Paper 1: Elementary Plant Biotechnology 1. 2. 3. 4. 5. 6. 7. Explain the methods and emerging trends of plant genetic transformations.

Describe designing of plant genotypes with enhanced resistance to salinity. Justify the statement Wild species of Oryza as important reservoir of useful alleles. Explain inducible gene expression systems and tool for plant function genomics. Explain the homozygous plant production through anther, ovule, pollen culture. Explain the concept of technology transfer, equipments and producers. Elaborate following terms: a) b) Somaclonal variation Artificial seed production

Paper 2: Plant

Tissue Culture Technology

1. 2. 3. 4. 5. 6.

Explain the historical background of plant tissue culture. Describe somatic embryogenesis and organogenesis in plants. Write down applications, micro propagation in agriculture, horticulture and forestory. Production of disease free plants by tissue culture methods. Explain the concept of totipotency. Elaborate following terms: a) b) Sterilization Somatic hybrids

7.

Explain cybrid formation and its application.

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Salinity is a global problem. The continuous loss of arable land due to irrigation in arid and semi-arid regions of the world, over-exploitation and mismanagement contribute to global change in a way which currently appears to draw much less concern in the media and the general public than the accumulation of green house gases carbon dioxide, methane and others in our atmosphere and putative temperature increase and climate changes associated with them. Nevertheless, the advancement of desertificaton by salinization and its threatening of global agriculture can be readily quantified. Agricultural losses caused by salinity are difficult to assess but estimated to be substantial and expected to increase with time.Secondary salinization of agricultural lands is particularly widespread in arid and semi-arid environment where crop production requires irrigation schemes. At least 20% of all irrigated lands are salt affected with some estimates being as high as 50% whereas the world's population continues to rise, the total land area under irrigation appears to have levelled off. Salinity is one of the most severe environmental factors limiting the productivity of agricultural crops. Most crops are sensitive to salinity caused by high concentration of salts in the soil. The cost of salinity to agriculture is estimated conservatively to be about US$ 12 billion a year, and is expected to increaseas soils are further affected (Ghassemi et al., 1995). In addition to this enormous financial cost of production, there are other serious impacts of salinity on infrastructure, water supplies, on soil structure and stability of communities. Responses to salinization have been of many general kinds;engineering the environment to manage increased salt in the soil irrigation anddrainage management; by "engineering" the plants to increase their salt tolerance. Salt tolerant plants may also ameliorate the environment by lowering the water table in salt affected soils. Salinization commonly occurs as an outcome of agricultural practices, either associated with irrigation or due to long term changes in water flow in the landscape that can follow land clearance or changed water management. Salinization associated with agriculture occurs when salts build up in the root zone, either because the soil is intrinsically saline, or because the drainage of water from the sub-soil is not sufficient to prevent saline waters rising into the root zone. It therefore tends to be common in arid and semi-arid regions where leaching of salt is poor due to lower rainfall; where there are strongly saline sub-soils formed from marine deposits or where irrigation changes water tables and salt flow.

Saline soil refers to a soil that contains sufficient soluble salts to impair its productivity. Similarly, alkali soils can be defined in terms of productivity as influenced by exchangeable sodium. So,alkali soils may or may not contain excess soluble salts. The soluble salts that occur in soils consist mostly ofvarious proportions of the cations sodium, calcium and magnesium, and the anions chloride and sulphate. Constituents that ordinarily occur only in minor amounts are the cation potassium and the anions bicarbonate, carbonate and nitrate.

Salinity is a major threat to irrigated agriculture because many of the soils and irrigation waters contain significant amount of dissolved salts. Due to excessive evapotranspiration in arid and semi-aridregions, the secondary salinization is becoming important factor for salinity.

oil salinity is widely reported to be a major agricultural problem, particularly in irrigated agriculture, and research on salinity in plants has produced a vast literature. However, there are only a handful of instances where cultivars have been developed which are resistant to saline soils. Reasons for the lack of success in developing salt-resistant genotypes, and for the low impact that plant physiological research has made, are explored. We conclude that soil salinity has not yet become a sufficient agricultural problem, other than on a local scale, to make salt resistance a high priority objective for plant breeders. The limited success of simple selection, where this has been practised in breeding programs, can be accounted for by the fact that research has consistently shown salt resistance is a complex character controlled by a number of genes or groups of genes and involves a number of component traits which are likely to be quantitative in nature. We also conclude that the results of physiological research have been poorly marketed by physiologists and, understandably, have failed to impress plant breeders. We anticipate that the importance of salinity as a breeding objective will increase in the future. Our assessment of reports of the degradation of irrigation systems, together with projections of the future demands of irrigated agriculture, is that enhancing the salt resistance of at least some crops will be necessary. Salinity resistance will both help provide stability of yield in subsistence agriculture and, through moderating inputs, help limit salinisation in irrigation systems with inadequate drainage. It is emphasised that plant improvement and drainage engineering should be seen as partners and not alternatives. We conclude with a personal view of one way forward for developing salt-resistant genotypes, through the pyramiding of physiological characters.

Recombination Strategy

The recombination strategy includes the i) grouping of the genotypes based on the predominant inherent physiological mechanism responsible for salinity tolerance, ii) intermating of the genotypes with high degree of expression of the contrasting salinity tolerance mechanism, and iii) identifying / screening of the recombinants for pooling of the mechanisms to enhance the further level of salt tolerance. Crosses are made between the groups (rather than within the groups) possessing contrasting physiological traits like tissue tolerance, Na+exclusion, K+ uptake and Clexclusion to pyramid the genes governing or contributing for salinity tolerance into one agronomically superior background. The ideal high yielding salinity tolerant variety should posses following traits: Able to withstand high amount of Na+ (tissue tolerant) Per day uptake of Na+ is minimum (takes more number of days for LC50 stage) High uptake of K+ per day Good initial vigour Agronomically superior with high yield potential

8.3 Recent Strategies to supplement the conventional breeding programs: a)Somaclonal Approach: Pokkali type of rice varieties are highly salt-tolerant and commonly grows in coastal areas of Kerala State, India. It is a traditional, tall, susceptible to lodging, photoperiod-sensitive rice variety with low tillering and long, broad, dark, and droopy leaves. It has red pericarp and poor grain quality. It is highly tolerant of salinity but yielding ability is low. Pokkali was subjected to cell culture for induction of somaclonal variation. Somaclonal variants of Pokkali with improved agronomic traits were identified. The variant isolated (TCCP 266-249-B-B-3) had desirable levels of all the characteristics tested. Its level of tolerance for salinity remains as high as the donor. It has vigorous growth and, unlike the parent Pokkali, is semi-dwarf, a trait essential in increasing the yield potential without lodging. The variant had white pericarp which is a distinct advantage. There was also improvement in cooked rice quality, with the semi-dwarf variant having medium consistency. These characteristics make the somaclonal variant TCCP 266-2-49-B-B-3 superior to Pokkali as a donor of salt tolerance in hybridization programs. It has become a popular donor parent and has produced new high-yielding salinity-tolerant lines, some of which were already released as varieties. b) F1 anther culture (AC)-derivatives Development of a promising salinity-tolerant line through hybridization and selection procedure could take atleast 8-10 years where more than one rice crop can be taken in a year, while it takes much more time where only one crop per year is grown. This period could be substantially reduced by applying the F1 anther culture technique. In 1996, some high-yielding salt-tolerant anther culture-derived lines were generated at IRRI. It only took 3 years for the tolerant lines to be isolated. These AC-derived lines were IR51500AC11-1, IR51500-AC17, IR51485-AC6534-4, IR72132-AC6-1, IR69997-AC1, IR69997AC2, IR69997-AC3, and IR69997-AC4. IR51500-AC11-1 was released as a salt-tolerant variety in the Philippines with the name PSBRc50 or Bicol. This is the first time for anther culture-derived lines from indica-indica cross to be released as a variety and also the first to be recommended for cultivation in adverse environments. IR51500-AC17 and IR51485-AC6534-4, named CSR21 and CSR28, respectively, were also identified as tolerant rice varieties in India.

c) Marker Assisted Selection (MAS) DNA MAS has been seen as a means of improving the speed and efficiency of plant breeding programs because it is growth stage independent, unaffected by environment; no dominance effect and efficient to use in early generations. Most widespread use of MAS to date has done in the marker assisted backcrossing (MAB) of major genes to into already established varieties, mega-varieties (which occupies a large area within the country on across the countries) or elite cultivars. These markers could reduce the linkage around the target gene, and also recover the recurrent parent background within less number of generation in comparison to conventional breeding. IRRI has already using marker assisted selection for saltol QTL, responsible for seedling stage salinity tolerance in rice, to facilitate the breeding for salt tolerant cultivars.

In a world where population growth is outstripping food supply agricultural -and especially plantbiotechnology, needs to be swiftly implemented in all walks of life. Achievements today in plant biotechnology have already surpassed all previous expectations, and the future is even more promising. The full realisation of the agricultural biotechnology revolution depends on both continued successful and innovative research and development activities and on a favourable regulatory climate and public acceptance. Biotechnology should be fully integrated with classical physiology and breeding: (1) as an aid to classical breeding, (2) for generation of engineered organisms, (3) for integration of microorganisms into agricultural production systems. Biotechnology is nowadays changing the agricultural and plant scene in three major areas: (1) growth and development control (vegetative, generative and reproduction/propagation), (2) protecting plants against the ever-increasing threats of abiotic and biotic stress, (3) expanding the horizons by producing specialty foods, biochemicals and pharmaceuticals.

Abiotic and biotic stress tolerance

The application of molecular genetics and plant transformation to the diagnosis and control of plant pests has become one of the major practical success stories of plant biotechnology in the past decade. The availability of dozens of transgenic crop plants which are resistant to a range of insects, viruses and herbicides, as well as to several phytopathogenic fungi and nematodes has been validated under both field and laboratory conditions, and is of great economic importance. Moreover, applying the principles of engineering plants for resistance to these pests to other plants of agricultural importance is now considered routine, although in practice still laborious, especially for new genotypes. Apart from a wider application to additional plants, the real challenges lying ahead include: 1. improved expression of the target genes in the plants, especially their spatial and temporal control, 2. the use of wide-spectrum and alternative target genes to circumvent the problem of pest resistance, 3. intensified integration of biological control via the use of selected and engineered microorganisms with a biocontrol potential. While plant biotechnology has been applied successfully to fighting a large number of pests, this is not yet the case for abiotic stress conditions such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress. Drought and salinization are the most common natural causes of lack of food and

famine in arid and semiarid regions, and the most serious environmental threats to agriculture in many parts of the world. Desertification, resulting from overexploitation by the local inhabitants, is often aggravated by regional climatic changes, and results in increased soil erosion and a decrease in land and agricultural productivity. It is estimated that increased salinization of arable land will have devastating global effects, resulting in 30 % land loss within the next 25 years, and up to 50 % in the year 2050. Although more difficult to control and engineer than the usually monogenic traits of resistance to biotic pests and herbicides, the genetically complex response to abiotic stress is globally and regionally far more important. Therefore, breeding for plant tolerance to drought and salinity stress should be given a high research priority in all future agbiotech programs. Strategies for the manipulation of osmotic stress tolerance in plants might include: expression of osmoprotectants and compatible solutes, ion and water transport and channels, expression of waterbinding and membrane-associated dehydrins and other proteins, transcription factors and DNA-binding proteins, etc. Also of specific interest are the intervening stages of stress perception, signal transduction (ABA and others), and protein modification. The discovery of new stress-related genes and the design of stress-specific promoters are equally important.

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