5D line and Agropyron. He was able to transfer segment from 3 Ag. (carrying Lr24 gene) and 7Ag (carrying Lr19) to chromosomes 3D and 7D respectively. Joshi and Singh (1978), Singh (1992) were able to transfer rust resistance genes from Secale cereale to hexaploid wheat through homoeologous recombination. In this process monosomic 5B of var. Pb.C591 (Fig.1) was crossed with a strain of rye (Fig.2) from Russia. Backcrossing the F 1 hybrid deficient for chromosome 5B (Fig.2), to hexaploid wheat. Further selfing and selection lead to the isolation of plants possessing rust resistance from Secale cereale.
Fig. 1
Wheat h
X Rye

Fig. 2

Meiotic Metap e pahse I F1 hybrid 5B x Rye h (deficie chromosome 5B) ent showing hig chromosome pairing gh g

speltoides and Ae. mutica which suppress the effect of Ph1. This method was used by Riley et al. (1968) for transfer of stripe rust (yellow rust) resistance from Ae. comosa to wheat. In this process, monosomic addition line isolated from the derivatives of the original cross (Wheat x A. comosa) was crossed to Ae. speltoides to get plants with 2n=29, where homoeologous pairing might have taken place due to suppression of 5B activity. It was backcrossed to wheat using selection for resistance at each stage. Finally, it gave a stock called Compair which had segment from chromosome 2M from Ae. comosa carrying yellow rust resistance. It was transferred to 2D chromosome of wheat. In Oats, a genotype of Avena longiglumis (CW 57) has been reported to carry genes suppressing chromosome pairing in the hybrids with Avena sativa, similar to that of Aegilops speltoides in wheat. Utilizing this species, mildew resistance was transferred from A.barbata to A. sativa. Mutants at Ph1 locus on 5BL chromosome arm of wheat have been successfully utilized for homoeologous recombination among wheatAgropyron and Wheat-rye chromosomes. Other approaches for wheat improvement. Aegilotricum

Homoeologous recombination can also be achieved by using certain strains of Aegilops


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Aegilotricum (2n = 56 = AABBDDNN) is the name given to an amphiploid derived from the cross Aegilops ventricosa (2n = 28 =

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DDNN) x Triticum turgidum (2n = 28 = AABB), and has been used for the transfer of resistance against the disease eyespot from Ae.ventricosa to wheat. By repeated crossing of this amphiploid with wheat, new resistant cultivar of wheat, namely Roazon was produced. However, Aegilotricum (2n = 70 = AABBDDDDNN), involving hexaploid wheat in the initial cross, could not be produced. Agrotricum Agrotricum was obtained as an amphiploid from a cross Agropyron intermedium (2n = 42 = E1E1E2E2NN) x Triticum aestivum (2n = 42 = AABBDD). Their vigor and fertility were however low. However, by backcrossing the F-1 hybrid with 6x wheat, a partial amphiploid, namely TAF 46, was obtained which had 2n = 56 AABBDDXX (where X is a new reconstructed genome having chromosomes from E), E2 and N genomes). This partial amphiploid was later used for getting alien addition lines for A. intermedium chromosomes to wheat, which were successfully utilized for transfer of resistance against the rusts to wheat. Triticales The raw amphidiploid triticales are often designated as primary triticales. As such raw triticales were not successful. Different approaches have been used to improve the triticales. 1. Crossing primary hexaploid triticales among themselves to get recominnant triticales. 2. Crossing primary triticales with either octoploid triticales or with hexaploid wheat to get secondary triticales. 3. Crossing of hexaploid triticales with rye and selecting superior types from segregating generation. 4. Crossing of hexaploid triticale with tetraploid wheat.
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5. Crossing hexaploid triticales with F1 hybrid, ABDR (6x wheat x 2x rye) 6. Induce mutations in primary triticale strains.Triticale Armadillo was a good success in 1970s. Late, amber seeded triticale DT 46 was developed from IARI, New Delhi. Genome reconstitution in Triticinae It is observed that the transfer of entire genome may not be desirable. All chromosomes in a genome may not be carrying useful genes. It, therefore, will be desirable to reconstruct a genome from two diploid species. It was done by Evans (1964). In this approach, Evans (1964) used Aegilops squarrosa (DD) and Agropyron elongatum (EE), two diploid species to produce two amphidiploids with the constitutions AABBDD and AABBEE, which were crossed to produce AABBDE (14II +14I). On selfing these hybrids, the plants with 21II, selected in the progeny, probably had AABB genomes intact and a constituted third genome having chromosomes from D and E genome. Availability aneuploids of hexaploid has greatly been enhanced because of its nature of polyploid. It has been possible to make use of these lines to identify, locate and transfer desirable genes not only from land races but also from alien sources for increasing the quantity and quality of wheat. The wheat aneuploids is therefore a model system to follow in other crop plants like cotton, sugarcane, sugarbeet, groundnut, tobacum, oats and many more. REFERENCES Driscoll, C.J. and N.F. Jansen. 1964. Characteristics of leaf rust resistance transferred from rye to wheat. Crop science 4: 372-374. Evans, L.E. 1964. Genome construction within the Triticinae I. The synthesis of

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hexaploids (2n=42) having chromosome of Agropyron and Aegilops in addition to the A and B genome of Triticum durum Can. J. Genet. Cytol.. 6: 19-28. Feldman, M. 1966 The effect of chromosomes 5B, 5D and 5A on chromosomal pairing in Triticum aestivum.. Proc. Natl. Acad. Sci. USA 55:1447-1453. Islam, A.K.M.R. Shepherd, K.W. and Sparrow, D.H.B. 1975. Addition of individual barley chromosomes to wheat. In: Gaul H. (ed.) Barley Genetics III. (Proc. 3rd Int. Barley Genet. Symp. Garching), pp. 260-270. Islam, A.K.M.R., Shepherd,K.W. and Sparrow, D.H.B. (1978). Production and characterization of wheat-barley addition lines. In: Ramanujam, S. (ed) Proc. 5th Int. Wheat Genet. Symp. New Delhi, pp.365371. Joshi, B.C. and D. Singh. 1978. Introduction of alien variation into bread wheat. Proc. Vth Intl. Wheat Genet. Symp. (India) 1: 342-348. Kruse, A. 1973. Hordeum X Triticum hybrids. Hereditas 73: 157-161. Matsumura, S. 1952. Chromosome analysis of the Dinkel genome in the offspring of a pentaploid wheat hybrid. III. 29 Chromosome D- haplosomics and their relation to nullisomuics. Cytologia. 17: 3549. Okamoto, M. 1957. Asyneptic effect of chromosome V. Wheat Info. Services. 5: 6. Riley, R. 1960. The diplodization of polyploid wheat. Heredity. 15: 407-429. Riley, R. 1966b. The genetic regulation of meiotic behaviour in wheat and its relatives. Mackey J. (ed). Proc. 2nd Int. Wheat Genet. Symp. Hereditas (Suppl.) 2: 395-408. Riley, R., and Chapman, V. 1958. Genetic Control of the cytologically diploid behaviour of
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hexaploid wheat. Nature (Lord) 182 : 713715. Riley, R., and Chapman, V. 1963. The effect of the deficiency of chromosome V (5B) of Triticum aestivum on the meiosis of synthetic amphidiploids. Heredity. 18(4):473-484. Riley, R., Chapman, V. and Belfield, A. M. 1966. Induced mutation affecting the control of meiotic chromosome pairing in Triticum aestivum. Nature 211: 368-369. Riley, R., Chapman, V. and Johnsen, R. 1968. The incorporation of alien disease resistance in wheat by genetic interference with regulation of meiotic chromosome synapsis. Genet. Res. 12: 199-219. Sears, E. R. 1947. The sphaerococcum gene in wheat. Genetics 32: 102-103. Sears, E.R. 1954. The aneuploids of common wheat. Messouri. Agri. Sta. Res. Bull. 572:1-58. Sears, E.R. 1956. The transfer of leaf rust resistance from Aegilops umbellulata to wheat. Brookhaven Symp. Biol. 9:1-22. Sears, E. R., Loegering, W.Q. and Rodenhiser, H.A. 1957. Identification of chromosomes carrying genes for stem rust resistance in four varieties of wheat. Agronomy Journal 49:208-212. Sears, E.R., Okamoto ,M. 1958 Intergenomic chromosome relationship in hexaploid wheat. Proceedings of the Xth International Congress of Genetics 2: 258-259. Singh, D. 1992. Mutation and recombination studies in wheat and rye. Ann. Wheat Newsletter, 38: 119-120. Wall, A.M., Riley, R. and Chapman, V. 1971. Wheat mutants permitting homoeologous meiotic chromosome pairing. Genet. Res. 18: 311-328.

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ROLE OF POLYPLOIDY IN COTTON


Khadi, B.M. and Vinita P. Gotmare

ABSTRACT
Polyploidy is common in plants and probably has been involved in the evolution of all Eukaryotes). The genus Gossypium L. is one of the best systems for examining genome evolution in polyploids, which comprises approximately 50 known species distributed in Arid to Semi-arid regions of the Tropics and Subtropics. Out of these 50 known species of Gossypium, 45 species are diploid and 5 allopolyploid falling into eight cytological groups designated as A, B, C, D, E, F, G and K while five allopolyploid species are designated with AD genome. A peculiarity among the 50 species identified and described so far in Gossypium, four species namely G. arboreum, G.herbaceum, G.hirsutum and G. barbadense are cultivated and remaining all are wild species.. Five natural polyploids of Gossypium species are recognized of which all are allotetraploid bearing A and D genomes (AADD, 2n = 52). These species originated following hybridization between an African or Asian diploid species (Genome AA, 2n = 26), as female with a diploid American pollen donor (Genome DD, 2n = 26). Molecular data suggests that allopolyploid Gossypium lineage arose about 1-2 million years ago (mya), with divergence of the two progenator diploid genomes occurring 4-8 mya. Recent studies have clarified many evolutionary aspects of Gossypium, relationship within and among the eight genome groups, domestication of the four cultivated species and the origin of the allopolyploid cottons. Also that chromosome translocation have not played a role in the divergence of polyploid cottons . The resulting genomic reunions have led to an array of genetic mechanisms and adaptive response that are not yet fully understood. An insight of the polyploidy of Cotton will help in understanding its contribution to the ecological success and agronomic potential for its improvement.

Introduction Polyploidy is common in plants and probably has been involved in the evolution of all eukaryotes ( Soltis & Soltis 1992) The cotton genus Gossypium L, is one of the ideal system for examining genome evolution in polyploids, which comprises of approximately 50 species distributed in arid to semi-arid regions of the tropics and sub-tropics. Out of these 50 known species of Gossypium, 45 species are diploid and 5 allopolyploid falling into eight cytological groups designated as A, B, C, D, E, F, G and K while five allotetraploid species are designated with AD genome. A peculiarity among the 50 species identified and described so far in Gossypium, four species namely, G. arboreum, G. herbaceum, G. hirsutum and G. barbadense are cultivated and remaining all are wild species (Endrizzi et al. 1985; Steward 1995 and Wendel
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et al. 2003). Five natural polyploid Gossypium species are recognized of which all are allotetraploid bearing A and D genomes (viz. AADD; 2N = 4x = 52). These species originated following hybridisation between an African or Asian diploid species (genome AA; 2n = 26), as female and with a diploid American pollen donor (genome DD; 2n = 26) (Percival et al.1999, Wendel 1995, Wendel et al.1999). Molecular data suggest that the allopolyploid Gossypium lineage arose about 1-2 million years ago, with divergence of the two progenitor diploid genomes occurring 4-8 million years earlier (Seelanan et al. 1997, Wendel and Albert 1992). Molecular data suggests that allopolyploid Gossypium lineage arose about 12 million years ago (mya), with divergence of the two progenator diploid genomes occurring 4-8 mya. Recent studies have clarified many

Central Institute for Cotton Research, Post Bag No 2, Shankar Nagar P. O., Nagpur-10

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evolutionary aspects of Gossypium, relationship within and among the eight genome groups, domestication of the four cultivated species and the origin of the allopolyploid cottons. Also that chromosome translocation have not played a role in the divergence of polyploid cottons ( Waghmare et al 2005). The resulting genomic reunions have led to an array of genetic mechanisms and adaptive response that are not yet fully understood. An insight of the polyploidy of Cotton will help in understanding its contribution to the ecological success and agronomic potential for its improvement. Origin and diversification of Cotton Cotton is unique among crop plants in that four separate species were independently domesticated for the specialised single-celled trichomes, or fibres, that occur on the epidermis of the seeds. This parallel domestication process involved four species, two from Americas, Gossypium hirsutum and Gossypium barbadense and two from Africa-Asia namely G. arboreum and G. herbaceum. Although all four cotton species spread beyond their ancestral homes during the last several millenia, one species G. hirsutum has dominated the world cotton scenario. G. hirsutum has spread from its original home in Mesoamerica to over 50 countries in both hemispheres. G. hirsutum (upland cotton) and G. barbadense (Pima cotton, Egyptian cotton) have polyploidy genomes resulting from a truly remarkable chance biological reunion among ancestral diploid genomes that are geographically restricted to different hemispheres. The cotton tribe, which includes eight genera (Fryxell et al. 1992) is different from other members of the Malvaceae on the basis of morphological features of the embryo, wool and seed-coat anatomy, and by the presence of gossypol glands also known as punctae or lysigenous cavities widely distributed on the plant body. Monoploidy of the tribe has been confirmed
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using comparative analysis of chloroplast DNA restriction site variation (La Duke and Doebley 1995) and DNA sequence data (Seelanan et al. 1997, Wendel et al. 2002, Wendel & Cronn 2003). Genus Gossypium in the tribe Gossypieae is largest and most widely distributed containing about 50 species (Fryxell 1992) including four domesticated species. These cultivated species are genetically diverse but this diversity is dwarfed by that included in the genus as a whole whose species belong to both tropical and sub-tropical regions of the world. Gossypium is distinguished from other related genera like Lebronnecia, Gossypioides, Cephalohibiscus, Kokia, Hampea, Cienfugosia and Thespeca by a combination of characters including - Undivided style, Coriacious capsule containing several seeds per locule, A somatic chromosome number of 26 and Presence of three foliaceous involucellar bracts subtending each flower Recent molecular phylogenetic analysis have demonstrated that the diverse group of species belonging to Gossypium constitute a single natural lineage (monophylectic group) despite their world-wide distribution and extraordinary morphological and cytogenetic diversity (Cronn et al. 2002b, Seelanan et al. 1997, Wendel et al. 2002) Using sequence divergence data from the chloroplast gene ndhF and published sequence divergence rates to calibrate a molecular clock, it was suggested that Gossypium branched off from Kokia and Gossypioides approx. 12.5 mya with Kokia and Gossypioides separating some 3 mya (Cronn et al. 2002b). These two genera which have now geographically isolated from one another by thousands of kilometers of open ocean (Kokia from Hawaii and Gossypioides from Madagaskar and East Africa) implies that trans-oceanic dispersal was evolved in the evolution of one or both genera. Thus, Gossypioides-Kokia examples represent only the long distance, oceanic dispersal as a factor

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in the evolution of the cotton Tribe and the genus Gossypium (De Joode and Wendel 1992; Wendel and Percy 1990). Species in the Genus Gossypium At present, Gossypium includes approximately 50 species, but new species continue to be discovered from the primary centres of diversity in tropics and sub-tropics and are grouped as Australian species , African Asian species and American diploid species. . The wild species of cotton represent an ample genetic repository for potential exploitation by the cotton breeders but still remain largely untapped genetic resource (Khadi et al. 2003, Kulkarni & Khadi 1998). Asian-African species Gossypium consists of fourteen species from Africa and Arabia. There are four subsections in Gossypium (Fryxell 1992). 1. Senata : G. turfacatum from deserts in Somalia. This species is poorly understood taxonomically and cytogenetically and there is a possibility that it may not belong to Gossypium but to Cienfulgosia due to its unusual feature of dentate leaves. 2. Sub-section Pseudopambak : E-genome, very limited material is available. G. benadirense, G. brichettii, G. volksenii. Cytogenetic characteristics or molecular phylogenetic affinities not yet studied. 3. Cultivated cottons of sub-section Gossypium, one G. arboreum and G. herbaceum : A genome 4 Sub section Anomalum : B genome species F-genome longicalyx. Australian species Australian species (Sub-genus: Strata) comprises of 16 named species as well as a new species that is yet to be named. They comprise of C- (Sturtia), G-(Hibiscoides) and K(Grandicalyx taxonomic section) groups with 2,
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3 and 12 species respectively.They are herbaceous perennials with a bi-seasonal growth pattern whereby vegetative growth dies back during the dry season. Grandicalyx have pedicels that recurve following pollination so that the capsules are pendent and open inverted at maturity; releasing seeds that bear laisomes, which attract ants. Many of these species are poorly represented in collections are incompletely understood taxonomically. Molecular phylogenetic analysis have yielded conflicting results regarding interspecific relationships in this group (Liu et al. 2000, 2001a; Seelanan 1999; Small & Wendel 2000, 2002b; Seelanan 1999) American diploid species These are the New World D-genome diploids (Table-1) and have been collected and studied than others and their taxonomy is well understood. Increasing evidences suggest that G. gossypioides is basal-most . The centre of diversity for 13 species of D-genome diploids is western Mexico, but long distance dispersals have led to the evolution of G. raimondii in Peru and G. klotzschianum in Galapogas Islands. Chromosomal evolution and the concept of genome group Cytogenetic investigations started as early as 1920s and revealed that species in Gossypium had haploid chromosome complement 13 and 26. Longley (1933) suggested that a duplication of the chromosomes of an ancestral type for this doubled chromosome number 26. Later, it was suggested that the species having 26 pairs are allotetraploids and that the ancestral donors involved both wild American species and Asiatic species. Allopolyploid nature of the American tetraploid cotton species emerged from the work of Beasley (1940) and Harland (1940) wherein allotetraploids from A genome (Asiatic) and D genome (American) diploids showed that

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these could form fertile hybrids. These classical cytogenetic studies demonstrated that American tetraploid species are true allopolyploids consisting of A and D genomes and that D genome is similar to those found in the American diploids. A number of additional findings support this hypothesis of allopolyploidy origin of the American tetraploid origin which include studies of duplicate factors controlling morphology, meoitic pairing behaviour in synthetic polyploid, phytochemical analysis, isozyme markers, comparative genetic mapping and comparative analysis of DNA sequences. These studies prove that the allotetraploid species formed from hybridization between A and D genomes ancestors, most nuclear genes are duplicated in the AD-genome cottons and when both copies are isolated and sequenced, they correspond phylogenetically and phenetically to those of A and D genome diploids (Wendel et al. 2002). Five known allopolyploid Gossypium species are diversified and distinct. G. darwinii is native to the Galapogas islands where large and continuous populations are formed. G. tomentosum has more diffuse population structure, occurring mostly as scattered individuals. G. mustelinium is restricted to NE Brazil and is an uncommon species. All above three are the true wild species whereas the remaining two G. hirsutum and G. barbadense have been domesticated over a period of time.G. hirsutum is distributed in Central and South America, the Carribean and islands in the Pacific. G. barbadense is distributed in the North of South America. Phylogenetic relationship among the species Recent molecular phylogenetic investigations have shown the genealogical lineages of the species and relationship within and among the different genomic group and geographical distribution. Each genome group corresponds to
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single natural lineage. (Cronn et al. 2002b) Phylogenetic history of Gossypium There exist four major lineages of diploid species corresponding to three continents Australia(C, G, K genome), America (Dgenome), Africa-Arabia (A, B & F genome in one group, and E genome in the other group). New world and Old world diploids are phylogenetic sisters. African F genome, which consists only one species G. longicalyx could be sister to A genome species. The wild forms are more closely related to the A genome species G. arboreum and G. herbaceum. At the time of allopolyploid formation, the A and D genomes have merged which represents the reunion of two genomes belonging to different hemispheres and diverged for millions of years in isolation from one another. The lineages of Gossypium were established in relatively rapid succession from Kokia Gossypioides. The phylogenetic studies suggest that all African Arabian Cottons comprise a single group. A lot of variation exists in the views for the divergence of Gossypium. Gossypium is relatively ancient and was thought to be evolved 60-100mya (million years ago). Recent molecular data suggest that the earliest split in Gossypium has taken place 12 mya (Seelanan 1997) and another Cp DNA sequence divergence data suggests that the genus originated 5-15 mya (Cronn et al.). While the maximum likelihood approach to estimate the lineage of diploid cottons suggest that their divergence occurred within a span of 2 mya. Long distance dispersal has played an important role in the diversification of Gossypium and atleast one dispersal between Australia and Africa and another to the Americas (probably Mexico) has lead to the evolution of the D genome diploids and later colonization of New World has taken place by the A genome ancestor of the AD genome allopolyploids.

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Table 1. Genome groups in Gossypium


Genome
A (2n=26) B (2n=26) C (2n =26) D (2n =26)

Total species
2 3 2 13

Species
G. herbaceum, G. arboreum G. anomalum, G. triphyllum, G. capitis-viridis G. sturtianum, G. robinsonii G. thurberi, G.armourianum, G. harknessii, G.klotzchianum, G.davidsonii, G.aridum,G.raimondii, G.gossypioides G. lobatumG.trilobum, G.laxum, G.turneri, G.schwendimanii G. stocksii, G. somalense, G. areysianum, G.incanum, G. benadirense, G.bricchettii, G.vollesenii, G. trifurcatum G. longicalyx G. bickii, G. australe, G. nelsonii G. anapoides, G. costulatum, G. cunninghamii, G.exiguum, G. enthyle, G.condonderriense, G.merchantii, G. nobile, G. pilosum, G. populifolium, G. pulchellum, G. rotundifolium, G. sp. nov G.hirsutum, G.barbadense, G.tomentosum, G.mustilinum, G.darwinii

Geographical origin
Africa possibly Asia Africa, Cape Verde Island Australia

Primarily Mexico, Peru, Galapagos Islands, Arizona

E (2n =26)

Arabian Peninsula, N.E. Africa, S.W. Asia

F (2n =26) G (2n =26)

1 3

E. Africa Australia

K (2n =26)

13

N. W. Australia

AD(2n=56)

New world Tropics & subtropics including Hawaii

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Long distance dispersal clearly has played an important role not only in the diversification of major evolutionary lines but also in speciation within Gossypium genome groups. Examples include dispersals from Southern Mexico to Peru (G. raimondii), from Northern Mexico to Galapagos Islands (G. klotzschianum), from Western South America to Galapagos Islands (G. darwinii), from Africa to Cape Verde Islands (G. capitis viridis). The origin of Kokia Gossypioides from a common ancestor suggest a common dispersal mechanism of Oceanic drift. In this respect it is found that seeds of many species of Gossypium are tolerant to prolonged periods of immersion in salt water. Seeds of G. tomentosum are capable of germination even after three years of immersions in artificial salt water. Seeds of some species may retain buoyancy for atleast two to three months, which may be insufficient for trans oceanic dispersal perhaps in some cases long distance dispersal, may have taken place through natural rafting on floating debris (Stephens 1966). Parentage of Alloployploids Which of the modern species of A and D genome diploids best serve as models for the progenitor genome donors? Over the decades a diverse arrays of tools have been used to solve this questions. Phylogenetic investigations using DNA sequencing of homologous genes is the latest tool. Allometric occurring allopolyploids G. hirsutum and G. darwinii (Stephens 1946) were the first two to explain the hypothesis of parentage and it states that either G. klotzschianum its closed relative of G. davidsonii or G. raimondii in combination with G. arboreum would produce a hybrid showing considerable similarity to present day New World Cottons. Addition support for the hypothesis of G.raimondii as D genome donor emerged from comparative analysis of plant habit and shape, floral features and extra floral bract morphology in synthetic A D amphiploids and from observations of lint characteristics and vigour of
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intergenomic hybrids (Hutchinson et al. 1945). The ployploid parentage could be explain with the help of cytogenetic data combined with the observations suggesting that the only known wild A genome cotton is African (G. herbaceum subsp. Africanum) and that polyploidization has occurred following a trans Atlantic introduction to the New World of species similar to G. herbaceum. As a member of the D genome diploids G. raimondii, from Peru, belongs to Mexican evolutionary lineage and appears to share cytoplasm with G. gossypoides. But G. gossypoides genome contains a number of repetitive DNAs that are shared with A genome species (Endrizzi et al. 1985). As G. gossypoides is the only D genome diploids that exhibits evidence of genetic contacts with an A genome plant, it must have acquired these introgressant genomic components after phylogenetic separation from the lineage leading to G. raimondii (Abdalla et al. 2001, Cronn et al. 1999) Hence, long distance dispersal of A genome in the New World may have occurred after G. gossypioides diverged from G. raimondii. This evolutionary history raises the possibility that the G. gossypioides lineage was involved in the origin of allopolyploid. A-genome introgression into G. gossypioides and initial polyploid formation may have been spacially temporarally-associated events (Wendel et al. 1995b) But recent analysis of nuclear genes place G. gossypioides as basal within the subgenus of, distant from D-genome of the allopolyploids. Thus, there are difference of opinion for the chloroplast and nuclear genomes with respect to the relationship between G. gossypioides and G. raimondii and a full understanding of the parentage of polyploids is yet not clear. Stabilization of Chromosomes and Genomic Interaction in Polyploids The A and D-genomes of allopolyploid

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Gossypium are more distinct from one another than their diploid progenitors (endrizzi et al. 1985) One possibility of genome stabilization after polyploidization could be reorganization of the two genomes so that they are no longer capable of homeologous pairing. Genetic map comparisons showed that two reciprocal translocations arose in the diploid lineage after allopolyploid formation. Allopolyploid Gossypium was not accompanied by chromosomal rearrangement (Paterson et al. 2000, Wendel & Cronn et al. 2003). Recent data shows that Chromosome arm translocations have not played a role in the divergence of polyploid Cottons and that one terminal inversion on Chromosome 3 appears to differentiate G.tomentosum from G. barbadense (Waghmare et al 2005).

ultimately degenerate as pseudogene. Duplicated genes may maintain their original function or their function may be distributed . The mechanism that affect gene expression , evolution and gene silencing for stabilization of allopolyploids have played a very important role. Ecological and Agronomical Consequences of Polyploidization Polyploidy is often associated with broader ecological amplitude and novel evolutionary opportunity, mediated by the increased buffering capacity afforded by duplicated genes and the enhanced vigor resulting from the fixed heterozygosity of their duplicated genomes. In Gossypium allopolyploidy led to the established of a new ecological niche. In contrast to majority of diploid species , allopolyploid species occur in coastal habitat and among the five allopolyploid species , two are completely restricted to near coastline ( G. darwinii & G.tomentosum ) and for G.barbadense and G . hirsutum , wild forms in litoral habitats from Gulf of Mexico, NW South America to Pacific Islands (Brubaker and Wendel 1994, Fryxell 1979). In case of allopolyploid Gossypium, this dispersal capacity was associated with specialization for establishment in coastal communities. Thus, the allopolyploid could exploit the fluctuating sea levels i.e a new ecological niche.

A - genome diploids have twice the DNA content per cell as D genome diploids, with a corresponding difference in chromosome size (Endrizzi et al 1985). These differences are maintained in allopolyploid Gossypium, although DNA content is not additive and A genome has chromosome size slightly smaller than those in diploids (Brubaker et al 1999b). Recombination rates are conserved between A & D diploid genomes and for a common set of markers, the genetic length of these two genomes differ by 6 % and at tetraploid level, the recombination in the two resident genomes differed by only The agronomic consequence of polyploidy by 5%. Polyploidy has promoted higher rates is important as it deals with the fibre. Four of recombination in Gossypium. separate species of Gossypium were The genomic consequences of domesticated for their seed hairs, which allopolyploidy in Gossypium could be correlated evolved only once in the progenitors of all four to duplication of all nuclear genes and this cotton species . Earlier Gossypium species had duplication is responsible for relaxation, seed with short seed trichomes and tightly divergence between the duplicated genes and adherent to the seed . Mature seeds from wild acquisition of new function (Lynch and Conery species exhibit great diversity in fibre length , 2000, Otto and Whitton 2000, Zhao et al. 1998). colour and other properties but the earliest The other possibility of outcome of gene developmental stages are similar among all duplication could be that one member of the species (Applequist et al. 2001) . The fibre duplicated gene pair will become silenced and elongation terminates about two weeks from
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anthesis in most of the wild species while in A & F genome diploids it is extended to three weeks. This prolonged elongation period represents a key evolutionary event in the origin of long fibre and it happened prior to domestication. Thus , domestication of the New World Cotton was first precipitated by a developmental switch that occurred millions of years ago in a different hemisphere. Fibre growth curves for wild AD genome allopolyploids are similar to those of the wild A genome species but the fibre of allopolyploids is superior than that of cultivated Old World diploids. The genome wide gene duplication caused by allopolyploidization provided the raw material necessary for the evolution of novel gene expression pattern, which subsequently were exploited by the modern plant breeders of G.hirsutum and G. barbadense (Jiang et al. 1998, Wright et al 1998) . Majority of the loci affecting the fibre yield and quality are found in the D genome rather than A genome which explains the superiority of the lint of the allopolyploids. Theses studies in turn suggest allopolyplodization provided novel opportunities for agronomic improvement. REFERENCES Abdalla, A. M., Reddy, O.U.K., El-Zik, K.M., and Pepper, A. E. (2001). Genetic diversity and relationships of diploid and tertraploid cottons revealed using AFLP. Theor. Appl. Genet. 102: 222-229. Applequist, W. L., Cronn, R.C., and Wendel, J. F. (2001). Comparative development of fiber in wild and cultivated cotton. Evol. Dev. 3:317. Brubaker, C.L., and Wendel, J. F. (1994). Reevaluating the origin of domesticated cotton (Gossypiun hirsutum; Malvaceae) using nuclear restriction fragment length polymorphisms (RFLPs). Am. J. Bot. 81: 1309-1326.
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Brubaker, C. L., Bourland, F. M., and Wendel, J. F. (1999a). The origin and domestication of cotton. In Cotton : Origin, History, Technology and Production (C. W. Smith and J. T. Cothren, Eds.), pp. 3-31. Wiley, New York. Brubaker, C. L., Paterson, A. H., and Wendel, J. F. (1999b). Comparative genetic mapping of allotetraploid cotton and its diploid progenitors. Genome 42: 184 203. Cronn, R. C., Zhao,X., Paterson, A. H., and Wendel, J. F. (1996). Polymorphism and concerted evolution in a tandemly repeated gene family : 5S ribosomal DNA in diploid and allopolyploid cottons. J. Mol. Evol. 42. 685-705. Cronn. R., Small. R. L., and Wendel. J. F. (1999). Duplicated genes evolve independently following polyploid formation in cotton. Proc. Natl. Acad. Sci. USA 96. 14406-14411. Cronn, R. C., Small. R. L., Haselkorn, T., and Wendel. J. F. (2002b). Rapid diversification of the cotton genes (Gossypium : Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes. Am. J. Bot. 89. 707-725. Endrizzi, J. E., Turcotte, E. L., and Kohel, R. J. (1985), Genetics, cytogenetics, and evolution of Gossypium. Adv. Genet. 23: 271-375. Fryxell, P. A., Craven, L. A., and Stewart, J. M. (1992). A revision of Gossypium sect. Grandicalyx (Malvaceae), including the description of six new species. Syst. Bot. 17: 91-114. Geever, R.F., Katterman. F. R. H., and Endrizzi, J. E. (1998). DNA hybridization analyses of a Gosssypium allotetraploid and two closelyrelated diploid species.

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Theor. Appl. Genet. 77: 553-559. Hang. C., Wright, R. J., Woo, S. S., DelMonte, T.A., and Patterson, A. H., (2000). QTL analysis of leaf morphology in tetraploid Gossypium (Cotton).Theor. Appl. Genet. 100: 409-418. Hanson, R. E., Zhao, X.-P., Islam-Faridi, M. N., Paterson, a. H., Zwick, M. S., Crane, C. F., McKnight, T. D., Stelly, D. M. , and price, H. J. (1998). Evolution of interspersed repetitive elements in Gossypium (Malvaceae). Am. J. Bot. 85: 1364-1368. Jiang, C., Wright, R., Ei-Zik, K., and Patterson, A.H. (1998). Polyploid formation created unique avenues for response to selection in Gosssypium (cotton). Proc. Natl. Acad. Sci. USA 95. 4419-4454. Khadi,B.M.,Kulkarni,V.N.,Ansighkar A.S.,Singh, V.V. and Gotmare Vinita, (2003) Genepool concept for diploid cultivated cotton. World Cotton Research Conference 3 held at Cape Town South Africa, 9-13 March 2003 pp, 213-225 Kulkarni,V.N. and Khadi, B.M. (1998).Long linted G.arboreum for meeting textile industry needs. Proceedings of world cotton research conference-2 held at Athens, Greece: 1074-77. Liu, B., and Wendel, J. F. (2000). Retroelement activation followed by rapid repression in interspecific hybrid plants. Genome 43: 874-880. Liu, B., Brubaker, C. L., Mergeai, G., Cronn, R. C., and Wendel, J. F. (2001a). Polyploid formation in cotton is not accompanied by rapid genomic changes. Gemone 43: 874880. Lynch, M., and Conery. J. S., (2000). The evoluationary fate and consequences of duplicate genes. Science 290: 1151-1155.
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Otto, S. P., and Whitton, J. (2000). Polyploid incidence and evolution. Annu. Rev. Genet. 34: 401-437. Paterson, A.H., Bowers, J. E., Burow, M. D., Draye, X., Elsik, C. G., Jiang, C. X., Katsar, C. S., Lan, Y. R., Ming, R., and Wright, R. J. (2000). Comparative genomics of plant choromosomes. Plant Cell 12: 1523-1539. Percy, R.G., and Wendel, J. F., (1990). Allozyme evidence for the origion and diversification of Gossypium barbadense L. Theor. Appl. Genet. 79: 529-542. Seelanan, T., Brubaker, C. L., Stewart, J. M., Craven. L. A., and Wendel, J. F. (1999). Molecular systematics of Australian Gossypium section Grandicalyx (Malavaceae). Syst. Bot. 24: 183-208. Small, R. L., and Wendel, J. F. (2000). Copy number liability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium). Genetics 155: 1913-1926. Small, R. L., and Wendel, J. F. (2000). Phylogeny, duplication, and intraspecific variation of Adh sequences in New World diploid cottons (Gossypium L. Malvaceae). Mol. Phyl. Evol. 16: 73-84. Small, R. L., and Wendel, J. F. (2002). Differential evolutionary dynamics of duplicated paralogous Adh loci in allotetraploid cotton (Gossypium). Mol. Biol. Evol. 19:597-607. Soltis, D. E., and Soltis. P. S. (1995). The dynamic nature of polyploid genomes. Proc. Natl. Acad. Sci. USA 92. 8089-8091. Waghmare V.N. , J Rong , C Rogers , G J Pierce, J F Wendel & AH Paterson (2005) Genetic mapping of a cross between G.hirsutum (Cotton) and the Hawaii endemic , G. tomentosum. Theor. App.

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Genet. 111: 665- 676. Wendel J F (2000). Genome evolution in polyploids. Plant Mol. Biol. 42: 225-249 Wendel J F and R. C. Cronn ( 2003) . Polyploidy and the evolutionary history of Cotton. Advances of Agronomy . 78: 139-186 Wright R. J. , Thaxton P.M , El Zik , K. M and Paterson A. H. ( 1998) D-subgenome bias of Xcm resistance genmes in tetraploid

Gossypium (cotton) suggests that polyploid formation has created novel avenues for evolution. Genetics 149: 1987-1996. Zhao. X.-P., Si, Y., Hanson, R. E., Crane, C.F., Price, H. J., Stelly, D. M. Wendel. J. F., and Paterson, A.H. (1998). Dispersed repetitive DNA has spread to new genome since polyploid formation in cotton. Genome Res. 8: 479-492.

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CRYPTIC GENOMIC EXCHANGE BETWEEN CULTIVATED SAFFLOWER (CARTHAMUS TINCTORIUS L.) AND WILD SPECIES, C. GLAUCUS M. BIEB. SUBSP. ANATOLICUS (BIOSS.)
Anjani, K1 and M. Pallavi

ABSTRACT
Phenotypic traits, meiosis and nuclear DNA (2C) content were investigated in partial hybrids between cultivated safflower (C. tinctorius L.) and the wild species C. glaucus M. Bieb. ssp anatolicus (Bioss.) to confirm partial hybridity About 2% of F1 plants were predominantly similar to the female parent C. tinctorius in morphological and phenological traits while the rest were intermediate to both the parents and female-sterile due to cytogenetic abnormalities. The tinctorius-type F1 plants were both male sterile and fertile. In fertile hybrids there were 24 chromosomes forming 10 to 12 bivalents. In F2 and F3 of tinctoriustype F1 plants, partial to fully male sterile and fertile progenies with few distinctly intermediate traits of both parents as well as fertile progenies with predominantly tinctorius phenotype have appeared. This indicated that the tinctorius-type F1 plants resulted from partial hybridization. Nuclear DNA content of parents and tinctorius-type hybrids was estimated using Partec-PA flow cytometry. DNA content of partial hybrids (2.8-3.08 pg) was close to that of C. tinctorius (2.33 pg) as corresponding to their phenotype. The nuclear DNA content of male parent C. glaucus ssp anatolicus was quite high (6.31 pg). The phenotype and 2C DNA content strongly suggest that the phenotype and genotype of partial hybrids were due to higher contribution of female parent to hybrid. The occurrence of plants with few intermediate traits among progenies of tinctorius-type hybrid suggests genome exchange between male and female genomes. The cryptic genome exchange between cultivated and wild species in partial hybrids would allow exploitation of wild species genome, when this interpecific cross was a failure due to female-male sterility.

Introduction Safflower is a multi-purpose crop with wide adoptability. It is now grown for its much-valued edible oil. Safflower (Carthamus tinctorius L.) belongs to the family Compositae or Asteraceae. The genus Carthamus contains approximately 25 valid species. Of which, C. tinctorius is the only cultivated species. Safflower is vulnerable to many diseases and insect pests. The resistance sources for many biotic stresses are either not available or extremely narrow within the cultivated species. Carthamus species remained unexploited and there has been little research on these species. The wild species Carthamus glaucus ssp anatolicus (2n=20) is not readily crossable to C. tinctorius (2n=24) due to chromosomal imbalance. It is a source of

resistance to Fusarium wilt, the major disease of safflower. In order to assay the feasibility of transferring the desirable trait from the wild species to cultivated one, the cultivated species (C. tinctoius) as a female was manually crossed to C. glaucus M. Bieb. ssp anatolicus. Herein we report on the remarkable features displayed by interspecific hybrid plants and their self-derivatives and backcross individuals with regard to phenotypic traits, meiotic chromosomal behaviour and nuclear DNA content. This paper will discuss the confirmation of partial hybridity and cryptic genomic exchange between the incompatible parental species. Material and methods Plant material: The wild species C. glaucus

1. Directorate of Oilseeds Research, Hyderabad-500 030, India. Email: anjani_kammili @rediffmail.com

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M. Bieb. ssp anatolicus (Bioss.), 2n=20, received from Institute of Plant Genetics and Crop Plant Research, Germany, was established in the field and self-pollinated since 1997 to have true-to-types. The recessive genetic male sterile lines MS 107 (R), MS 6 (O) and MS 9 (O) belong to cultivated species C. tinctorius were used as male sterile female parents in the crossing programme. The initial crosses between male sterile C. tinctorius lines and C. glaucus ssp anatolicus were made by hand pollination under controlled conditions. The two types of phenotypes observed in F1 were backcrossed to PI 259994-1, a non-genetic male sterile line of C. tinctorius. Each capsule of female and male parents was covered with butter paper bag prior to anthesis as well as after pollination to prevent cross contamination through honeybee. For nuclear DNA (2C) estimates, tinctorius-type F1 and F2 plants, self-pollinated progeny of the plants of C. glaucus ssp anatolicus and of the female parents used in the initial crosses and of PI 259994-1 used in backcross were taken along with three genotypes of C. tinctorius (A1, SFS 9943, SFS 2032). A1 is a variety and SFS 9943 and SFS 2032 are highly stabilized pure lines. Diploid chromosome number of each species was confirmed prior to interspecific hybridization. Male sterility was assessed in main, primary, secondary and higher order capitula by visual observation for pollen presence, and by squashing anthers prior to anthesis in acetocarmine for stainability of pollen grains under microscope. Unstained pollen grains were considered sterile. To study meiosis, floral buds were fixed in Piennars fluid. PMC smears were stained with aceto-carmine. Preparation of nuclear samples: Approximately 0.5 cm square slice of freshly picked leaf of a plant at rosette stage was used for each sample. Crude samples containing nuclei were prepared from leaf material by chopping it very finely with a sharp razor blade in 300l of CyStain UV Precise P nuclei extraction buffer
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and left the suspension for 10 minutes. Then the suspension was filtered through filer CellTrics and 1200l of CyStain UV Precise P staining buffer containing 4-6-diamidino-2phenylindole (DAPI), was added to the filtrated suspension. After 20 to 30 minutes, the stained nuclei were analyzed using Partec-PA flow cytometer equipped with a mercury arc lamp. Fluorescence intensities were registered over 500 channels and displayed as histograms. At least 5000 nuclei were analyzed per run and each sample was repeated three to four times. To minimize variation due to runs on different days, two samples of A1 nuclei were analyzed each day. The first A1 sample was used to set the fluorescence intensity at channel 50. The unknown samples as well as the remaining A1 were then analyzed. The second sample of A1 was to determine accuracy. The data were, therefore, collected and compared as fluorescence intensity relative to A1. Samples giving coefficient of variation (CV) less than 6% were considered for nuclear DNA count. Since CV of DNA peaks says nothing about the reproducibility of DNA content replicate measurements were taken for each genotype. Florescence ratios, relative to the standard, were used to calculate DNA content (pg) according to the formula 2C DNA content/ sample (pg): [Sample Peak mean x Standard DNA content]/Standard Peak mean (Lysak and Dolezel 1998). The symbol C corresponds to the haploid nuclear DNA content. 2C value represents the DNA content of a diploid somatic nucleus. Results and Discussion Morphological and Chromosomal studies: C.tinctorius (2n=24) and C. glaucus ssp anatolicus (2n=20) belong to two different chromosomal groups of the genus Carthamus and do not cross readily (Ashri and Knowles1960). The F1 plants of the cross C. tinctorius x C. glaucus ssp anatolicus exhibited intermediate-type and tinctorius-type

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phenotypes. About 98% of F1 plants were intermediate to both parental species in their morphological and phenological traits and were female-male sterile. At the same time, about 2% of interspecific hybrid plants predominantly resembled C. tinctorius and were both male sterile and fertile. Intermediate-type plants did not produce seed upon self-pollination, backcrossing to C. tinctorius, sib crossing to fertile tinctorius-type sister plants thus indicating their male-female sterility. The fertile tinctoriustype plants were advanced to F2 and F3 through self-pollination and sib crossing to sister plants. Sterile tinctorius-type plants were backcrossed to PI 259994-1 (C. tinctorius). In sib-cross, backcross, F2 and F3 generations, about 98% of the plants resembled C. tinctorius and about 2% showed a few intermediate traits of both parents. Tinctoius-type plants were observed among backcross progeny. Pollen stainability of sterile interspecific hybrids and the sterile backcross progenies has ranged from 0-3.6%. Variation in pollen size and presence of multiple microspores were noticed in sterile plants. Pollen grains of fertile plants were well stained, uniform in size and round in shape. Pairing of chromosomes in intermediate-type F1 plants was not complete. The chromosome number in pollen mother cells ranged from 20 to 22. The mean number of bivalents per cell was 6 with a range from 0 to 9 whereas the mean number of the univalents per cell was 11 with a range from 10 to 22. Occurrence of multipolar division, laggards and bridges were also observed. The variation in pollen size and presence of multiple microspores in sterile intermediate-type plants was in full accord with the irregular and multiploar disjunction of chromosomes at anaphase. These chromosomal aberrations were responsible for sterility in intermediate-type F1 plants. The intermediate-type phenotype and formation of bivalents and chiasmata in these F1 plants indicate genomic exchange between parental species. Furthermore, chromosome analysis of
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tinctorius-type hybrid plants revealed presence of 24 chromosomes at meiosis instead of expected 22 chromosomes. There was perfect pairing of chromosomes in tinctorius-type F1 plants and 12 bivalents at diakinesis. There was an indication of loose pairing between one pair of heteromorphic chromosomes forming rodshape bivalent, which is an indication of partial non-homology. In some of the cells, asynchronization of meiotic divisions was seen, where anaphase-II and telophase-II were found simultaneously in the same cell. Similar chromosomal number and behaviour was observed in tinctorius-type F2 and F3 plants and in recombinant plants that possessed a few distinctly intermediate traits of both parents. The uniform pollen size in them is in full accord with regular chromosome disjunction at anaphase. Chromosome number and behaviour suggest progressive elimination of chromosomes of C. glaucus ssp anatolicus and spontaneous doubling of chromosomes of C. tinctorius during hybrid embryo development, which configured tinctorius-type plants in F 1. Appearance of plants possessing intermediate traits with 24 chromosomes in F2 and F3 of tinctorius-type F1 plants indicates that C. glaucus ssp anatolicus genome was distributed during zygote formation but only a part of it was able to mix with the entire female genome prior to elimination of its chromosomes leading to partial hybridization. Genomic exchange between parental species was supported by presence of 20-22 univalents, laggards, loops and clumps in (F1-tinctoius-type x C. tinctorius) backcross progenies. If there was no genome exchange between parental species and the only genome of C. tinctorius persisted in tinctorius-type F1 hybrids, there should be perfect meiosis in backcross progenies when they were backcrossed to C. tinctorius. But the meiotic abnormalities in backcross progenies clearly indicate presence of genomic variation in tinctorius-type F1 hybrid

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that appeared from cryptic genomic exchange between parental species. Elimination of complete set of chromosomes of one species in interspecific hybridization was also observed in the interspecific cross between Hordeum vulgare and H. bulbosum (Kasha and Kao 1970) and Crepis capillaries x C. neglecta (Wallace and Landgridge 1971). Only very few well-documented cases of chromosome doubling have been reported; for example, the amphidiploid hybrid between Nicotiana glutinosa L. and N. tabacum L. (Clausen and Goodspeed 1925) and chromosome doubling in vine cacti hybrids (Tel-Zur et al 2003). Partial hybridization was reported in rice with introgressed traits from Zizania latifolia (Liu et al 1999), wheat and oat pollinated by maize, between cultivated sunflower and Helianthus tuberosus (Natali et al 1998, Faure et al 2002) and cotton interspecific hybrids (Wendel et al 1995). Partial hybridization was interpreted as a consequence of genomic shock (McClintock 1984). Why genomic shock was seen only in a few zygotes that produced tinctorius-type partial hybrids is not understood. This is still to be analyzed but this sort of mild genomic shock is also possibly important in interspecific hybridization of incompatible species to isolate partial hybrids having introgressed hidden genomic part of the eliminated wild species. Nuclear DNA content: Flow cytometry was used to determine nuclear DNA content of the parental species and the hybrid plants as it was found to be a useful and highly sensitive tool for determining nuclear DNA content in many plant species (Armuganathan et al.1999, Asif et al. 2001, Moscone et al. 2003, Thiem and Sliwinska 2003). Flow cytometric analysis of nuclei isolated from leaves showed one peak that corresponds to the G0/G1 phase (2C level) of the cell cycle. Peaks corresponding to the G2+M (M=mitosis) phase or beyond were not detected, indicating absence of dividing cells in the material (Figure1). This also validates that flow cytometry
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had unambiguously determined the 2n ploidy level of material studied. The signals occurring in the lower-channel region (0-50) are resulted from disrupted nuclei and/or non-specific staining of other cell constituents. Table1 gives the nuclear DNA (2C) contents of parental species and partial interspecific hybrids. The nuclear DNA content varied remarkably between parental species. The men nuclear DNA content of C. tinctorius was 2.33 pg and of C. glaucus ssp anatolicus was 6.31 pg. Significant variation was not observed in DNA content among various C. tinctorius genotypes. A1 samples were used for determining the accuracy. Its DNA content was in close agreement with that of other genotypes of C. tinctorius. Nuclear DNA content of tinctorius-type partial hybrids was very close to that of C. tinctorius, ranging from 2.8 to 3.08 pg. The CV of analyses was from 4.1 to 5.6, which was in acceptable limits for accuracy of the measurements. The close harmony between DNA contents of C. tinctorius and partial hybrids supports the cytogenetic findings of elimination of chromosomes of C. glaucus ssp anatolicus and doubling of C. tinctorius chromosomes during hybrid embryo development. Because of occurrence of plants with a few intermediate traits expressed by recombinants in F2 and F3, it can be attributed that the minute genomic exchange between parental species was responsible for the slight deviation of DNA content of partial hybrids (2.8-3.08 pg) from C. tinctorius (2.33 pg). The 2C DNA content variability among individual partial hybrids could be due to variation in the amount of genome exchanged between parents. In conclusion, the present investigation indicates that under controlled pollination, partial hybridization had taken place between C. tinctorius and C. glaucus ssp anatolicus leading to occurrence of interspecific partial hybrids. Appearance of a few recombinants in F2 and F3, proximity of their DNA content to

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C. tinctorius with slight deviation and the DNA content variability among partial hybrids support cryptic genomic exchange between parental species as a result of partial hybridization and elimination of chromosomes of wild species as a consequence of genomic shock. The closeness of phenotype as well as DNA content of tinctorius-type F1 partial hybrids to C. tinctorius and occurrence of intermediate-type plants in F2 and F3 point out that C. tinctorius genome was contributed maximum and only a part of C. glaucus ssp anatolicus genome was able to mix with it. The imprecise meiotic behaviour of backcross progenies also support the existence of cryptic genomic exchange that caused partial homology among backcross progeny chromosomes. The proposition of partial hybridization was conclusively proved in this investigation. Partial hybridization phenomenon in many crops when crossed with their wild relatives was overlooked because most of the plants did not display the expected hybrid pattern but instead resembled the female parent, which led to the conclusion that the crosses had failed. However, in our experiment this phenomenon could be established evidently at morphological, cytological and basic nuclear DNA content level. Partial hybridization had allowed us to exploit a part of the wild species genome, when the cross between cultivated safflower and wild species had apparently failed. Some of the partial hybrids exhibited resistance against Fusarium wilt in wilt sick plot in two contiguous years, indicating introgression of wilt resistant genomic part of C. glaucus ssp anatolicus into cultivated species. So it was possible to transfer the desirable trait of C. glaucus ssp anatolicus to cultivated safflower through partial hybridization. Work is underway to analyse DNA of these plants to spot the introgressed or recombined fragments using various DNA markers, which would further support the cryptic genomic exchange between parental species at molecular level.
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REFERENCES Armuganathan, K., Tallury, S.P., Fraser, M.L., Bruneau, A.H., Qu, R.1999. Nuclear DNA content of thirteen turfgrass species by flow cytometry. Crop Sci. 39: 1518-1521. Asif, M.J., Mak, C., Othman, R.Y. 2001. Characterization of indigenous Musa species based on flow cytometric analysis of ploidy and nuclear DNA content. Caryologia. 54: 161-168. Ashri A and Knowles PF.1960. Cytogenetics of safflower (Carthamus L.) species and their hybrids. Agron. J. 52: 11-17. Clausen, R.E., Goodspeed, T.H. 1925. Interspecific hybridization in Nicotiana. II. A tetraploid glutinosa-tabacum hybrid, an experimental verification of Winges hypothesis. Genetics.10: 279-284. Faure ,N., Serieys, H., Cazaux, E., Kaan, F., Berville. 2002. Partial hybridization in wild crosses between cultivated sunflower and the perennial Helianthus species H. mollis and H. orgyalis. Ann of Bot. 89: 31-39. Kasha, K.J., Kao, K.N. 1970. High-frequency haploid production in barley (Hordeum vulagare L.). Nature. 225: 874-876. Liu, B., Piao, H.M., Zhao, F.S., Zhao, J.H., Zhao, R. 1999. Production and molecular characterization of rice lines with introgressed traits from a wild species Zizania latifolia (Griseb.). J Genetics and Breeding. 53: 279-284. Lysak, M.A., Dolezel, J.1998. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia. 51: 123-132. McClintock B. 1984. The significance of response of genome to challenge. Science. 226: 792-801. Moscone, E.A., Baranyi, M., Ebert, I., Greilhuber, J., Ehrendorfer, F., Hunziker,

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A.T. 2003. Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry. Ann. of Bot. 92: 21-29. Natali, L., Giordani, T., Polizzi, E., Pugliesi, C., Fambrini, M., Cavallini, A. 1998. Genomic alterations in the interspecific hybrid Helianthus annuus x Helianthus tuberosus. Theoretical and Applied Genetics. 97: 1240-1247. Tel-Zur, N., Abbo, S., Bar-Zvi, D., Mizarhi, Y. 2003. Chromosome doubling in vine cacti hybrids. J Heredity. 94 (4): 329-333. Thiem, B., Sliwinska, E. 2003. Flow cytometric

analysis of nuclear DNA content in cloudberry (Rubus chamaemorus L.) in vitro cultures. Pl. Sci.164: 129-134. Wallace, H., Landgrige, WHR. 1971. Differential amphiplasty and the control of ribosomal RNA synthesis. Heredity. 27: 1-13. Wendel, J.F., Schnabel, A., Seelanan, T. 1995. Bi-directional interlocus concerted evolution following alloploid speciation in cotton (Gossypium). Proc. National Academy of Science of the USA. 92: 280284.

Figure 1. Histograms of nuclear DNA content of parents and partial hybrid (X-axis: Channel number; Y-axis: Nuclei count)

(a) C. glaucus ssp anatolicus

(b) C. tinctorius

(c) Tinctorius-type partial hybrid between C. tinctorius and C. glaucus ssp anatolicus

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Table 1. Nuclear DNA (2C) content of parents and partial hybrids between C. tinctorius and C. glaucus ssp anatolicus Parental species and partial hybrid C. tinctorius (Female parent) C. glaucus ssp anatolicus (Male parent) C. tinctoius x C. glaucus ssp anatolicus Chromosome number (2n) 24 20 24 2C DNA content (pg) 2.33 6.31 2.8 - 3.08 CV (%) 4.1 5.1 4.2-5.6

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MORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF PLOIDY VARIANTS IN COFFEE FOR GENETIC IMPROVEMENT
Mishra, M.K.1, M. Violet DSouza, N. Sandhyarani, S.B. Hareesh, Anil Kumar, S. R. Mythrasree, R.K. Sabir, A. SantaRam and Jayarama

ABSTRACT
The genus Coffea consists of more than hundred species of which only C. arabica (known as arabica coffee) and C. canephora (known as robusta coffee) are commercially cultivated. All the species of the genus Coffea are diploids and self incompatible except C. arabica, which is an allotetraploid and self-compatible species. In C. arabica, a series of ploidy variants of spontaneous origin were recovered and documented. In C. canephora, which is a diploid species, colchicine was used successfully to induce tetraploidy. In addition, a few spontaneous triploid plants conforming to either arabica or robusta phenotype were also recovered. All the ploidy variants were studied for morphological characters such as leaf shape, leaf venation pattern, leaf anatomy, and stomatal features. The biochemical characters such as chlorophyll, starch, carbohydrate, phenol, sugar, amino acid content and flavonoid profile pattern also have been investigated. Molecular characterization of all the ploidy variants was carried out by using RAPD and PCR-RFLP and polymorphism was recorded. These results along with the utilization of the ploidy variants in coffee breeding program is discussed.

Introduction The genus Coffea belongs to the family Rubiaceae and consists of over 100 species (Bridson and Verdcourt 1988). All the coffee species are diploid except C. arabica, which is a putative allotetraploid. The spontaneous occurrence of different ploidy levels such as haploids, hexaploids and octoploids in C. arabica and other species has been reported by various workers (Sybenga 1960; Vishveshwara 1960; Sreenivasan et al1982). Techniques are also devised to obtain spontaneous haploids in C. canephora . ( Couturon 1982). In addition, a few spontaneous triploid plants conforming to either arabica or robusta phenotype were also documented. Due to the ubiquitous occurrence, polyploidy in higher plants has received maximum attention (Grant 1981; Masterson 1994). For exploiting genetic potentialities, polyploids and their diploid progenitors have been compared for diverse aspects like photosynthetic rate, fertility, yield,
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biochemical constituents and molecular diversity. A perusal of literature reveals that duplicated genes caused by polyploidy retain their original or similar functions or one copy may become silenced (i.e. mutational and epigenetic interactions) and polyploidization will affect DNA structure, allowing greater diversity at higher ploidy levels (Wendel 2000). Genetic diversification in polyploids, can therefore, lead to increased polymorphism in nuclear and cytoplasmic markers. In the present study, several morphological, biochemical and molecular aspects of ploidy variants in coffee were analysed with an objective to better understand the influence ploidy on these characteristics. Materials and Methods The details of plant materials used in the present study are given in Table.1. Ploidy status of all these materials was confirmed through chromosome counting (Chinnappa

1. Central Coffee Research Institute, Coffee Research Station 577117 Dist- Chikmagalur, Karnataka, INDIA

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1968; Sreenivasan et al1982; Mishra et al. (unpublished). For stomatal measurements the first pair of fully expanded leaves were used. A strip of lower epidermis from the middle portion of the leaf was peeled off and mounted in glycerol after staining with safranin. To determine stomatal guard cell length, 25 randomly selected stomata from five leaves per plant were measured microscopically using an ocular micrometer. Similarly, 25 randomly selected microscopic field areas from five leaves were counted per plant to obtain stomatal and epidermal cell frequency. For counting plastids in guard cells the epidermal peels were stripped and stained in a saturated solution of potassium iodide iodine (I2 K + I) and mounted in glycerol. Counts were made on plastids present in two guard cells of sixty stomata per plant in single plant samples and thirty stomata per plant in other samples. To determine pollen grain diameter, pollen samples were stained in saturated solution of potassium iodideiodine (I2 K + I) and darkly stained fertile pollen grains were measured by ocular micrometer. Leaf area measurements were carried out using PT area meter. (Delta T Devices). For anatomical studies, leaf samples were fixed in FAA and processed following the conventional paraffin methods of Johanson (1940). Serial transections were cut using a rotary microtome and sections were stained in crystal violet Erythrosin and mounted in DPX mountant. For leaf venation patter studies, third pair leaves were collected and immersed in 70% ethanol with several changes. Further the leaves were treated with sodium hydroxide at 400C for 12 hours and cleared with a thin brush. Leaves were further treated with saturated choral hydrate solution. For detail microscopic studies, cleared leaf pieces were stained in safranin and mounted in DPX. For flavanoid studies, the protocol adapted by our previous study (Mishra et al.,1998) was
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followed. The flavanoid similarity was computed for pairwise comparison between different ploidy variants using the formula a/a+b where a= number of spots common to both, and b= collective number of spots exhibited individually. Chlorophyll extraction was carried out from 100 mg samples of fully expanded leaves (4th pair) by using DMSO following the method of Hiscox and Israelstam (1979). Chl a, chl b and total chlorophyll content were calculated using the formula of Arnon (1949). Total Carbohydrate was determined in the leaf samples of all the ploidy variants by Anthrone method following the procedure of Hedge and Hofreiter (1962). Sugars and starch were estimated following the method of Nelson (1944) and Patel (1970) respectively. The method of Moore and Stein (1948) was used for calculating the total free amino acids and phenolics were analysed following the method of Malick and Singh (1980). Genomic DNA was isolated from the fresh young leaves using the extraction protocol by Murray and Thompson (1990) with modifications. PCR amplification using random primers PCR was carried out in a total volume of 25Pl Reaction mixture containing in 1x Taq assay buffer with 1.5mM MgCl2,100 oM dNTP,0.3 o M of the primer (Operon Technologies) ,50 ng of template DNA. 1U Taq DNA polymerase (Bangalore Genei, 3units/ol). PCR reaction was performed in Palm Cycler (Corbett Research) using the following amplification profile: 1 cycle of 950C for 4 min followed by 40 cycles of 950C for 1 min 380C for 1min, 720C for 2 min 1 cycle: and lastly 1 cycle of 720C for 10 min. Amplified PCR products were electrophoresed on an agarose gel (1.5%) pre stained with Ethidium bromide in 1xTAE buffer and visualized by Syngene Gene snap (UK).

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PCR amplification using mitochondria and chloroplast specific primers Intergenic regions of the chloroplast genome (trnL intron ) were amplified using conesenses primers (Taberlet et al 1991) . And PCR was done according to Dane et al (2004). PCR amplicons were separated on an agarose gel (1.5%) pre-stained with Ethidium bromide in 1xTAE buffer and visualized by Syngene Gene snap UK. Restriction digestion of The Genomic DNA and PCR fragments PCR products (5-10ol) were digested in a reaction mixture (20ol) containing: 2.0ol of 10x Assay buffer, 1mg/ml BSA, Restriction enzyme (Fermentas) 4U at 370C for 2-3 hours. The digested DNA was electrophored on 1.5% agarose gel and visualized as described earlier. Results and Discussion Leaf area and Pollen grain diameter Leaf area and pollen grain diameter associated with various ploidy variants were analysed. Distinct differences were observed between the diploids and tetraploids of C. canephora and dihaploids and tetraploids of C. arabica (Table 1). However, there is no regular relation between leaf area and other ploidy levels. In contrast it is interesting to note that there was a progressive increase in pollen grain diameter with the increase in ploidy level (Table.1). In dihaploid of C. arabica and diploid of C. canephora, the pollen grain diameter was found to be almost same indicating the less importance of genomic constitution affecting this trait in coffee (Sreenivasan et al., 1992). Gould (1957) and Speckmann et al. (1967) also observed a positive correlation with pollen grain diameter and ploidy level in Andropogon and Brassica respectively and the present observation lends support to their views. Stomatal characteristics The mean stomatal and epidermal cell
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frequency, stomatal guard cell length and stomatal plastid number were calculated at different ploidy levels and data are presented in Table1. In both C. arabica and C. canephora, the stomatal and epidermal cell frequency decreased while stomatal guard cell length increased with an increase in ploidy level. However, no significant difference in stomatal frequency could be found between the hexaploid and octoploid levels although the mean epidermal cell frequency was significantly different. The reduction in stomatal and epidermal cell frequency at higher ploidy level was attributed to the larger stomatal and epidermal cell size as well as reduced stomatal differentiation at higher ploidy level (Mishra et al. 1991; Sreenivasan et al. 1992; Mishra, 1997). The mean number of plastids in stomatal guard cells at different ploidy evels was counted and progressive increase in plastid number was observed with the increase in ploidy level (Fig1.J-K) except at the octoploid level where the plastid number decreased compared to the hexaploid level (Table1). No significant differences in plastid number were observed between dihaploid of C.arabica (9.05) and natural diploids of C. canephora (8.75) as well as tetraploids of C.arabica (15.96) and C. canephora (15.29). This clearly suggested that the plastid number is directly related to the chromosome number rather than genomic constitution (Sreenivasan et al.1992). The same conclusion was also drawn by Bingham (1968) in alfalfa and by Krishnaswami and Andal (1978) in Gossypium. Foliar anatomy and venation pattern Various anatomical characters were analysed in the different ploidy groups and presented in table 2. From the table it is apparent that although the basic anatomical structure is same at different ploidy levels, there is a progressive increase in majority of the

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anatomical features viz., lamina thickness, vertical extent of palisade tissue, diameter of palisade cells, thickness of spongy tissue, number of layers in spongy parenchyma and diameter of spongy cells. In C. arabica and in C .canephora the lamina thickness, vertical extent of palisade tissue, spongy tissue increased progressively with the increase of ploidy level (Fig1.A- 1I ) Suryakumari et al. (1989) observed similar tendency of increase in thickness of palisade, diameter of palisade, thickness of spongy parenchyma and lamina thickness in polyploids compared to diploids and the present observation is in agreement with their report. The leaf venation pattern was studied at different ploidy levels of Coffea. In general, the thickness of the 1, 2, and 3 veins increased with the increase of ploidy level (Fig1C, 1F, 1I). At higher ploidy level, the areoles were small and the vein islets inside the areoles are extensively branched and very close to each other. The small areoles with extensive vein islets is proposed to be advantageous in water conductions during stress conditions (Mishra et al. communicated). In field conditions, generally coffee polyploids (Hexaploids and octoploids of arabica and tetraploids of robusta) retain the full leaf complements and remain green during the drought conditions. Therefore this anatomical feature could be of adaptive significance in combating drought conditions. Biochemical characteristics Chlorophyll content In coffee, total chlorophyll content generally decreased with the increase of ploidy level although significant differences were not seen in few cases (Mishra et al.1996). Dihaploid arabica contain the highest chlorophyll (1.76 mg/ g) where as octoploid manifested the least chlorophyll (1.18 mg/g) among the ploidy variants. (Table 3a) However, in C. canephora, chlorophyll content of diploid (1.785 mg/g) and tetraploid (1.497mg/g) shows significant
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differences. Contrasting reports are available regarding the effects of polyploidy on chlorophyll content in many plant species. In tall fescue, chlorophyll/ mg/g fresh leaf tissue increased whereas in winter rye chlorophyll content decreased (Bordyugova, 1987) with increase of ploidy level. In contrast to the foregoing, an ascending order in chlorophyll content per unit leaf area was registered with increase in ploidy level (Table 3b). Leaf thickness was found to be strongly correlated with per unit area chlorophyll content (Leverenz, 1987). In Coffea increase in leaf thickness from dihaploid to octoploid level was reported (Prakash et al. 1993). Hence the observed increase in chlorophyll value per unit leaf area could be due to the influence of leaf thickness. Starch, carbohydrate and sugar content The percentage of total carbohydrate, starch and sugar content in different ploidy level of Coffea was studied and the same is given in table 4. However no consistent pattern was observed among all the ploidy variants. In C. arabica, the percentage of total sugar, starch and carbohydrate increased from dihaploid to tetraploid level but decreased at the hexaploid and octoploid level. Among arabica ploidy variants, except in triploid, which is of hybrid origin, maximum percentage of sugar, carbohydrate and starch were observed at tetraploid level. However in C. canephora, the per cent of total sugar and carbohydrate decreased and starch content increased from diploid to tetraploid level (Table 4). This clearly suggests that not only ployploidy affect different physiological parameters differently but also depend upon the genotype. Phenols and Amino Acids The phenol and amino acid content was studied at various ploidy levels and data is presented in table 4. It is observed that among ploidy variants of arabica octoploid contains highest phenol and lowest amino acids where

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as in C. canephora, phenolic content increased and amino acids content decreased from diploid to tetraploid level. In C. arabica, tetraploids contain the maximum amino acid content than any other ploidy variants. This observation further strengthens the contention that in plants polyploidy affect different physiological and biochemical constituents differentially in various genotypes. Flavonoids Leaf flavanoids were isolated from various ploidy levels of coffee. A qualitative difference in flavanoid pattern was observed among the members. Among arabica ploidy variants, dihaploids exhibit the minimum and tetraploids exhibit the maximum flavanoid profile pattern compared to any other group (data not shown). Most of the flavanoid spots observed in the dihaploid were encountered in tetraploid. In addition to the above, tetraploid group contain additional spots those are not seen in dihaploid. The hexaploid and octoploid group displayed less number of spots compared to tetraploid group. Mishra et al. (1993) explained that flavanoid biosynthesis efficiency reached its maximum at tetraploid level and beyond that there is no increase. Interestingly, most of the flavanoid spots observed in hexaploids were also encountered in tetraploid group, which supports the hypothesis of autoallopolyploid origin of hexaploid. Based upon the flavonoid similarity index value, dihaploids of arabica was found to be closer to the octoploid group (Table 3a). Loss of duplicate gene expression has been demonstrated in polyploid crop including Triticum and Chenopodium (Hart 1983; Wilson et al. 1983) and if so there may be a chance of gene silencing involving the loss of duplicate gene expression at higher ploidy level. In C. canephora, variation in flavanoid profile pattern was observed between the diploid and tetraploid group. Based on the similarity index value diploid was found close to colchicines
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induced tetraploid than natural tetraploid (Table 3b). This could be ascribed to the origin of tetraploid and their genomic constitution (Saraswathi et al .1991). Molecular analysis RAPD Random amplified Polymorphic DNA analysis was initiated to find out the polymorphism among ploidy variants. Using Operon primer (GGGTAACGCC) polymorphism was noticed among the samples. (Fig 2A). In C. canephora, two bands which are present in diploid were missing in tetraploid. This missing bands in tetraploid could be explained either due to the alteration or the elimination of the particular sequence during genome duplication. The RAPD data further revealed the close similarity between the tetraploid and hexaploid of S.795 and Sarchimor and thereby further strengthening our contention that hexaploids in coffee are of autoallopolyploid in origin. In addition, a close similarity was observed between the two triploids of coffee and thereby supporting the close affinity between them. However more primers need to be screened for molecular evaluation of ploidy variants in coffee. Chloroplast and Mitochondrial DNA analysis The consensus primer pair of the trnL intron (Taberlet,1991) successfully amplified the corresponding cpDNA region in all the ploidy variants (Fig 2B). Although the amplified DNA fragment seemed to be the same in size, the intensity of the bands were less in Sarchimor tetraploid and hexaploid and natural octoploid indicating the possibility of low copy number in these ploidy variants. Restriction digestion of the PCR product with enzyme AluI revealed polymorphism among the ploidy variants (Fig 2C). Variations in cpDNA are known to have evolutionary significance and therefore the

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observed variation in cpDNA among the ploidy variants could be associated with ecological adaptation during evolutionary process. The consensus primer pair of the mt-nad1B nad 1C region of the mitochondrial genome (Demesure et al. 1995; Dumolin et al. 1997) amplified the corresponding mtDNA region in all the ploidy variants except the tetraploid plants of Kents, S.795 and Sarchimor and hexaploid of Sarchimor (Fig 2D). Repeated attempts to amplify the mtDNA in these samples failed which indicate the possibility of sequence divergence in mitochondrial genome in those ploidy variants. Although the PCR amplified DNA fragments seemed to be the same in size but there were differences in copy number, which further suggests the gene diversification during evolution. Polyploids and coffee breeding Unlike other crops, the utilization of polyploids in coffee breeding is meagre. This is probably because of the perennial nature of the plant and enormous time that usually takes to release a coffee variety. However recently, research was focussed on utilizing the polyploids in coffee breeding. As a first step, a natural triploid of Coffea canephora was crossed with arabica tetraploid and hybrid plants with both arabica and canephora phenotypes were recovered (Amaravenmathy et al. 2004). Individual F1 plants were selfed and progenies were currently evaluated for various characteristics. Conclusion Polyploidy or the doubling of chromosome is a wide spread phenomenon in plants. Due to its ubiquitous nature, polyploidy is the subject of intense research by various workers. In plants, polyploidy may be advantageous or disadvantageous depending on their effects. In the foregoing as we have observed, in coffee, polyploidy has differentially affected several features in different genotypes. In coffee (C.
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arabica) tetraploid level seems to be the optimum level as revealed by various morphological and biochemical characteristics. Our hypothesis is supported by the fact that tetraploids are commercially cultivated wherein the physiological and biochemical functions have reached their efficiency. However haploids in coffee are important as they can be exploited to obtain the homozygous plants for both breeding and molecular studies. Similarly, hexaploids and triploids are also important as they form the important breeding material for coffee genetic improvement. A complete analysis of organelle DNA will probably give more information on the nature of ploidy variants in coffee and give insight to their evolutionary and adaptive significance. REFERENCES Amaravenmathy, V.S., Kumar, A., Santaram, A., Srinivasan, C.S . 2004. Robusta-like Coffee plants with Arabica-like Cup qualityMyth or Possibility? ASIC 20th Bangalore 1165-1170. Arnon, P.I.1949. Copper enzymes in isolated chloroplasts, Polyphenol oxidase in Beta vulgaris L. Plant Physiol. 24:1-15 Bingham, E.T.1968. Stomatal chloroplast in alfalfa at four ploidy levels. Crop Science 8: 509-511. Bordyugova, E.D. 1987. Effect of polyploidy on chlorophyll content of the leaves in winter rye. In: Novoe V Selektsii i. Seminovodstve selskokokhozyaibrst vennykh kul tur. Kamennaya step; USSR 84-90. Bridson, D.M. and Verdcourt, B. 1988. Flora of tropical east Africa- Rubiaceae (part2) Polhill R M (Ed) 727p. Chinnappa, C.C. 1968. Interspecific hybrids of Coffea canephora x C. arabica Current Science. 37: 676-677.

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Couturon, E. 1982. Obtention d haploids spontanea de Coffea canephora Pierre par utilisation du greffage d embrynos Caf Cacao The (Paris) 30: 155-160. Dane, F., Lang, P., Bakhtiyarova, R. 2004. Comparative analysis of chloroplast DNA variability in wild and cultivated Citrullus species. Theor and Appl Genetics. 108: 958966 Demesure, B., Sodzi, N., Petit, R. J. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 4: 129-131. Dumolin, S., Pemonge, M., Petit, R. 1997. An enlarged set of consensus primers for the study of organelle DNA in plants. Mol Ecol. 6: 393-397. Gould, F.W. 1957. Pollen size as related to polyploidy and speciation in Andropogon saccharoides A barbinodis complex. Brittonia. 9: 71-75. Grant, V. 1981. Plant speciation, 2nd edition. Columbia University Press, New York. Hart, G.E . 1983. Genetics and evolution of multilocus isozymes in hexaploid wheat. In isozymes Current topics in Biological research. Vol.10: 365-380, M.C. Rattazi et al(eds.) Liss, New York. Hedge, J.E, Hofreiter, B.T. 1962. Carbohydrate Chemistry 17 (Eds Whistler. R. L. and Be Miller, J.N) Academic Press New York. Hiscox, J.D., Israelstam, G.F.1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot.57: 13321334. Johanson, D.A. 1940. Plant Microtechnique. McGraw-Hill, New York. Krishnaswamy, R., Andal, R. 1978. Stomatal
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chloroplast number in diploids and polyploids of Gossypium. Proc. Indian Academy of Science 87B . Plant Science.109-112. Leverenz, J.W. 1987. Chlorophyll content and the light curve of shade adapted conifer needles. Physiol. Plant. 71: 20-29. Malick, C.P., Singh, M.P. 1980. Plant Enzymology and Histo Enzymology Kalyani Publishers, New Delhi p 286. Masterson, J. 1994. Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science. 264: 421-424. Mishra, M.K. 1997. Stomatal characteristics at different ploidy levels in Coffea L. Annals of Botany. 80: 689-692. Mishra, M.K., Padmajyothi, D., Prakash, N.S., Sreenivasan, M.S.1993. Leaf flavanoid profiles in different cytotypes of Coffea arabica L . Journal of Plantation Crops 21 ( supplement): 258-263. Mishra, M.K., Padmajyothi, D., .Prakash, N.S., Srinivasan, C.S., Naidu, R. (1998) Comparative leaf flavonoid profiles of natural and induced purpurascens mutants of Coffea arabica . Journal of Plantation Crops 127-132. Mishra, M.K., Prakash, N.S., Sreenivasan, M.S. 1991. Relationship of stomatal length and frequency to ploidy level in Coffea L. Journal of Coffee Research. 21: 32-41. Mishra, M.K., Ram, A.S., Prakash, N.S., Jyothi, D.P., Sreenivasan, M.S. 1996. Polyploidy and chlorophyll content in Coffea L. Indian Journal of Forestry. 19(3): 241-243. Moore, S., Stein, W.H. 1948.: Methods in Enzymology (Eds, Colowick, S. P and Kaplan, N.D) Academic press New york 3468. Murray, M., Thompson, W.F. 1980. Rapid isolation of high molecular weight plant

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DNA . Nucleic Acids Res. 8: 4321-4325. Nelson, N. 1944. A Photometric adaptation of the Somogyie method for determination of Glucose. J.Biol. Chem. 153: 375-380. Patel, R.Z. 1970. A note on seasonal variation in starch content of different parts of arabica coffee. East African Agricultural and Forestry Journal. 36:1-6 Prakash, N.S., Padmajyothi, D., Mishra, M.K., Ram, A.S. and Sreenivasan, M.S. 1993. Ploidy level- its influence on leaf anatomical features of Coffea L. Journal of Coffee Research. 23: 75-83. Saraswathi, P., Mishra, M.K., Prakash, N.S., Sreenivasan, M.S. 1991. Flavanoid profiles in diploid and tetraploid cytotypes of Coffea canephora Pierre ex. Froechner. J. Coffee Res. 21: 119-126. Speckman, G.J., Post, J., Dijkstra, H. 1965 The length of stomata as an indicator for polyploids in Rye grasses. Euphytica. 14: 225-230. Sreenivasan, M.S., Prakash, N.S., Mishra, M.K. 1992. Evaluation of some indirect ploidy indicators in Coffea L Cafe Cacao The Vol XXXVI No.3 199-205.

Sreenivasan, M.S., Ramachandran, M., Sundar, K.R. 1982. Frequency of polyploids in Coffea arabica Proceedings of PLACROSYM IV on Genetics, Plant Breeding and Horticulture. Kasargod. Indian society for Plantation crops. 23-28. Suryakumari, D., Seshavataram, V., Murthy, U.R. 1989. Leaf anatomical features of some interspecific hybrids and polyploids in the genus Arachis L. J. Oil seeds Res. 6:75-84. Sybenga, J. 1960. Genetics and cytology of coffee. A literature review. Bibliograhica Genetica ( Wageningen).19: 217 316. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J. 1991. Universal primers for amplification of three non coding regions of chloroplast DNA. Plant Mol Biol. 17: 1105-1110. Vishveshwara, S. 1960. Occurrence of a haploid in Coffea arabica L. Cultivar Kents. Indian Coffee. 24: 123-124. Wendel, J.W. 2000. Genome evolution in polyploids. Plant Mol Biol. 42: 225-249. Wilson, H.D., Barber, S.C., Walters, T. 1983. Loss of duplicate gene expression in tetraploid chenopodium. Biochem. Syst. Ecol. 11: 7-13.

Table1. Leaf area, pollen grain and stomatal features at different ploidy levels Mean Stomatal Pollen guard Stomatal no. Mean grain Ploidy level Leaf plastid stomata epidermal cell areacm2 diameter 2 cells length number 0.10mm m m 0.10mm 2 Dihaploid 28.58 23.33 33.35 75.40 17.32 9.05 Triploid 75.56 21.78 25.50 82.65 20.14 12.20 Tetraploid 57.24 27.13 17.90 50.45 29.47 15.96 Hexaploid 87.43 33.88 10.30 36.30 35.46 21.27 Octoploid 58.05 32.24 9.55 28.00 33.45 20.33 Diploid 166.16 19.84 39.65 69.75 19.64 8.75 Tetraploid 239.15 26.51 20.90 62.00 26.81 15.29
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Sl. No 1 2. 3. 4. 5 6 7

Variety

Arabica Arabica Arabica Arabica Arabica Robusta Robusta

Table 2. Anatomical characteristics of ploidy variants

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Ploidy level

Lamina thickness

Thickness Thickness Average Average Thickness of No.of No. of of diameter of diameter of Thickness spongy spongy cell palisade epidermis epidermis of palisade spongy cells of palisade tissue layers layers upper cells lower 19.23 28.31 29.36 33.13 31.60 20.98 33.00 19.92 62.68 2 11.82 290.52 13.70 16.46 17.13 18.04 16.59 13.32 36.88 37.48 49.22 55.70 75.50 36.23 1 1 1 1 1 1 9.38 9.15 10.94 11.40 11.36 9.64 139.14 146.83 172.93 257.45 259.18 116.35 7.38 7.91 8.07 9.53 10.33 7.30 11.23 16.35 19.95 21.82 22.69 23.70 15.80 25.46

220.60 241.96 281.29 375.07 396.63 195.40 418.35

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Dihaploid(C. arabica) Triploid(interspecific) Tetraploid(C. arabica) Hexaploid Octoploid Diploid (C.canephora) Tetraploid (C. anephora)

Table 3 a .Index of flavanoid similarity for pair-wise comparison of ploidy level of Coffea arabica

Table 3b. Index of flavanoid similarity for pair-wise comparison of ploidy level of Coffea canephora

1 1 2 3 X 0.50 X

5 1 Diploid X Tetraploid natural 0.30 Tetraploid colchicine 0.36 2 X 0.36 3

Dihaploid

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2 3 4 5 X 0.54 0.58

Triploid Tetraploid Hexaploid Octoploid

0.75 0.66 0.52 0.87

X 0.66 0.57 0.72

Table 4. Biochemical characteristics at different ploidy levels of coffee

Sl. No

Variety

Ploidy

Total Starch % Total Phenols Total free chl a Chl b Chl a/ Total chl Total chl Reducing Non 2 mg/g Chl b amino % carbohy mg/g mg/cm sugar % Reducing sugar mg/g acids % sugar % % drate %
0.66 0.56 0.62 0.59 0.43 0.69 0.52 1.87 1.49 0.033 2.12 5.74 1.58 1.78 0.026 0.79 8.05 1.72 1.18 0.042 1.28 8.05 1.56 1.52 0.040 0.975 6.84 7.81 9.33 8.84 7.86 1.75 1.70 0.035 1.29 8.32 9.62 1.74 1.53 0.025 1.39 9.77 11.16 1.87 2.56 1.83 1.85 1.54 1.70 1.65 1.76 0.028 1.39 5.28 6.68 1.91 8.76 13.04 12.18 9.65 11.18 10.38 9.56 0.41 1.52 0.76 0.52 1.40 0.37 1.49 0.0332 0.085 0.200 0.033 0.004 0.180

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C.arabica

Dihaploid

1.10

2 C. arabica

Triploid

0.98

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3 C. arabica

Tetraploid

1.08

4 C. arabica

Hexaploid

0.93

5 C. arabica

Octoploid

0.74

6 C. canephora

Diploid

1.08

7 C. canephora

Tetraploid

0.975

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Fig. 1A 1K C. arabica Fig. 1A. T.S of dihaploid leaf lamina Fig. 1B. T.S. of dihaploid leaf blade Fig. 1C. Dihaploid leaf venation pattern Fig. 1D. T.S of tetraploid leaf lamina Fig. 1E. T.S. of tetraploid leaf blade Fig. 1F. Tetraploid leaf venation pattern Fig. 1G. T.S of octoploid leaf lamina Fig. 1H. T.S. of octoploid leaf blade Fig. 1I. Octoploid leaf venation pattern Fig. 1J. Plastids in stomatal guard cells of tetraploid Fig. 1K.Plastids in stomatal guard cells of hexaploid
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Figure 2 a M 1 2 3 4 5 6 7 8 9 10 11

Figure 2b M 1 2 3 4 5 6 7 8 9 10 11

Figure 2c M 1 2 3 4

M 5 6 7 8 9 10 11

Figure 2d 1 2 3 4 5 6 7 8 9 10 11 M

Fig.2a 2d ( 1-11) Coffee Ploidy variants Fig. 2a RAPD profile in ploidy variants Fig.2b Chloroplast DNA amplification of ploidy variants Fig.2c Restriction digestion pattern of cpDNA with AluI enzyme Fig.2d Amplification of mitochondrial DNA Lane details M- DNA ladder
1. S.274 ( Robusta ) Diploid 2. S. 274 ( Robusta) Tetraploid 3. Arabica haploid ( c.v. Kents) 4. Arabica tetraploid ( c.v Kents) 5. Arabica tetraploid ( c.v. S.795) 6. Arabica hexaploid (c.v.795) 7. Triplod ( Natural. 1) 8. Triplod ( natural.2) 9. Arabica Octoploid 10. Arabica tetraploid ( c.v. Sarchimor) 11. Arabica hexaploid ( c.v. Sarchimor)
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CYTOLOGICAL STUDIES ON SUGARCANE INTERGENERIC HYBRIDS


Babu, C1., K.Koodalingam1, U.S. Natarajan2, R.M. Shanthi2 and S. Thangasamy1

ABSTRACT
Modern sugarcane varieties (Saccharum spp., 2n=100-130) are derived from interspecific crosses, between Saccharum officinarum and wild relatives mainly S. spontaneum in which only a few parental clones were involved resulting in narrow genetic base. To broaden this genetic base, use of other genera from the Saccharum complex, mainly the cane forming Erianthus arundinaceus which is known for high tillering, high fibre, high biomass, resistance to drought, red rot, borers and good ratooning ability was attempted. Despite several attempts, during the recent past, to introgress E. arundinaceus characters in sugarcane varieties, no conclusive success has been achieved. The first difficulty appears to be the identification of true hybrids using morphological traits and other difficulty in getting seed fertility and the lack of recombination between the chromosomes of the two genera. In order to overcome the problem of sterility, S. spontaneum could be used as bridge species. In one such cross IK 76-092 (E. arundinaceus) x SES 286 (S. spontaneum), five intergeneric progenies were examined cytologically to confirm their hybridity. The expected chromosome number from the cross IK 76-092 (2n=60) x SES 286 (2n=64) for n+n transmission is 62. The root tips were examined and mean of two observations was taken as the chromosome number. The results showed that none of the hybrids had 62 chromosomes for n+n transmission indicating that chromosome elimination could be a common phenomenon in such crosses. Chromosome elimination in these progenies ranged from 4 to 12.

Introduction Sugarcane belongs to the genus Saccharum, a complex genus which comprises six species all characterized by a high ploidy level. Modern sugarcane varieties (Saccharum spp., 2n=100130) are derived from interspecific crosses, performed early this century, between sugarproducing Saccharum officinarum (2n=80) and wild relatives mainly S. spontaneum (2n=40128). Only a few parental clones were involved in these crosses (Arceneaux, 1965; Price, 1965). Thus, the genetic base of modern varieties appears to be very narrow and could be the reason for the present slow progress in sugarcane breeding. To broaden this genetic base, interest has turned to utilize other genera from the Saccharum complex, mainly Erianthus arundinaceus (Berding and Roach 1987; Roach

and Daniels 1987; Walker 1987). Cane forming E. arundinaceus (2n = 60) is known for high tillering, high fibre, high biomass, resistance to drought, red rot, borers with multiratooning ability. Despite several attempts, during the recent past, to introgress E. arundinaceus characters in sugarcane varieties, no conclusive success has been achieved. The first difficulty appears to be the identification of true hybrids using morphological traits. Two other factors suggested to be responsible for this lack of success are difficulty in getting seed fertility and the lack of recombination between the chromosomes of the two genera. Many classical cytological studies have been undertaken to provide general information about the cytogenetics of sugarcane and to support breeding programmes (Sreenivasan

1. Centre for Plant Breeding and Genetics,Tamil Nadu Agricultural University, Coimbatore 2. Sugarcane Breeding Institute, Coimbatore

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et al., 1987). The occurrence of 2n gamete transmission in hybrids between S. officinarum and S. spontaneum and their first backcross with S. officinarum has been demonstrated (Bremer 1922 and 1961). But unlike in S. spontaneum nobilisation, S. officinarum fails to transmit 2n gametes when it is crossed with E. arundinaceus. Another obstacle is that there is manifestation of sterility in the intergeneric hybrids. To overcome these two difficulties, attempts have been made to use S. spontaneum as bridging species between S. officinarum and E. arundinaceus (Natarajan, 2002). Towards this objective crosses have been made (Natarajan, 2002). In one such cross between IK 76-092 (E.arundinaceus) as female and SES 286 (S. spontaneum) as male, the resultant intergeneric progenies (013501, 013502, 013504, 013505 and 013102) were subjected to cytological studies to confirm their hybridity. The expected chromosome number from the cross IK 76-092 (2n=60) x SES 286 (2n=64) for n+n transmission is 62. Materials and Methods Single bud setts of the intergeneric hybrids viz., 013501, 013502, 013504, 013505 and 013102 derived from the cross IK 76-092 (2n=60) x SES 286 (2n=64) were planted four each in mud pots containing pure sand in order to facilitate early growth of roots on 07.01.2003. After twenty days, the germinated plants were carefully depotted and the roots were thoroughly washed in fresh running water and the healthy root tips (as indicated by white portion at the tip) of 0.5 cm in length were collected. The procedure for the root tip squash technique was followed as per the method suggested by Jagathesan and Rathnambal, 1967. The root tips were pretreated with a-1 bromonaphthalene and stored in refrigerator at 10C for 1-1.30 hrs. The root tips were washed free of a-1 bromonaphthalene in running water and rinsed with distilled water. The washed root tips were then fixed in 6:3:2 (methanol: chloroform: distilled water) fixative
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and kept in refrigerator for overnight (Ostergren and Haneen, 1962). Then, the root tips were washed and hydrolyzed with 1N HCl at 60C for 13 minutes. The root tips were washed free of HCl in running water and rinsed with distilled water. The root tips were treated with 1:1 citrate buffer: pectinase mixture and kept in dark for 1 hr. Finally, the root tips were stained with basic fuchsine and kept in dark for 1.30 hrs. The actively dividing portion at the root tip get stained with basic fuchsine and that portion was carefully excised with a sharp blade in a clean glass slide and squashed with 1% acetocarmine and examined under microscope. The salient features of the parental clones viz., IK 76-092 and SES 286 (Kandasamy et al., 1983; Sreenivasan et al., 2001) are given in the Table 1. Results and Discussion Out of the five test entries viz., 013501, 013502, 013504, 013505 and 013102 root tips from four entries viz., 013501, 013502, 013504 and 013505 were collected, processed and chromosome number was observed. Sufficient number of roots was not formed from the root eyes of the intergeneric progeny 013102. The mean of two observations was taken as the chromosome number and are given in the Table2.
The results showed that, none of the hybrids between E. arundinaceus (IK 76-092; 2n=60) and S. spontaneum (SES 286; 2n=64) observed by cytological methods had the expected 62 chromosomes for n+n transmission; between 4 and 12 chromosomes were eliminated. Similar results of chromosome elimination have been obtained by Piperidis et al. 2000 using GISH technique when S. officinarum (2n=80) was crossed with E. arundinaceus (2n=60). One hybrid (013505) had chromosome number 58 (Fig.1) almost equivalent to the expected number 62 and this could be the result of n+n transmission followed by elimination of chromosomes to a lesser degree. Although

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slight inaccuracy in chromosome count (due to the large number and small size of chromosomes) cannot be ruled out completely (Piperidis et al., 2000), this probably reflects that chromosome elimination has occurred.

and genetics retrospects and prospects, pp 42-48. Ostergen, G. and Haneen, W.K. 1962. a squash technique for chromosome morphology studies. Heriditas. 33 : 261-269. Piperidis, G., Christopher M.J., Carroll, B.J., Nils Berding and DHont. 2000. Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus. Genome. 43: 1033-1037. Price, S. 1965. Interspecific hybridization in sugarcane breeding. Proc. ISSCT, 12: 1021-1026. Roach, B.T., J. Daniels. 1987. A review of the origin and improvement of sugarcane. In: Coperscular Int. Sugarcane Breed. Workshop, Coperscular, Sao Paulo. pp 131. Sreenivasan, T.V., Ahloowalia, B.S. and Heinz, D.J. 1987. Cytogenetics. In Heinz D.J (ed.) Sugarcane improvement through breeding. Elsevier Press, Amsterdam. pp 211-253. Sreenivasan, T.V., Amalraj, V.A. and William Jebadhas, A. 2001. Catalogue on Sugarcane Genetic Resources IV Erianthus species. Sugarcane Breeding Institute (ICAR). pp 54. Walker, DIT. 1987. Manipulating the genetic base of sugarcane. In: Coperscular Int. Sugarcane Breed. Workshop, Coperscular, Sao Paulo. pp 321-334.

REFERENCES Arceneaux, G. 1965. Cultivated sugarcane of the world and their botanical derivation. Proc. ISSCT, 12: 844-854. Berding, N., Roach BT. 1987. Germplasm collection, maintenance and use. In: Heinz D.J (ed.) Sugarcane improvement through breeding. Elsevier Press, Amsterdam. pp 143-210. Bremer, G. 1922. Ned. Indie. pp. 1-112. [English translation in Genetica, 5: 97-148, 273-326 (1923)]. Bremer, G. 1961. Problems in breeding and cytology of sugarcane. Euphytica, 10: 5978. Jagathesan, D. and M.J. Rathnambal. 1967. Karyotype analysis in Saccharum officinarum. The Nucleus. 10: 159-167. Kandasamy, P.A., Sreenivasan, T.V., Ramana Rao, T.C., Palanichami, K., Natarajan, B.V., Alexander, K.C., Madhusudana Rao, M. and Mohan raj, D. 1983. Catalogue on Sugarcane genetic resources I S. spontaneum. Sugarcane Breeding Institute (ICAR). pp 72. Natarajan, U.S. 2002. Nobilisation a pivotal procedure in sugarcane breeding. In: Winter school training manual, Sugarcane breeding

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Table 1. Salient features of Parental clones Parental clones


Sl. No

Particulars Year and source of collection Chromosome number Stalk length (cm) Stalk diameter (cm) Flowering time Pollen fertility (%)

IK 76-092 (E.aundinaceus) 1976 (IndonesiaKalimantan) 60 300 1.5 November 0.0

SES 286 (S. spontaneum) 1951 (Spontaneum Expedition Scheme) 64 135 1.2 November 100

1 2 3 4 5 6

Table 2. Results of the chromosome number observed in Intergeneric progenies Sl No. Observed Intergeneric chromosome hybrid number progeny 013501 013502 013504 013505 52 50 54 58 Expected chromosome number for n+n transmission 62 62 62 62 Chromosomes eliminated

1 2 3 4

10 12 8 4

Fig. 1. Chromosome number in the intergeneric progeny 013505

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CYTOLOGICAL OBSERVATIONS IN COLCHICINE INDUCED HEXAPLOIDS AND THEIR TRIPLOIDS OF CROSS BETWEEN GOSSYPIUM HIRSUTUM [2N = 4X = 52, (AD1)] AND GOSSYPIUM RAIMONDII [2N = 2X = 26, D5]
Saravanan, N.A., T.S. Raveendran and M. Kumar

ABSTRACT
Two interspecific hexaploid [2n=6x=78, 2(AD1)D5] fertile hybrids between two varieties of cultivated tetraploid species G. hirsutum [2n=4x=52, (AD1)] viz., MCU 5 and MCU 7 and wild diploid species G. raimondii [2n=2x=26, D5] were synthesized by doubling the chromosome number of their respective F1 sterile triploid [2n=3x=39, (AD1)D5] hybrids using aqueous colchicine solution. Morphological and cytogenetic analysis confirmed the true nature of triploid and its hexaploid hybrids. The F1 triploid plants were intermediate in morphological characters and they were highly pollen sterile as well as ovule sterile. A maximum of 18 bivalents per PMC was recorded in G. hirsutum var. MCU 5 X G. raimondii triploid [2n=3x=39, (AD1)D5] but 13 I + 7 II + 4 III was the most frequent meiotic configuration. In the triploid of G. hirsutum var. MCU 7 X G. raimondii [2n = 3x = 39, (AD1)D5], maximum of 15 bivalents was recorded with an average of 11.35 bivalents, but 10 I + 13 II + 1 III was the most frequent meiotic configuration. The hexaploids [2n=6x=78, 2(AD1)D5] showed the expected features of the colchiploidised plants such as large sized flowers than triploids, increased pollen grain size, fertile pollen grains, boll and seed set with fibres as compared to the F1 sterile triploid plants. The hexaploid G. hirsutum var. MCU 5 X G. raimondii [2n=6x=78, 2 (AD1)D5] recorded maximum of 31 bivalents per PMC with most frequent meiotic configuration of 7 I + 28 II + 5 III. The maximum of 25 bivalents with an average of 19.68 bivalents per PMC was recorded in G. hirsutum var. MCU 7 X G. raimondii hexaploid [2n=6x=78, 2 (AD1)D5], but 25 I + 19 II + 5 III was the most frequent meiotic configuration. The morphological and meiotic behavior of these hexaploid hybrids provided valuable information for their practical utilization in a cotton breeding programme.

Introduction The cotton genus Gossypium contains about 50 diploid and tetraploid species which are distributed throughout the arid and semiarid regions of Africa, Central and South America, Indian Subcontinent, Arabia and Hawai (Fryxell et al. 1992). The diploid species (2n=2x=26) are divided into eight different cytological groups designated by A, B, C, D, E, F, G and K based on the chromosome pairing relationships (Beasley, 1940; Beasley, 1942; Gorham and Young 1996), while the five allotetraploid species are designated with AD genome. Cultivated types belong to four species, of which
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Gossypium herbaceum and Gossypium arboreum are diploids, while Gossypium hirsutum and Gossypium barbadense are tetraploids. The tetraploid species (2n=4x=52) contains two distinct sub genomes which are related to the A genome of the Asiatic cultivated diploid species and the D genome of the American wild diploid species (Geever et al. 1989). The wild species of Gossypium are rich with rare desirable attributes that are not available in the germplasm of cultivated species. Many interspecific hybrids have been made which provided (i) useful information for understanding species relationship in the genus

Centre for Plant Breeding and Genetics,Tamil Nadu Agricultural University, Coimbatore

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and (ii) new sources of germplasm to be incorporated into breeding programme. Without doubt, most hybridization programmes utilizing wild species of Gossypium, have their primary objective of the transfer of disease and insect resistance. The work of transferring bollworm resistance from, G. thurberi and G. armourianum to sakel cotton was reported by Knight et al. (1953) and Thombre and Mehetre (1981). Jassid resistance from G. tomentosum and boll weevil resistance from G. armourianum was transferred to G. hirsutum (Narayanan et al., 2004). Black arm resistance has been transferred from G. arboreum to G. barbadense and rust resistance from G. raimondii to G. hirsutum. Besides pest and disease resistance some of the fibre quality traits have also been introgressed from wild species of Gossypium to cultivated cottons. In texas, hybridization works involving thurberi gave successful results in the transfer of the high lint strength to upland cotton (Guany, 1952). Marappan (1960) reported the transfer of fineness from G. anomalum to the back ground of G. arboreum. Muramoto (1969) synthesized hexaploid cottons by crossing G.hirsutum and G. sturtianum and showed the possibilities of producing spinnable yarn with very high yarn strength. With this background the present investigation was attempted for introgression of desirable genes from G. raimondii into cultivated G. hirsutum cotton varieties to synthesize new breeding lines with in-built resistance to biotic and abiotic stresses coupled with desirable fibre quality traits in addition to desired economic characters. Materials and Methods The experimental materials used for this study consisted of two Gossypium hirsutum cultivated tetraploid [2n=4x=52, (AD 1)] genotypes viz., MCU 5 and MCU 7 (used as female parents), wild diploid species Gossypium raimondii [2n=2x=26, D5] (used as male parent), their two F1 sterile triploid [2n=3x=39, (AD1)D5] hybrids viz., G. hirsutum var. MCU 5 X G.
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raimondii and G. hirsutum var. MCU 7 X G. raimondii and their colchicine induced fertile hexaploids [2n=6x=78, 2(AD1)D5] viz., G. hirsutum var. MCU 5 X G. raimondii and G. hirsutum var. MCU 7 X G. raimondii. The triploids and hexaploids used for morphological and meiotic behaviour studies were grown in the species garden maintained at the Department of Cotton, TNAU. Doaks method was followed for hybridization. Simultaneously, selfing of parents was also carried out and selfed bolls were collected, ginned and seeds were secured. The seeds of the F1 triploid hybrids were sown in the poly bags for colchicine treatment. One week after germination when the seedlings attained two leaf stage, a thin wad of absorbent cotton was spread over the apical meristems of the seedlings and sufficient drops of 0.1 and 0.2 per cent aqueous solution of colchicine were applied to soak the cotton wad to enable the efficient penetration of the chemical into the apical meristem. The treatment was given twice a day for five consecutive days. Slides were prepared for cytological examination following usual procedures. Stainability of pollen grains was also studied using 1 per cent acetocarmine. Results and Discussion The present investigation was carried out to transfer the genes resistant to biotic and abiotic stresses combined with fibre quality characters from the wild diploid species G. raimondii to susceptible and high yielding adapted genotypes. Two triploids viz., G. hirsutum (MCU 5) x G. raimondii [(AD1)D5] and G. hirsutum (MCU 7) x G. raimondii [(AD 1 )D 5 ] developed by interspecifc hybridization were found to be characterized by vigorous healthy and rapid growth, profuse branching and tolerance to jassids during both the seasons. Evidently, these characters were transmitted from the wild parent G. raimondii to the triploid hybrids. The observations made

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by Deodikar (1949) involving G. anomalum were also similar to those obtained in the present hybrids with 2n = 39 chromosomes. These two triploids were found to be intermediate between both the parents in plant height, growth habit, number of monopodial branches, number of anthers per flower, size of anther, and length of pistil and bracterial teeth number which concur with G. hirsutum and G. raimondii. The leaf shape, flower shape, petal spot, flower colour, flower size, stem and leaf hairiness etc., of G. raimondii were dominant as the F1 triploid hybrids and their hexaploids exhibited these characters. Mehetre et al. (2003) also reported that the flower shape, flower size, petal spot, pollen colour, filament colour, stem and leaf hairiness of G. raimondii were dominant in the F1 triploid hybrids of G. hirsutum x G. raimondii. Memon and Ahmed (1970) described similar interspecific triploid hybrid in terms of vigour, tallness, petal spot and hairiness and reported that these characters were being transmited from G. anomalum. However the hybrids had flower shape and petal colour of G. hirsutum indicating the dominance nature of these characters in cultivated upland hirsutums and recessive in wild relatives. The same author further reported that such differences at diploid and triploid levels might be due to the presence of two Ah Dh chromosome complements carrying genes or modifier complex or both inhibiting the expression of the characters of the diploid parent G. anomalum in the triploid hybrid. Mehetre and Thombre (1982) reported that in F1 triploid; the bract shape of G. hirsutum was dominant while leaf shape, petal colour, petal spot and gossypol glands of G. anomalum were dominant. The triploids did not produce fertile pollen grains obviously due to chromosomal differences. A number of strategies have been proposed for overcoming the ploidy barrier, but all involve the synthesis of a sterile intergenomic F1 and
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doubling chromosome complement to achieve the fertility (Stewart, 1995). Umbeck and Stewart (1985) also suggested that the doubling of interspecific hybrids is necessary to restore pollen fertility and it enabled to continue back crossing with cultigens. Thus polyploidy has been used as the main tool to overcome the sterility of interspecific hybrids. In the present study also two triploid plants G. hirsutum (MCU 5) x G. raimondii [(AD)1 D5] and G. hirsutum (MCU 7) x G. raimondii [(AD)1 D5] were polyploidised using colchicine. The morphological expressions like slower growth, abnormal branching, stunted growth, large sized flowers, increased pollen grain size, fertile pollen grains, boll and seed set as compared to normal F1 triploids suggested that the chromosome complements of these colchicine treated plants were successfully doubled. Stephens (1942) reported that doubled tetraploid showing gigas characters, slower growth, coarser and fleshier vegetative parts, increased size of pollen grains, and broader leaves as compared with normal diploid. Brown and Menzel (1952) also observed that amphidiploids were distinguished from their corresponding F1 hybrids by larger, thicker, less lobed leaves, larger and broader bracteole, flower parts, larger anther, more regular shedding of pollen and often by more irregular, ruffled, some what lobed petal margins. Similar observations have also been made by Harland (1940) and Amin (1941). Brubaker et al. (1999) also confirmed the doubling of G. sturtianum x G. hirsutum and its reciprocal hybrid G. hirsutum x G. sturtianum by increased flower size. The hexaploids G. hirsutum (MCU 5) x G. raimondii [2(AD) 1D 5] and G. hirsutum (MCU 7) x G. raimondii [2(AD)1D5] obtained by chromosome doubling of their respective triploids were also intermediate between the tetraploid Gossypium hirsutum and their wild diploid parent G. raimondii and conformed to the findings of Brown and Menzel (1952) who

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reported that in general, the hexaploids, like F1 triploid hybrids, are intermediate between G. hirsutum and the wild diploid parent. Influence of the wild genome on individual plant parts varies markedly from species to species, but both the diploid and the hirsutum components are clearly discernible in all. In these two hexaploids, G. hirsutum (MCU 5) x G. raimondii [2(AD1)D5] and G. hirsutum (MCU 7) x G. raimondii [2 (AD)1 D5] normal boll formation was observed occassionally with well developed seeds (Plate I-Fig.13 & 14). Brown and Menzel (1952) concluded that the triploid hybrids, from which the hexaploids are derived, are almost completely sterile while all the hexaploids, are more or less fertile, the degree of fertility being different with the varying diploid species involved. The E1 (Stocksii) hexaploids are most prolific and readily set many selfed bolls with a high number of seeds. The A1 (herbaceum), A2 (arboreum), B1 (anomalum) and D1 (thurberi) hexaploids are also rather highly self-fertile, while in the D5 (raimondii) hexaploids boll set was occasional (Brown, 1951). The bolls produced in the two hexaploids involving D5 genome were smaller in size than the hirsutum but larger than the diploid raimondii. The fibres produced on the hexaploid seeds are light brown in colour, shorter in length and less dense than the fibres on seeds of the upland cotton probably due to dominant influence of G. raimondii (Plate I-Fig.17 & 20). Beasley (1940) also reported similar results, in the hexaploids [2 (AD)1 D2], G. hirsutum x G. harknessii and [2 (AD)1 C1], G. hirsutum x G. sturtianum. In the development of intergenomic hybrids for resistance, a thorough knowledge about the chromosomal behaviour in hybrids is essential as it forms the basic information over which the breeding programme is formulated. Cytological study helps in establishing desired forms in a more precise way within a shorter span of time. In the present study, in diploid and tetraploid
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species, normal orientation, association and disjunction of chromosomes were observed while in triploids and hexaploids, low frequency of trivalents, quadrivalents and pentavalents besides univalents and bivalents were observed. The cytological analysis in the F1 hybrids, G. hirsutum (MCU 5) x G. raimondii [(AD)1 D5] and G. hirsutum (MCU 7) x G. raimondii [(AD) 1 D5] showed the mean pairing association of 11.50 I + 9.97 II + 2.31 III + 0.16 IV and 13.50 I + 11.35 II + 0.96 III respectively (Table 1). The chromosome association observed in these hybrids did not markedly differ from earlier report (Barducci and Madoo, 1940). Mehetre et al. (2002) observed on an average of 12.78 I 11.13 II + 1.32 III at metaphase I of F1 hybrid involving G. hirsutum CMS x G. raimondii. Similar results were also noticed by Mehetre et al. (2003). A high frequency of trivalents (2.31) and as high as 4 trivalents in majority of PMCs (18.75 %) observed, indicate partial homology of D 5 chromosomes with A and D chromosomes of cultivated tetraploids. The hexaploids G. hirsutum (MCU 5) x G. raimondii [2(AD) 1 D 5 ] showed a mean association of 4.05 I + 26.91 II + 4.55 III + 1.23 IV + 0.32 V (Table 2). Iyengar (1944) and Brown (1951) also recorded similar results of mean association 2.32 I + 32.25 II + 0.86 III + 1.75 IV + 0.02 V + 0.04 VI and 1.26 I + 25.81 II + 1.18 III + 5.19 IV respectively. But the other hexaploid G. hirsutum (MCU 7) x G. raimondii [2(AD) 1D5] showed a mean association of 24.55 I + 19.68 II + 3.73 III + 0.73 IV (Table 2) indicating diverse genetic constitution of parents. The formation of trivalents and higher chromosome associations in triploids and hexaploids indicate the pairing affinities between the genome involved. Endrizzi (1962) reported that the main force, which controls regular pairing behaviour in Gossypium, is the differences in the degree of chromosome

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condensation. Homeologous chromosome of differently sized Gossypium genomes rarely pair. The univalents observed in this study can be attributed to asynapsis because of lack of homology between the different sets of chromosomes or to the failure of the chromosomes to remain associated (desynapsis). Cytological analysis of the hybrids revealed that they were true interspecific crosses. Observations of meiotic metaphase chromosomes indicated the degree of relatedness between species. As expected, chromosome pairing indicated a close homology of G. raimondii (D5) with D subgenome of the G. hirsutum. In the present study, greater homology observed between A and D genomes aid in production of desirable recombinants despite minor cytological disturbances as there are successful boll setting and viable seed production in hexaploids. Hence, those species with D genome can be used successfully in gene

transfer if fertilization barriers are overcome by novel techniques. The successful utilization of wild species in breeding programme is often restricted by the operation of either prefertilization or post fertilization barriers during wide hybridization. Hence success in transfer of characters in such interspecific crosses has been limited because of the sterile nature of their F1s though the allohexaploids of these triploids were fairly fertile. Thus, development of stabilized lines from such an interspecific gene transfer has been a long-term programme with comparatively low probability of success. Nevertheless, it is worthwhile to go in for backcrossing of the hexaploids with respective cultivated tetraploids repeatedly and observe for the cross over segments carrying resistant genes for jassid resistance so that high yielding resistant genotypes can be achieved in due course.
G. hirsutum (MCU 7) x G. raimondii I 16 17 11 15 14 14 9 10 18 351 13.50 II 10 8 11 12 8 12 15 13 9 295 11.35 III 1 2 2 3 1 1 1 25 0.96 IV -

Table 1. Chromosome association in triploid hybrids: G. hirsutum x G. raimondii AD) 1 D5 G. hirsutum (MCU 5) x G. raimondii FreqFreqI II III IV uency uency of PMC of PMC 1 16 10 1 4 2 6 12 3 1 4 13 8 2 1 2 3 10 13 1 5 1 18 9 1 2 2 8 8 5 1 3 14 11 1 3 5 13 10 2 5 2 10 10 3 3 1 13 8 2 1 2 3 18 6 13 7 4 Total 368 319 74 5 26 32 Mean 11.5 9.97 2.31 0.16 Associ ation
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Table 2. Chromosome association in hexaploid hybrids: G. hirsutum x G. raimondii 2(AD) 1 D5

G. hirsutum (MCU 5) x G. raimondii G. hirsutum (MCU 7) x G. raimondii FreqFreqII III IV V I II III IV V uency uency I of PMC of PMC 1 3 17 7 5 3 34 12 4 2 5 7 28 5 5 25 19 5 3 5 24 4 2 1 1 21 22 3 1 2 3 31 3 1 2 18 20 4 2 1 2 27 3 2 1 2 17 24 3 1 4 2 27 6 1 1 22 25 2 1 4 23 4 4 4 25 22 3 3 4 27 5 1 3 24 19 4 1 2 2 31 2 2 1 28 22 2 Total 89 592 100 27 7 23 540 433 82 16 Mean 4.05 26.91 4.55 1.23 0.32 24.55 19.68 3.73 Associ ation

REFERENCES Amin, K. C. 1941. Interspecific hybridization and colchicine induced polyploidy in cotton. Indian Central Cotton Committee, Genetics and Plant Breeding. 5: 1-14. Beasley, J. O. 1940. The production of polyploids in Gossypium sp. Hered. 31: 39-48. Beasley, J.O. 1942. Meiotic chromosome behavior in species, species hybrids, haploids and induced polyploids of Gossypium. Genetics. 27: 25-56. Brown, M.S. 1951. The spontaneous occurrence of amphiploidy in species hybrids of Gossypium. Evolution. 5: 25-41. Brown, M.S. and M.Y. Menzel. 1952. Polygenomic hybrids in Gossypium. I. Cytology of hexaploids, pentaploids and hexaploid combinations. Genetics. 37: 242263. Brubaker, C.L., A.H.D. Brown, J.M. Stewart, M.J. Kilby and J.P. Grace. 1999. Production
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of fertile hybrid germplasm with diploid Australian Gossypium species for cotton improvement. Euphytica. 108: 199-213. Deodikar, G.B. 1949. Cytogenetic studies on cross of G. anomalum with cultivated cotton. I. (G. hirsutum x G. anomalum) doubled x G. hirsutum. Indian J. Agric. Sci.19: 389-399. Endrizzi, J.E. 1962. The diploid like cytological behaviour of tetraploid cotton. Evolution. 16: 325-329. Fryxell. P.A., L.A. Craven and J McD. Stewert. 1992. A revision of Gossypium sect. Grandicalyx (Malvaceae), including the descriptions of six new species. Syst. Bot. 17: 91-114. Geever, R.F., F.R.H. Katlermand and J.E. Endrizzi. 1989. DNA hybridization analysis of a Gossypium allotetraploid and two closely related diploid species. Theor. Appl. Genet. 77: 553-559.

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Gorham, J. and E.M. Young. 1996. Wild relatives of cotton and rice as sources of stress resistance traits. Proc. Meet. Tropical Plants. 13-15 march. 1996.Montpellier, 39 52. CIRAD, Montpellier Guany, R. L. 1952. Impressions of American Cotton Research. Emp. Cott. Gr. Rev. XXIX, 171-181. Harland, S.C. 1940. New polyploids in cotton by the use of colchicines. Trop. Agric. 17: 53-54. Knight, R.L., S.O.S. Park and R.L. Euany. 1953. Progress report from experimental stations (1951-52), E.C.G.C.10-16. Marappan, P.V. 1960. Cotton improvement through interspecific hybridization: Behaviour of arboreum anomalum back crossing. Dissertation submitted to the University of Madras as part fulfillment for the award of Masters degree. Mehetre, S.S. and M.V. Thombre. 1982. Cyotmorphology of haploid Gossypium hirsutum x G. anomalum. Indian J. Genet. 42: 144-149. Mehetre, S.S.,V.L. Gawande, A. R. Aher, and G.C. Shinde. 2003. Cytomorphology of interspecific hybrids between Gossypium hirsutum L., its haploid and Gossypium raimondii. Indian J. Genet. 63 (4): 319 324. Memon, A.M. and M.Ahmed. 1970. Morphological, cytological and fertility studies in interspecific hybrid G. hirsutum L. Var. M4 x G. anomalum, Waw. Et. Peyr. Pak. Cott. 14: 253-265. Muramoto, H. 1969. Hexaploid cotton: Some plant and fiber properties.Crop Sci, 9: 2729.

Narayanan, S.S., V. V. Singh, Punit Mohan and Vinita Gotmare. 2004. Germplasm and its utilization in cotton improvement retrospect and prospects. In: recent advances in cotton research and development in India-Lead Papers presented at the National Symposium on Changing world order Cotton research, development and policy in context, Acharya N. G. Ranga Agricultural University, Hyderabad, August 10-12, 2004. pp, 3-24. Stephens, S.G. 1942. Colchicine produced polyploids in Gossypium. I. An autotetraploid Asiatic cotton and certain of its hybrids with wild diploid species. J. Genet. 44: 272-295. Stewart, J. M.C. D. 1995. Potential for crop improvement with exotic germplasm and genetic engineering. In: Challenging the future. G.A. Constable and N.W. Forrester. (eds.) Proceedings of the world cotton research conference-1, Brishbane, Australia, February 14-17, pp. 313-327, Melborune, Australia. Thombre, M.V. and S.S. Mehetre. 1981. interspecific hybridization in Gossypium. II. In hybrid between G. hirsutum, L. haploid (2n=2x=26, AhAh) x G. thurberi, Tod. (2n=2x=26, 2D1D1). Cytologia. 46(1/2): 291-299. Umbeck, P.F. and J.M. Stewart. 1985. Substitution of cotton cytoplasms from wild diploid species of cotton. Crop Sci. 25: 1015-1019.

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STUDIES ON THE EFFECT OF PRE CONDITIONING OF POLLEN AND DYNAMICS OF POLLEN TUBE GROWTH IN GOSSYPIUM SPP.
Gunasekaran, M. and T.S.Raveendran

ABSTRACT
The effect of pre-conditioning of pollen grain in in vitro and pollen tube dynamics following selfing as well as cross pollination under in vivo of seven diploid wild species and two cultivated tetraploids of the genus Gossypium was studied. Pollen grains of G. trilobum and G. gossypioides did not require pre- conditioning for good germination where as other species required 15 to 45 minutes of pre-conditioning to attain more than 90 per cent of pollen germination. With regard to pollen tube growth under in vivo, the dramatic reduction in the number of pollen tubes occurs as they grow along the style both in selfing and crossing and the percentage on number of pollen tubes grow on the style and entry into the ovary was found to be independent of the initial pollen load during selfing. This suggests that genetic interaction may play a major role in the regulation of pollen tube attrition. The rate of pollen tube attrition is more or less the same along the selfed pistils of different ploidy levels as well as the pistils of tetraploid and diploid crosses. The greater percentage of pollen tube number reduction in diploid and tetraploid crosses due to the presence of inhibitory substances besides physical and physiological barrier was observed. The arrest of pollen tube growth in stylar regions of the majority of diploid and tetraploid crosses suggested that stylar pollination could not be an effective method to overcome the prefertilization barriers in cotton.

Introduction In plants, the post pollination success is based on two mechanisms, the competition among the male gametophytes (pollen competition) and the other, inter sexual mechanism, consisting in female mate choice that could interact with the male male competition. These two mechanisms have been intensively studied in numerous animal species but only recently have their implications in plants started to be considered. (Hormaza and Herrero, 1994). In general, evolutionarily advanced trinucleate pollen grains have fully developed mitochondria at dehiscence, allowing for a rapid germination when it contacts with the stigma (Hoekstra, 1979). In contrast, many binucleate pollen species like Gossypium have far less developed mitochondria. These are further assembled during a lag phase prior to emergence of the pollen tube, showing prolonged germination. Hence pollen of such species show low germinability and the maximum could be obtained only when it was
Tamil Nadu Agricultural University, Coimbatore 3

pre-conditioned in a humid atmosphere. In most plant species the number of pollen grains deposited on the stigma greatly exceeds the number of ovules available for fertilization (Plitmann, 1993) and consequently numerous male gametophytes are lost inside the pistil during the process that extends from pollination to fertilization. This kind of studies have been extensively made in self incompatible species (Lewis, 1994). However, it is not clear to what extent pollen-pistil interaction may play a role in determining the success of fertilization in distant hybridization particularly, hybridization among the species of different ploidy levels. The present investigation was undertaken to estimate the requirement of the preconditioning period and to evaluate extent of pollen tube attrition after selfing and crossing and to ascertain the role of stigma and style in restricting or preventing the pollen tube to reach the ovary in incompatible species.

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Materials and Methods Seven wild diploid species viz., G.thurberi, G.armourianum G.davidsonii, G.gossypioides, G.trilobum, G. triphyllum and G. barbosanum (2n-26) and two cultivated tetraploid species G.hirsutum (Variety MCU 5 and MCU 9) and G.babadense (variety Suvin) (2n-52) were subjected to investigation. In vitro pollen grain studies The pre-conditioning period for pollen grains for different species of the genus Gossypium was studied with pollen grains from freshly dehisced anthers and those after 15, 30 and 45 minutes of dehiscence. The above pollen grains were dusted on a drop of medium placed on a cavity slide and observed under microscope. The materials were replicated three times with five slides per replication. The duration of preconditioning period for each species was assessed by recording the percentage of pollen germination. The percentage values were transformed into angles and analysed in Completely Randomised Design (CRD) and transformed means were compared with Duncans Multiple Range Test. In vivo pollen tube growth studies Self pollinated pistils and cross pollinated pistils of direct and reciprocal combinations of above parents were collected at 2,10,12 and 24 hours after pollination, fixed in 6:3:1 (ethanol : chloroform : acetic acid) fixative and stored at 4-10oC for 24 hours. The pistils were then transferred to 70 per cent ethanol and stored in refrigerator till further use. For in vivo studies, the pistils taken out from fixative were washed with distilled water three to four times and then macerated in 10 N NaOH for 10 hours (when diploid was used as female parent). Then they were thoroughly washed in distilled water and stained for four hours to 12 hours in 0.3 per cent aniline blue prepared in 0.1 N K3PO4. The stained pistils were placed on a glass slide
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containing a drop of glycerol and covered with 23 x 30 mm coverslip and gently pressed. The slides were observed under Nikon Microphot Fx microscope with fluorescence attachement, illuminated with 200w high pressure ultraviolet lamp. The observations were taken with B (380 490 nm) and BG (650 nm) excitation filters in combination with BA 520 barrier filters. The number of pollen tubes on stigmatic surface, middle of the stylar region and at the entry of ovary were recorded from 10 pistils per cross combination per replicate and observations were recorded thrice. The data were analysed in CRD to find out the statistical significance. Results and Discussion In vitro pollen germination studies Among the seven wild and two cultivated species studied, the freshly dehisced pollens of G.gosssypioides and G.trilobum recorded more than 90 per cent germination in artificial medium and indicated that these two species are evolutionarily advanced and pollen grains of these binucleate type equiped with fully developed mitochondria, required active respiration and quick pollen germination (Table1). The pollens of other species viz., G. thurberi, G.armourianum, G.davidsonii, G.hirsutum var. MCU 5 and MCU 9 required 15 minutes of pre-conditioning to give more than 90 per cent of pollen germination. The percentage of pollen germination was more than 90 per cent in the species G.triphyllum, G.barbosanum and G.barbadense when their pollens were placed in the medium for 45 minutes after anther dehiscence. There were also significant differences between the different duration of pre-conditioning. Except G.trilobum and G. gossypioides, all the species had less than 90 per cent germination. G. thurberi, G. armourianum, G. davidsonii and the cultivated G.hirsutum had higher pollen germination of more than 90 per cent which

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sustained during subsequent treatments also. G.barbadense required minimum period of 30 minutes per-conditioning while the two species G.triphyllum and G.barbosanum provided higher percentage only after 45 minutes of preconditioning. The existence of variation in the duration of pre-conditioning in both ploidy levels indicates that ploidy level did not have any influence on the above phenomena. Stanley and Linkens (1974) also observed that in Petunia freshly shed pollen showed low germinability and pre-conditioning improved the germination. According to Bar-Shalom and Mattsson (1977) the pollen from plants of wet stigma type is often found to germinate readily in liquid media with appropriate osmotic balance. In contrast, pollen from species possessing dry stigma often requires special condition to establish something near a natural hydration rate. The present study reveals the existence of species variation for the pre-conditioning in the genus Gossypium. It also indicated that the level of phylogenetic advancement of the male gametophyte is characterized by the overall state of metabolic development at dehiscence rather than by the number of generative cells or ploidy level of the pollen. In vivo pollen tube growth studies The number of pollen tubes at stigmatic, stylar and at the entry of ovary in selfing of parents, diploid x tetraploid and tetraploid x diploid cross combinations is presented in the Tables 2, 3 & 4 respectively. The results showed dramatic reduction in the number of pollen tubes as they grew along the style both in selfing as well as crossing in Gossypium species and this phenomenon appears to be consistent upon selfing. However, significant differences among the number of pollen tubes on the entry of ovary in different species showed the presence of genetic variability among the species studied. Different number of pollen tubes grew in the style depending on the initial number of pollen
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grains deposited. However, the present reduction in the number of pollen tubes was independent of the initial load during selfing, with the number of pollen tubes reaching the ovary being more or less similar. The reduction in the number of pollen tubes passed on the stylar and entry of ovary region in diploid x tetraploid and tetraploid x diploid cross combinations was found to be higher than in selfing and suggested that the genetic interactions may play a role in the regulation of the pollen tube attrition. Although many studies in other species have shown (Herrero and Dickinson, 1980; Pimienta et al. 1983; Herrero 1992) reduction in the number of pollen tubes growing down the style, only in few cases the pattern has been studied. The main bottleneck seems to be the upper portion of the style in Brassica oleracea and Cucurbita pepo (Ockendon and Gates, 1975 and Winsor and Stephenson, 1995). Penetrabily is limited to one pollen tube irrespective of more pollen tubes formed in Triticum durum (Rudramuniappa and Panchaksharappa, 1974). In the present study, gradual reduction in the number of pollen tubes occur along the style and only 6.9 (G.thurberi) to 26.11 (G.hirsutum var. MCU 9) per cent of total pollen tubes reached the ovary in self pollinated pistils. This suggested that the selection pressure in Gossypium species appears to occur along the entire style length. It is believed that two main forces could determine such a reduction of male gametophyte in the pistil. One force could be the differences in competitive ability of the pollen and the other could be a modulation of these differences by the pistil. This modulation would comprise both physical and physiological constraints and genetics of pollen pistil interaction as was reported by Hormaza and Herrero (1994). According to Herrero (1992) the width of the transmitting tissue of the style would be a physiological limitation while physical

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Plant Breeding in Post Genomics Era

limitation include restriction in the nutrient supply. The bionuleate pollen, as in the case of crop under investigation, after an initial period of autotrophic growth, grows heterotrophically consuming large amount of nutrients required to produce cell wall from stylar tissues. This trophic dependence allows the pistil to influence the pollen tube growth rate and dynamics. In the present investigation, in diploid x tetraploid crosses out of 21 crosses, pollen tube reached the entry of ovary only in nine crosses. Such greater percentage of reduction in pollen tube number can be attributed to the presence of inhibitory substances besides the physical and physiological barriers. It is also observed from the present study that the fertilization does not depend uniquely on passive physical or physiological constraints by the pistil, but also on the genetic interactions among the male gemetophytes and between the male gemetophytes and female tissues and appears this type of interactions are probably super imposed. REFERENCES Bar-Shalom, D. and Mattsson, O. (1977). Mode of hybridization as an important factor in the germination of trinucleate pollen grains. Bot. Tiddskv. 77 : 254 257. Herrero, M and Dickinson, H.G. (1980). Pollen tube growth following compatible and incompatible interspecific pollinations in Petunia hybrida. 148 : 217-221. Herrero, M. (1992). From pollination to fertilization in fruit trees. Plant Growth Regul, 11 : 27-32. Hormaza, J.I. and M. Herrero. (1994). Gametophytic competition and selection. I. Williams E.G., Clarke, A.E., Knox R.B. (eds.). Genetic control of incompatibility and reproductive development in flowering plants. Kluwer, Dordrecht. PP 372 400.
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Hoekstra, F.A., (1979). Mitochondrial development and activity of binucleate and trinucleate pollen during germination in vitro. Planta, 145 : 25-26. Lewis, D. (1994). Gametophytic sporophytic incompatibility. In : Williams E.G., B.R. Know and A.E. Clarke (eds). Genetic control of incompatibility and reproductive development in flowering plants. Kluwer, Dordrecht. Pp 88-107. Ockendon, D.J. and Gates, P.J. (1975). Growth of cross and self pollen tubes in the style of Brassica oleracea. New Phytol. 75 : 155 160. Pimienta, E., V.S. Polito and D.E. Kester (1983). Pollen tube growth in cross and self pollinated Nonpareil almond. J. Ann. Soc. Horti. Sci. 108 : 643 647. Plitmann, U. (1993). Pollen tube attrition as related to breeding systems in Brassicaceae. Plant. Syst. Evol. 188 : 6572. Rudramuniyappa, C.K. and Panchaksharappa, M.G. (1974). Histochemistry of pollen tube growth in vivo in Triticum durum. Desf. Cytologica. 39 : 665-671. Stanley, R.G. and H.F. Linkens (1974). Pollen : Biology, Biochemistry and Management. Springer Verlag, Berlin. Winsor, J.A. and Stephenson, A.G. (1995). Demographics of pollen tube growth in Cucurbita pepo. Can. J. Bot, 73 : 583 : 589.

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Plant Breeding in Post Genomics Era

Table 1. Pre-conditioning of pollen grain of parents


Species G. armourianum G. armourianum G. davidsonii G. gossypioides G. trilobum G. triphyllum G. barbosanum G. hirsutum var. MCU 5 G. hirsutum var. MCU 9 G. barbadense var. Suvin SE CD (5%) Pollen germination (%) min. after dehiscence of anther 15 30 45 Freshly shed pollen 68.62 b(86.71) 68.62 b(86.71) 64.97 c(82.09) 80.33 a(96.95) 81.12 a(97.61) 45.66 e(51.15) 47.22e(53.87) 66.09(83.56) 69.15b(87.31) 58.62d(72.88) 0.829 2.447 77.54 b(95.3) 77.54 b(95.3) 80.12 ab(97.0) 80.12 ab(97.0) 80.64 a(97.3) 56.17 c(69.0) 57.42 c(71.0) 80.27 ab(97.0) 78.10 ab(95.7) 68.87 b(87.0) 0.899 2.696 78.06 bcd(95.7) 78.06 bcd(95.7) 79.04 a-d(96.3) 80.11 ab(97.) 81.25 a(97.7) 67.49 e(85.3) 68.60 e(86.7) 80.64 a(97.3) 79.50 abc(96.7) 77.54 cd(95.3) 0.788 2.324 78.00 b(95.7) 78.00 b(95.7) 79.50 ab(96.7) 80.91 ab(97.3) 80.64 ab(97.3) 74.34 c(92.7) 78.52 b(96.0) 80.64 ab(97.3) 82.05 a(98.0) 78.98 b(96.3) 0.887 2.617

In a column, means followed by common letters are not significantly different at the 5% level by DMRT. Percentage values are in parentheses
Table 2. Number and percentage (in brackets) of pollen tubes at stigmatic and stylar regions and entry into ovary in parents upon selfing
No. of pollen tubes Middle of the style Range Mean 133-172 106-159 127-175 127-173 138-170 116-192 102-157 135-182 123-177 175-212 7.98 24.88 160(69.3) 110(51.6) 158(65.5) 140(59.1) 151(59.4) 129(55.8) 139(59.1) 179(64.3) 160(60.4) 198(63.9)

Parents G. thurberi G. armourianum G. davidsonii G. gossypioides G. trilobum G. triphyllum G. barbosanum G. hirsutum var. MCU 5 G. hirsutum var. MCU 9 G. barbadense var. Suvin SE CD (%)

Stigmatic region Range Mean 197-247 206-216 198-258 219-278 229-265 180-250 212-247 190-290 202-271 296-318 8.94 28.16 231(100) 213(100) 240(100) 237(100) 254(100) 231(100) 235(100) 278(100) 265(100) 310(100)

Entry into ovary Range Mean 37-64 40-52 50-55 41-61 32-55 46.65 42.55 55.82 56.79 73.95 5.14 16.19 16(6.9) 45(21.3) 53(22.1) 46(19.4) 47(18.5) 52(22.5) 46(19.6) 68(24.5) 69(26.1) 80(25.8)

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Table 3. Number and percentage (in brackets) of pollen tube at stigmatic and stylar and entry of ovary in diploid x tetraploid crosses No. of pollen tubes Middle of the style Range Mean

Parents

Stigmatic region Range Mean

Entry into ovary Range Mean

G. G. G. G. G. G. G. G.

thurberi x MCU 5 armourianum x MCU 5 davidsonii x MCU 5 gossypioides x MCU 5 trilobum x MCU 5 triphyllum x MCU 5 barbosanum x MCU 5 thurberi x MCU 9

212-250 100-147 42-68 152-206 190-226 10-63 15-50 190-227 115-163 77-115 165-221 181-210 12-51 34-50 22-43 160-231 86-132 29-73 18-71 20-65 17.30 50.70

232(100) 128(100) 53(100) 183(100) 215(100) 22(100) 20(100) 209(100) 142(100) 92(100) 180(100) 191(100) 16(100) 39(100) 29(100) 178(100) 112(100) 40(100) 55(100) 37(100) -

77-125 15-43 25.82 52-94 82-152 19-62 31-78 66-96 32-91 8.44 27.52

G. armourianum x MCU 9 G. davidsonii x MCU 9 G. gossypioides x MCU 9 G. trilobum x MCU 9 G. triphyllum x MCU 9 G. barbosanum x MCU 9 G. thurberi x Suvin G. armourianum x Suvin G. davidsonii x Suvin G.gossypioides x Suvin G. trilobum x Suvin G.triphyllum x Suvin G.barbosanum x Suvin SE CD (%)

90(38.8) 12-34 21(9.0) 19(14.8) 44(24.0) 7-19 11(6.0) 77(35.8) 15-26 19(8.8) 96 22-35(12.9) 27 (45.9) 41(28.9) 7-19 11(7.7) 50(27.7) 12-17 13(7.2) 75(39.3) 19-25 22(11.5) 69(38.8) 11-21 14(7.9) 2.07 6.96

- denotes no pollen tube growth.

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Table 4. Number and Percentage (in brackets) of pollen tubes at stigmatic and stylar regions and entry of ovary in tetraploid x diploid crosses
No. of pollen tubes Parents Stigmatic region Range Mean Middle of the style Range Mean Entry into ovary Range Mean

MCU 5 x G.thurberi MCU 5 x G.armourianum MCU 5 x G.davidsonii MCU 5 x G.gossypioides MCU 5 x G. trilobum MCU 5 x G.triphyllum MCU 5 x G.barbosanum MCU 9 x G.thurberi MCU 9 x G.armourianum MCU 9 x G.davidsonii MCU 9 x G.gossypioides MCU 9 x G. trilobum MCU 9 x G.triphyllum MCU 9 x G.barbosanum Suvin x G.thurberi Suvin x G.armourianum Suvin x G.davidsonii Suvin x G.gossypioides Suvin x G. trilobum Suvin x G.triphyllum Suvin x G.barbosanum SE CD (%)

82-135 62-75 94-141 102-138 152-195 37-72 38-59 98-152 55-69 66-137 165-206 159-200 44-65 37-68 47-65 76-146 40-75 56-62 96-135 19-69 28-70

105(100) 71(100) 120(100) 128(100) 180(100) 65(100) 51(100) 131(100) 63(100) 107(100) 186(100) 175(100) 57(100) 51(100) 52(100) 115(100) 64(100) 60(100) 117(100) 51(100) 56(100) 9.92 14.64

58-69 40-53 42-73 70-95 68-100 19-65 22-46 74-96 26-58 46-71 76-115 73-120 27-39 16-41 13-27 59-90 15-32 12-19 57-95 10-60 25-53

66(52.9) 47(66.2) 65(54.2) 84(65.6) 92(51.1) 40(61.5) 34(64.2) 83(63.4) 46(73.0) 62(57.9) 89(47.8) 95(54.2) 31(54.3) 29(56.9) 19(36.5) 78(67.8) 23(35.9) 17(28.3) 65(55.6) 37(72.5) 32(57.1) 5.63 16.60

16.32 18-26 9-12 35-53 31-45 19-37 15-29 21-40 19-37 14-22 35-50 33-46 8.22 12-24 7-10 23-44 5-10 10-18 23-35 5-26 12-32

20(19.0) 22(30.9) 10(8.3) 40(31.2) 39(21.7) 27(41.5) 21(41.2) 32(24.4) 29(46.1) 15(14.0) 39(21.0) 42(24.0) 15(26.3) 17(33.3) 8(15.4) 26(22.6) 7(10.9) 14(23.3) 29(24.8) 16(31.4) 21(37.5) 2.34 6.90

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CYTOLOGICALANALYSIS VIGNA RADIATA X V. UMBELLATA L. HYBRIDS


Pandiyan, M., B. Subbalakshmi, AR. Muthiah and M. Kumar

ABSTRACT
Among the twelve interspecific crosses attempted, seed set was observed in the entire cross combination except V.radiata x V.umbellata cross. The reason for no seed set could be attributed to complete pollen sterility. To asses the reasons for the high pollen sterility in the F1, the cytogenetic. analysis of pollen mother cells (PMCs) was carried out. In the sterile interspecific hybrid, all types of abnormalities were observed. Out of 25 PMCs studied at Anaphase 1., only one cell revealed 11 bivalents. The univalents and quadrivalents were frequently observed. The number of univalents ranged from 0 to 14 while the number of quadrivalents ranged from 0 to 5. The average chromosome association per cell was IV (1.28) + II (4.96) + I (6.96). Precarious separation of chromosomes and formation of anaphase bridges was commonly observed in many PMCs. The sporad count for parents and direct crosses were also studied. The highest number of dyads (8 each) was observed both in V. radiata var. sublobata and V. radiata x V. radiata var. sublobata cross. The lowest number was observed in V. radiata x V. vexillata and V. radiata x V. hainiana. The triads and tetrads were observed in the all the cross combinations except V. radiata x V. mungo var silvestris and V. radiata x V. hainiana. There was no seed set in back cross with both parents. To asses the reasons for the high female sterility in the F1, the cytogenetic analysis of megaspore cells was carried out. All types of abnormalities were observed in the megaspore cells. The hybrid cell megaspore completely degenerated. The occurrence of abnormal megaspore was frequently observed which leads to female sterility.

Tamil Nadu Agricultural University, Coimbatore - 641 003

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TECHNICAL SESSION IV HYBRID BREEDING IN CROPS

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

TRANSGENIC HYBRID COTTON TECHNOLOGY AND SOME GENETIC OBSERVATIONS


Narayanan, S.S.1

ABSTRACT
Heterosis has been effectively used in cotton to increase yield through expansion and harmonization in the expression of various yield influencing parameters and fibre quality, while concurrently broadening the adaptability to larger areas. The genetic engineering advances and thereby the incorporation of B.t gene (from Bacillus thuringiensis) technology for bollworm control into hybrid technology of cotton have not only enabled to achieve a quantum jump in yield, but also accelerated the process of privatization of R&D and seed distribution on a more globally competitive scale. If heterosis exploitation is one method to rapidly increase the stagnating cotton yields, by incorporating Bt Cry 1Ac gene (transgenic hybrid), the hybrid will yield double benefit of yield improvement through heterotic vigour for yield as well as consolidating the yield gains through bollworm control at a reduced cost of production. Genotype deployment strategy, gene outsourcing technique, in-house germplasm development strategy for use as parents of hybrids, molecular evaluation strategy, transgenic-conversion strategy, parental purity maintenance techniques, mass scale hybrid evaluation and selection techniques at initial and advanced testing stages and in multi-location and market acceptability tests, strategy for developing competitive hybrids and placement in farms and meeting the expectations of spinners, technology for production management for maximizing the genetic potential for yield and fibre quality in transgenic hybrids, scientific seed production practices for transgenic hybrids and systems for ensuring cost reduction and assured planting quality etc., independently and collectively represent the primary factors influencing the success and sustainability of the transgenic hybrid technology. Extra-long staple cottons are needed with 2.5% span length of >34 to 38mm, tensile strength of >28-32 with 0.90 and above strength-length ratio, Micronair value of 3.6 to 4.4 with ginning outturn >33-35 per cent. Parallel development of hirsutum and barbadense germplasm using superior sources in global gene pool and identifying the best combinations for fibre quality and wide adaptation are suggested. Genetics in seed cost reduction especially GMS, CMS-R or gametocide systems have not become successful nor made any recognizable impact on seed production cost. Self-incompatibility induction through biotech approaches was indicated as alternatives and enable harvesting of crossed bolls from both parents projected, but still not turned into practical use. Private seed companies and their R&D face the pressures from competition in the market based on grower acceptance of the technology and generate partnerships with necessary agreements to advance in this area. If the public sector R&Ds can intensify their basic researches in this area, it would help the Indian seed industry to depend more on local technology and provide service at lower cost to farming community. In-house germplasm development in private seed industry has gained momentum and is the primary source of parents in successful hybrids rather than acquired germplasm. Development of marker systems such as RFLP, AFLPs, SSRs and SNPs considerably facilitated the estimation of genetic diversity. The use of molecular marker Restriction Fragment Length Polymorphism (RFLP) and coefficients of parentage for identifying heterotic effects in cotton had been beneficial. Such study has been helpful in detecting
Retired Scientist, CICR, Nagpur & Research Advisor (cotton), JK Agri Genetics, Hyderabad

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significant heterosis for total and first harvest yields, boll weight, lint percentage, and span length of fibres. A large leaf area during seedling growth allows the F1 hybrids to absorb more light than their parents, potentially resulting in increased photosynthetic activity per plant in cotton. Earliness in cotton should allow the maximum usable growing period within the given seasonal limitations for effective lint production and expression of fibre quality attributes. A mismatch between Farmers and Breeders perception of genotype product for a given area, i.e., a variety genotype or hybrid is sometimes responsible for the lapses in commercial realizations in the field as well as at the end-user level. During 2005-06, Australia growing only variety has reported 2100 kg / ha average lint yield, while that of India it is only 460 kg / ha even with hybrids. Regarding Plant Varieties Protection as per TRIPs, a system for protection has been established in India through the Patents Act and the Plant Variety and Farmers Rights Protection Act. The most relevant proprietary technologies and materials being developed all over the world are: (1) transformation systems (2) promoter systems (3) insect resistance genes (4) disease resistance genes (5) selectable marker genes (6) genetic markers (7) drought resistance genes (8) fibre quality-enhancing genes especially for strength, elongation and length (9) diagnostic probes and (10) others. Certain frequently asked questions for which more analytical genetic and breeding researches are needed have been listed in the paper. The transgenic hybrid technology after facing initial resistance from certain farmers and others has now created a great demand from farmers as well as textile industry as it has helped to improve the production to more than 4 million tonnes and productivity to over 460kg/ha within 3 years of its introduction in India. In the world, several major cotton-growing countries have adopted this technology on a large scale as a result of which global cotton output has reached 26 million tonnes and average yields to 730 kg lint per hectare. The International Cotton Genome Initiative (ICGI) is gradually providing guidelines for the study of structural genomics, functional genomics, evolutionary relationships among Gossypium species and related members of the plant kingdom, genetic resources and germplasm stocks and bio-informatics in cotton for a better appreciation and understanding of the molecular basis of differentiation in the genera. Once such a project takes full shape at the global level and the various scientific laboratories come together for the mapping of the cotton genomes on a coordinated basis, the era of cotton genetics and breeding would undergo further metamorphosis. The potentiality for revolutionary improvements in the diploid cottons as well as barbadense and hirsutum may be expected to touch new horizons in the history of cotton improvement.

Introduction Cotton crop yields the most versatile fibre, the purest form of cellulose in nature and also a substantial quantity of oil and protein in the cotton seed on which the spinnable lint hairs are borne, influencing the industrial economy and global markets. The goal of cotton breeding is to primarily increase the (1) productivity and profitability to the cotton farmers and enable them to produce what the spinners need (2) to improve the combinations of quality parameters desired by the textile industry and trade and to
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face inter-fibre as well as global competition (3) to increase the oil output primarily through enhancement of seed yields with reasonable optimization of oil and protein on the one hand and the spinnable lint fibre on the other and (4) to ensure reasonable employment opportunities and sustainable livelihoods for a large section of the population representing various sectors. Systematic cotton improvement through the intelligent application of Genetics and Plant Breeding has proved to be the most practical means for achieving these primary objectives

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and now the new tool of biotechnology and genetic engineering has provided further impetus to crop improvement in cotton. Transgenic hybrid technology When a shift from traditional breeding methods adopted up to the latter half of 1960s occurred in favour of hybrid breeding technology in cotton in India, both the seed cotton yield and fibre quality improvements witnessed higher growth rates. Heterosis has been effectively used in cotton to increase yield through expansion and harmonization in the expression of various yield influencing parameters and fibre quality, while concurrently broadening the adaptability to larger areas. The genetic engineering advances and thereby the incorporation of B.t gene (from Bacillus thuringiensis) technology for bollworm control into hybrid technology of cotton have not only enabled to achieve a quantum jump in yield, but also accelerated the process of privatization of R&D and seed distribution on a more globally competitive scale. In addition, the higher boll numbers per plant produced and retained through heterosis and transgenic effects has given a boost to transgenic heterotic cultivars. If heterosis exploitation is one method to rapidly increase the stagnating cotton yields, incorporating Bt Cry 1Ac gene (transgenic hybrid), the hybrid will have added advantage of double benefit of yield improvement through heterotic vigour for yield as well as consolidating the yield gains through bollworm control at a reduced cost of production. Multiple strategies for success Genotype deployment strategy, gene outsourcing technique, in-house germplasm development strategy for use as parents of hybrids, molecular evaluation strategy, transgenic- conversion strategy, parental purity maintenance techniques, mass scale hybrid evaluation and selection techniques at initial and advanced testing stages and in multi-location and market acceptability tests, strategy for
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developing competitive hybrids and placement in farms and meeting the expectations of spinners, technology for production management for maximizing the genetic potential for yield and fibre quality in transgenic hybrids, scientific seed production practices for transgenic hybrids and systems for ensuring cost reduction and assured planting quality etc., each of which represent a critical factor influencing the success and sustainability of the transgenic hybrid technology. Further, the testing of fibre harvested in the hybrids should be based on proper samples from well-maintained crop plots. The testing of transgenic presence in grow-out test lots is a critical requirement. Measures to prevent the ginned seeds from commercial crops of transgenic hybrids going to the planting seed market as transgenic F2 should be undertaken to ensure the supply of homogeneous lint parameters to the textile industry in the interest of real long-term benefits. Existing genotype analysis Product analysis of cotton genotypes of proprietary and non-proprietary kinds in India in the current context of global competition and mill industry needs is urgently needed to tone up breeding research in both sectors for ensuring a 4 per cent higher growth rates. In hybrid cotton breeding technology, major casualties include lower ginning outturn, unimpressive fibre strength improvements, wide variations in micronaire values due to genetic and management deficiencies, some increase in seed coat fragments and motes. Seed rate has been drastically curtailed and boll load per plant has been increased to five to six folds in India in the use of hybrid technology both in Bt and non-Bt versions. Yet in most advanced countries especially in ten top ranking countries, far higher yields per hectare are obtained from superior straight variety cultivars, whether transgenic or non-transgenic. While admitting that India has a large rain-fed area unlike those

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top cotton countries, the scope for India to accelerate its yield levels by effective transgenic hybrid deployment and cooperative contractual farming type crop management cannot be underestimated. Potential of technology Hybrid and Bt cotton hybrid technology have enabled substantial increases in cottonseed yields resulting in higher contribution to edible oil output, protein, gossypol, linters and other by-products. Though 100 percent seed replacement rate is advocated for cotton Bt or non-Bt hybrids and use of labeled seeds year after year, it is being widely misused by certain traders and misguided farmers by enabling the supply of F2 seeds. This is also a major reason for our shortfalls in production and deficiencies in fibre quality. Hybrid technology has also become a ploy in the hands of certain unscrupulous farmers and traders by floating of numerous versions of hybrids with low genetic diversity and causing harm to genuine private seed companies with R&D with a vision and mission to help the growers and textile industry. Potential of this powerful technology may be ruined by such unscientific activities. New efforts needed Bt cotton transgenic technology has become a boon to farmers, traders and textile industry, but superimposed on the hybrid technology with the popular hybrids in vogue in last ten years has caused innumerable deficiencies in the field level. For a superb performance of Bt plus hybrid technology, superior parental choice and efficient painstaking breeding methods are advocated that was not effectively taken care of in the first generation Bt cotton hybrids and a realization has come after costly mistakes have been committed. Introgressive breeding has been revived after a long gap in India and the breeding material reported developed under NATP and TMC projects appear impressive. But the purification process needs to be done to the core
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to carry out genetic enhancement of parental lines developed through introgressive breeding to get the best results through superior expression for known and novel, but valuable attributes. Genomic tools & diversity Genomic tools like MAS have a significant new role in parental line development with enhanced fibre quality attributes like high fibre strength, higher elongation per cent and appreciable micronaire value apart from drought tolerance and certain critical factors that influence length uniformity in fibre even under rain-grown conditions. Gene x Environment (G x E) interaction in hybrids is an important fact that cannot be ignored in genotype deployment and hence special attention to multi-location testing with special precision and attention and critical evaluation of performance a must for ensuring the success of the selected hybrids. The spectrum of genetic and phenotypic diversity in cotton is critical for understanding the complex processes in plants, inferring the effects of selection based on very efficient targeted molecular selection practices in hybrid breeding programme. Appreciation of the molecular basis of complex traits will provide genetic solutions and remove the misconceptions currently inhibiting an understanding and improvement of the performance of hybrids. Analysis of molecular variance has assumed importance in detecting polymorphism, to map the genome to identify the traits of interest, for critically studying the quantitative trait loci (QTL) and also to detect linkage with undesirable genes. Breeding for quality ELS cottons Extra-long staple cottons are needed with 2.5% span length of >34 to 38mm, tensile strength of >28-32 with 0.90 and above strengthlength ratio, micronaire value of 3.6 to 4.4 with ginning outturn >33-35 per cent. This requires priority efforts in the next three years and R&D

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Units should focus on this need. After Varalaxmi and DCH32, which are over 25 years old and TCHB 213 also similar, still superior inter-specific hybrids or even a more adaptable pest resistant Suvin-like (3 decades old barbadense cultivar), superior genotypes with acceptable fibre quality standards have not been developed and the above mentioned genotypes have lost their charm and preference, but for the absence of any alternative. Importing such type quality cottons are dictated by ruling international prices, politics and constant escalations. Parallel development of hirsutum and barbadense germplasm using superior sources in global gene pool and identifying the best combinations for fibre quality and wide adaptation are suggested. Bt gene technology with stacked genes would be helpful in effecting early maturity, good quality cotton and pest resistance. Genetics in seed cost reduction Forecast or continued efforts on new technology to reduce the cost of hybrid seeds produced especially GMS, CMS-R or gametocide systems have not become successful nor made any recognizable impact on seed production cost. Self-incompatibility induction through biotech approaches was indicated as alternatives and enable harvesting of crossed bolls from both parents projected, but still not turned into practical use. Irrespective of various mechanisms planned, transfer of pollen in cotton hybridization is a problem requiring manual work and an issue requiring molecular approaches to fix heterosis. Innovative approach on diploid cottons Desi or diploid cotton area expansion especially under rain-fed cotton areas is advocated using the released hybrids and recent straight variety cultivars in Maharashtra state. The desi cotton hybrids/varieties are no match in production, handling and processing to the H x H or H x B hybrids on account of the various advantages derived by growing the latter. The desi hybrids with GMS system are also not
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making much headway. By and large, boll features and fibre quality are unattractive. Attention is needed to improve these aspects through the genetic engineering route, if desi cotton has to make a higher progress in superior spinning uses. More innovative approaches rather than standard breeding methods are required to make it useful in the global competitive scenario. Bt cotton Performance enhancement Performance appraisal all over the country on Bt with hybrid technology indicated that there is need for much more R&D in agronomy and IPM technologies to maximize potential output and fibre quality for the entire crop duration. The public breeding programme has fallen by nearly half during the last one decade. In the private seed R&D sector also constant changes in personnel have resulted in lower breeder retention rate. Earlier cotton improvement resorted to pedigree selection, backcross, reselection, bulk, recurrent and single seed descent systematically for developing cultivars, but now short cuts have been employed with hybrid development, which often tells on the stability and fibre quality. Adoption of strict selection criteria for genes controlling yield, stability, resistance to drought and pests, pubescence, fibre properties etc., is essential. Public and private sector R&D cooperation Public cotton breeders in India spend very little efforts in transgenic breeding since access to transgenic by public breeders has been limited. Private seed companies and their R&D face the pressures from competition in the market based on grower acceptance of the technology and generate partnerships with necessary agreements to advance in this area. If the public sector R&Ds can intensify their basic researches in this area, it would help the Indian seed industry to depend more on local technology and provide service at lower cost

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to farming community. In-house germplasm development In-house germplasm development in private seed industry has gained momentum and is the primary source of parents in successful hybrids rather than acquired germplasm. Even in the public R&D centers, pre-parental germplasm development for hybrids and also line development in different cultivated species particularly in CICR (Nagpur, Coimbatore and Sirsa), UAS, Dharwad, MAU, Parbhani etc., have been accelerated rather than mere acquisition from other countries and this has been practiced in a systematic manner. The large array of hybrids floated in the market also can provide ample scope for new germplasm development by all participants by undertaking pedigree selection coupled with panmixis, selective intermating and other methods. Cotton breeders are also no longer unduly concerned about segregating populations and assumed that heterogeneity and heterozygosity are beneficial to commercial cotton production. But there is a limit to the level up to which this may be acceptable. Choice of transgenic hybrid The search for any unique genetic combination average more than 100 combinations in a well planned crossing programme each year involving over a thousand or more twin parental crosses (single cross) and depends on distinct parental development and choice of the right material. A magnificent look at the phenotype of the hybrid is an important factor for farmer acceptance besides a set of prominent agronomic attributes related to field performance in identifying the best hybrids. Other factors most important from end-user point of view like seeds per boll, lint per seed, lint per cent, fibre length and fibre fineness are measured after harvesting. These are very important to decide on the suitability to meet consumer industry requirements. The hybrid cotton release and
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replacement process in the competitive scenario is a delicate decision making process and is an important factor in private seed scenario for survival and brand image. Need for higher genetic orientation The principal genetic models in heterosis are dominance, over-dominance, pseudodominance, epistasis and also interaction between genetic and environmental and also physiological / biochemical factors which explain the causes of heterosis and hybrid vigour. By pedigree data, morphological data and agronomic performance information, biochemical data and DNA data, one can analyze parental genetic distance. The vast genomic and technological resources available in model species like Arabidopsis etc could be used to rapidly advance our understanding of the underlying physiological and molecular processes and a precedence could be established that may support the analysis of heterosis in cotton to make further breeding advances Genetics of biomass and harvest index In crop plants, habitability for biomass production ranging from 60-85 per cent has been reported. Cotton is a predominantly selfpollinated crop and often cross-pollinated to varying extents in different environments and may mimic crosses between inbred lines and out-breeders, if a large number of crosses are tested. In well-documented breeding lines, relatedness and consequently genetic distance can be deduced from pedigree selection. Quality improvement & MAS Development of marker systems such as RFLP, AFLPs, SSRs and SNPs considerably facilitated the estimation of genetic diversity between genotypes in various centres. The relationship between molecular markers heterozygosity and heterosis depends on germplasm used and the characters analyzed.

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Hence use of detailed characteristics of germplasm and an in-depth comprehension of the genetic basis of heterosis would be needed to develop strategies for utilization of molecular markers in pre-breeding parental lines exclusively for a hybrid programme and prediction of hybrid performance. Quantitative traits being the most important for improvement of yield and fibre attributes, the focus on genetic factors underlying them should be given importance along with heritable epigenetic factors influencing gene expression. The use of molecular marker Restriction Fragment Length Polymorphism (RFLP) and coefficients of parentage for identifying heterotic effects in cotton had been beneficial. Such study has been helpful in detecting significant heterosis for total and first harvest yields, boll weight, lint percentage, and span length of fibres. Genetics of hybrids for earliness & quality A large leaf area during seedling growth allows the F1 hybrids to absorb more light than their parents, potentially resulting in increased photosynthetic activity per plant in cotton. Attention on biomass productivity, root biomass, R/S ratio at seedling stage coupled with hybrid vigour and harvest index can be useful to identify better hybrids at initial stage of testing. Earliness in cotton should allow the maximum usable growing period within the given seasonal limitations for effective lint production and expression of fibre quality attributes in full. Some breeders are attempting shifting of photosynthates to lint at the expense of seed to increase the lint per cent by decreasing the seed size. But if it is unduly smaller seeds, it can cause problems in ginning with seed coat fragments to the detriment of textile processing. Fragile seed can affect planting seed quality also. Seed oil development requires higher amount of energy than lint development and lint being the primary commodity for which cotton is grown may be given the priority for lint attributes. Any increase
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in seed cotton yield and thereby increased seed yield can provide the higher oil output and also protein. Hence in identifying the best hybrids, primary attention may be focused on higher seed cotton yield with good quality fibre and then avail the benefit of resulting higher seed yield and therefore higher amounts of seed oil and protein there from. Benefits & side effects of technology India is a pioneer in hybrid cotton technology and has made great impact on R&D, trade, textile industry and by-product utilization, farmers ways and means of cultivation and profitability, fibre quality improvement to meet textile needs, large scale privatization of seed industry, employment potential, commercial seed production practices, high quality seed with 100 per cent seed replacement rate and many other spin-off benefits. Since its introduction in 1970s to 2002, the total production increased substantially and spinning potential of Indias cottons from 40s to 80-120 counts. No other country could adopt this technology successfully on such a large scale for various reasons including the difficulty of hybrid seed production. Though they have retained the straight variety-based cultivation, still managed to far exceed the average productivity level of India. Hybrid technology has a special role in several situations in India. In the continued special emphasis on hybrid cotton technology, certain casualties and deficiencies had occurred such as low volume seed rate of 1.125 kg per hectare (15-20kg in variety technology in India and 20-70 kg in certain countries) with high cost value of seeds, exploitation of the potential for high boll load (80-150 bolls per plant), opting for high pesticide application, increase in cost of production and facing other problems. The farmers have realized the advantage of hybrid vigour on yield potentials and quality up-gradation and millers got the produce they wanted. But the primary

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loss from bollworms caused high environmental pollution from large-scale use of chemicals, escalation in cost of production, indebtedness, emergence of resistance in insects, serious emergence of whitefly, damage to beneficial insect population and other harmful effects. It also far reduced the realizable benefits from heterosis and hybrids, because the bollworms took a heavy toll on the high boll loads. However the Bt transgenic hybrid technology has come in handy from 2002 to ward off these deficiencies. Perception of commercial genotype In hybrid breeding, success depends on (1) high mean performance and a large genetic variance in the hybrid population, (2) high per se performance and good adaptation of the parent populations to the target regions and (3) low inbreeding depression, if hybrids are produced from inbred/homozygous lines. Analysis of the existing products in the country would indicate only partial attention to these aspects and a better adherence to these criteria may help to improve the performance of cotton hybrid genotypes in future. A mismatch between Farmers and Breeders perception of genotype product for a given area, i.e., a variety genotype or hybrid is sometimes responsible for the lapses in commercial realizations in the field as well as at the end-user level. Under-use of diverse germplasm is responsible for the genetic diversity remaining untapped and in recent years, this process is hampered by the non-availability of genetic resources from various sources to users easily. Australias experience Under the very high yield conditions in Australia, the heterosis for yield was not consistent enough to warrant the production of hybrid seeds in cotton for achieving higher yields. During 2005-06, Australia has reported 2100 kg / ha average lint yield, while that of India is only 460 kg / ha even with hybrids. However, a study has shown that in certain combinations of G.
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hirsutum x G. barbadense hybrids, the fibres were stronger and finer appearing to be better suited to cotton spinning equipment now being used in their textile industry. In the USA, in upland cottons, it was found that in a hybrid, one parent should be from the targeted region of adaptation and the other parent from any other source provided it is a good combiner. In hybrids with one parent having above average fibre quality combined with high yield and yield combining ability, the F2s of such hybrids can be expected to yield good fibre and high yield. IPR and genetic engineering Regarding Plant Varieties Protection as per TRIPs, a system for protection has been established in India through the Patents Act and the Plant Variety and Farmers Rights Protection Act. The most relevant proprietary technologies and materials being developed all over the world are: (1) transformation systems (2) promoter systems (3) insect resistance genes (4) disease resistance genes (5) selectable marker genes (6) genetic markers (7) drought resistance genes (8) fibre qualityenhancing genes especially for strength, elongation and length (9) diagnostic probes and (10) others. In the next one decade, cotton research would be witnessing expanded and amazing activities in cotton breeding with the help of the new gene technologies. FAQs & research investigations Certain frequently asked questions on the subject include the following for which more and more analytical genetic and breeding researches are needed and to provide solutions for major deficiencies in the future: * The genetic behaviour of hybrids with transgenic (Bt) in hemizygous locus and homozygous dominant loci condition * Bt gene incorporation in FP or MP or both parents and effects and advantages. * Differential potentials in different Cry

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1Ac versions and ability to keep all the three bollworms and Spodoptera under control. * Constitutive Bt genes in Bollgard I and II and effect on plant metabolism, protein interaction and yield level in cotton in differing environments. * Bt Cry 1Ac in different character backgrounds like medium okra leaf, smooth leaf, hairy leaf, nectarilessness, naked seed, red-plant body pigmentation and effects on major expressions for agronomic and technological attributes, beside pest resistance * Role of MAS approach with various tools and techniques for drought tolerance and fibre quality parameters in Bt cotton and manner of combining them * Randomness of bombarded Cry 1Ac gene incorporation in cotton genome/ chromosomes through genetic analysis and the differential genetic behaviour. * Combining CLCV/whitefly resistance with Cry 1Ac Bt gene by genetic engineering. (whitefly is normally a minor pest that assumed serious proportions with nonjudicious use of synthetic pyrethroids and now with Bt cotton and reduction in such pesticide use, whether both whitefly and induced CLCV would come down) * Complete backcrossing and limited backcrossing and pedigree selection in Bt gene incorporation into hybrid parental genotype. * Bt transgenic based GMS and CMS-R hybrids and effect on simultaneous conversions for male sterility and Bt gene. * Genetic purity assessment techniques / tools and their relative efficiencies in detection for planting seed quality * Comparison of fibre quality and bollworm
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resistance efficacy in F2, F1 and straight varieties and dilution of effects if any * Genetic disturbances in Bt Cry 1Ac gene inserted genotypes on oil content, ginning outturn, boll size and boll weight and seed weight if any, due to linkages on other factors * Gossypol in cottonseed may lose the effective role as a deterrent against bollworm in Bt cotton and the effects thereon. * Hybrid performance as influenced by incomplete conversion into original parental genotype in cry 1Ac Bt genotypes * Plant age increase and diminishing gene expression in prolonged crop periods through plant management techniques for greater effectiveness * Genotype-phenotype relations and Cry 1Ac Bt protein expression variations in cotton hybrids * Bt Cry 1Ac in hirsutum and barbadense parents either in one or both parents on Bt gene effectiveness in hybrids on bollworms and influence on fibre quality. * Genomic explanations for earliness by 50% flowering, 50% boll bursting and Bartletts earliness index in Bt Cry 1Ac cotton hybrids. * Whether the Bollgard II versions nearing the release stage is really superior in bollworm management in Indian conditions and how much superiority can be expected in yield. In the same genotype hybrids with BGII versions, and BGI versions and what are the yield advantages likely. * There are many other points raised by NGOs, scientists with different perceptions and such numerous queries

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also need a scientific study and answer (e.g., super weeds, out-crossing and transgene flow, itching of fingers and palm during cotton picking in Bt cotton etc) by undertaking well-planned studies. Conclusion The transgenic hybrid technology after facing initial resistance from certain farmers and others has now created a great demand from farmers as well as textile industry as it has helped to improve the production to more than 4 million tonnes and productivity to over 460 kg/ha within 3 years of its introduction in India. In the world, several major cotton-growing countries have adopted this technology on a large scale as a result of which global cotton output has reached 26 million tonnes and average yields to 730 kg lint per hectare. The technology is powerful and to make it safer and sustainable as well as making new innovations for improving fibre quality and drought/disease resistance rests in

the hands of geneticists, genetic engineers and cotton breeders. The International Cotton Genome Initiative (ICGI) is gradually providing guidelines for the study of structural genomics, functional genomics, evolutionary relationships among Gossypium species and related members of the plant kingdom, genetic resources and germplasm stocks and bioinformatics in cotton for a better appreciation and understanding of the molecular basis of differentiation in the genera. Once such a project takes full shape at the global level and the various scientific laboratories come together for the mapping of the cotton genomes on a coordinated basis, the era of cotton genetics and breeding would undergo further metamorphosis. The potentiality for revolutionary improvements in the diploid cottons as well as barbadense and hirsutum may be expected to touch new horizons in the history of cotton improvement.

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EXPRESSION OF BRIX IN TOMATO INTERVARIETAL HYBRIDS


Panagiotis A. Michalakopoulos and S. R. Sree Rangasamy

ABSTRACT
Total soluble solids [TSS] measured as brix is an important fruit quality trait in tomato adding to the flavour, taste and nutrition. TSS consists of fructose and glucose sugars and acids. Brix shows a continuous phenotypic range in tomato varieties. Inheritance studies of brix in tomato made previously have indicated that it is quantitative in nature and in general intermediate between parents. Expression of brix in interspecific hybrids and inbreds developed from such crosses also has indicated that brix is governed by several quantitative trait loci. In this paper, the inheritance pattern of brix in fruits in intervarietal hybrids between genetically diverse tomato varieties and inbred lines showing brix values ranging from 3.0 to 5.4 is presented and discussed. Forty four genetically diverse tomato entries comprising 32 processing tomato varieties and 12 breeding lines were utilized as parents. A total of 182 intervarietal hybrids have been generated by crossing between diverse parents. The hybrids were evaluated for brix from fruits collected from replicated trials. The parents were grouped into low [3.00 to 3.50], medium [3.51 -4.50] and high brix [4.51 and above] phenotyes and hybrid between the three groups were also grouped into low, medium and high for brix expression. The pattern of variation in the parents and hybrids was continuous and similar. The coefficient of variation was very low (1.52). The differences between the parents and hybrids were highly significant. Among the hybrids, 39, 47 and 28 were showing over dominance, dominance and partial dominance respectively. In 36 hybrids brix was equal to either parent [Pt=P2=Ft] and in 22 of them brix was equal to the lower parent. Nine had brix lower than the lower parent. Significant and positive and negative heterosis was estimated. Significant positive heterosis was evident in 39 hybrids ranging from 7.5 to 30.0%. Different kind of gene actions such as dominance, dominance x dominance, dominance x additive and additive x additive and recessive epistasis governing the brix trait were evident connoting differences in alleles and different directions of non allelic interactions within the polygenic system for brix trait. Overdominance and dominance that were not reported in the hybrids between interspecific inbred lines x variety have been inferred in this study with intervarietal hybrids and this may be due to differences existing in the alleles between species. Preponderance of dominant gene action more in proportion in large number of hybrids suggests recurrent selection for further enhancement of brix levels.

Research and Development Divison,Agroaxon S.A.,Almyros 37100.Greece 237

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DEVELOPMENT OF MALE LINES RESISTANT TO FUSARIUM WILT IN CASTOR (RICINUS COMMUNIS L.,)
Lavanya, C.1 and Raoof, M.A

ABSTRACT
Castor, a non edible oilseed crop is an important income generating crop of several small / marginal farmers of rainfed regions of Andhra Pradesh, Karnataka, and Tamilnadu. Early or medium (150-180 days) duration varieties so as to complete their maturity within the maximum rainfall period with less node number to primary spike (1214), high branching, long effective spikes (25-40 cm) are preferred. Latest developments of breeding for high yielding male lines with in built resistance to Fusarium wilt at Directorate of Oilseeds Research is reviewed in the present paper.

Introduction India accounts for nearly 55% of the world castor area (11.63 lakh ha) and 51% of world castor production (11.44 l. t) and ranks first in both area and production (2002-03). It is cultivated in 7.32 lakh ha area with 8.01 l. t production and 1094 kg/ha productivity earning nearly Rs.614 crores through the export of castor oil (2003-04). In India, 52% of castor area is under rainfed conditions in states like Andhra Pradesh (40%), Karnataka, Tamilnadu, Orissa, Maharashtra, Madhya Pradesh (<10%) and 48% is under irrigated conditions. Production and productivity of castor is limited by wilt complex involving Fusarium wilt predisposed by Reniform nematodes and Macrophomina root rot in rainfed regions of Gujarat. Botrytis is another major biotic stress limiting the productivity in rainfed regions of castor cultivation especially during high humidity, continuous rainfall, and cloudy weather. The research efforts launched within and outside the All India Coordinated Research Project (AICRP) on Oilseeds resulted in the development of a number of high yielding varieties of regional and multi-regional importance with relatively early maturity habit (90-150 days). The most significant achievement was the release of wilt resistant varieties like 48-1 and DCS-9 for Southern India

(DOR, 2003). Successful cultivation of castor hybrids viz., GCH 4, DCH 32 and DCH 177 even in rainfed regions emphasized the need for development of high yielding, short duration and wilt resistant male lines or combiners. Breeding programmes initiated at the Directorate of Oilseeds Research, Hyderabad to develop high yielding male lines with in built resistance to Fusarium wilt and its importance are reviewed in the present paper. Materials and Methods A systematic and stream lined programme of activities is involved in collection of germplasm, purification, convergent breeding programme combined with stringent selection pressure for early and medium duration high yielding varietal/male lines with resistance to pests and diseases. A total of 800 single, 45 double, 30 triple, 10 multiple and 20 back crosses were effected since 1998. Special emphasis has been given to incorporate resistance to pests and diseases like Fusarium wilt, Macrophomina root rot, Reniform nematode, Botrytis, leaf miner and diversity of wilt resistance and parental material based on geographical diversity (Table 1). Using 700 crosses, 2500 individual plant selections, 750 bulk selections were made. Several selections were generated through hybridization involving

1. Directorate of Oilseeds Research, Rajendranagar, Hyderabad - 500 030.

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wilt resistant male lines viz., DCS 33, DCS 41, DCS 43, DCS 57, DCS 74, early duration, branching types like REC 2, REC 116 with wilt resistant, high yielding pistillate lines like DPC 9, DPC 11, M 574, M 584 followed by back cross and pedigree method of breeding. Such selections were bulked as advanced lines after attaining homozygosis for morphological characters like stem color, bloom and capsule nature and evaluated for their seed yield and yield components in an augmented randomized block design (ARBD). About 128 bulk selections were evaluated along with 3 checks replicated after every 20 rows in an Augmented RBD. Promising lines in the trial were further evaluated in preliminary station trials in three replications and multi location trials. Results and Discussion At the Directorate of Oilseeds Research, Hyderabad, several sources of wilt resistance have been identified through extensive screening of germplasm lines in the wilt sick plot. Varieties like 48-1, DCS 9, Co-1 and accessions like Baker 239, JM 6 have been identified as resistant sources to this pathogen (AICRP Castor, 1992, 1993 and 1994). Studies on inheritance of wilt resistance indicated that Fusarium resistance is controlled by recessive genes (Moshkin, 1967; Sviridov, 1967; Podkuichanko, 1989), interaction of duplicate genes (Sviridov, 1988) and polygenes (Desai et al., 2001). Wilt resistance was governed by a single dominant gene in Baker and by two complementary genes in 48-1 (Rao et al., 2003). Among different phenotypic traits, solid stem without hollowness in the central portion (pith) of the stem may be associated with wilt resistance and the character is being used in visual selection proceduresby breeders. However, other morphological characters viz., stem and petiole color, bloom, nature of stem and capsules are not linked with wilt resistance (Rao et al., 2003). Development of cultivars or
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lines with improved disease resistance is associated with the breeding methodologies and selection procedures depending on the mating systems of the plant, and heritability of the trait i.e. disease resistance. In the absence of any authentic information on inheritance of Fusarium resistance in castor, initially, the breeding methodology was confined to hybridization followed by selection of individual plants in the early segregating generation in the field condition and finally testing the lines in the wilt sick field conditions. About 200 crosses were generated using the available pistillate lines, high yielding male lines and hybrids with the above sources of resistance and geographically diverse germplasm accession. Pedigree method of selection is used to generate high yielding, wilt resistant lines. Screening of early and advanced generation material in wilt sick plot Ninety-six entries comprising early generations from F2 to F4 stage were screened under wilt sick plot conditions for resistance to Fusarium wilt (Annual Castor Report, 19992000). Nineteen entries were found resistant with less than 20% wilt infection. Among them the entries with zero wilt incidence viz., 9-2, 119-2, 122-3, 122-5, 125-1, 294-2, 299-2, 3921, 398-1, 544-3 and 544-7 were further advanced by pedigree method ofselection for selection of resistant lines (Table 2). Among 128 bulk selections evaluated in augmented black design, about 35 lines were found promising for seed yield and yield components (Table 3). These lines were screened for wilt incidence in a wilt sick plot established at DOR. Six lines were found wilt free while three lines recorded <15% wilt incidence (Annual Report, Castor, 2001-02). Seventeen promising entries were evaluated in the wilt sick field along with 5 checks for

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confirmation of wilt resistance. Three advanced lines Kh-2k- 505, 660, 1264 were wilt free while Kh-2k-1262, 1267, 507 recorded <10% wilt incidence (Annual Report, Castor, 2001-02). During the last decade about 50 advanced lines recorded high seed yield ranging from 8-62 % over the best check with low node number varying from 8.9 to 17.1 (Table 4). Among them, advanced lines 2K 1262 (37%), and 2K 1253 were high yielding than DCS 9 (833 kg/ha) and resistant to Fusarium wilt with 8 and 17% wilt incidence in wilt sick plot in preliminary station trials. Evaluation of varieties/ male lines in AICRP multi location trials Among six inbreds viz., DCS 99, DCS 100, DCS 101, DCS 102, DCS 103, DCS 104 evaluated in AICRP multi location trials (200405), DCS 102 evaluated in 11 rainfed centres and 4 irrigated centres recorded 5% increase over the best check 48-1 (1604 kg/ha) and resistant to Fusarium in wilt sick plots of Directorate of Oilseeds Research, SK Nagar and Palem (Table 5). Several wilt resistant sources are available both in the germplasm and male lines or varieties due to the net working of common resources. Researchable gaps like screening procedures for combined infection of root rot, nematode, Fusarium and their interactions, studies on variability of the pathogen and resistant sources to isolates need to be thoroughly examined. Emphasis need to be given for thrust areas like studies on variability of wilt pathogen considering the susceptibility of released wilt resistant varieties and hybrids like GCH 4, GCH 5 etc., Molecular tagging of genes for resistance to Fusarium wilt resistance may increase the efficiency of screening of large number of diverse germplasm in addition to already available field screening techniques and artificial root dip techniques. REFERENCES
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Annual Progress Report Castor. 1992-93. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. PP 17p. Annual Progress Report Castor. 1993-94. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. PP 33p. Annual Progress Report Castor. 1994-95. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 165p. Annual Progress Report Castor. 1999-2000. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 198p. Annual Progress Report Castor. 2001-02. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 181p. Annual Progress Report Castor. 2001-02. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 16p. Annual Progress Report Castor. 2002-03. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 132p. Annual Progress Report Castor. 2003-04. Directorate of Oilseeds Research. Rajendranagar, Hyderabad. 133 p. Chattopadhyay, C., Reddy,MCM. 1995. Wilt Complex of Castor (Ricinus communis L.): Role of reniform (Rotylenchulus reniformis Linford and Oliveira) nematode. J. Oilseeds Res.12: 203 - 207. Desai, A.G., Dange,S.R.S. and Pathak, H.C. 2001. Genetics of resistance to wilt in castor caused by Fusarium oxysporum f.sp. ricini Nanda and Prasad. J. Mycol. Pl. Pathol. 31: 322-326. DOR (Directorate of Oilseeds Research). 2003. Castor in India. Research achievements. 17 p. Rao, Hanumantha, C., Raoof, M.A., Lavanya, C. 2003. Study on segregation patterns and linkages between morphological characters

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and wilt resistance in castor ( R i c i n u s communis L). J. of Oilseeds Res. 22: 114118. Moshkin, V.A. 1967. Castor. Oxonian Press Pvt. Ltd. New Delhi. 132-145 . Nanda, S., Prasad, N. 1974. Wilt of Castor a new record. Indian J. Mycol. and Plant Path. 4: 103-105. Podkhichenko, G.V. 1989. Donors of resistance to Fusarium in castor oil plant. Instituta Maslichnykh Kullar.1:24-26. Sviridov, A.A. 1967. Breeding for resistance to Fusarium. In: Moshkin, VA editor. Castor.

Oxonion Press Pvt. Ltd. 157-163. Sviridov, A.A. 1988. Results of improving the parent forms of the castor hybrid Kranodarskit 3 for resistance to Fusarium. Institute Maslichnykh Kultur. 2: 16-19. Varaprasad, K.S. 1986. Rotylenchulus Reniformis Linford and Oliveria, 1940 A comparative account of systemics, biology and management. In : Swarup G, Dasgupta DR, editors. Plant parasitic nematodes of India-Problems and Progress. New Delhi (India): Indian Agricultural Research Institute.194-210.

Table 1. Sources of resistance used in crossing programme Resistance to


Leaf miner Fusarium wilt Germplasm RG 1472, RG 1647, RG 1648, RG 1938, RG 1941, RG 1944, RG 2127, RG 2178, RG 2445, RG 2529, RG 2602, RG 2612, RG 2661 DCS 33, DCS 41, DCS 43, DCS 57, DCS 59, DCS 69 DPC 9, DPC 11, M 574, M 571, M 619, M 584 RG 297 RG 1713, 1719, 1726, 1741, 2040, 2377, 2559, RG 1582, 1514, 1586, 1526, 1523, 30, 2300, 2426 RG 1930

Source

Advanced lines Pistillate lines Fusarium wilt, root rot and nematode Botrytis Diversity of parental lines (based on geographic diversity)

Table 2. Promising advanced generation material resistant to wilt Entry


623-1 617-1 607-2 586-1 597-1 556-2 306-1 376-1 407-1 14 entries
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Pedigree
DPC 11 x DCS 43 DPC 11 x DCS 43 DPC 11 x DCS 43 DPC 11 x DCS 43 DPC 11 x DCS 43 DPC 11 x DCS 33 DPC 11 x DCS 9 DPC 9 x DCS 23 DPC 9 x DCS 59

Wilt incidence in wilt sick plot (%)


0 0 0 0 0 0 15 11 8 < 20 %

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Table 3. Seed yield and components of promising advanced lines evaluated in ARBD S. No. Selection number Number Effective Effective of nodes spike length spikes to primary (cm) per plant spike
13.6 14.2 15.2 14.2 16.2 19.4 14.8 15.6 17.4 11.8 12.5 12.7 12.5 15.3 13.1 13 14.3 11.1 13.5 13.1 15.1 11.9 14.7 12.7 13.7 12.1 11.7 1.97 3.94 4.55 3.6 29.4 29.0 36.6 43.0 31.2 36.6 37.6 39.2 42.8 31.4 25.7 33.5 22.5 39.9 22.7 28.3 30.5 20.7 36.7 24.3 31.7 30.3 27.5 35.5 24.9 33.7 22.9 8.13 16.27 18.8 14.9 5.9 6.7 7.5 7.4 4.5 3.1 4.9 5.9 3.9 3.7 6.4 6.4 7 6.8 12.8 11 7.7 6.7 7.1 5.9 7.3 1.8 2.8 5.4 4.8 11 4 3.1 6.21 7.17 5.7

Oil (%)

Seed yield (g/plot)

I set 1 2 3 4 5 6 7 8 9 10 II set 1 2 3 4 5 6 7 III Set 1 2 3 4 5 6 7 8 9 10 CD between Two checks 2 varieties in same block 2 var. in different blocks Check and variety 1.56 3.12 3.61 2.9 436 871 1006 795 DCS 9 GCH 4 DCH 32 743 793 801 804 DCS 9 GCH 4 DCH 32 44 46.3 44.8 43.5 44.3 45.8 43.1 42.8 48.0 47.2 412 1189 617 856 515 759 1321 472 730 655 DCS 9 GCH 4 DCH 32 681 713 715 741 43.4 48.6 46.2 49 39.8 41.6 47.1 747 797 674 638 1028* 1123* 920 DSC 9 582 592 600 617 623 639 644 670 672 43.8 43.1 40.7 45.3 44.0 38.0 47.2 47.7 45.2 46.1 581 806 1086* 837 859 909 1060 1088* 1087 987

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Table 4. Promising advanced lines evaluated in Preliminary Varietal Trials (1998-2004) Year Advanced line Seed yield (kg/ha) 1560 1320 1239 2194 2060 1140 1159 2127 2537 2030 1659 1542 1545 1973 1638 1597 2952 2148 2067 No. of nodes to primary spike 8.9 10.9 15.9 11.9 10.2 12.1 9.0 11.2 12.1 13.1 16.1 17.1 11.6 14.0 13.1 12.0 18.1 14.0 13.3 Increase over best check (%) 68 42 34 16 9 37 39 21 45 16 39 29 29 49 24 20 60 16 12

1998

2000 2001 2002

2003

2004 (Set I)

Set II

Kh-96-461-2 Kh-96-458 Kh-96-545 Kh-98-338-1 Kh-98-393-1 2K 1262 2K 1253 PVT-25 PVT-26 PVT-27 PVT 25 PVT 26 PVT 29 PVT 17 PVT 8 PVT 12 PVT 37 PVT 33 PVT 43

Table 5. Promising varieties for seed yield and wilt resistance in AICRP trials (2004-05)

S. No.

Entry

Pedigree

Seed yield (kg/ha)


1685 1572 1316 1471 1604

% of best check
105.0 98.0 82.0 91.7 100

Wilt incidence (%) Palem DOR SK Nagar


27.5 33.3 41.2 31.4 25.5 7.2 3.4 6.7 10.8 7.7 24.2 18.9 0.0 0.0 3.3

1 2 3 4 5

DCS 100 DCS 102 DCS 103 DCS 9 (C) 48-1 (c)

DPC 11 x DCS 43 DPC 11 x DCS 43 M 571 x REC 2

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DEVELOPMENT OF SUPERIOR INBREDS AND SELECTION OF EFFICIENT RESTORERS FOR DIVERSE CMS SOURCES IN SUNFLOWER
Ranganatha, A.R.G.1, V. Vijay, C. Lavanya and K. Rukminidevi

ABSTRACT
Development of diverse and superior inbreds for seed yield and yield attributes, oil content and autogamy is required to develop new hybrids with higher heterotic potentials. In this direction, the inbreds are synthesised following recurrent selection from diverse gene pools. The superior inbreds identified morphologically were tested for the maintainer and restorer reaction. The identified maintainer inbreds are converted to develop diverse CMS lines under the background of different CMS sources. The identified superior hybrids are tested in the station and multilocation trials to save the time of conversion, utilizing the Gibberlic acid technique, to produce the male sterile inbred line. The results of the experiments conducted during kharif, rabi and summer seasons of 2004 and 2005 are discussed in this paper.

Introduction Sunflower is becoming one of the important oilseed crops in various agro-production situations. However, the productivity levels of sunflower are continued to be low (Virupakshappa and Ranganatha, 1998 and Virupakshappa and Ranganatha 1999). Hence, to increase the productivity levels and to diversify the inbred base to develop superior hybrids the following investigations were taken up in sunflower. Material and methods The morphologically superior inbreds were tested for the maintainer and restorer reaction. The maintainer inbreds were back crossed to develop new CMS lines under the back ground of different CMS sources. Further, the paralelly identified promising hybrids were tested in the station and multi-location trials to save the time of back crossing, utilizing Gibberlic acid technique to produce the male sterile inbred line. The newly developed inbreds were regularly tested further for maintainer and restorer reaction. The new inbreds and crosses synthesized were evaluated in the station and in

the multi location trials during kharif, rabi and summer seasons of 2004 and 2005. Results and discussion The inbreds were synthesized following recurrent selection from different gene pools (Ranganatha et al., 2000 and Ranganatha et al., 2003). The superior inbreds were tested for their maintainer and restorer reaction. The inbreds and crosses were evaluated for seed yield and yield attributes. Evaluation of new inbred lines Newly developed inbreds were evaluated during kharif and rabi seasons and a number of promising inbreds were identified for seed yield, oil content and other attributes. The identified inbreds possessed 4-5% higher oil content. Two inbred selections (GP 9-472-7-2 and GP 9-38-C-2-1) recorded higher oil per cent (>45%) and also exhibited higher necrosis resistance. The inbreds developed through the inbreeding generations and the superior stabilized types were evaluated under different sets and the performance of superior inbreds are given in tables 1 to 3. GP9-322-1 exhibited highest oil content (45%) followed by GP9-839-

1. Directorate of Oilseeds Research, Rajendranagar, Hyderabad 500 030

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5-1 (44.8%). In set-2, GP317-3 exhibited maximum oil content (45.5%) followed by GP2150-5 (44.8%). For seed yield, GP325-3 recorded highest (38.7g) followed by GP2166-5 (31.7g). In set-3, GP1-1044 (45.7%) recorded maximum oil content followed by GP1-10 (45.3%). For seed yield, GP1-69 (35.5g) recorded highest followed by GP1-2210 (32.9g). Development of new restorer lines The GP line inbreds were evaluated for the maintainer and restorer reaction during summer and kharif seasons and identified 22 new restorers for diverse CMS lines (Table-4). The studies resulted in identifying the following effective restorers in another set for diverse CMS sources viz., inbred GP201 restored fertility in ARM 243A, ARM 247A and PET-2-7-1A; line RARM-241 restored in 234A and ARM 247A; DRM-34 restored in ARM 247A, PF853A, PET-2-7-1A and PET2-89A; GP9-163 restored in ARM 247A, PET2-7-1A, IMS-400A, IMS-WG and IMS-IB-4; GP9-472-5 restored in ARM-247A, IMS-400A, PET2-7-1A and PFMRA and PF-853A. Further, P356 restored fertility in 9 lines; 3376R restored in 8 lines; R 856 restored in 8 lines and R-272-1 restored fertility in 3 lines. Evaluation of inbreds for combining ability A total of 150 experimental hybrids are synthesized and evaluated. Identified the new crosses DSC-20, 6, 35, 23, 27, 4 and 245 as superior hybrids compared to the popular checks KBSH-1 and MSFH-17 hybrids (Table-5). The hybrid DRSH-102 exhibited superiority over

popular checks KBSH-1, PAC-1091, MSFH17 and KBSH-44 for seed yield; KBSH-44, MSFH-17 and PAC-1091 for oil content and KBSH-44, MSFH-17 and PAC-1091 for oil yield in the multilocation evaluation of 14 centres (Table.6). Another hybrid DRSH-103 also exhibited seed yield and oil yield superiority over KBSH-1 (Progress Report, 2005) with zero per cent downy mildew in the multilocation evaluation. REFERENCES Ranganatha, A.R.G., Pradeep Kumar, P and Chattopadhyay, C, 2000. Evaluation of new CMS and inbred lines in sunflower, J. Oilseeds Research. 17: 385-386. Ranganatha, A.R.G., Tirumala Rao, V. and Rukmini Devi, K. 2003. Development of diverse maintainer and restorer inbreds and populations in sunflower. In: Advances in Genetics and Plant Breeding Impact of DNA revolution, UAS, Dharwad, p. 67. Virupakshappa, K. and Ranganatha, A.R.G. 1998. Approaches for enhanced exploitation of heterosis in sunflower. Lead paper presented in Heterosis national seminar, PKV, Nagpur Progress Report 2005. NATP-Sunflower Hybrid Project, DOR, Hyderabad Virupakshappa, K. and Ranganatha, A.R.G. 1999. Heterosis and hybrid seed production in sunflower. In: Heterosis and hybrid seed production in agronomic crops. ed. A.S. Basra, Food Products Press, New York, pp. 185-216.

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Table 1. Performance of top inbreds, Set-1 Entry Days to maturity 90 90 95 97 97 94 88 89 96 96 89 96 94 Plant Head height diameter (cm) (cm) 100 110 80 130 90 90 127 94 116 126 77 112 121 10.2 9.3 8.0 12.2 8.6 10.2 10.6 11.4 10.3 12.2 12.4 11.2 12.4 Seed yield (g/Plant) 26.6 20.3 10.8 33.1 16.9 18.1 17.4 24.0 19.3 21.1 25.4 30.0 31.0 Oil content (%) 43.3 44.6 43.8 41.9 44.1 44.8 45.0 44.1 43.0 43.4 39.0 40.0 39.5 Oil yield (g/Plant) 11.5 9.0 4.7 13.8 7.4 8.1 7.8 10.5 8.3 9.1 9.9 12.0 12.2

GP9-33-E-4-2 GP9-116-5-1 GP9-472-1-5 GP9-472-4-1 GP9-472-5-3 GP9-839-5-1 GP9-322-1 GP9-414-5-3 GP9-472-5-4 GP9-472-7-5 Morden (c) TNAUSUF-7 (c) GAUSUF (c)

Table 2. Performance of top inbreds, set-2 Entry Days to maturity 97 90 88 95 87 104 93 97 90 96 96 Plant Head height diameter (cm) (cm) 78 7.0 124 13.6 100 9.6 100 9.8 100 11.1 100 6.9 88 9.4 126 14.3 76.0 12.2 113.0 11.1 116.0 12.0
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GP-317-3 GP-325-3 GP-557-3 GP-1159-1 GP-2150-5 GP-224-1 GP-886-1 GP-2166-5 Morden (c) TNAUSUF-7 (c) GAUSUF-15 (c)

Seed yield (g/Plant) 13.2 38.7 15.0 28.1 14.5 32.1 27.3 31.7 23.8 26.7 31.4

Oil content (%) 45.5 44.6 44.7 42.8 44.8 43.8 43.3 42.2 37.4 38.9 38.3

Oil yield (g/Plant) 6.0 17.2 6.7 12.0 6.4 14.0 11.8 13.3 8.9 10.3 12.0

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Table 3. Performance of top inbreds, Set-3 Entry Days to maturity 91 95 100 97 95 93 97 90 91 95 100 Plant Head height diameter (cm) (cm) 108 13.3 100 12.4 120 12.0 111 8.3 85 7.3 119 7.8 87 9.0 100 6.0 77 13.2 108 9.7 125 13.2 Seed yield (g/Plant) 29.5 35.5 22.0 10.6 13.3 30.1 32.9 30.2 30.4 26.9 36.9 Oil content (%) 45.3 43.0 44.9 45.7 45.1 44.8 42.9 43.6 35.6 37.0 37.5 Oil yield (g/Plant) 13.3 15.2 9.8 4.8 5.9 13.4 14.1 13.1 10.8 10.0 13.8

GP1-10 GP1-69 GP1-737 GP1-1044 GP1-1986 GP1-2086 GP1-2210 GP1-2283 Morden (c) TNAUSUF-7(c) GAUSUF-15 (c)

Table 4. New Restorers for different CMS lines S.No.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Genotype (Restorer)
GP9-856-3 GP9-755-1 GP9-53-2 GP9-30-1 GP9-58-4 GP9-1932 GP9-220-1 GP9-846-4 GP9-733-5 GP9-556-7 GP9-811-4 GP9-811-5 GP9-201-1 GP9-58-5 GP9-1446 GP9-163-8 ARM-239 ARM-247 ARM-243 VND-5 (NB) LIB-02-M3 DRM-71-2
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CMS line
ARM-245A ARM-245A PF-274A PET2-7-1A PF-274A ARM-245A PET2-7-1A, ARM-245A PET-2-7-1A PET-2-7-1A PET-2-7-1A PET-2-7-1A ARM-245A PF-274A, PET2-89-A, PET2-7-1A PF-274A PET2-7-1A IMS-WGA, PF-400A PET2-89A, PET2-7-1A IR-265A I-IB4, I-852A, I-WGA, PF-400A PF-274A, ARM-245A ARM-245A PET2-89A, PET2-7-1A

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Table 5. Performance of superior new crosses in Sunflower Entry Days to 50% flowering 58 54 60 60 61 67 59 65 64 64 53 58 57 55 Days to maturity 93 90 96 94 96 99 92 97 96 95 90 94 94 91 Plant height (cm) 151.5 117.6 143.8 150.0 137.6 129.6 133.0 162.8 149.1 143.3 108.3 150.3 130.5 130.7 Head dia-meter (cm) 13.9 12.1 12.7 12.4 11.5 12.4 14.0 12.9 13.6 12.5 11.1 10.8 11.7 11.9 Seed yield (kg/ha) 1614 1398 1403 1165 1035 1008 1137 1265 1337 1465 1117 1331 1165 1150 Oil con-tent (%) 38.0 36.3 37.9 35.2 36.4 35.5 31.7 35.7 33.5 33.3 33.1 32.9 25.0 35.5

DSC-20 DSC-35 DSC-6 DSC-18 DSC-243 DCS-53 DSC-29 DSC-27 DSC-4 DSC-23 DSC-56 DSC-245 MSFH-17(c) KBSH-1 (c)

Table 6. Performance of DRSH-102 (Mean of 14 locations) Entry PAC-309 DRSH-102 KBSH-44(c) MSFH-17 (c) KBSH-1 (c) PAC-1091 (c) Seed yield(kg/ha) 1716 1517 1505 1468 1462 1446 Oil content(%) 33.1 35.1 26.9 29.4 37.6 33.3 Oil yield(kg/ha) 568 532 406 431 549 481

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RESTORER IDENTIFICATION FOR CMS LINE (IR 66707A) WITH ORYZA PERENNIS CYTOPLASM
Banumathy, S., K. Thiyagarajan and K. Siddeswaran

ABSTRACT
In order to identify restorer for O. perennis cytoplasm, the CMS line (IR 66707A) with O. perennis cytoplasm was crossed with 50 wide cross derivatives. The F1 seeds were obtained only for 26 cross combinations. Of the 26 F1s, germination was observed only in 20 crosses which were analysed for fertility restoration ability. Observations were recorded on pollen fertility, days to 50 per cent flowering, plant height, number of productive tillers per plant, panicle length, panicle exsertion and spikelet fertility. Pollen and spikelet fertility studies revealed partial restoration in eight crosses involving IR 66707A with WC 3, WC 4, WC 7, WC 9, WC 20, WC 35, WC 36 and WC 53. The range for pollen fertility was between 6.0 and 48.0 per cent and that of spikelet fertility varied from 10.3 to 67.33 per cent. Maximum pollen (48%) and spikelet fertility (67.33%) were observed in IR 66707A /WC 20 followed by IR 66707A /WC 53 with 30 and 40 respectively. Five crosses which had more than 20 per cent spikelet fertility were advanced to F2 generation . Pollen fertility studies of F2 progenies of IR 66707A/WC 4, IR 66707A/WC 20 and IR 66707A/WC 53 showed segregation for fertility and sterility. More than 50 percent of plants in IR 66707A/WC 20 expressed 70-80 per cent spikelet fertility. Seeds of IR 66707A were subjected to various doses (10, 20, 30, 40 and 50 kr) of gamma irradiation. Seeds were sown after 20 hours of soaking along with the untreated seeds of IR 66707A. Fertile revertants were observed in the population irradiated with 50kr. The revertants were morphologically similar to the maintainer line IR 66707B. Crossess were effected between IR 66707A and the newly identified fertile revertants. Pollen fertility studies of F1 progenies revealed that the revertants had no fertility restoring ability although they became fertile.

Introduction Most of the commercial rice hybrids released in India depend on few IRRI bred CMS lines. To achieve the desired productivity and sustainability of rice hybrids such an overdependence on few CMS lines is undesirable. Hybrid rice research must therefore broaden the nuclear diversity of parental lines through the conversion of agronomically superior genotypes into CMS lines. Exploitation of available sources of CMS is necessary to overcome the problem arising due to the overdependence of single CMS source (WA). Among various sources, CMS lines possessing Oryza perennis cytoplasm express stable sterility and good agronomic characters. A stable
TNAU, Coimbatore E-mail: mathysakthi@yahoo.com

CMS line IR 66707A possessing the cytoplasm of O. perennis and nuclear genome of IR 64 was developed by Dalmacio et al. (1995). Utilisation of this CMS line in hybrid rice development has been hampered due to nonavailability of restorers. Fertile revertants are the fertile plants isolated from any CMS line either naturally or by induced means. The revertant has the ability to restore the fertility of a CMS line from which it is originated. Burton (1977) discovered fertile revertants from a CMS line possessing A cytoplasm in pearl millet. These revertants had no fertility restoring ability although they became fertile. However, the fertile revertant isolated by Shen et al. (1996) from male sterile

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indica line II-32A was able to restore the fertility of CMS line II-32A. Research efforts in many countries attempted towards the identification of restorer for O. perennis cytoplasm. Hence, in the present study two approaches involving the use of wide cross derivatives and fertile revertants were adopted for the identification of restorers for O. perennis cytoplasm. Materials and Methods Crossing with Wide Cross Derivatives The CMS line IR 66707A with O. perennis cytoplasm was crossed with 50 wide cross derivatives (Table 1). The F1 seeds obtained from 20 cross combinations were raised during summer 2000 by adopting a spacing of 20 x 20 cm. Single seedling per hill was planted. Five plants were randomly selected in each hybrid at the time of flowering for recording biometrical observations viz., days to 50 per cent flowering, plant height, panicle length, percentage of panicle exsertion and percentage of pollen and spikelet fertility. Development and Use of Fertile Revertants Fertile revertants are the fertile plants identified in the M1 generation of any CMS line. These revertants are used to restore the fertility of the parental CMS line from which it was originated. In the present study, dry seeds of IR 66707A were subjected to gamma irradiation at 10, 20, 30, 40 and 50 kr at the gamma chamber 60 Co at Sugarcane Breeding Institute, Coimbatore. For each treatment 250 seeds were used. The untreated seeds were kept separately. The treated and control seeds were immediately soaked in distilled water for 24 hours. Excess water was drained and then sown in sowing trays. All the seedlings were transplanted by adopting a spacing of 20 x 20 cm during kharif 2000. Based on pollen fertility studies, fertile plants were identified in M1 generation and crossed with IR 66707A to analyse the restoration ability of fertile revertants.
250

Results and Discussion Diversification of cytoplasmic base is also very essential as that of widening the genetic base. Since, more than 90 per cent of commercial hybrid production involves only WA cytoplasmic source for male sterility (Yuan, 1993) diversification of the types of available cytoplasmic male sterile sources is essential to reduce the genetic uniformity and resulting vulnerability of hybrids to pests and diseases. Various CMS lines possessing different types of sterile cytoplasm can be used to overcome the problem arising due to overdependence on WA cytoplasm. Of this, the CMS line IR 66707A possessing the cytoplasm of O. perennis and nuclear genome of IR 64 (Dalmacio et al., 1995) was identified as the best due to its stable sterility along with desirable agronomic characters. Earlier reports (Ganesan et al., 1998) indicated that most of the cultivable varieties were unable to restore the fertility of IR 66707A. Keeping in view, the present study was directed towards the use of wide cross derivatives and development and use of fertile revertants for identification of restorers for IR 66707A. Use of Wide Cross Derivatives In order to identify restorers for O.perennis cytoplasm, the CMS line (IR 66707A) with O. perennis cytoplasm was crossed with 50 wide cross derivatives (Jayamani,2000). The F1 seeds were obtained only for 26 cross combinations. Of the 26 F1s, germination was observed only in 20 crosses, which were analysed for fertility restoration ability. Observations were recorded on pollen fertility, days to 50 per cent flowering, plant height, number of productive tillers per plant, panicle length, panicle exsertion and spikelet fertility. Pollen and spikelet fertility studies revealed partial restoration in eight crosses involving IR 66707A with WC 3, WC 4, WC 7, WC 9, WC 20, WC 35, WC 36 and WC 53 (Table 2). The

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range for pollen fertility was between 6.00 (IR 66706A/WC 3) and 48.00 per cent (IR 66707A/ WC 20) and that of spikelet fertility varied from 10.30 to 67.33 per cent. Maximum pollen (48 %) and spikelet fertility (67.33 %) were observed in IR 66707A/WC 20 followed by IR 66707A/WC 53 with 30 and 40 per cent respectively. The pedigree of WC 20 (IR 64/O. longistaminata 101221) and WC 53 (CO 43/ O. longistaminata 101221) revealed that the wild species O. longistaminata might have possessed the restoring gene which in turn resulted in partial restoration of IR 66707A. Five crosses viz., IR 66707A/WC 4, IR 66707A/WC 7, IR 66707A/WC 20, IR 66707A/ WC 35 and IR 66707A/WC 53 had more than 20 per cent spikelet fertility. The selfed seeds of these crosses were advanced to F2 generation in order to identify and select plants with high percentage (>80%) of spikelet fertility. The F2 progenies of IR 66707A/WC 4, IR 66707A/WC 7 and IR 66707A/WC 35 did not exhibit plants with high spikelet fertility. However, F2 progenies of crosses IR 66707A/WC 20 and IR 66707A/ WC 53 showed segregation for fertility and sterility. Pollen and spikelet fertility studies of F2 progenies of the cross involving WC 20 as male parent showed that 70 per cent of plants recorded 70-80 per cent fertility indicating the improvement of fertility in advanced generation. After 2-3 selfings the fertile lines obtained may be used as restorer parents for the restoration of fertility of IR 66707A. The F2 progenies of IR 66707A/WC 53 segregated for fertility and sterility and the fertile plants recorded 40-50 per cent spikelet fertility. This implies that there was a slow progress in fertility improvement as compared to the other F2 progenies of IR 66707A/ WC 20. As compared to these F2 populations, the advanced generation progenies of the cross involving IR 66707A and WC 20 may have better chance to restore fertility of O. perennis.

Development and Use of Fertile Revertants Fertile revertants are the fertile progenies obtained from any CMS line either spontaneously or by induced means. Fertile revertants were isolated successfully from CMS lines in a number of crops (Burton, 1977; Umbeck and Gangenbech, 1983; Nawa et al., 1987; He et al., 1989). Radiation and chemical mutation are the suitable ways to produce fertile revertants especially in the case where restoration for certain types of CMS lines is difficult to obtain, as suggested by Shen et al. (1996). These revertants were classified into B and R types, in terms of their fertility restoration ability. The B type was similar to maintainer and R was similar to restorer. Seeds of IR 66707A were subjected to various doses (10, 20, 30, 40 and 50 kr) of gamma irradiation. Seeds were sown after 24 hours of soaking along with the untreated seeds of IR 66707A. Fertile revertants were observed in M1 generation of the population irradiated with 50 kr. The revertants were morphologically similar to the maintainer line IR 66707B and recorded more than 70 per cent pollen and spikelet fertility. These revertants were crossed with IR 66707A to identify the restoration ability. The study of pollen and spikelet fertility of F1 progenies showed that the revertants had no fertility restoration ability, although they became fertile. Similar results were also observed by Nawa et al. (1987). They obtained fertile revertants from BT CMS line when treated with ethyl methane sulphonate. These revertants had no fertility restoration ability. Studies of Rottmann et al. (1987), Nawa et al. (1987) and Smith (1987) showed that this type of reversion might have resulted from recombination and or deletion of mitochondrial DNA. Shen et al. (1996) isolated a fertile

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revertant from the male sterile indica line II-32A when treated with 60Co gamma rays at a dose of 290 GY. The acquired revertant, T 24 was morphologically and agronomically similar to II32B, the maintainer of II-32A. The revertant was able to restore the fertility of a number of CMS lines besides II-32A and was identified as a near isogenic line with II-32A for restorer gene. REFERENCES Burton, G.W. 1977. Fertility sterility maintainer mutants in cytoplasmic male sterile pearl millet. Crop Sci. 17: 635-637. Dalmacio, R.D., Brar, D.S., Ishii, T., Sitch, L.A., Virmani, S.S., Khush, S. 1995. Identification and transfer of a new cytoplasmic male sterility source from Oryza perennis into indica rice (O. sativa). Euphytica. 82: 221225. Ganesan, K.N., Thiyagarajan, K., Amarlal, M.K., Rangaswamy, M. 1998. Restorers and maintainers for CMS lines of rice. Oryza. 35: 163-164. He P, Li Z, Li T. 1989. Fertility restoring mutants in T type wheat cytoplasmic male sterile line irradiated with 60Co gamma rays. Acta Genet. Sinica. 16: 1-6. Jayamani ,P. 2000. Wide hybridisation to transfer cytoplasm and floral traits in rice (Oryza sativa L.). Ph. D. Thesis submitted to Tamil Nadu Agricultural University, Coimbatore (Unpublished).

Nawa, S., Sano, Y., Yamada, M., Fuji, T. 1987. Cloning of the plasmids in CMS rice and changes of organisation of mitochondrial and nuclear DNA in cytoplasmic reversion. Jpn. J. Genet, 62: 301-314.Rottmann WH, Brears T, Hodge, T.P. 1987. The mitochondrial gene is lost via homologous recombination during reversion of CMS-T maize to fertility. EMBO J. 6: 1541-1546. Shen, Y., Cai, Q., Gao, M., Wang, X. 1996. Isolation and genetic characterisation of fertility restoring revertant induced from cytoplasmic male sterile rice. Euphytica 90: 17-23. Smith, R.K. 1987. Mitochontrial DNA rearrangements in Pennisetum associated with reversion from CMS to fertility. Plant Mol. Biol. 9: 277-286. Umbeck, P.F., Gangenbach, B.G. 1983. Reversion of male sterile T-cytoplasm maize to male fertility in tissue culture. Crop Sci. 23: 583-588. Yuan, L.P. 1993. Advances and constraints in the use of hybrid rice varieties. In: Wilson KJ, editor.International workshop on Apomixis in rice, 13-15 Jan 1992, Hunan Hybrid Rice Research Centre, Changsha, Peoples republic of China. p 1-4.

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Table 1. Wide cross derivatives used in the study


Sl. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Wide cross derivatives WC O3 WCO4 WC O5 WC O6 WC O7 WC O8 WC O9 WC 10 WC 11 WC 12 WC 13 WC 14 WC 15 WC 16 WC 17 WC 18 WC 19 WC 20 WC 21 WC 22 WC 23 WC 24 WC 25 WC 26 WC 27 WC 28 WC29 WC30 WC 31 WC 32 WC 33 WC 34 WC 35 WC 36 WC 37 WC38 WC 39 WC 41 WC 42 WC 43 WC 44 WC 45 WC 46 WC 47 WC 48 WC 49 WC 50 WC 51 WC 52 WC 53 Parentage ASD16 x O.nivara 105343 ASD16 x O.nivara 105343 ASD16 x O.nivara 105343 ASD16 x O.nivara 105343 ASD16 x O.nivara 105343 ASD16 x O.nivara 105343 CO43 x O.nivara 101871 CO43 x O.nivara 101871 CO43 x O.nivara 101871 CO43 x O.nivara 101871 CO43 x O.nivara 101871 CO43 x O.nivara 101871 IR64 x O.longistaminata 101221 IR64 x O.longistaminata 101221 IR64 x O.longistaminata 101221 IR64 x O.longistaminata 101221 IR64 x O.longistaminata 101221 IR64 x O.longistaminata 101221 IR58025 B x O.longistaminata 101221 IR58025 B x O.longistaminata 101221 IR58025 B x O.longistaminata 101221 IR58025 B x O.longistaminata 101221 IR58025 B x O.longistaminata 101221 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 IR50 x O.rufipogon 103305 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 ASD 18 x O.rufipogon 100916 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 CO43 x O. longistaminata 101221 253

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Table 2. Data on quantitative characters of crosses involving IR 66707A and wide cross derivatives

Cross Pollen Days to combinations fertility 50 % (%) flowering


IR 66707A/WC 3 IR 66707A/WC 4 IR 66707A/WC 5 IR 66707A/WC 7 IR 66707A/WC 9 IR 6707A/WC 10 IR 6707A/WC 13 IR 6707A/WC 14 IR 6707A/WC 20 IR 6707A/WC 25 IR 6707A/WC 26 IR 6707A/WC 29 IR 6707A/WC 33 IR 6707A/WC 35 IR 6707A/WC 36 IR 6707A/WC 37 IR 6707A/WC 43 IR 6707A/WC 46 IR 6707A/WC 47 IR 6707A/WC 53 6.0 20.0 0.0 20.0 10.0 0.0 0.0 0.0 48.0 0.0 0.0 0.0 0.0 20.0 10.0 0.0 0.0 0.0 0.0 30.0 95.0 90.0 95.0 98.0 92.0 90.0 95.0 94.0 95.0 98.0 90.0 100.0 102.0 96.0 93.0 98.0 97.0 95.0 95.0 92.0

Plant height (cm)


63.0 62.0 61.0 70.5 69.0 113.0 72.5 65.0 71.7 73.0 71.0 63.0 64.0 69.0 72.0 73.7 64.3 63.2 64.0 54.0

No. of Panicle Panicle Spikelet productive length exsertion fertility tillers per (cm) (%) (%) plant
21.0 23.0 20.3 18.5 20.5 22.5 22.5 18.3 13.7 25.0 22.7 21.7 23.5 19.0 24.3 23.0 24.7 20.2 25.5 19.0 24.3 23.0 22.0 21.5 22.5 27.5 24.5 23.3 21.5 25.0 23.0 23.3 23.0 23.0 24.3 24.0 23.7 23.4 25.5 22.5 87.7 88.4 87.3 83.7 86.7 89.1 85.7 87.0 89.8 90.0 89.9 88.4 91.3 91.3 89.1 88.6 88.7 90.6 94.1 93.3 10.3 30.0 0.0 28.4 12.9 0.0 0.0 0.0 67.3 0.0 0.0 0.0 0.0 22.1 18.3 0.0 0.0 0.0 0.0 40.0

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EVALUATION OF ISONUCLEAR ALL OPLASMIC HYBRIDS IN CHILLI (Capsicum annuum L)


Nanda, C1., A. Mohan Rao, S. Ramesh and R. S. Kulkarni

ABSTRACT
Chilli is, one of the major spice cum vegetable crops, specially consumed as food additives for its unique color, pungency and aroma. Most of the cultivars in chilli are conventional hybrids. CMS based hybrids were of little success in the hands of the private seed companies. Exploitation of CGMS in any crop species needs high nicking parents with perfect restoration which could be one of the limitations in chilli. In this background, 80 hybrids, synthesized using two CMS lines and their corresponding maintainers (as lines/ females) and 20 testers (as males) were evaluated along with their parents and commercial check (NS 1101) in RBD with two replications during summer 2005 at the experimental plots of the Department of Genetics & Plant Breeding, Main Research Station, UAS, Hebbal, Bangalore. Combined analysis of variance of isonuclear-alloplasmic lines and their hybrids revealed significant mean sum of squares due to and within cytoplasm for days to flowering, plant spread, fruit length and green fruit yield per plant suggesting the involvement of cytoplasm in the expression of these traits. Variance between cytoplasms was found to be significant for days to flowering, plant spread, fruit length, number of seeds per fruit and green fruit yield per plant. Significant mean sum of squares due to interaction between cytoplasm and male lines for all the traits studied was indicative of interaction of cytoplasm and the nucleus in the inheritance of these traits. Of the 80 hybrids, more number of hybrids which were based on sterile cytoplasm expressed significant sca effects in the desired direction compared to hybrids based on their fertile counterparts for days to flowering (18,14), plant height (14,8), green fruit yield per plant (7,5) and red fruit weight per plant (12, 6). Similar results were also obtained with respect to standard heterosis for days to flowering (12, 7), plant height (14 8) plant spread (7, 2) and number of green fruits per plant (3,2). However, more number of hybrids based on fertile cytoplasm expressed positive standard heterosis for green fruit yield per plant (2, 0), number of seeds per fruit (2, 1), red fruit number (3,1) and red fruit yield per plant (9,7) than hybrids based on sterile cytoplasm. The results revealed favourable effect of sterile cytoplasm on specific combining ability and standard heterosis for many traits suggesting the need to exploit CGMS system to develop heterotic hybrids in chilli.

1. Department of Genetics & Plant Breeding, Agriculture College, UAS, GKVK, Bangalore

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COMBINING ABILITY STUDIES FOR QUALITY TRAITS IN INDIAN MUSTARD


Mahak Singh1 and R.K.Dixit

ABSTRACT
Indian mustard (Brassica juncea) is an important oilseed crop and important source of edible oil in the country. The mustard oil is preferred in the kitchen in entire northern belt of the country. Not only oil but fat free meal is an important source of protein. Fat free meal is an important ingredient not only for internal consumption but also a good source of protein for export and earning of foreign exchange. Genetical studies on seed yield/ plant, 1000 seed weight, oil content and protein content were conducted with 9 parental diallel excluding reciprocals. 36 FIS, 36Ff2 s and 9 parents viz., Varuna, KR5610, RK1467, KRV-tall, T6342, RLM198, YRT-3, RC781 and PR-15 were evaluated in Randomized Complete Block Design with three replications at Oilseed Research Farm, Kalyanpur, Kanpur. General combining ability and specific combining ability variances were highly significant for almost all the traits. On the basis of gca effects, parents KRV-tall and RLM-198 for seed yield/plant, Varuna, KRV-tall and PR-15 for 1000 seed weight, KRV-tall, PR-15 and varuna for oil content and KR-5610 and RLM-198 for protein content were found good general combiners. However, on the basis of sca effects, out of 36 crosses only eight crosses (KR5610xPR-15, YRT-3xPR-15, RK1467x T6342, VarunaxYRT-3, KRV-tallxT6342, RLM-198xYRT-3, Varuna x RLM-198 and KR5610xKRV-tall) were significant for seed yield/plant. Two crosses (YRT-3xPR-15 and Varuna x YRT-3) for 1000-seed weight, three crosses (YRT-3xPR-15, RK1467xT6342 and Varunax RLM 198) for oil content and only one cross (KR5610 x PR-15) for protein content were the best crosses on the basis of specific combining ability effects. The above study suggested that these lines/crosses can be successfully utilized for improving particular traits in Indian mustard.

Introduction Rapeseed-mustard are important oil seeds crops and are next to groundnut in the country. Among the different state, Uttar Pradesh is one of the leading state covering an area of 1.12 million hectares with the production of 1.07 million tonnes. There are several species of rape seed-mustard viz. Brassica campestris, Brassica juncea, Brassica napus, Brassica carinata and Brassica nigra but Indian mustard [Brassica juncea (L.) Czern and Coss] is widely cultivated. It has higher yield potential and is suitable for sole cropping as well as intercropping. In planning of an efficient breeding programme in any crop, selection of parents plays a crucial role and combining ability analysis serves as a very handy tool for the

selection of parents. Information on the relative importance of general and specific combining abilities are also helpful in the analysis and interpretation of the genetic basis of important traits. The present study was therefore, undertaken to gather such informations in Indian mustard regarding seed yield and its component traits. Materials and Methods Nine strains of Indian mustard [Brassica juncea (L.) Czern and Coss] namely, Varuna, KR-5610, KR-1467, KRV-Tall, T-6342, RLM198, YRT-3, RC-781 and PR-15 were crossed in a diallel fashion (excluding reciprocal crosses) to obtain 36 F1 hybrids. The F1s were grown at Oilseed Research Farm, Kalyanpur

1. Department of Genetics and Plant Breeding, C.S.Azad University of Agriculture & Technology Kanpur- 208 002 (UP)

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of C.S.Azad University of Agriculture and Technology, Kanpur to obtain F2 progenies. The parents were maintained by selfing. The experimental material comprising eightyone viz., 9 parents, 36 Ffs and 36 F2s were grown in a randomized complete block design with three replications in rabi. The parents and F1s were grown in one row and F2s in four rows of fivemeter length and 45 cm apart and 15 cm distance from plant to plants within a row was maintained by thinning. All treatments were given equal doses of fertilizers @ 80 kg N, 40 kg P2O5 and 40 kg K2O per hectare with two irrigations. Spraying of Ekatin and Dithane M-45 was done for protecting the crop from aphids and Alternaria blight respectively. Recommended agronomic practices were followed for raising a good crop. Data were recorded on 5 competitive plants in each of parents and F1s and 20 plants in F2s for each replication selected at random in all the three replications. Days to maturity were recorded by counting the days from seedling to turning of the plant yellowish i.e. at physiological maturity. The oil content was estimated by N.M.R. and protein content analysis was done in cake. The oil was removed and residue left was taken for protein analysis. The protein analysis was done by biuret method of Williams (1961). Data were analyzed for randomized complete block design and mean squares due to general combining ability (GCA) and specific combining ability (SCA) was calculated by Griffings (1956) model I, method IV used for diallel analysis. Results and Discussion Combining ability studies help in selection of best combiners and provide opportunity for the use of these combiners in hybridization programme. General combining ability is primarily a function of additive gene action and additive x additive inter action whereas specific combining ability is due to non-allelic gene interaction. The analysis of variance for general combining ability
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was found significant for all the characters in both the generations except 1000-seed weight in F2 generation. The results are presented in Table-1. The estimate of variance due to gca and sca indicated that magnitude of s2s was higher than s2g for all the traits in both the generation. The ratio of s2g/s2s were less than unity for all the characters in both the generations except for number of secondary branches, length of main raceme, number of siliquae on main raceme and oil content in F1 generation. The average degree of dominance expressed as (s 2 s/s 2 g) suggested over dominance for all the traits in both the generations except primary branches and yield per plant in both the generations and protein content in F1 which indicated no dominance. The yield per plant was found to be controlled by non-additive gene effects. The findings were in conformity with findings of Singh and Srivastava (1986) and Jain et al. (1988). General Combining Ability The estimate of gca effects of the parents for all the characters are presented in table 2. It is revealed from the table that the parents which had good per se performance were also good general combiners for yield and its main yield components. Ranking of desirable parents in order of merit, on the basis of gca effects in F1 and F2 generations are presented in table 3. Based on this, promising combiners for earlines are YRT-3 and KRV-Tall and for dwarfhess PR-15 and Varuna as they had desirable gca effects. Parents RC-781, Varuna and PR-15 for primary branches; RC-781 for secondary branches, RK-1467 and KR 5610 for length of main raceme; RC-781 for number of siliquae on main raceme; RLM-198, PR-15 and T-6342 for early maturity; RC-781, KRV-Tall and RLM-198 for yield per plant appeared to be good general combiners. Parents Varuna, KRV-Tall and PR-15 for 1000-seed weight and

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KRV-Tall, T-6342, PR-15 and Varuna for oil content were most desirable combiners in both the generations. However, Varuna, KR 5610 and RLM-198 are good combiners for protein content. These lines can be successfully utilized for improving particular characters for which improvement is desired because these parents had high general combining ability effects and thus has fixable components of variance like additive and additive x additive epitasis. These lines may be utilized for producing the intermating population in order to get desirable recombinants of Indian mustard. The findings were in conformity with findings of Dixit et al (1983), Thakral and Singh (1995) and Sachan et al. (1996). Specific Combining Ability Effects The estimates of sca effects of crosses for all the characters are presented in table 4. The specific combining ability is the important parameter for judging the specific combinations for exploiting it through heterosis breeding. The good and promising cross combiners for seed yield are presented in table 5. A perusal of the table indicated that out of 36 crosses only eight crosses KR-5610 x PR-15; YRT-3 x PR-15; RK-1467 x T.6342; Varuna x YRT-3; KRV-Tall x T.6243; RLM-198 x YRT-3, Varuna x RLM198 and KR-5610 x KRV-Tall had desirable specific combining ability effects for yield and the same crosses had desirable sca effects in F2 generation also. These crosses involved all the three possible combinations between the parents of high and low gca effects i.e. high x high, high x low and low x low. Poor inbred parents although lacked the additive effects of the good inbred yet they were highly responsible to heterozygosity in the way of non-additive effects (Darrah and Hallauer, 1972). Crosses involving high x high and high x low combiners may give rise transgressive segregants in the next generation (Langhum, 1961). In general, additive and nonadditive type of gene action were predominant
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in the population for all the traits. Therefore, for harnessing the maximum yield potential, a certain degree of heterozygosity should be maintained in the population. The reciprocal recurrent selection will be most suitable breeding procedure for mopping up the desirable additive gene actions through selection of desirable segregants. Breeding methods such as biparental mating followed by reciprocal recurrent selection may increase frequency of genetic recombination and hasten the rate of genetic improvement. Mass selection with concurrent random mating would be another breeding methodology for breaking the bottle neck of seed and oil yield in Indian mustard. From practical point of view, high sca effects of crosses alone will not lead to much improvement unless it is coupled with high per se performance. Therefore, selection of crosses for further breeding programme may be based on higher values of both of these parameters. In the present investigation crosses (KR 5610 x PR-15; YRT-3 x PR-15; RK-1467 x T. 6342; Varuna x YRT-3; KRV-Tall x T-6342; RLM-198 x YRT-3, Varuna x RLM-198 and KR-5610 x KRV-Tall) apart from having high sca effects and per se performance for various yield components also had both the parents as good general combiners. Hence, these crosses are expected to throw transgressive segregants in the later generations. Formation of a new heterotic group It has now become established that there is a good association between sca effects and mean performance of crosses. However, a few crosses appeared to have high mean value but non-significant sca effects and vice-versa. Keeping these facts in view, eight best crosses were composited to form a new heterotic group for grain yield. It is obvious from the data that the ranking of crosses based on the mean performance and sca effects was not always

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the same; however, there was a significant association between these two criteria of ranking. This new group of crosses justifies the development of commercial hybrids in Indian mustard. In order to exploit hybrid vigour at commercial level attempts should be made to convert high heterotic parents into cytoplasmic male sterile lines and search for fertility restorer lines in the germplasm to develop mustard hybrids. REFERENCES Darrah, L.L. and Hallauer,A.R. (1972). Genetic effect estimates from generation mean in four diallel set of maize in breeds. Crop Sci. 12 : 615-621. Dixit, R.K. Prasad, K. and Srivastava, A.N. (1983). Combining ability for quality character in Indian mustard. Indian J. Agric. Sci. 53 : 776-778. Griffing, B. (1956). Concept of general and specific combing ability in relation to diallel crossing systems. Australian J. Biol. Sci. 9 : 463-493.

Jain, A.K. Tiwari, A.S.; Kushwaha, V.S. and Hirve, CD. (1988). Genetics of Quantitative traits in Indian mustard. Indian J. Genet. 48: 117-119. Langhum, D.C. (1961). The high-low method of improvement. Crop Sci. 276-378. Singh, R.N. and Srivastava, A.N. (1986). Combining ability for yield and its components in Indian mustard. Farm Sci. J. 1 : 52-57. Sachan, S.P., Srivastava, A.N. and Singh, P. (1996). Combining ability studies in Indian rapeseed.Farm Sci.J.6: 31-35. Thakral, N.K. and Singh, H. (1995). Combining ability for yield components and oil content over saline environment in Indian mustard. J.Oilseeds Res. 12: 74-82. Williams, P.C. (1961). The determination of protein in whole wheat meal and flour by the biuret procedure. J. Sci. Food. Agric.12: 59-60.

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Table 1. ANOVA for combining ability for eleven characters in a 9 parent diallel in [Brassica juncea (L.) Czern and Coss].
Primary Secondary branches branches Length of main raceme No. of siliquaon main raceme Days to maturity Yield/ plant 1000-seed Oil weight content in (g) (%) Protein content (%)

Source of variation

d.f

GeneDays Plant ration to flowering height

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gca

88

sca

36

Error

88

260

V2g

V2s

(V2gs/V2gf5

F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2

7.20** 45.79** 14.74** 57.18** 11.21** 35.74 6.00** 39.51 0.37 0.32 0.43 0.52 0.36 0.91 0.79 1.60 10.84 35.42 5.56 38.99 5.49 6.24 2.65 4.93

0.29** 0.17** 0.15** 0.23** 0.02 0.01 0.01 0.00 0.13 0.22 3.60 0.00

9.13** 5.24** 7.81** 3.94** 0.03 0.07 0.12 0.11 7.78 * 3.86 8.55 5.92

17.32** 12.27** 7.54** 7.90** 0.11 0.14 0.88 0.39 7.43 7.76 2.90 4.46

19.62** 10.87** 15.18** 8.83** 0.16 0.20 0.40 0.18 15.01 8.62 6.12 6.92

18.87** 18.94** 16.56** 8.83** 2.54 1.47 0.21 0.62 14.01 10.61 8.17 4.14

7.51** 5.58** 6.23** 5.58** 0.13 0.17 0.11 0.00 6.10 5.41 7.45 0.00

0.32** 0.45 0.19** 0.20 0.01 0.00 0.01 0.02 0.18 0.19 4.24 3.08

2.66** 1.99 1.33** 0.70** 0.07 0.14 0.12 0.11 1.25 0.55 3.23 2.23

0.61* 1.40** 0.68* 0.66** 0.28 0.21 0.00 0.06 0.39 0.44 0.00 2.71

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* significant at 5 per cent level ** significant at 1 per cent level

Table 2. Estimates of gca effects of parents for eleven characters in a 9 parent diallel cross in [Brassica juncea (L.) Czern and Coss].
Primary branches
F1
1 2

Parents

Days to flowering
F2 0.05 -0.17" 0.31" -0.1 1** 1.98** 0.84** -0.15** -0.19" -0.01 -0.01 0.14** 0.02 0.04 0.06 0.10 0.07 0.05 0.09 0.15 0.17 0.19 0.21 0.07 0.12 0.20 0.23 0.25 0.28 0.30 0.23 0.04 0.07 0.12 0.14 1.16 0.17 0.19 0.68 1.16 0.88 0.03 0.05 0.08 0.09 0.10 0.11 0.12 0.45 0.09" -0.49** 0.07 -0 88" -0.28" -0.71" 0.36* 006 -0.75* 0.34 0.51 0.14" 0.75" 0.05 2.28" 1.54" 1.79" 1.05** 1.40** 0.06 0.10 0.15 -0.05 -0.38** -0.19** -0.17 -0.54" -2.04" -1.35* 0.34 -0.14** -0.03 -1.17" .0.62" -0.96" -1.14" -0.71" -0 08 -2.05" -0.32" 0.02 -0.12" -0.62" -0.72" -1.04** -1.49" -0.49** -0.32** -0,17 -0.03 -0.88** -0.32** -1.40" 0.06 0.85** -0.05 2.09** 1.30" 0.02 1.21" 0.08" 1.53" 1.56" 1.29" 0.51** 0.12 1.33" 0.20" 0.19** -0.65** 0.74" 0.74** 0.57" 1.22** 1.64"* -1.72" 0.51 -0.33" -0.34 -0.18* 0.12" 0.33** 0.69** -0.45" -0.45** 0.95** -1.12** -2.05" 1.76" -0.62" -0.16 0.32" 0.22" 0.26" 0.59" -0.15" -0.07" -0.|0 0.11 0.17 0.01 002 0.04 0.10" -0.17" -0.10" 0.02 -0.03 0.16* 0.07 0.11 F2 F1 F2 Fraceme2. F 1 F1 F2 F1 F2 F1 F2 F1

Plant height main raceme F F


F2 0.76" 0 15 0.15 -0.36" 002

Secondary branches

Length of main

No. of siliqua on

Days to maturity

Yield/plant

1000-seed weight

Oil content (%)


F1

Protein

content
F2 0.12 -0.24 028 -0.01 -0.11 -0.71" 0.23 028*

F1

F2

F1

VARUNA

0.25

-0.36*

-1.79"

-2.60

KR-5610

1.28** 2.40"*

1.20*

-0.17

Second National Plant Breeding Congress 2006

RK-1467

0.01

-1.52** 1.18** -0.67"*

KRV-Tall

-0.07

-1.14" .0 78**

0.44"

T-6342

-0.06

0.02** -1 18** 2.21"*

-1.05** -1.03** -1.17" -0.19** -0.14**

261

RtM-198

0.00"

0.22

-0.88** 1.69**

YRT-3

-0.18

-0.37* 0.80**

0.26

-0.00** -0.42** -0.40"

R.C-781

-0.99**

0.15

4.04**

2.00"

PR-15

-1.22**

-0.30

-2.68** -4.15"

SE (gi)

0.17

0.18

0.16

0.20

SE (gi-gj)

0.26

0.28

0.24

0.30

CDat l%

0.43

0.46

0.41

0.51

CD at 5%

0.33

0.35

0.31

0.30

* significant at 5 per cent level

Plant Breeding in Post Genomics Era

** significant at 1 per cent level

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

Table 3. Ranking of desirable parents in order of merit, on the basis of gca effects in F1 and F2 generations for eleven characters in a 9 x 9diallel cross of [Brassica juncea (L.) Czern and Coss].

Characters

Best parents based on Best parents based on per se performanceon gca effects
F1 F2 F1 F2

Best common parents in F1, F2 and in both F1 and F2


F1 F2 In both F1 & F2

Days to flowering

PR-15 RC781 YRT-3 KRV-Tall T-6342 PR 15 Varuna T6342 RLM198 KRVTall RK1467 RC781 Varuna PR-15 YRT3 RK 1467 RC781 Varuna RLM 198 YRT3 RC781R K1467 KR5610 YRT3 Varuna RC781R K1467 KR5610 Varuna T6342 Varuna KR5610 T6342 RLM 198 PR 15

RK1467 KRVTall YRT3 Varuna PR 15 PR-15 Varuna RK1467 KR5610 YRT-3 KR5610 RC781 Varuna PR-15 KRV-Tall RK 1467 RC781 KR5610 KRVTall PR 15 RK 1467 RC781 KR5610 KRV-Tall PR 15 KR5610 RK781 RK 1467 PR-15 KRV-Tall RLM 198 T6342 YRT3 PR 15 RC781

RK1467 RC 781 KRV Tall Varuna YRT3 PR 15 T6342 Varuna KRV-Tall YRT3 Varuna KR5610 RC781 PR-15 YRT3 KR5610 PR 15 RLM 198 KRVTall RC781 KRV-Tall PR-15 YRT-3 KR5610 RK1467 PR 15 KRV-Tall Varuna T6342 RC781 T6342 RLM 198 YRT3 PR 15 KR5610

59.23 59.80 60.86 63.46 63.93 152.13 164.80 165.90 166.50 172.33 3.93 3.90 4.06 4.30 4.53 10.10 10.26 10.60 12.50 12.56 0.46 53.36 53.76 55.86 56.76 29.66 29.90 30.23 30.43 33.90 121.33 121.66 123.66 125.66 127.00
262

RC781 YRT3 KRV-Tall

RK1467 KRVTall YRT3 Varuna

YRT3 KRVTall

Plant height (cm)

PR-15 T6342 Varuna KRV-Tall

P7R-15 Varuna YRT-3

PR-15 Varuna

Number of primary branches

RC781 Varuna PR-15 YRT3

KR5610 RC781 Varuna PR-15

RC781 Varuna PR-15

Number of secondary branches

RC781 RLM 198

RC781 KR5610 KRVTall PR-15

RC781

Length of main raceme (cm)

RK 1467 KR5610 YRT3

RK 1467 KR5610 KRVTall PR 15

RK1467 KR5610

Number of siliquae on main raceme

RC781 Varuna T6342

RC781PR 15KRVTall

RC781

Days to maturity

RLM 198 PR-15 T6342

RLM 198 R6342 YRT3 PR 15

RLM 198 PR 15 T6342

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

Characters

Best parents based Best parents based on per se performanceon on gca effects
F1 F2 KRV Tall RC781 RK 1467 RLM 198 Varuna KRV Tall Varuna RL ml98 RK 1467 PR 15 Varuna KR5610 KRV-Tall T6342 PR 15 T6342 PR 15 RLM 198 KR5610 Varuna F1 Varuna PR 15 RLM 198 RC7 81 KRV Tall PR 15 RLM 198 KR5610 KRV-TaU Varuna PR 15 T6342 KRV Tall KR5610 Varuna RC781 Varuna PR 15 KR5610 RLM 198 F2 9.36 9.36 11.63 14.26 14.36 2.80 3.43 3.70 3.73 3.76 38.93 39.93 40.16 40.20 40.40 31.56 32.20 32.50 32.60 32.80

Best common parents in F1, F2 and in both F1 and F2


F1 RC 781 KRV Tall RLM 198 PR 15 F2 KRV Tall RC 781 RLM 198 Varuna In both F1 & F 2 RC 781 KRV Tall RLM 198

Yield per plant (g)

RK 1467 RC781 PR 15 KRV Tall RLM 198 Varuna KR 5610 KRV Tall RK 1467 PR 15 KRV Tall T6342 Varuna PR 15 KR5610 Varuna T 6342 KR5610 RC781 RLM 198

1000-seed weight (g)

Varuna KR 5610 KRV Tall PR-15

KRV Tall Varuna RLM 198 PR-15

Varuna KRV-TaU PR-15

Oil content (%)

KRV-TaU T6342 PR-15 Varuna KR5610 Varuna KR5610 RC781 RLM 198

Varuna KR.5610 KRV-Tall T6342 PR-15 PR-15 RLM 198 KR5610 Varuna

KRV-Tall T6342 PR15 Varuna KR5610 Varuna KR5610 RLM 198

Protein content (%)

263

TabIe 4. Estimates of sca effects of parents for eleven characters in a 9parent-diallel cross in [Brassica jundea (L.) Czern and Coss].
0.14 0.26* -0.16 0.16 0.75" 0.29" -1.42** -2.90 -0.91" -4.74" 2.86** 1.37** 0.62* -1.12" -0.09 1.13" 0.78" 2.26" 0.96" 4.37" -4.25" 4.46" 2.45" 1.35" 0.30 6.21 -0.71 9.39" 2.06" 1.50" 10.71** -0.96" 5.22 6.27** 2.00** -2.31" -1.93" 0.18 -0.73** 1.01" -3.35" -0.46 -1.05 -2.46" -4.60" -4.88" -3.23" 0.80* 2.03" 0.75* 2.06** -1.19** 0.43 2.34* 1.22** 2.37* -3.15** -1.29 0.64 -0.53 4.46" -0.41 0.40 1.40" 3.70** 2.98** 3.07** 0.52 2.94** 3.18* 3.89** 3.68" -0.77" 2.21" 2.37** 1.93** 2.43* 6.55** 1.63** 2.62" -2.25** -2.11" -4.26** -4.39** -2.26" 2.98** -6.22" 2.54" 1.00" 0.46 -1.33" -1.35" -1.56 2.14" 0.54 1.90" -2.55 -1.14" -6.30** -1.79** -4.78" -3.77** -0.53 2.16* 2.17** 0.78" -0,89" 2.14" 0.27 -0.20 2.46* -0.82" 0.69* -0.78" 0.02 -2.14** -0.76* 5.94** 2.64" -0.14 4.73** 1.86" 3.57" -2.04" 1.30" -2.60** 0.61* -1.12** 1.63** 0.61 3.13** 1.26" 1.95" -0.57 4.18** -1.71" -2.04** -2.45" -0.87"* -3.02" -2.29* 0.13 -0.25 0.02 -0.10 -1.07** 0.40 0.01 0.74" -1.61** 0.92" -2.81" 3,72" -1.59** . -2.23* -1.10 -0.04 0.69* 0.08 0.25" -0.41 0.10 -0.51" -0.69** -1.78" 1.44" -1.74** 1.27** -2.29" -0.26 -2.92" 4.60** -0.63 0.30 -0.29** 0.75** 1.28" 0.86" -0.45 -0.65* 0.38 0.31 -0.17 0.18 -0.06 -0.08 -0.07 -0.19* -0.30 -0.01 0.46 -0.36" -0.75" -0.34" -0.54" -0.65 0.76** -0.14* -0.87** -0.39 0.16 0,33* -0.43 -0.21 -0.50* 0.31 0.45 0.23 0.39 -0.46" 0.83** -1.01" -0.07 0.56" 0.07 0.16* 0.05 0.26 0.23 -0.01 -0.12 0.26 0.86" 0.77 0.04 0.41 -0.33 -0.27 -1.16" 0.39 0.23 0.06 0.10 -1.06* 0.91** -0.85* 1.01" 0.94** 1.48" 2.60" 0.24 1.14" -0.49 -3.41"* -6.10" 0.64* -0.67" 0.05 -0.46" -0.94** -0.26 -0.21 0.20 -1.33" 0.26 0.09 -0.48 0.49 -0.58 1.23" 0.24 -0.06 -0.05 0.15 -0.45 0.15 -0.89 1.39 -0.16 0.11

Varuna xKR 5610

-2.19"

-0.80

1.30"

4.61**

Varuna xRK-1467

4.38"

-2.73"

8.69"

-0.05

Varuna x KRV-Tall

-1.63"

0.94

-4.13"

-6.44"

VarunaxT-6342

1.39**

-0.92

3.16" -1.97** 0.59"*

Varuna xRLM 198 -0.62" 0.46** 0.29** 0.51" -0.08 0.01 -0.03 0.01 0.35"

136**

-0.38

-0.83

7.17** 0.37**

0.15* 1.34** 0.68*

Varuna xYRT -3

-3.45**

0.45

-5.75** 1.24*

0.21" -0.80** -1.21** -0.58 0.19*

Second National Plant Breeding Congress 2006

Varuna x RC 781

3.48"

3.98" -1.93** 1.94"

Varuna x PR-15 -0.97"

-3.41** -2.88** -3.57** -8.64** -0.30"

-3.15" -2.42** 0.72** 0.85** 0.55*

KR56IOxRK-l467

-3.65** 1.29* -2.54** -5.61"

264
0.05 0.06 0.33** 0.30** 0.42** 0.68** 0.41** -0.02 0.30** 0.41** -0.27* 7.83" -2.45" 0.07 5.06" 3.59" 0.44** 1.11" 2.76** 0.15 2.80** 1.47** -1.58" 5.10** 2.18*

KR5610xKRV-Tall

2.12** -4.75" -2.90** -1.90** -0.30"

KR5610xT-6342

-1.61"

4.10" -9.53** -0.60

KR56IOxRLM 198

-1.83** -3.95"

1.96

-2.95**

KR56IOxYRT-3

1.97"

3.41** -6.65"

-1.08

KR5610xRC-781

0.88

3.54"

5.13"

3.71"

KR5610xPR-l5

1.68"

-0.76 13.29** 2.53"

RK 1467xKRV-Tall

1.75"*

4.90"

1.38**

6,43"

RKI467xT-6342

4.23"

-0.46

9.45"

2.19"

Plant Breeding in Post Genomics Era

RK [467xRl..M-l98

2.48"

-1.18*

-0.54

-0.42

Table 4. Contd..,
0.02 -0.02 3.60" 0.37 -1.01 -2.81" 0,22 -0.92" -4.11** -0.08 -0.24 -2.23" -2.06** -1.80" 0.58 -1.36" 0.82 2.49* 1.06 -0.26 0.44 0.30 0.45 0.43 0.50 0.47 0.32 0.54 0.52 2.95** 0.36 0.61 0.58 0.67 1.29 2.15 2.04 -0.41 -0.68 -2.63" 6.91" 2.61" 1.94" 2.98" 1.30 1.55 -2.69" -0.35 -0.23 -1.68" 3.61" -1.71 0.98 -2.77** 0.23 0.07 0.01 3.23 3.98" -0.58 2.72" -0.08 0.16 -3.39** 4.06** 1.95* -1.06" -3.23 -3.47** 3.64** -3.38** -1.33" -0.01 0.14 -0.26" -0.16 -0.25" 0.02 0.49" 0.07 -0.17 2.92** 3.30** 2.19** 0.72** 1.31 0.98 1.63 1.55 -0.71** 1.15** 0.25** 0.29 0.48 0.46 0.33 0.55 0.52 0.08 0.13 0.13 1.44" 0.58 -2.10"* -2.66" 1.64 -0.74 -1.20" 1.830.45** 0.28" -0.12 -2.52" -1.58" -3.54" -2.19" 2.37" 3.86** -3.91" -4.75 0.18* 0.15* 0.62" 0.93** 0.12 -0.78" -0.51* -0.01 0.19* 0.28* 1.26" 0.33 1.09" 0.55" -0.85** 0.25" 0.48" 1.85" 0.45 4.05" -2.54" 5.36 0.17" 3.79" 2.92** -3.11" -3.19" -0.50" -1.51" 4.89" 0.14 2.97** 3.55" 0.92 1.59" 1.36" -0.23" -0.18* -0.66" -0.68* 0.58** -0.73* -0.54 -0.24 1.30" -0.33 0.29 -0.37 -0.38 0.50 1.17" -0.20 0.36** 1,13** 1.27** -0.04 -0.01 0.07 0.12 0.12 1.79** 0.23 0.22 0.37 0.35 0.42 2.05** 0.30 0.51 0.48 -1.08 -4.78" 3.67 -0.37 0.61 -1.07 1.04* -0.27 0.15 -0.33 -2.96" -1.27" -0.33 -0.18 -0.33 l.ll 1.15" -0.14 0.06 -0.53" 2.37" -2.08" -2.73** -3.08 -3.93" -4.19" -0.38 -2.77** -1.32" -4.12" -0.41" -0.62" -2.62"* -0.35 0.50 -0.48 -0.36 -1.10" -0.85* -0.97" 0.07 0.19 -0.48 -1.58** -1.16" 1 -0.78 -0.28 -0.77 0.49 0.49 -0.68 -0.68 -0.74 0.43 0.72 0.68 -0.15 -1.30 -0.48 1.09" 1 -0.08 0.38 0.66 0.31 0.37 0.63 0.60 -0.20* -0.41" -3.06" 2.56" 0.51 -2.06" -3.05 4.33" 1.83 1.73** 0.75* 0.14 0.07 0.09 -0.42 -0.45 -0.80*

RKI467xYRT-3 5.18"

-2.87"

-2.58"

-0.63 -8.01**

RLI467xRC-78l

7.66"

1.68**

-0.58

RXI467XPR-I5 1.74" 5.35" 0.28 -0.32" -2.03 -0.15 0.15 -1.33"

-3.09

0.57

-7.05" 5.19** -0.26* -0.40** -2.14"

KRV-TallxT6342

-2.28** -1.44"

9.46"

KRV-TallxRLM 198

-0.87

0.66

-5.40"

K.RV-TallxYRT-3 1.76" 6.34" -0.28 0.20 -3.08" -0.16 -0.02 -2.01" -0.12 -0.05 1.27"

3.16** -1.40" -0.99* -4.43** -0.64 -0.54** -3.87"

KRV-TaUxRC-781

-0.25

-0.10

-4.63"

KRV-TallxPR-15

5.34"

0.95

6.95"

Second National Plant Breeding Congress 2006

T-6342xRI.M 198 -7.89" -0.41** -0.05 -3.13** -1.18 -2.28** -0.36 0.95 0.01 0.33* -0.36** 0.37" -0.77** -0.74** -0.99" 0,21 0.11 -0.13 0.23 1.54** -0.33 0.50 0.19* 1.47" 1.19" 1.46" -1.42** -0.84** -2.21" -0.53** -0.12 -1.99** 1.06" 1.52" 0.60" -0.17 -1.62" -1.88** 3.96" 3.61 0.24** 0.61" -0.55" 0.12 -3.08** -0.33 -0.26* -0.19 -3.20" -0.81" -0.08 -3.65"

0.95

0.22

-3.17" 13.81"

. T-6342xYRT-3

0.92

-0.94

-3.89"

265
res1.06** 1.58** 5.15** 2.29** 1.58** 1.52** 0.03 0.11 0.19 0.18 0.14 0.22 0.15 0.23 0.37 0.35 0.09 0.14 0.22 0.27 0.46** 1.88** -0.77** -1.37** 5.03** 0.58 0.97 0.92

T-6342xRC-78l

-2.29 -1.37"

-4.90"

T-6342xPR-15

-7.12"

-0.81

0.95* -4.69**

RLM 198xYRT-3

0.33

-2.20** -5.32** -9.36"

4.88" -4.44** 1.36"

RLM-l98xRC-78l

-5.78" -1.96** 6.35** -5.72"

RLM-l98xPR-l5

-2.08*

0.45

1.98"

YRT 3 x RC 781

-2.31** 1.59** 7.43** -4.95** 0.25*

YRT 3 x PR 15

-0.54 -1.64** 10.63** 15.16** 0.05

RC 781 x PR 15

0.89

-0.30

-2.31* 1.62**

SE (sij)

0.49

0.53

0.46

SE (sij-sik)

0.82

0.89

0.76

Plant Breeding in Post Genomics Era

SE (sij-skl)

0.78

0.84

0.72

TabIe 5. Ranking of desirable crosses based on per se performance, sca and gca effects in F1 generation for eleven characters in a 9 x 9 diallel cross of [Brassica juncea (L.)
Days to flowering Plant height Primary Secondary Length of branches branches main raceme
0.41 2.80** 2.26** 6.21** 4.46**

Cross

sca for yield


0.06 13.29** 1.68**

Per se Performance No. of Days to 1000-seed siliqua maturity weight on main in(g) raceme

gca of P1 gca of P2

Oil Protein content content (%)


0.06

KR5610

3.68**

16.10

-0.33**

X PR 15 0.06 10.63** -0.54 1.06** 5.15** 1.58** -0.26 0.72** 1.79**

Second National Plant Breeding Congress 2006

YRT-3 X PR 15 -1.03** 9.45** 4.23** 0.30** 5.06** 4.37** 9.39** 3.18**

3.30**

15.63

-0.42**

RK 1467 X T 6342 -0.42** -3.45** -5.75** 5.94**

2.21**

15.43

1.57**

0.23

266
-1.03** 9.46 -2.28** -0.42** 0.33 -0.32** 1.36** 0.37 0.02 -2.90** 2.12** 2.86** 0.62 2.06

VarunaX YRT-3

1.86**

13.50

-0.62**

-0.14

0.21**

KRV-Tall X T 6342

1.59**

13.26

0.02

3.55**

RLM 198 X YRT3

1.36**

13.30

-0.32**

4.88**

Varuna X RLM 198

1.26**

13.00

-0.62**

0.61

1.34**

KR5610 XKRV-Tall

1.22**

13.60

-0.33**

0.43

* significant at 5 per cent level

Plant Breeding in Post Genomics Era

** significant at 1 per cent level

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

TECHNICAL SESSION V IN VITRO BREEDING TOOLS IN GENETIC ENHANCEMENT OF CROPS

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

COMBINED EXPRESSION OF CHITINASE AND -1,3 - GLUCANASE GENERATES HIGH LEVELS OF SHEATH BLIGHT RESISTANCE IN HOMOZYGOUS TRANSGENIC RICE LINES
Sridevi, G1., C. Parmeswari, N. Sabapathy and K. Veluthambi

ABSTRACT
Sheath blight disease caused by Rhizoctonia solani is the second major fungal disease in rice next to blast. A major limitation in the management of sheath blight disease is the lack of complete resistance in the known cultivars of rice. Many plant genetic engineering efforts to develop fungal resistance involve PR (pathogenesis-related) protein genes. Among the PR proteins, chitinase (PR-3) and b-1,3-glucanase (PR-2) are efficient in the lysis of chitin and glucan polymers in the fungal cell wall. Agrobacterium-mediated transformation of rice was performed using a binary vector (pNSP3), harbouring rice chitinase (chi11) gene under maize ubiquitin promoter and tobacco b-1,3-glucanase gene under CaMV 35S promoter. Nine transgenic rice plants harbouring both the chitinase and b-1,3-glucanase genes were generated. Five T0 plants carried single T-DNA copies, one had two linked TDNA copies and a long-transfer event, one had a head-to-head dimer and the remaining two had complex integration patterns. Seven T0 lines showed a segregation pattern of 3:1. Northern and western analysis of T1 plants of single copy lines showed constitutive expression and accumulation of chitinase and b-1,3-glucanase. All the single copy lines, which were made homozygous, showed 10-fold higher chitinase enzyme activity as compared to nontransgenic control rice plants. Interestingly, two T1 plants of T0 line 57 (a head-to-head dimer) expressed chitinase constitutively but showed silencing of the b-1,3-glucanase gene. Bioassay of homozygous T2 plants of three single copy transgenic lines against Rhizoctonia solani revealed a 60% reduction of sheath blight disease index. By combining the genetic analysis by segregation and molecular analysis by semi-quantitative Southern hybridization, a method was developed to achieve genetic separation of two unlinked transgenic loci and to identify homozygous transgenic lines in the T1 generation.

1. School of Biotechnology, Madurai Kamaraj University, Madurai 625021

267

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

TRANSFORMATION OF THREE ANTIOXIDANT GENES FROM A HIGHLY SALT TOLERANT GRAY MANGROVE, AVICENNIA MARINA FORSK. (VIER H.) IN INDICA RICE
Ajay Parida, S. R. Prashanth, M.N. Jithesh and KR. Sivaprakash

ABSTRACT
Salinity is one of the major threats in decreasing crop productivity worldwide. It leads to a reduction in photosynthesis and the unused excess light energy results in over-reduction of molecular oxygen. The transfer of electrons to molecular oxygen results in the production of reactive oxygen species (ROS). Under high light and CO2 limiting conditions caused by environmental stress like salinity, the antioxidative enzymes (SOD, CAT and FER) play an important role in scavenging toxic radicals in different organelles of the plant such as chloroplasts, cytosol, mitochondria and peroxisomes. To investigate the functions of antioxidative enzymes in the abiotic stress responses in a mangrove plant, we isolated three cDNAs encoding cytosolic Cu/Zn SOD (Sod1), catalase (Cat1) and ferritin (Fer1) from Avicennia marina cDNA library. We studied the expression of these antioxidant genes in response to different abiotic stress factors like salt, iron, and light stress, an osmoticummannitol and direct oxidative stress factor-hydrogen peroxide by mRNA expression analysis. Cat1, Fer1 showed short-term induction while Sod1 transcript was found to be unaltered in response to NaCI stress. A decrease in mRNA levels owas observed for Sod1, Cat1 while Fer1 mRNA levels remained unaltered with osmotic stress treatment. Sod1, Cat1 and Fer1 mRNA levels were induced by iron, light stress and by direct H2O2 stress treatment. These results reveal the importance of these three genes in abiotic stress responses. All the three genes have been cloned in binary constructs for transformation in indica rice using Agrobacterium and particle bombardment methods

M.S.Swaminathan Research Foundation, Chennai - 600 113

268

Second National Plant Breeding Congress 2006

Plant Breeding in Post Genomics Era

IN VITRO GENETIC TRANSFORMATION FOR THE HELICOVERPA RESISTANCE USING CRY1 A(B) IN PIGEON PEA ( CAJANUS CAJAN L. CV MARUTI)
Sandhyarani.N1, Mukund Shiragur. Sumangala Bhat and M.S.Kuruvinshetti

ABSTRACT
The present study was undertaken to standardize in vitro plant regeneration and Agrobacterium mediated transformation procedure for pigeonpea (Cajanus cajan L. cv Maruti). A direct regeneration protocol was employed using cotyledonary node (CN), half cotyledon with cotyledonary node (1/2 CNC) and cotyledon with cotyledonary node (CNC). These were cultured on various levels of benzyl amino purine (BAP) viz., 1,2,3 and 4 mg/l and thidiazuron (TDZ) 0.01,0.05,0.1,0.5 mg/l. CNC was found to produce highest average of 1.69 shoots per explant, and average of 3.5 buds per explant. Among different levels of cytokinin, BAP 2 mg/l was found to be better for the multiple shoot and shoot bud induction . Elongation of shoot buds was achieved at reduced level of the cytokinins and among all the cytokinins tested TDZ (0.05 mg/l) showed better response. Sufficiently elongated shoots were rooted on the half strength of MS medium with various levels of IBA of which 0.2 mg/l gave the good healthy roots. Rooted plants were transferred to pots for acclimatization in the green house before sending to field . For transformation, Agrobacterium strain EHA105 harboring pBIN bt1 plasmid with nptII as selectable marker, which confers kanamycin resistance was used. Initially kanamycin sensitivity of the control explants was tested at different growth stages. Inhibitory levels at different stages were used for selection of the transformants. Precultivation of the explants on MS with 2 mg/l BAP for two days prior to cocultivation resulted in increased transformation frequency. PCR with nptII specific primer revealed that approx. 1.4% transformants were obtained. This protocol can be further used to transfer many other useful genes in pigeonpea for genetic improvement.

Introduction The importance of the grain legume is multipurpose as source of protein for both human and animal consumption. Nutritionally they are richer in proteins than cereals. Pigeonpea popularly known as redgram ( Cajanus cajan L. Millsp) is one of the major grain legumes of tropics and subtropics, where it provides a large proportion of the dietary protein requirements. It also improves soil fertility by fixing atmospheric nitrogen. In Karnataka, it is grown in an area of 4.4 lakh hectares with a production of 2.2 lakh tonnes. In the northern part of Karnataka, especially in Gulbarga, it is grown as a commercial crop. However in Gulbarga so called

redgram bowl of Karnataka, it is grown in an area of 1.67 lakh hectares with a production of 0.57 lakh tonnes and an average productivity of 359 kg ha-1 which is very low (Singhal, 1999), compared to the overall productivity of Karnataka state (494 kg/ha) and the country (500 kg/ha). However, pigeonpea or redgram production is threatened by many insects, diseases and weeds. Pigeonpea cv ICPL 8863 (Maruti), a popular variety of Karnataka is of medium duration and wilt resistant, but highly susceptible to pod borer. The lower productivity of pigeonpea is mainly because of wilt disease and pod borer damage by lepidopteran insect Helicoverpa armigera which causes yield

1. Institute of Agri Biotechnology, UAS Dharwad 580005 Coffee Board CRS,CCRI,Chikmagalur dist-577117 ssnishani@rediffmail.com

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losses to the extent of 46.6 to 63.6 per cent (Anon, 1978). Attempts to obtain pest resistant genotypes of pigeonpea species by conventional breeding methods have not been successful because of the limited available genetic variation among cultivated species and presence of incompatibility with wild , pest resistant species ( Nene et al., 1990). Because of its usefulness for human and animal consumption in Karnataka, we were interested in biotechnological methods to improve this important variety. With the advent of the genetic engineering techniques , it is now possible to transfer the useful genes across species (Nayak et al., 1997). High frequency in vitro regeneration compatible with gene delivery is a prerequisite for genetic transformation. As in vitro regeneration in pigeonpea is highly genotype dependent , an attempt has been made to induce shoot buds and regeneration of complete plants from different explants of pigeon pea cv ICPL 8863 ( Maruti) and a genetic transformation protocol is described. Materials and methods Plant material Uniform seeds of pigeonpea cv ICPL-8863 (Maruti) were obtained from National seed project and Breeders Seed Project BSP- NSP unit University Of Agricultural Sciences, Dharwad, Karnataka, India. The seeds were surface sterilized in 70% ethanol, followed by 0.1% aqueous mercuric chloride solution for 10 min and then rinsed five times with sterile distilled water. Thereafter, they were cultured in test tubes/ culture bottles containing germination medium. The seed germination medium consisted of half strength of mineral salts and vitamins of Murashige and Skoog (1962), 3% sucrose and 0.9% agar pH 5.7 supplementsd with 2 mg/l 6benzylaminopurine (BAP). Seeds were germinated in dark for six days. Shoot apices (ST), cotyledonary nodes(CN), cotyledonary node with cotyledon (CNC) cotyledonary node with half cotyledon (1/2 CNC) were used as explants for regeneration.
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The culture medium used for direct shoot organogenesis consisted of MS basal medium (Murashige and Skoog, 1962) containing 3% sucrose, 0.9 % agar (Hi media,Bombay) and supplemented with either 6-benzyl amino purine (BAP) (1.0,2.0,3.0,4.0mg /l) or Thidiazuron (TDZ)(0.01,0.05,0.1 and 0.5 mg/l) at various concentrations . All media were adjusted to pH 5.7 before autoclaving at 1210C (1kg/sq.cm) for 15-20 min. All the cultures were incubated at 25_+ 20 C with light intensity of ca 1000 lux provided by fluorescent tubes ( 7200 0 K) over a light/dark cycle of 16/8 hours. Explants obtained with MS with or without 2mg / l BAP were used in studies on shoot regeneration for 30 days. Each regeneration treatment had 20 replications and each replication with 2 cultures were arranged in a completely randomized block design. Observations on cultures showing regeneration and number of shoot buds and shoots per responding culture were recorded after 3 weeks at each sub culture. Shoots and shoot buds were subcultured on medium with lower levels of cytokinins for shoot elongation. To promote rooting, excised shoots were cultured on MS and half strength MS medium with 3% sucrose, 0.8% agar and supplemented with either naphthalene acetic acid (NAA) or Indole butyric acid (IBA) at 0.0, 0.1, 0.2 and 0.5 mg /l. Significance was determined by analysis of variance (ANOVA) using the randomized block design. Differences between means were compared by Duncans multiple range test using MSTAT-C computer program (Michigan State University). Data given in percentages were subjected to arcsine(sin 1 p) transformation (Gomez and Gomez, 1984) for statistical analysis and analysed as factorial experiment. Bacterial strain and vector The disarmed and hyper virulent

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Agrobacterium tumefaciens strain EHA 105 (Hood et al., 1993) harboring binary vector pBinBt1 was used for transformation experiments. This construct contains the cry1A(b) gene linked to the cauliflower mosaic virus (CaMV) 35 S promoter, pAOcs terminator and neomycin phosphotransferase (npt II) gene under the control of nopaline synthase (nos) promoter and terminator. npt II was used as a selectable marker (Fig.1).The Agrobacterium EHA 105 containing pBinBt1 culture was maintained on solid MSY medium containing 100 mg l-1 of kanamycin. Single Agrobacterium colony was taken from the LB plate and inoculated into 100 ml LB liquid medium containing 100 mg l-1 kanamycin and was incubated on shaker for 48 hrs at 280Cand at 200rpm( Sambrook et al.,1989) and fresh culture was used for transformation. Transformation Kanamycin sensitivity test was carried out to find out the concentration of kanamycin required to be used for the selection of the transformed plants and was carried at 0, 25, 50, 75 and 100 mg l-1 concentration and lethal dosage was identified at different growth stages. Further, to know the minimum level of cefotaxime that will completely eliminate the excess bacteria after cocultivation, experiment was conducted at 100 mg l-1, 200 mg l-1, 300 mg l-1 and 400 mg l1 cefotaxime along with control. Cocultivation was done just before the formation of shoot buds. Injury was made near the place of bud formation with a sterile scalpel blade and the following protocol was followed. Six to eight days old seedlings were taken from in vitro germinated seeds for explant preparation. Cotyledonary nodes with cotyledon (CNC) were put on regeneration medium for preculturing for two days. Precultured explants were immersed in Agrobacterium suspension for 15 minutes with gentle shaking. The explants were taken out of bacterial suspension and
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excess bacteria were blotted dry using sterile blotting paper. Explants were placed on regeneration medium for different days i.e. 1, 2, 3, and 4 in dark for cocultivation. After cocultivation, explants were regenerated and shoots were transferred to selection medium after thorough washing with MS broth along with 100 mg l-1 kanamycin and cefotaxime ( 300 mg/l) and blotting with sterile blotting paper. Subculturing was done at every 15 days on same medium to avoid escapes. The resistant shoots obtained were separated and transferred individually to rooting medium (MS+0.2 mg l-1 IBA + 25 mg l-1 kanamycin). Confirmation of the transformants using specific PCR To confirm the transformation, plasmid DNA was extracted according to Sambrook et al., (1989). and plant DNA was extracted according to Edwards et al., (1991). DNA was quantified using saranwrap method of quantification ( Sambrook et al., 1989) To test the integration of npt II) gene in transformed plants, PCR amplification of npt II gene was carried out using specific primers. Forward 51 GAG GCT ATT CGG CTA TGA CTG 31Reverse 51 ATC GGG AGG GGC GAT ACC GTA 31. PCR was performed in 25 mL solution mix containing Taq pol 1U , 1XTaq assay buffer, 100mM dNTP mix , 0.3 M of each forward and reverse primer. 50ng of the template DNA in a Hybaid omn-E thermal cycler according to Geetha et al., (1999). Separation of amplified products was done on 1.2 per cent agarose gel using 1 x TAE buffer and amplified products were photographed using gel documentation system (UVI-Tec Cambridge, England). Results and Discussion Regeneration Pulses in general are recalcitrant to regeneration under in vitro conditions and

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response is highly genotype specific. Thus there is a need to develop an efficient regeneration protocol for a given genotype. Initially shoots and shoot buds derived from seedlings germinated on half strength MS were used, but explants derived from seedlings germinated on half strength MS supplemented with 2 mg/l BAP produced more number of shoots and shoot buds. Bulged nodal region was seen in the later seedlings.(Fig A.) Thus differences in the germination medium influences multiple shoot production from explants. These findings are in agreement with previous investigations of Shivaprakash et al., (1994). Thus in the present investigation seeds were germinated on half MS with 2mg/l BAP for further analysis. This pretreatment with BAP during seed germination is also practiced in micropropagation of sugarbeet cultivars (Grieve et al., 1997, Zhong et al., 1993 and Zhang et al., 2001) Pigeonpea seeds germinated within six days and attained the explant extractable stage on both plant growth regulator free and BAP supplemented MS media. There were no apparent deviations in either the rate or frequency of seed germination attributable to with or without growth regulator. However, the growth of the seedlings was significantly altered in the presence of BAP. Seedlings grown on BAP supplemented media showed poor root development with enlarged cotyledons, thick stout stem and bulged node region. A limited elongation and development of the shoot apex was observed and subsequently many axillary buds developed on this. When explants were detached from BAP treated seedlings and cultured on fresh regeneration medium (MS supplemented with BAP or TDZ) shoots were initiated 15 days after the incubation. Explants derived from seedlings grown on plant growth regulator free medium produced very few shoots.(Data not shown). Identification of most suitable growth regulator and its concentration is crucical for morphogenesis. Shivaprakash
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et al. (1994) compared the relative effectiveness of cytokinins for multiple shoot formation and the order of effectiveness was BAP> Kinetin> Zeatin>Adenine in pigeonpea. Superiority of BAP over Kinetin was also demonstrated by Geetha et al. (1998). In the present investigation, comparison was made between different levels of BAP and TDZ. BAP was found to give better response to multiple shoot organogenesis, in Maruti cultivar. Among the four different levels of BAP, 2mg l-1 BAP induced highest mean number of multiple shoot (1.47) and at higher levels of BAP, shoot numbers decrease significantly. When CNC was cultured on MS+2 mg l-1 BAP, both per cent explant responding (100%) and mean number of shoots (3.0) and shoot buds (6.225) per explants were higher (Table 1 and Fig b,c,d). In general lower levels of cytokinins in the medium supplemented with auxins enhances shoot elongation (Franklin et al. 1998,Geetha et al. 1998). In the present investigation, explant containing shootbuds were subcultured on MS supplemented with 2 levels of BAP (0.1 mg and 0.2 mg l-1) and 2 levels of TDZ (0.01 mg and 0.05 mg l-1) for shoot elongation. MS supplemented with 0.2 mg l-1 BAP was found to be better in terms of per cent explant responding (94%). However, when mean number of shoots elongated per explant was considered MS with 0.05 mg l-1 TDZ was (5.4) found better. Therefore MS+0.05 mg l-1 TDZ was considered for shoot bud elongation in further experiments (Table2 and Fig e). Further, when shoot initials were cultured without cotyledon on elongation medium, callus was observed at the cut end and shoot elongation was very slow. On the other hand when shoot initial were cultured along with cotyledon, shoot elongation was faster and callus was not observed. In the present study, MS medium was found more responsive for rooting (88%) but

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shoots had less number of roots and no rootlets. Thus various levels of indole butyric acid (IBA) or naphthalene acetic acid (NAA) (0.1, 0.2, 0.5 mg l-1) were tried with MS basal (Table 3). Among the various levels of IBA and NAA used 0.2 mg l-1 IBA was found better in inducing roots (75% response with well developed roots and rootlets). The roots produced on MS supplemented with NAA were short, thick but per cent response was very low (25 to 35%). The roots were often associated with the callus. In contrast to this when IBA was used in the medium roots were thin, long and with many rootlets. Superiority of IBA over NAA and IAA was also reported by Geetha et al. (1998) and Lawrence and Koundal (2001). Transformation Development of Agrobacterium mediated in vitro transformation protocol required efficient regeneration protocol. In our laboratory it has been noticed that routine protocols standardized for multiple shoot regeneration in pigeonpea do not work as such when explants are cocultured with A. tumefaciens. Therefore, modification in the standardized regeneration protocol is necessary. In view of this certain modification are usually made in the standardized regeneration medium to have efficient regeneration after cocultivation (Geetha et al., 1999; Lawrence and Koundal, 2001). Cocultivated CNC cultured on MS+2 mg l-1 BAP+300 mg l-1 cefotaxime produced mean number of 2.55 shoots and 3.4 shoot buds per explant. However non cocultivated CNC produced mean number of 3.44 shoots and 6.225 shoot buds. Trend of CNC producing more multiple shoots and shoot buds on MS+2 mg l-1 BAP remained same in both conditions. Therefore, this combination was chosen for cocultivation. The test was conducted to know the effective lethal level of kanamycin on different explants during shoot bud initiation and further growth stages (Table 4). This will allow
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preferential growth of the transformed tissues containing nptII gene. The sensitivity to kanamycin differs with genotypes (Geetha et al. 1999; Lawrence and Koundal, 2001) and levels ranged from 25 to 100 mg l-1. In the present study shoots and shoot buds were transferred to selection media containing 50 mg l-1 kanamycin and 75 mg l-1 kanamycin, respectively. The surviving shoots in the first step were subcultured on the same medium and elongated shoot buds were shifted to 50 mg l-1 kanamycin for second level of selection. The level of kanamycin was reduced according to growth stage of explant. Repeated selection on kanamycin was done to eliminate escapes and resistant shoots were analysed further. Among four levels of cefotaxime tested (100, 200, 300 and 400 mg l-1) , 300 mg l-1 was the minimum level to control Agrobacterium effectively. This finding is in agreement with Geetha et al. (1999), Arundhati (1999) and Lawrence and Koundal (2001), who observed effective inhibition of Agrobacterium at 300 mg/l in all the explants. Although cefotaxime is supposed to be non toxic to plant tissues it inhibited root growth in the present study. Thus, in the rooting media cefotaxime was eliminated. This finding is in accordance with Geetha et al. (1999) and Lawrence and Koundal (2001) who used selective rooting media (MS + 25 mg l-1 kanamycin + 0.2 mg l-1 IBA) without cefotaxime. Coculviation period influences survivability of explants. In the present investigation, survival of cocultivated explants with Agrobacterium was maximum upto two days beyond which explants did not survive because of excessive growth of Agrobacterium. In pigeonpea, Arundhati (1999) reported increased frequency of transformation from 4 day cocultivated leaf discs (47.8%) over 2 day cocultivated leaf discs. Pre-culture of explants on regeneration medium prior to cocultivation

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may play an important role in transformation. Preincubation for 24 hr was followed in pigeonpea (Geetha et al., 1999; Lawrence and Koundal, 2001), chickpea (Kar et al., 1996). In the present investigation preculturing of explants increased the survivability after cocultivation over direct cocultivated explants. Explants after cocultivation were cultured on MS along with 2 mg l-1 BAP and 300 mg l-1 cefotaxime without kanamycin for 15 days. Such regenerated shoot and shootbuds were transferred to selection media viz., MS supplemented with 0.2 mg l-1 BAP, 75 mg l-1 kanamycin and 300 mg l-1 cefotaxime for shootbuds and only 50 mg l-1 kanamycin for shoots in two sub cultures(Table 5 and Fig. J). Surviving shoots in first selection were selected once again in the second sub culture to avoid escapes Several technique are being used to confirm the presence of transgene viz., Southern blotting, Dot bloting, Northern blotting, Immuno blotting PCR etc. Of these PCR based techniques with transgene specific primers are the easiest methods to detect the presence of transgene. Survived shoots were kept for rooting and DNA extracted from leaf sample at this stage was used for PCR analysis. Shoots were selected on elongation medium at 75 mg l-1 kanamycin (Fig . I) (Table5). In two cycles of subculture only two shoots survived and were transferred to rooting medium (MS + 0.2 mg l-1 IBA + 50 mg l-1 kanamycin) (Fig . k). Leaf samples from these shoots were used for DNA extraction and PCR analysis. Presence of the npt II specific band 700bp in transformants, positive control and its absence in negative control and in untransformed plant confirmed the presence of transgene in the tested plants(Fig. L).Frequency of transformants was more among tested plants when selection started at bud initiation stage, (66%) followed by only at elongation stage (50%). And in total tranformation efficiency is 1.4% (Table 6). The authors thank Dr. M. K. Mishra, Seed production officer Coffee Board, and Dr.
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Jayarama DR Coffee Board, Dr. Santharama, HDB, Coffee Board for their encouragement and critical evaluation of the manuscript. REFERENCES Anonymous. 1978. The pulse pigeonpea. In Annual Report for 1978-79, International Crop Research Institute for Semi Arid Tropics, Patancheru, Hyderabad, Andhra Pradesh, India, pp.105 Arundhati, A. 1999. Agrobacterium mediated transformation of pigeonpea (Cajanus cajan L. Millsp.) by using leaf disks. International Chickpea and Pigeonpea Newsletter. 6: 62-64. Edwards, K., Johnstone, C., Thompson, C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research. 19: 1349 Franklin, G., Jeychandran, R., Melchias, G., Ignacimuthu, S. 1998. Multiple shoot induction and regeneration of pigeonpea (Cajanus cajan (L.) Millsp) Cv. Vamban 1 from apical and axillary meristem. Current Science. 74: 936-937. Geetha, N., Venkatachalam, P., Prakash, V., Lakshmisita, G. 1998. High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeonpea (Cajanus cajan L.). Current Science. 75: 1036-1041. Geetha, N., Venkatachalam, P., Lakshmisita, G. 1999. Agrobacterium-mediated genetic transformation of pigeonpea (Cajanus cajan L.) and development of transgenic plants via direct organogenesis. Plant Biotechnology. 16: 213-218. Gomez, K.A., Gomez, A.A. 1984. Statistical Procedures for Rice Research Workers. Publication by International Rice Research Institute, Manila, Philippines, pp.196-211.

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Grieve,T.M., Gartland,K.M.A, Elliot, M.C. 1997. Micropropagation of commercially important sugarbeet cultivars. Plant growth Reg. 22: 15-18. Hood, E.E., Gelvin, S.B., Melchers, L.S., Hoekema, A. 1993. Agrobacterium helper plasmid for gene transfer to plants. Transgenic Research. 2: 203-289. Kar, S., Johnson, T.M., Nayak, P., Sen, S.K. 1996. Efficient transgenic plant regeneration through Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Plant Cell Reports. 16: 32-37. Lawrence,P.K., Koundal, K.R. 2001 Agrobacterium tumefaciens mediated transformation of pigeonpea (Cajanus cajan L. Millsp) and molecular analysis of regenerated plants. Current Science. 80: 1428-1432. Murashige, T., Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiologia Plantarum. 15: 473479. Nayak, P., Basu, D., Das, S., Basu, A., Ghosh, D., Ramakrishan, N.A., Ghosh, M., Sen, S.K. 1997. Transgenic elite indica rice plant expressing cry1A(c) AC-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Seripophaga intertulou). In Proceedings of National Academy of Science. 94: 2111-2116.

Nene, Y.L., Hall, S.D., Sheela, V.K. 1990. The Pigeonpea. CAB International, Cambridge, United Kingdom, pp.120-130. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989. Molecular Cloning A Laboratory Manual, Second Edition. Cold Spring, Harbor Laboratory Press, pp.103-107 Singhal, V. 1999. Handbook of Indian Agriculture. Vikas Publishing House Private Limited, New Delhi, p.10. Shivaprakash, N., Pental, D., Sarin, N.B. 1994. Regeneration of pigeonpea (Cajanus cajan) from cotyledonary node via multiple shoot formation. Plant Cell Reports. 13: 623-627. Zhong, Z., Smith, H.G., Thomas, T.H. 1993. In vitro culture of petioles and intact leaves of sugarbeet ( Beta vulgaris) Plant growth Reg. 12: 59-66. Zhang, C. L ., Chen, D.F., Elliot, M .C. and Slater, A. 2001, Thidiazuron induced organogenis and somatic embryogenesis in sugarbeet ( Beta vulgaris.L) In vitro cell. Dev. Boil-plant. 37: 305-310.

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Table 1. Effect of various levels of BAP and TDZ on per cent response , shoot and shoot bud differention from different explants in pigeonpea.
Shoots per explant(average) Mean Shoot tip CN CNC CNC Mean Shoot tip CN CNC CNC Shoot buds per explant(average) Mean

Per cent response CNC

Media

Shoot tip 97.5 0.00 0.00 0.900 1.850 1.90 1.162


b

CN

CNC

MS 0.470 0.20 0.975 1.70 1.500 1.80 0.00 0.675 2.20 100.0 65.00
b

0.0

25.0

85.0

51.87

0.942 e

3.375 4.500

1.562d 1.843 c

MS + 1.0 mg l-1 BAP 100.0 75.625 a 0.00 1.400 1.950 3.00 0.40 1.475 a 2.050

10.0

55.0

95.0

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MS + 2.0 mg l-1 BAP 92.5 52.50


d

25.0

80.0

97.5

3.175

6.225

2.962 a

MS + 3.0 mg l-1 BAP 0.00 0.325 1.825 2.20 87.5 52.50


d

0.0

25.0

92.5

1.087 c

0.00

0.400

2.175

3.070

1.411 e

276
0.00 0.375 1.375 1.62 1.186 90.0 55.62
cd

MS + 4.0 mg l-1 BAP 0.00 0.225 1.225 1.57

0.0

30.0

92.5

0.00

0.375

1.650

1.750

0.943

MS + 0.01 mg l-1 12.5 TDZ 92.5 58.75 c 0.05 0.330 0.950

25.0

95.0

1.091 f

0.37

0.275

2.425

2.175

1.311 e

MS + 0.05 mg l-1 30.0 TDZ 100.0 56.05 c 0.00 2.250

25.0

87.5

0.92

1.073 f

0.63

0.125

1.225

2.475

1.088 f

MS + 0.1 mg l-1 TDZ 90.0 50.625


cd

9.2

25.0

90.0

1.400

2.17

1.450 e

0.315

0.500

2.275

4.050

1.424

MS + 0.5 mg l-1 TDZ 0.00


b

10.0

7.5

95.0

0.000

0.175

0.07

0.061

0.160

0.225

1.575

2.075

1.007

fg

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Mean 94.44 57.48

10.74

33.05 c

92.22

0.005

0.697 c

1.361

1.69 a

0.938

0.230

0.622 c 2.286

3.299 a

1.500

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Table 2. Effect of lower levels of BAP or TDZ on shootbud elongation

Media

Average number of elongated shoots per explant

Per Cent

MS + 0.1 mg l-1BAP MS + 0.2 mg l BAP MS + 0.01 mg l-1TDZ MS + 0.05 mg l-1TDZ


-1

3.200 d 3.920
c

80.00 95.00 85.00 87.50

4.500 b 5.746 a

Table 3. Effect of different levels of IBA or NAA on rooting

Media Half MS MS MS + 0.1 mg l-1 IBA MS + 0.2 mg l-1 IBA MS + 0.5 mg l-1 IBA MS + 0.1 mg l-1 NAA MS + 0.2 mg l-1 NAA MS + 0.5 mg l-1 NAA

Per cent respose 42.85 e 88.00 a 65.00 d 75.00 b 70.00 c 35.00 f 25.00 g 35.00 f

Table 4. Effect of kanamycin on inhibition of growth at various stages

Level of kanamycin (mg l-1) 0 25 50 75 100

Shoot bud

Elongation

Rooting

+++ +++ +++ ++ +

+++ +++ ++ + -

+++ + -

Figures are average of 20 explants in each treatment +++ No inhibition of growth + More inhibition of growth ++ Less inhibition of growth
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Table 5. Selection of putative transformants during shoot elongation on selective medium (MS + 0.2 mg l-1 + 300 mg l-1 cefotaxime + 75 mg l-1 kanamycin)

Explants
CNC Control CNC CNC Control CNC

Cultured
96 (196) 10 (30) 108(250) 15 (20)

Responded
17 2 7 0

shoots survived shoots survived After 30 days After 60 days


25 3 16 0 6 0 4 0

Figures in parenthesis show total shoot buds (visible) Table 6. Conformation of the transformants using specific PCR
Explants cultured 300 Selection at various stages Further elongation (Kan 50) Elongation (Kan 75) Shoot bud ( Kan 100) Control Putative transformants 2 nptII positive shoots rooted 2 Number of of growth 0

500 200 50

10 6 2

8 4 0

1 0 0

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Fig 1. Regeneration of Pigeonpea

Legend of the Fig. 1 (Regeneration of Pigeonpea)


A.. One week old seedlings a .germinated on half strength MS + 2 mg l-1 BAP b. germinated on half strength MS Shoot B. Shoot bud induction from different explant on MS+ 2 mg l -1 BAP 30 days after culture a. cotyledonary node.b shoot tip. c. half cotyledon with cotyledonary node d. cotyledon with cotyledonary node (CNC) C. Shoot and shoot bud induction from CNC on MS media with different levels of BAP 30 days after culture a. 1 mg l-1 b. 2 mg l-1 c 3mgl-1 d. 4 mg l-1 D Shoot and shoot bud induction on MS media with different levels of TDZ 30 days after incubation a. MS basal b. 0.01 mg l-1 c. 0.05 mg l-1 d. 0.1 mg l-1 e.0.5 mg l-1 E. Shoot bud elongation on MS media with TDZ 0.05 mg l-1 30 days after incubation

F. Rooting of elongated shoots on different levels of IBA and NAA a.0.2 mg l-1 IBA b.0.5 mg l-1 IBA c.0.1 mg l-1 NAA d.0.2 mg l-1 NAA e.0.5 mg l-1 NAA G. Hardened rooted plants H. Plants transferred to big pots I. Putative transformants selected during shoot elongation stage on MS + Kan75 + 0.05 mg l-1 TDZ + cef300 30 days after incubation a and b.Putative transformant c.Untransformed d.Control J. Putative transformants selected during shoot and shootbud initiation stage on MS + Kan100 + 2 mg l-1 BAP + Cef300 20 Days after incubation a and b.Putative transformant c.Control K. Rooting of putative transformants on MS + 0.2 mg l-1 IBA + Kan50 after 20 days after incubation a.Root initiation b.Root elongated L. Agarose gel electrophoresis showing nptII fragment in the transformed plants, a b c Transformed plants. d untransformed plant e control plant f. plasmid g. marker Hind III digest.

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DIRECT ORGANOGENESIS AND SOMATIC EMBRYOGENESIS IN PIGEONPEA (CAJANUS CAJAN L. MILLSP.)


Josnamol Kurian, K Ramakrishnan, R Gnanam and A Manickam

ABSTRACT
Pigeonpea (Cajanus cajan L. Millsp.) is an important grain legume crop, and a good source of dietary protein in the tropics and subtropics. One of the major problems in pigeonpea cultivation is pod borer (Helicoverpa armigera) which causes extensive damage to the crop. Attempts to obtain pest-resistant genotypes of pigeonpea by conventional breeding methods have given limited success due to narrow genetic variation, and sexual incompatibility with wild relatives. The availability of a reliable in vitro regeneration protocol is a pre-requisite for the application of most biotechnological techniques such as production of transgenic plants with suitable gene(s). Attempts were made to develop protocols through somatic embryogenesis and organogenesis pathways. In direct organogenesis approach, mature, aseptic seeds showed numerous green adventitious shoot initials from the swollen cotyledonary nodal region within 3 days of culturing. The presence of seed coat, in both imbibed and non-imbibed seeds, drastically affected the differentiation of shoot-buds. Seedlings developed from imbibed or non-imbibed decoated seeds exhibited stout seedlings but with stunted growth. Seedlings raised from non-imbibed seed, with seed coat intact, failed to produce multiple shoot-initials; instead, differentiated clusters of leafy structures appeared which suppressed further morphogenesis. However, imbibed seed with intact seed coat differentiated to form maximal adventitious shoot buds in the cytokininsupplemented medium. For callus induction, different combinations of hormones were tried with 2, 4-D (1.0, 1. 5, 2.0 mg/l), NAA (1.0, 1.5, 2.0 mg/l), TDZ (1.0, 1.5, 2.0 mg/l) etc. Application of 2, 4-D (2.0 mg/l) alone was found ideal. Suspension cultures of calli derived from10-day-old primary leaves of in vitro grown Cajanus cajan L. (var. Vamban 2) produced somatic embryos. The highest embryogenesis frequency was observed on MS+B5 medium supplemented with 2, 4-D 2mg/l, casein hydrolysate 100mg/l and L-Glutamine 50mg/l. Maximum somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with reduced amount of 2, 4-D (0.5mg/l). Studies on ontogeny of somatic embryos showed that the cells determined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos.

Tamil Nadu Agricultural University, Coimbatore 641003

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SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE INFLORESCENCE AND LEAF EXPLANTS OF SORGHUM CULTIVARS
Kumaravadivel, N1., M.Umadevi1 and Susan Eapen2

ABSTRACT
Sorghum is one of the important grain and fodder crops in many parts of the world. In the present study, in vitro response of immature inflorescence and young leaf in sorghum genotypes was evaluated. The basal medium used for callus induction and regeneration was MS with different levels of auxins and cytokinins. Two types of calli were identified after 15 days of inoculation. One was white, compact, embryogenic nodular callus and another one was yellow, non-embryogenic unorganized callus. Out of the different levels of 2,4 -D and kinetin used for callus induction, the combination of 2,4-D 2.0 mg/l and kinetin 0.5 mg/l was found to be suitable for callus induction. The callus induction frequency was up to 80% for immature inflorescence and was up to 86% for young leaf explant when cultured in vitro. The percentage regeneration efficiency of embryogenic calli from Co 27 was found to be greater (85%) than that of calli derived from other genotypes. For regeneration, MS medium with NAA 0.1 mg/l, BAP 2.0 mg/l and casein hydrolysate levels of 500 mg/l was found to be the best combination. The effect of various factors such as growth regulators, charcoal, iron, proline, nitrogen source, etc., were also studied. The regenerated plants were transferred to green house condition.

Introduction Sorghum the great millet is an important crop, which has a special agronomic importance because of its multi product and diversified usage as food, feed and fuel. It is well adapted to a wide range of soil types and environmental conditions, especially drought. Using conventional breeding techniques, much progress has been made in developing superior cultivars (Smith and Bhaskaran, 1986). To supplement these efforts, tissue culture in four sorghum cultivars (CO 25, CO 26, CO 27 and COS 28) including both grain and fodder sorghum has been initiated. High frequency plant regeneration from cultured tissues is a pre-requisite for successful application of in vitro culture for crop improvement. In vitro derived plants will be useful to generate somaclonal variation. The different hormonal combinations, the type of explants, effect of activated charcoal, proline and type of genotypes are the critical factors that influence the in vitro plant regeneration. Wen et
1. Tamil Nadu Agricultural University, Coimbatore 641 003 2. Bhabha Atomic research centre, Mumbai 400 085

al. (1991) and Cai and Butler (1990) used immature inflorescence as explants. Young shoot was used as explants for plant regeneration by Devi and Sticklen (2001). The present investigation was therefore undertaken (i) to assess the comparative performance of the hormonal combination and levels, (ii) to find out the suitable explant size and age, (iii) to study the effect of activated charcoal and proline and (iv) to find out the genotype showing maximum frequency of callus induction and regeneration. Materials and Methods Selection of expla]nts and surface sterilization Immature inflorescence and young leaf of sorghum genotypes Co.25, Co 26, Co 27 and CoS 28 were used for callus induction and regeneration. The explants were collected from 60 70 day old plants. After all the outer leaves were removed, the explants were surface sterilized with 0.1% (v/v) HgCl2 for 2-3 min
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and then rinsed thrice with sterile double distilled water. The surface sterilization steps were done aseptically. Experiments conducted Effect of hormonal levels, explants and genotypes Five different hormonal combinations were used for callus induction and three different hormonal combinations were tried for plant regeneration. Different expants (young leaf and immature inflorescence) of genotypes viz., Co 25, Co 26, Co 27 and CoS 28 were cultured for callus induction on Murashige and Skoog (1962) medium and incubated in darkness at 26oC for callus induction. They were subcultured every two weeks and maintained at 26oC for callus induction. For initiation of callus, the explants were cultured on MS medium with 2,4-D (1.0, 2.0, 2.5, 3.0 and 4.0 mg/l) and kinetin (1.0 mg/l). For regeneration of plants, the MS medium was supplemented with a combination of 6benzylaminopurine (1.5, 2.0 and 2.5 mg/l) + 0.01 mg/l NAA and incubated in the light under a 16 h photoperiod under white fluorescent light. Effect of activated charcoal and Fe EDTA Activated charcoal is a well known absorption agent. The positive effect of activated charcoal includes: 1. absorbing phytotoxins released by the tissue in culture (Brettell et al., 1980) and 2. absorbing inhibitory substances in the media and 3. absorbing gases such as ethylene that is present in cultures. Sorghum somaclones generally have a low establishment rate due to their underdeveloped root systems. Considering these effects, media have been developed for routine use with the incorporation of activated charcoal. Immature inflorescence was cultured on MS medium supplemented with 2 mg/l 2,4-D, 1 mg/l kin with different concentrations of activated charcoal (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 g/l) and double the doses of Fe-EDTA.
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Effect of proline and nitrogen source The development of tissue culture protocols for embryogenic callus induction and plant regeneration is imperative for successful application of tissue culture technology for crop improvement. In the present study, we have investigated the influence of proline on plant regeneration from immature inflorescence of two different genotypes of sorghum namely Co 26 and Co 27. The various levels of praline (1g, 2g and 3g/l) were tried to find out the optimum concentration for callus induction and regeneration. Immature inflorescence was cultured on MS (four different levels) and ratios of NH4+ (370, 1130 mg/l) and NO-3 (2500, 4500 mg/l), ions with without organic nitrogen (proline 2 mg/ l). The differences in NH 4 + and NO 3 concentrations were obtained by changing the conc. of KNO3 and NH4NO3 in the basal MS medium. Result and Discussion Callus induction The immature inflorescence and young leaf segments showed a general expansion followed by appearance of visible callus within 10-15 days of culture (Table 1). In all sorghum genotypes, callus induction was found to be high with young leaf in all treatments. Nature of the embryogenic calli is highly dependent on explant age, size and medium composition. The immature inflorescence was collected from plants before the emergence of the inflorescence at the boot leaf stage. The immature inflorescence was dissected and those less than 2 cm long were selected and cut into 3-5 mm, 5-10 mm and 1015 mm segments. Embryogenic callus formed after 25-40 days of culture. The callus induction was very much dependent on age of immature inflorescence. The callus was creamy white and compact in the initial stage and became very nodular in later stage.

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When the length of the inflorescence ranged from 3-5 mm, callusing was extremely poor (Table 1). When inflorescence length varied between 5-10 mm, maximum callus was obtained. Inflorescence of 10-15 mm produced non-embryogenic, friable callus from the cut end of rachis. Somatic embryogenis was strictly dependent on the age of the inflorescence and embryos were induced only when inflorescence length ranged from 5-10 mm. No somatic embryogenesis was observed when inflorescence was younger or older (Brettel et al., 1980).
Table 1. Effect of various levels of 2,4-D and kin (0.5 mg/l) on callus induction (%)

Regeneration The experiments conducted to study the hormonal levels on regeneration are presented in Table 3. MS medium with NAA (0.1 mg/l), BAP (2.0 mg/l) and casein hydrolysate (500 mg/l) showed the highest per cent of shoot regeneration. In the medium supplemented with BAP, embryoids turned green and produced a huge clump of shoots and roots. The highest frequency of regeneration was obtained from Co 27 immature inflorescence (86%) than the other genotypes. The rooted plantlets grew satisfactorily in soil in paper cups and also in glass house condition. Forty out of 83 transplanted plants were transferred to field condition and regenerants were successfully established.
Table 3. Response of calli for regeneration in various combination of media

Genotypes Explant

2,4-D (mg/l)

1.0 2.0 2.5 3.0 4.0 20 45 10 39 50 70 50 76 60 86 68 80 15 40 20 35 65 72 50 70 71 73 20 35 30 40 15 43 48 40 50 50 25 12 60 47 35 13 40 10

CO 25

L I

CO 26

L I

Regene- % of Explants ration regenemedium ration

Growth response

CO 27

L I

CO 28

L I

Young leaf callus Co 25 RGM 1 67 Co 26 RGM 1 75 Co 27 Co 25 RGM 2 RGM 2 83 70

L Young leaf I Immature inflorescence Table 2. Influence of inflorescence length on callus induction, somatic embryogenesis and plant regeneration in sorghum

Green plantlets were noticed after 20 25 ays Green plants were observed along with rooting

No. of Length of Intensity cultures Average of the showing number of callusing somatic plants/ inflorescence (mm) embryog- culture enesis 3-5 5-10 10-15 *** ** 0 15-20 5 0 5 0

Immature inflorescence Complete plants Co 25 RGM 1 67 were derived Co 26 RGM 2 60 from compact Co 27 RGM 2 85 calli Co 28 RGM 1 75 Effect of charcoal and Fe-EDTA Immature inflorescence of sorghum varieties viz., CO 26, CO 27 were used as the source material. The explant showed signs of callusing after 15 days after incubation on MS medium supplemented with 2,4-D 2.0 mg/l and
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*** Excellent callus initiation ** Moderate callus initiation - No callus initiation

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kinetin 1.0 mg/l. The addition of activated charcoal to the callus induction medium increased the percentage of callus in two genotypes. The per cent of callus was maximum at 0.4 g/l activated charcoal and double dose of Fe-EDTA in CO 26 inflorescence explant (Table 4). Regeneration was achieved by incubating calli on MS medium with 0.1 mg/l NAA, 2 mg/l BAP with 0.4 g/l activated charcoal. The maximum regeneration (80%) was observed both the genotypes of immature inflorescence calli.
Table 4. Effect of activated charcoal and Fe-EDTA on callus induction (%)
Activated charcoal (g/l) Geno-Explant types Co 26 Immature inflorescence Co 27 inflorescence 0.1 0.2 0.3 0.4 0.5 Fe-EDTA Single dose 48 Double dose 86

Table 5. Influence / Effect of nitrogen sources on callus induction (%)


Medium NH4+ (mg/l) NO 3 - O r g a n i c Callus (mg/l) nitrogen induction g/l (%) (Proline) 2500 2500 4500 4500 1 1 2 2 25.02 29.42 45.42 51,58

MS 1 MS 2 MS 3 MS 4

370 370 1130 1130

field. The effect of various factors viz., genotype, explant and tannin exudation should be taken utmost care. In vitro culture studies will enable the generation of somaclones, basic genetic studies, in vitro selection and in vitro mutagenesis. In vitro studies enhance genetic diversity and helps in the development of transgenic plants. REFERENCES

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83

80

60

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Influence of nitrogen sources on callus induction We found that addition of amino acids to basal MS medium was more effective than increasing the concentration of inorganic nitrogen for embryogenic callus induction (Table 5). Compared with MS, the medium with double the concentration of NO3- (4500 mg/l), treble the concentration of NH4+ (1130 mg/l) and the addition of organic nitrogen (proline 2 g/l) was more effective in stimulating embryogenic calli growth. The increase in concentration of NH4+ (or) NO3- in the media supplemented with organic nitrogen was also less effective. This shows that both sources of inorganic nitrogen and optimal NH4+ : NO3- ratio are important. The necessity of an optimal NH4+ : NO3- ratio for the induction and growth of embryogenic calli has also been shown in the tissue culture of other plant species (Mordhorst and Cortz, 1993). The above study clearly indicated the scope of in vitro culture in sorghum towards successful regeneration of plants and establishment in the
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Brettell, R.I.S., Wernicke, W. and Thomas, E. 1980. Embryogenesis from cultured immature inflorescence of Sorghum bicolor. Protoplasma. 104: 141-148.
Cai,T., and Butler, L. 1990. Plant regeneration from embryogenic callus initiated from immature inflorescence of several high tannin sorghums. Plant Cell Tissue Organ Culture. 20: 101-110. Devi, P.B. and Sticklen, M.B. 2001. Culturing shoot tip clumps of Sorghum bicolor and optimal microprojectile bombardment parameters for transient expression. J. Cytol. Genet. 2: 89-96. Mordhorst, A.P. and Cortz, H. 1993. Embryogenesis and development of isolated barley microspore are influenced by the amount and composition of nitrogen sources in culture media. Journal of Plant Physiology. 142: 485-492. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tissue cultures. Physiol. Plant. 15: 473-497.

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Smith, R.H. and Bhaskaran, S. 1986. Sorghum (Sorghum bicolor K. Moench). In: Biotechnology in agriculture and forestry. (ed.) Y.P.S.Bajaj (Berlin: Springer Verlag), Vol.2, pp.220-233. Wen, F.S., Sorensen, E.L., Barnett, F. and Liang, G.H. 1991. Callus induction and plant regeneration from anther and inflorescence culture of sorghum. Euphytic. 52: 177-181.

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ENGINEERING SHEATH ROT RESISTANCE IN RICE


Rajesh,T., K. Kalpana, S. Maruthasalam, K. Poovannan, R. Samiyappan, D. Sudhakar and P. Balasubramanian

ABSTRACT
The study was aimed at engineering resistance against sheath rot (ShR) caused by Sarocladium oryzae Gams and Hawksworth in a local elite indica rice cultivar, ASD16, using a rice chitinase (chi11) gene. Transgenic rice lines were generated using Agrobacterium-mediated transformation. In T2 generation, three homozygous lines viz., KL-ASD16-4-1-1, KL-ASD16-5-2-1 and KL-ASD16-6-1-1 expressing chitinase were identified through western blotting analysis. These homozygous lines were evaluated for ShR resistance in T2 generation. A small lesion surrounding the brown border was observed after three days of inoculation (D AI) in non-transgenic ASD16 control plants. These lesions slowly spread and developed into characteristic large size oblong greyish lesions within 12 D AI. In the above three lines, no such lesions formed on the third day and small brownish lesions started appearing only on six DAI. These brownish lesions enlarged in size and irregular extensive browning was observed within 12 D AI. A sudden and extensive browning was noticed only in transgenic plants whereas the characteristic oblong greyish lesions were produced in non-transgenic control plants without brownish lesions. The significant reduction of per cent sheath infection was observed in three homozygous lines (2.48, 2.71 and 2.95) over non-transgenic control (18.56). Among the three homozygous ASD16 lines tested, KL-ASD16-4-1-1 performed better and gave an enhanced protection against ShR than the other two lines. The present study suggested that these lines could be utilized as a resistance source against ShR pathogen in rice breeding programmes.

Tamil Nadu Agricultural University, Coimbatore - 641 003 286

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TECHNICAL SESSION VI CONTRIBUTIONS OF GENOMIC TOOLS IN CROP IMPROVEMENT

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Plant Breeding in Post Genomics Era

MOLECULAR BREEDING FOR BROWN PLANTHOPPER (BPH) AND BLAST RESISTANCE IN RICE
Kshirod K. Jena and David J. Mackill

ABSTRACT
The last three decades of the twentieth century have made significant progress in developing semi dwarf, high yielding rice cultivars and have achieved food security in many countries of Asia. However, brown planthopper (BPH) and blast (Bl) disease of rice are a continuous threat to rice production due to changes in biotypes of BPH and pathotypes of Bl. The plant breeders of the 21st century have to combine modern tools of biotechnology with conventional plant breeding tools to identify novel genes that can express broad spectrum of resistance to BPH and Bl. We have identified new sources of BPH resistance in the breeding line, IR65482-7-216-1-2 and Bl resistance in the breeding line, IR654824-136-2-2 using the Korean biotype of BPH and pathotypes of Bl. The new BPH resistance gene, Bph18(t) has been tagged to STS marker through e-landing and high-resolution mapping approach using Nipponbare genome sequence information and BPH bioassay. A major blast resistance gene, Pizau(t) has been identified using e-landing and high-resolution mapping and bio-informatics tools. We have developed advanced breeding lines using marker-assisted backcross (MAB) breeding. The breeding lines possessing resistance to BPH and Bl combining high yielding and superior grain quality traits would be valuable breeding materials for rice improvement.

International Rice Research Institute, Los Banos,4031 Laguna, Philippines 287

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QUANTITATIVE TRAIT LOCI, DNA MARKERS AND CANDIDATE GENES - WHAT DO WE DO WITH THESE?
Shashidhar, H.E.

ABSTRACT
QTL mapping has been a very popular activity among molecular biologists. Hundreds of papers deal with this subject across crops and traits. There are innumerable quantitative trait loci (QTLs) reported for different traits on different chromosomes. Hundreds of molecular maps are being developed in certain crops and thousands of markers being discovered or added. Several of simple sequence repeats, expressed sequence tags and candidate genes are being added to the databases. This is mind boggling to a breeder who is committed to improve the traits he handles. The magnitude of situation is explored and analysed to propose a way out to maximize utility of all these new tools