CHARACTERIZATION OF SESAME GENOTYPES THROUGH MORPHOLOGICAL, CHEMICAL AND RAPD MARKERS

Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfilment of the requirements for the Degree of

Master of Science (Agriculture)

In

SEED SCIENCE AND TECHNOLOGY

By

Ms. SUHASINI K.S.

DEPARTMENT OF SEED SCIENCE AND TECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005

JULY, 2006

ADVISORY COMMITTEE
DHARWAD JULY, 2006 (N.K. BIRADARPATIL) MAJOR ADVISOR Approved by : Chairman : ________________________ (N.K. BIRADARPATIL) Members : _________________________ 1. (K.G. PARAMESHWARAPPA)

_________________________ 2. (H.L. NADAF)

_________________________ 3. (M.N. MERWADE)

CONTENTS

Chapter No.

Title

INTRODUCTION

II

REVIEW OF LITERATURE

III

MATERIAL AND METHODS

IV

EXPERIMENTAL RESULTS

DISCUSSION

VI

SUMMARY

VII

REFERENCES

VIII

APPENDIX

IX

ABSTRACT

LIST OF TABLES

Table No. 1.

Title

Monthly meteorological data for experimental year 2005-06 of Main Agricultural Research Station, University of Agricultural Sciences, Dharwad Identification and grouping of sesamum genotypes based on plant height, number of primary branches per plant and number of nodes per plant

2.

3. 4. 5. 6. 7. 8. 9. 10. 11 12 13 14 15

Identification and grouping of sesamum genotypes based on internodal length, stem pigmentation and number of leaves per plant Identification and grouping of sesamum genotypes based on leaf length, leaf shape, leaf colour and leaf petiole pigmentation Identification and grouping of sesamum genotypes based on days to 50 per cent flowering and days to maturity. Identification and grouping of sesamum genotypes based on flower petal colour and flower hairiness Identification and grouping of sesamum genotypes based on number of pods for axil, number of pods per plant and pod length Identification and grouping of sesamum genotypes based on pod shape, pod beak, pod pubescence, number of locules per pod and pod dehiscence Identification and grouping of sesamum genotypes based on seed colour, thousand seed weight and oil content Identification and grouping of sesamum genotypes based on germination, seedling length and seedling vigour index. Identification and grouping of sesamum genotypes based on NaOH and KOH tests. Identification and grouping of sesamum genotypes based on coleoptile growth response to GA3 Identification and grouping of sesamum genotypes based on coleoptile growth response to 2, 4-D Analysis of RAPD banding pattern for sesamum genotypes Genetic similarity coefficient based on RAPD data among the ten sesamum genotypes

LIST OF FIGURES

Plate No.

Title Dendrogram obtained from pooled data of RAPD profile of sesamum genotypes Sesamum genotypes identification key on the basis of stem pigmentation and leaf characteristics Sesamum genotypes identification key on the basis of flower characteristics Sesamum genotypes characteristics Sesamum genotypes characteristics identification key on the basis of pod

1.

2.

3.

4.

5.

identification

key on

the

basis

of

pod

6.

Sesamum genotypes identification key on the basis of Seed characteristics

LIST OF PLATES

Plate No.

Title Stem pigmentation, leaf length, leaf colour of sesamum genotypes

1.

2.

Leaf petiole pigmentation, flower petal colour, flower hairiness of sesamum genotypes Number of pods per axil, pod length, pod shape, pod beak, pod pubescence of sesamum genotypes Number of locules per pod, pod dehiscence, seed colour, NaOH test of sesamum genotypes KOH test, seedling response to GA3 , seedling response to 2,4-D of sesamum genotypes RAPD profile of ten sesamum genotypes

3.

4.

APPENDIX
Appendix No. 1. Title Physical and chemical properties of the soil of the experimental site

I. INTRODUCTION
Sesamum (Sesamum indicum L.), a member of the order Tubiflorae, family Pedaliaceae is perhaps the oldest oilseed known and used by human beings (Weiss, 1971). It is an important annual oilseed crop in the tropics and warm subtropics, where it is usually grown in small plots (Bedigian and Harlan, 1986). Sesamum is described as the Queen of oilseeds because it contains high oil (3854%), protein (18-25%), calcium, phosphorous, oxalic acid and excellent qualities of seed oil and meal (Prasad, 2002). Sesamum seed oil has long shelf life due to the presence of lignans (Sesamin, Sesaminol, Sesamolinol), which have remarkable antioxidant function, resisting the oxidation. India is the world leader in sesamum production. In India, sesamum is cultivated on an area of 2.18 m.ha with a production of 0.73 million tons annually (Anon., 2004). It is predominantly grown in Uttar Pradesh, Rajasthan, Orissa, Gujarat, Andhra Pradesh, Tamil Nadu, Karnataka, West Bengal, Bihar and Assam. India ranks first in area and production among the sesamum growing countries. However, the productivity is low in India (312 kg/ha) as compared to worlds average (389 kg/ha) and it is far below as compared to Egypt (1175 kg/ha) being the highest. This evidently indicates the potentiality of the crop for improvement in yields. To meet the need for seed certification and to obtain optimum yield, the seed material should be of high quality, i.e., seed should be genetically and physically pure. The production of quality seed involves a number of multiplication stages. But many factors play an important role in affecting the quality of the seed such as cross pollination, admixture and genetic drift as affected by drought, frost, temperature, soil chemical reaction and seed borne diseases. In order to maintain the required genetic purity standards in the seed fields, field inspection, seed and seedling inspection and grown out test are required. With the introduction of Indian legislation on Protection of Plant Varieties and Farmers Rights (PPV&FR), the release of new crop varieties is possible if it is distinct (D) from other varieties, uniform (U) in their characteristics and generally stable (S) over the years (DUS). Farmers and seed growers need an assurance that they are being supplied with correct seed material known identity of a specific variety and assured quality. Thus, there is a need to search for rapid and reliable methods of varietal identification and genetic purity testing of seed. Identification of varieties based on morphological characteristics of seed, seedling and plant is the most widely used method. According to International Union for Protection of New Plant Varieties (UPOV), any new characteristic used in varietal characterization should be clearly defined, accepted and should have standard method of observation, least or not affected by environment, accessible to breeders, associated with reasonable costs and efforts. For identification of varieties through morphological characters and conduct of GOT, the plant and seed characters need to be studied and thoroughly documented. The National Test Guidelines are to the developed for conduct of DUS testing. Such characterization studies are lacking in sesamum. The interesting fact is that most of the currently used morphological characteristics do not fulfill all these criteria. Moreover, varietal characterization using morphological characters possess several undesirable features like seasonal dependence, large space requirement, time consuming, tedious and environmental influence. The alternative ways to overcome these limitations and to speed up the testing procedures is to use chemical tests or electrophoresis in addition to morphological markers. The chemical tests are spot tests and useful in identification by change in seed colour as well as solution due to added chemicals. Simple biochemical tests viz., phenol colour reaction, NaOH test, KOH test, seedling response to various chemicals eg. growth regulators, herbicides etc., have also been proved quite useful in detecting varietal mixtures as well as grouping large number of genotypes into distinct classes (Chakrabarthy and Agrawal, 1990).

While electrophoresis is the most widely used biochemical test for identification and characterization of genotypes for routine purpose, the use of DNA profiling techniques (RAPDS, RFLPS, AFLP etc.) will help to establish the varietal identity, to measure the genetic relationship and to assess genetic diversity with. The right choice of a technique, proper sampling procedure and judicious interpretation, these laboratory methods can provide reliable and accurate results for variety identification and genetic purity testing in a considerably short period of time (Silvanacristae et al., 2005). The research on cultivar identification in sesamum is limited and only seed morphological characters cannot distinguish the closely related cultivar. Therefore, more emphasis is needed to develop a system of varietal identification in sesamum. Hence, the present investigation entitling characterization of sesamum genotypes through morphological, chemical and RAPD marks was undertaken with the following objectives. 1. 2. 3. Identification and grouping of varieties through morphological characters. Identification and grouping of varieties through chemical tests. Identification and grouping of varieties through molecular or RAPD markers.

II. REVIEW OF LITERATURE

Variety identification and varietal purity assessment are very important for varieties, hybrids and their parents. This is essential for maintenance, multiplication and seed certification. The development of new and improved plant varieties or hybrids is a continuous process. It is of critical importance for sustained increase in agricultural productivity. Under the New Seed Policy Act, 2001, all the new varieties have to be registered based on the criteria of novelty, distinctness, uniformity and stability (DUS). Hence, there is a need to develop and identify the gene markers of the variety/hybrid and also to standardize the laboratory based techniques for genetic purity testing in support of the grow-out test. The grow-out test is tedious, laborious and time consuming and also the marketing of seeds is hindered due to late receipt of results. While the differential response of seeds or seedlings to various chemical solution and bio-chemical test (PCR based markers) can be used as a tool to identify the hybrids/varieties, which is less time consuming, simple and reproducible. The literature on identification of crops through morphological characteristics, response of seeds and seedlings to various chemicals and PCR-based markers (RAPD) has been reviewed here.

2.1 MORPHOLOGICAL CHARACTERS

Seeds, seedlings and plants of various cultivars exhibit a wide range of morphological distinctness which is helpful in varietal identification and genetic purity testing.

2.1.1 Seed morphology

Morphological characters of seeds viz., seed shape, size, colour, test weight are useful traits for varietal identification of number of crops. Elsaeed (1967) reported varietal differences in Beladi and Rebya-34 of broad beans, based on seed weight which was 192.7g and 317.1g, respectively per 500 seeds. Paukens (1975) reported that the kernel colour (white, light yellow, bright yellow, dark yellow, red, etc.) length (short, medium and long) width (narrow, medium and wide) and thickness (thin, medium and thick) were used for determining the cultivar trueness and purity in maize. Shivasubramanian and Ramakrishnan (1978) reported that the shape and colour of kernel were useful in identifying rice varieties. Paramesh (1983) classified 24 soybean genotypes based on the number of seeds per pod and 100 seed weight. He grouped the seeds into small, medium and large seeded groups based on test weight. Tiwari et al. (1978) grouped twenty one soybeans varieties based on 100 seed weight into three groups viz., small, medium and bold. Mikhailov and Travyank (1987) revealed that the inheritance of seed coat colour in soybean is due to dominant genes. Vanagamudi et al. (1988b) grouped the rice varieties based on colour of silk integument (yellow, grey, brown, slightly reddish, red or violet), colour of hulled grain (white, brown, reddish brown, red yellow or orange), vitrous characters, length of hulled grain (short, medium, long very long), shape, 1000 seed weight and presence and absence of pearl spot. Chakrabarthy and Agrawal (1989a) developed seed keys for the identification of 16 black gram varieties on the basis of seed size and colour of the seeds. The seed size varied from small to medium and colour from shiny black to dull black.

Agrawal and Pawar (1990) revealed that soybean varieties could be distinguished from each other on the basis of morphological characters of seed viz., seed size (bold, medium, small) and seed coat colour (black, and yellow). Twenty two pea cultivars were grouped based on their variation in seed size (small, medium and large) seed colour (white, pale green, mottled green and grey) and seed shape (round and flattened) (Anon., 1992) Nagapadma et al. (1996) characterized 23 maize inbred lines based on seed colour (yellow and white) and 100 seed weight (bold, medium and small). For identifying soybean cultivars, seed and seedling characters viz., seed coat colour, seed shape, seed size, 1000 seed weight and seedling length could be used (Anon., 1998) Ponnuswamy et al. (2003) reported that 22 cotton genotypes showed wide variation for seed and seedling characters viz., seed colour, seed shape, seed size, fuzzy nature of seed, 100 seed weight, seedling pigmentation, pigmentation of leaf petiole, number of gossypol glands and angle of petiole. Arunkumar et al. 2004 reported that 45 cultivars of pearlmillet including 14 hybrids and their parental lines were characterized using qualitative morphological characters of seed (seed colour as green, gray, gray brown and brown), seedling characters and plant morphological characters. Karivartharaju (2005) presented a list of seed characters viz., fuzz colour, size (100 seed weight), density of fuzz and fibre characters like colour, strength, fineness, uniformity, maturity and ginning per cent in cotton for identification of varieties.

2.1.2 Seedling characteristics

Lirindae (1986) and Terao (1986a) classified the rice genotypes based on seedling characters viz., seedling length, coleoptile and sheath colour and mesocotyl colour. Chakrabarthy and Agrawal (1989b) developed seed keys for identification of 16 blackgram genotypes using seedling charactes like pigmentation (strong, moderate, weak), stem hairiness (glabrous, pubescent), leaflet shape (lanceolate, obovate), hypocotyle and radicle length. Venkat Reddy (1991) reported that soybean shoot length, root length and seedling length were used as criteria for distinguishing genotypes under laboratory condition.

2.1.3 Morphological characters of plant

Kashiram (1930) isolated the thirty four types of Punjab sesamum on the basis of number of flowers in each axil, seed colour, corolla colour (purple, light purple, deep purple, very pale purple), amount of hairiness of stem and capsule, colour of stem and capsule and maturity (early, medium and late). Mohammed and Alam (1933) reported on the isolation of 34 different types of sesamum in Punjab alone. They used characters viz., number of flowers in leaf axil, colour of corolla, degree of hairiness on stem and capsule, maturity, arrangement of leaves on the main shoot and seed colour for distinguishing different types. Rhind and Thein (1933) classified the Burmese cultivated sesames based on the branching habit, seed coat colour, number of capsules per axil, number of locules per capsule and hairiness of capsule into 34 early (monsoon) types and 15 late (winter) types. Kooistra (1964) revealed that morphological characters of plant viz., shape of leaflets, leaf serration, and number of tendrils, shape of flower, pod size and pod shape could be used for distinguishing pea varieties. Edgar et al. (1970) studied soybean morphological characters for growth habit, nature of stem hairiness, flower colour and pubescence colour. Shrivastav and Kaushal (1972) in their study identified 132 distinct types of sesame from samples collected from various regions of Madhya Pradesh. Morphological variability in characters like days to flowering and other plant habit characters was more among the kharif season.

Ramachandra et al. (1972) presented the relationship between the morphological characters such as plant height, number of branches, capsule size and stem girth. A study with thirty one sesame lines by Sanjeevaiah and Joshi (1974) implied that environment has little effect on plant height, number of capsules and number of branches. Rosta (1975) grouped the rice varieties based on leaf blade length, width (short, medium, long), leaf colour, colour of ligule, colour of auricle and colour of flowers. Chaudhary et al. (1977) observed phenotypic variability for the number of days to initial flowering, number of branches per plant and plant height in sesamum crop. Gupta and Gupta (1977) observed differences between 34 varieties of sesame for characters like 1000 seed weight, plant height, capsules per plant, seeds per capsule and capsule length. Nakagawa et al. (1978) characterized the cultivars of sesame based on days to flowering, plant height and number of branches. Stahi and Pandey (1981) evaluated 21 soybean varieties and grouped them on the basis of nature of maturity and number of days to 50 per cent flowering. Agrawal (1984) classified soybean varieties based on spreading type, pubescence presence on stem, leaf shape and size, flower colour, pod colour at maturity, days taken for maturity and days to 50 per cent flowering. Bahrenfus and Fehr (1984) observed differences in two soybean cultivars viz., Cumberland and Harper. Harper has purple flower, tawny pubescence, brown pods at maturity and higher seed yield compared to Cumberland. But both cultivars were similar in days to maturity and plant height. From the french bean varietal trial, ten cultivars were grouped based on morphological characters viz., plant height, number of branches and leaves per plant, seeds per pod, 100 seed weight and pod length (Anon., 1985) Kandaswamy (1985) used different morphological characters like branch number, capsule number per branch, seed number per capsule in nine varieties of sesamum. Bhagat et al. (1985) evaluated groundnut germplasm collection of 4030 accessions for nine morphological characters. At maturity 191 accessions of var. vulgaris, 128 of var. fastrigiata, 831 of var. hypogaea (bunch) and 1061 accessions of var. hypogaea (runner), had no stem pigmentation. While, 1099 accessions of var. vulgaris, 523 of var. fastrigiata, 209 of var. hypogaea (bunch) and 33 of var. hypogaea (runner) showed stem pigmentation. Rasaily et al. (1986) tabulated 20 soybean genotype characters based on plant height, number of branches, pods per plant and seed yield per plant. Virupakshappa and Sindagi (1987) characterized 429 sunflower germplasm accessions and reported that most of the collections (394 out of 429) possessed cordate leaves, while only 35 were deltoid types with respect to leaf margin except one accession with entire margin. About 325 accessions were without branches while, 34 were fully branched. Basal and top branching were recorded in 23 and 35 accessions respectively. The seedling vigour was low in 57, intermediate in 251 and high in 121 accessions. Chakrabarthy and Agrawal (1989b) suggested that morphological characters such as stem pigmentation (weak, strong and moderate) hairiness, leaflet shape (lanceolate and obovate) and cotyledonary leaf shape could be used for classifying black gram varieties. Reddy et al. (1989) recorded variation in five soybean cultivars for days to maturity, period from flower initiation to maturity, plant height, seeds per plant and yield per plant. From 150 soybean germplasm accessions based on useful characters viz., plant height, days to flower, days to maturity, pods per plant, seeds per pod, number of branches and seed yield per plant and other varietal identification characters were recorded (Anon., 1990) Muralikrishna et al. (1990) attempted to screen the cotton varieties based on morphological characters viz., pigmentation, gossypol glands and plant height.

Luan and Han (1990) analyzed the oil content of 379 different genotypes of groundnut, out of which 38 had the oil content of more than 50 per cent and six had more than 53 per cent. Ashwanikumar et al. (1993) studied varietal identification of six pearlmillet varieties through morphological characters and presented list of key characters on the basis of seed shape, leaf characters and head shape at field level. Jayaramaiah et al. (1993) observed that most of the sunflower germplasm studied were with triangular leaf shape, moderate leaf margin, dark green leaves, medium petiole length, internodal length and medium seedling vigour. Jawaharlal (1994) and Ezilkumar (1999) differentiated cotton genotypes using field parameters such as leaf colour, leaf shape, hairiness on the leaf and stem, leaf nectaries, flower characters, petal colour, pollen colour, petal spot, boll size, boll shape and leaf petiole pigmentation. Mudzana et al. (1995) reported that the morphological characters such as plant height, foliage colour, number of days to 50 per cent flowering, flower length, pod length, pod breadth and number of seeds could be used for variety discrimination of faba beans. Satisha (1995) evaluated 352 sunflower germplasm accessions and observed that most of the accessions possessed cordate leaf shape, leaf margin with medium serration and medium green coloured leaves. Shadakshari et al. (1995) evaluated 225 indigenous and exoitic sesame genotypes and observed a wide range of variability for the characters like number of branches, days to first flowering, capsule length and oil content. Bonetti et al. (1995) reported that 17 bean cultivars were grouped based on leaf colour (very light green, light green, medium green, dark green, very dark green), pod length (very short, short, medium, long, very long), maturity (early, medium, late) and time of flowering (early, medium and late). Patil et al. (1996) developed the keys to characterize and identify cotton varieties and hybrids on the basis of growth habit, leaf shape, hairiness, floral characteristics and boll surface. Surendra Prakash and Singal (1997) reported that seven grain and six vegetable pea cultivars were grouped based on plant height (short, medium, tall, very tall), leaf length (short, medium, long), time of flowering (early, medium) pod length (short, medium, long) and leaf colour (yellow green, blue green, green). Jain (2001) characterized 15 mungbean varieties based on days to maturity, plant height, days to flowering, seed coat colour, stem and petiole pigmentation. Upadhyaya et al. (2002) evaluated 1956 chickpea accessions for flower colour (white, light flower), plant colour, seed colour (orange, yellow orange) and other characteristics. Yadav and Shrivasthava (2002) characterized chickpea varieties based on seed colour (brown, light brown, dark brown, reddish brown, salmon white, green), seed size (bold, medium, small), stem pigmentation (strong, medium, absent), flower colour (white, deep pink, pink, light pink), foliage colour (dark green, green, light green), plant height (tall, medium, dwarf), podding habit (single, double), pod number per plant (low, medium, high), number of locules per pod (one, tow, three) and duration (early, medium and late). Sankarapandian (2002) reported that four cowpea varieties were grouped based on pod shape, seed colour and leaf shape. Rajendra Prasad et al. (2003) characterized the 10 sunflower varieties, hybrids and their parental lines based on the leaf petiole pigmentation (absent, present), stem pigmentation (weak, medium, dense), number of leaves per plant (low, medium, high), time to 50 per cent flowering (early, medium, late) and their seed characteristics. Muralimohan Reddy et al. (2004) characterized the castor genotypes based on the seed colour (white, maroon, brown, dark chocolate, black), 100 seed weight (low, medium, high), capsule dehiscence (non dehiscent, partially dehiscent, dehiscent), number of nodes

on main stem (low, medium, high, very high), leaf shape, leaf colour, leaf length (small, medium and large) and other morphological characteristics. Tarasatyavathi et al. (2004) reported that 75 released soybean varieties were characterized based on leaf shape (lanceolate pointed ovate, rounded ovate, triangular), leaf colour intensity (light, medium, dark), flower colour (white, violet), pod pubescence (absent, present), plant height (short, medium, tall), days to flowering (early, medium and late) and days to maturity (early, medium, late). Kumar et al. (2005) reported that 27 jute varieties were grouped based on leaf shape (ovate, ovate- lanceolate, elliptical, palmate), pod dehiscence (absent, present), time of 50 per cent flowering (early, medium, late), plant height (short, medium, tall), maturity days (early, medium, late), seed size on 1000 seeds weight basis (small, medium, large) and seed colour (blue, steed grey, chocolate brown, black). In Jabalpur, 11 niger varieties were characterized based on leaf colour (light green, green), days to 50 per cent flowering (medium early) flower colour (yellow, orange), days to maturity (early, medium), plant height (tall, medium) and seed colour (light black, dark black golden black, black, brown). (Anon., 2005) Thangavel et al. (2005) grouped 12 sorghum cultivars based on seed colour (white, dull white, brown, red), seed size (small, medium, bold), seed luster, seed shape (narrow elliptic, elliptic, circular) and other seed morphology. Mate and Shelar (2006) characterized 16 sorghum hybrids based on plant height (very short, very tall), 50 per cent flowering (very early, late) anther colour, stigma anthocyanin, colour of straw and glume colour at maturity.

2.2 CHEMICAL TESTS

Studies on characterization of cultivars based on response of seed and seedling to various chemicals viz., sodium hydroxide, potassium hydroxide, GA3 and 2, 4-D etc., offer wide variability and can be used in characterization of genotypes.

2.2.1 Sodium hydroxide test

Agrawal (1987) observed the colour change in wheat varieties after immersing in NaOH. After 15 minutes, the seeds of red and white varieties became dark orange to orange brown and yellow to straw colour, respectively. Chakrabarty and Agrawal (1990) reported that sodium hydroxide (0.5% and 0.1%) could be used to group the seeds of black gram into five groups as red, brown, greenish yellow, light green and green. The sodium hydroxide chemical test is simple, quick and cheap. Based on the secondary metabolites present in the seed coat, the seed coat produces distinct colour pattern (Vanderburg and Vanzood, 1991). At Akola, 10 jute genotypes were classified as black, dark brown, brown and no change in colour when soaked in five per cent sodium hydroxide for six hours (Anon., 2002). Sambasiva Rao et al. (2002) categorized 58 rice genotypes into two groups as light yellow and dark yellow based on sodium hydroxide test. Based on the 22 cotton genotypes response to five per cent sodium hydroxide test, the genotypes were classified as dusky red, dark red, red, reddish yellow, yellow, alive yellow, yellowish red and strong brown types (Ponnuswamy et al. 2003). At Jabalpur, 18 sesame varieties were categorized into three groups using sodium hydroxide at five per cent as reddish brown, dark brown and light brown. (Anon., 2005). Biradarpatil et al. (2006) grouped 20 genotypes of safflower as light brown, brown and dark brown based on their response to two per cent NaOH.

2.2.2 Potassium hydroxide test

Mckee (1973) suggested that five or ten per cent potassium hydroxide solution could be useful for separating white grain wheat varieties from red grain wheat varieties.

Rosta (1975) reported that treating rice seeds with five per cent aqueous potassium hydroxide solution could be useful for grouping red grain varieties from white grain varieties. Agrawal (1987) differentiated sorghum cultivars through KOH bleach test on the basis of presence or absence of darkly pigmented seed coat. Vanagamudi et al. (1988 a) classified 85 rice varieties based on colour development by treating them with five per cent potassium hydroxide solution for three hours and solution was observed for deep wine red staining. Jawaharlal (1994) reported that, cotton genotypes can be classified based on the colour development in five per cent potassium hydroxide as dark red, red, yellowish brown and brown. Palaniswamy et al. (1998) opined that KOH test could be useful for detecting red rice cultivar (TKM 09) which turns to deep red colouration within one hour. Twelve varieties of pigeonpea could be distinguished based on the colour development in KOH test. Four varieties showed no response, four varieties showed dark tan, while rest were brown in colour. (Anon., 1998) Sambasiva Rao et al. (2002) categorized 37 groundnut genotypes into light brown and dark brown based on based on seed coat response to KOH solution. Biradarpatil et al. (2006) grouped 22 safflower genotypes as light brown and brown by using five per cent KOH solution.

2.2.3 Seedling growth response to GA3

Prakash and Lal (1968) reported that cotton seeds soaked in acid for 12 hours exhibited morphological and biochemical changes. 100ppm gibberllic

Robert et al. (1980) reported that application of gibberllic acid showed the greatest stimulation in coleoptile length in slow emerging wheat varieties and little or no response in the case of rapid emerging wheat varieties. Pain and Basu (1985) observed wide variation in shoot and root length due to different concentration of gibberllic acid among different rice genotypes. Kurdikeri and Kurdikeri (1988) reported that gibberllic acid soaked seeds produce more vigorous seedlings. Further, cotton genotypes showed varied response to gibberllic acid treatment and the cultivars were grouped as high and low response types. Based on the response of seedlings to GA3 and DDT the mungbean genotypes were categorised into different groups (Agrawal and Sharma, 1989). Agrawal and Pawar (1990) categorised soybean genotypes into long, medium and short types based on the seedling response to 15ppm GA3 and developed seed key for identification of soybean varieties. Singh and Afria (1990) showed that gibberllic acid at 200mg/litre enhanced seedling growth and emergence and promoted cent per cent germination in Bikaneri Nerma cotton cultivar than in any other cultivars. Chakrabarthy and Agrawal (1990) classified 16 black gram varieties based on seedling response to growth hormones and herbicides. Lee et al. (1992) grouped 15 Korean soybean cultivars based on seedling response to GA3. The seedling length varied with genotypes. Bansal et al. (1992) distinguished tall and dwarf genotypes of rice based on the variations in root and shoot length in response to GA3 treatment at 50ppm. Jawaharlal (1994) studied the effect of gibberllic acid at 100ppm in cotton and grouped the genotypes based on hypocotyls length as long, medium and short. Nagapadma et al. (1996) studied the response of seedlings of 23 maize inbred lines to gibberllic acid at 15ppm. Based on the per cent increase in seedling length, the inbreds were grouped into high (> 20%), medium (10-20%) and low (<10%) response groups.

Sambasivarao et al. (2002) classified the 37 groundnut genotypes into three groups as low, moderate and high response in coleoptile length to GA3. Ponnuswamy et al. (2003) grouped 22 genotypes of cotton into high, medium and low response groups based on the seedling response to 100ppm gibberllic acid. Kirankumar (2004) classified cotton genotypes as very high, high, medium and low response varieties based on their response to GA3. At Jorhat 20 sesamum varieties were grouped based on GA3 growth response test as low, medium and nil response (Anon., 2004). Thangavel et al. (2005) grouped 12 sorghum cultivars into low, medium and high response based on relative seedling length when sprayed with 100ppm of GA3 solution. Biradarpatil et al. (2006) grouped 20 safflower genotypes by using GA3 at 25ppm as moderate response, low response and very low response types.

2.2.4 Seedling growth response to 2, 4-D

Wax et al. (1974) observed 338 soybean cultivars response to 2, 4-D herbicide. Among them 11 cultivars were highly sensitive to 2, 4-D. Chakrabarty and Agrawal (1990) classified 16 black gram varieties based on seedling response to added 2, 4-D (5ppm). Ashwani Kumar et al. (1995) grouped 14 genotypes of pearlmillet including six elite hybrids and their eight parental lines based on 2, 4-D test at 5ppm based on coleoptile stimulation and root inhibition. Nagapadma et al. (1996) studied the response of seedling of 23 inbred lines to 2, 4-D at 5ppm and reported the effectiveness in identifying and differentiation the maize inbreds. Shivakumar (2000) grouped 26 cultivars of rape seed and mustard into susceptible, moderate and resistant types to 2, 4-D test. Sambasivarao et al. (2002) reported that thirty seven groundnut genotypes were grouped into low, moderate and high response by using 2, 4-D. Ponnuswamy et al. (2003) indicated that seedling response to 2, 4-D at 0.5ppm moistened germination towels was proved to be a futile exercise as it could not distinguish the cotton genotypes. Kirankumar (2004) reported that it was not possible to categorise the cotton genotypes into different groups based on 2, 4-D at 5ppm as all genotypes had fallen into the same category of highly susceptible group. Biradarpatil et al. (2006) classified 20 genotypes of safflower based on response to 2, 4-D at 5ppm into three groups namely highly susceptible, susceptible and less susceptible.

2.3. RAPD MARKERS

Variation in morphological traits, geographical distribution, cytogenetic relationships, breeding system, cross compatibility and biochemical markers though used extensively to elucidate the relation among the species are restricted in their resolving power mainly because of small number of variables available and some of them are developmental specific. In contrast, molecular approaches provide genetically interpretable variability with extensive genomic coverage and are becoming immensely important in studies on population biology and systematic. Among the PCR based marker techniques, randomly amplified polymorphic DNA (RAPD) technology is widely used, as it is easy and simple. A brief review of literature has been presented regarding RAPD marker technology here under. Using RAPD analysis, Lawson (1994) studied a collection of cultivars, breeding lines, wild and distantly related species for assessing the genetic diversity. A considerable amount of polymorphism was revealed. Over all, 33 per cent dissimilarity was detected with an average of 27 per cent among the hybrids and breeding lines. Multan et al. (1995) used RAPD markers generated by 30 random primers to fingerprint twelve cultivars and a breeding line of Gossypium hirsutum and one cultivar of

Gossypium barbedense. Amongst a total of 453 developed markers, 69 (15.2%) were present only in Gossypium barbedense cultivar Pima S-7. Of the remaining markers, 128 (33.3%) were fixed in all 15 G. hirsutum cultivars. In pair wise comparisons of the degree of band sharing, nine closely released cultivars showed 92.1-98.9 per cent genetic similarity. Ten of the Gossypium hirsutum cultivars could be characterized individually based on cultivar specific RAPD markers, thus making it possible to differentiate closely related cultivars by molecular markers. Doldi et al. (1997) used RAPD technique to evaluate genetic diversity among 18 soybean genotypes selected for a breeding programme to increase the protein content of varieties adopted for central European growing conditions. Out of 33 random primers used in RAPD locations, only 12 showed polymorphism useful for characterization of these genotypes. The resulting dendrograms, from similarity measures and cluster analysis were compared with each other and with the available pedigree information as a control. The dendrogram derived from RAPD data showed some divergence from the pedigree information available for the lines. Parani et al. (1997) reported that with the objective of transferring phyllody resistance, hybridization has been done between Sesamum alatum and Sesamum indicum. Identical chromosome number of the species ambiguous morphological characters and lack of segregation in F2 necessitated to look for molecular markers to established the hybridity. SDS-page of seed protein revealed transfer of five male-specific proteins to the putative hybrid. Further, inheritance of the establish locus of esterase, peroxidase and loci of peroxidase and RAPDS of decamer random primers have not only established the hybridity, but were also found to be useful as markers to distinguish the hybrids from the selfed progenies. Bhat and Suman Lakhanpaul (2000) reported that a total of 48 sesame cultivars were characterized using RAPD technique. Sixty-two primers were screened and data from twenty one selected primers were used for molecular profiling of the cultivars. The total number of amplification products scored was 116. The extent of polymorphism among the cultivars was low indicating narrow genetic diversity among the released sesame cultivars. Subramanian et al. (2000) studied 70 selected groundnut genotypes for polymorphism employing RAPD assay with 48 oligonucleotide primers out of the 48, only 7 (14.6%) yielded polymorphic amplification products. The total number of bands from the 7 primers was 408 of which 27 were polymorphic. Based on this they concluded that this approach would be useful for developing marker-assisted selection tools for genetic enhancement of groundnut for desirable traits. Gulhanercan et al. (2004) obtained the bands through RAPD technique for all sesamum populations and 78 per cent of which were polymorphic. According to ANOVA and shannons index that were performed separately for each region, the highest value of genetic variation was observed among North West region populations and lowest in the South East region populations. Nei and Lis similarity index was calculated and phylogenetic true was established using the neighbour joining algorithm. This phenotypic analysis grouped 35 of 38 accessions in six groups leaving three highly diverse accessions outside. These results indicate that RAPD technique is useful for sesame systematics, and valuable for the maintenance of germplasm banks and the efficient choice of parents in breeding programmes. Encheva et al. (2005) reported that the method of direct organogenesis has been successfully used for overcoming the inability for crossing between Helianthus annuus (V. albena) and verbesina helianthoides (genus verbesian). As hybrid materials, fertility restorer lines were produced in the R10 generation. The applied molecular method (RAPD) indicated an introgression of verbesina helianthoides. DNA into some of the hybrid progenies produced and concluded that RAPD could be used for characterization of intergeneric hybrid progenies in sunflower at a later stage of selection (F9) in which an increased genetic variation was discovered. Silvanacreste et al. (2005) studied the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty seven primers were tested, of which nine produced unique fingerprinting for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per

primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n=18 chromosomes. The bootstrap analysis divided the genotypes into 2 significant clusters. The first cluster contained all the section Arachis species and the accessions within it were grouped based on the presence or absence of the A pair and the number of chromosome. The second cluster grouped all the accessions belonging to section Heteranthae. Deepamala et al. (2005) reported that 14 sunflower cultivars have been fingerprinted by RAPD, ISSR and AFLP markers utilizing 361, 21 and four primer combinations, respectively. On an individual assay basis, AFLP was proven to be the best marker system as compared with the other two markers. To understand genetic relationships among these cultivars, Jaccards similarity coefficient and UPGMA clustering algorithm were applied to the 3 marker data sets. However, strong correlation was observed between RAPD and ISSR marker systems.

III. MATERIAL AND METHODS

The field experiment was conducted at the Main Agricultural Research Station, University of Agricultural Sciences, Dharwad during kharif season of 2005 for morphological identification of sesamum genotypes. Further the laboratory studies were carried out at Seed Research Laboratory, National Seed Project, University of Agricultural Sciences, Dharwad. The details of the materials used and methods adopted are presented here.

3.1 LOCATION OF EXPERIMENTAL SITE

The field experiment was conducted at H block of Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad, situated in Northern 0 0 Transitional Zone of Karnataka state and located at 15 26 North latitude, 75 7 East longitudes with an altitutude of 678m above mean sea level.

3.2 CLIMATIC CONDITIONS

The monthly meteorological data pertaining to rainfall, temperature and relative humidity prevailed during experimental period at Main Agricultural Research Station, University of Agricultural Sciences, Dharwad are presented in Table 1. The mean annual rainfall of 773.5mm was fairly distributed from April to November. 0 0 The mean maximum temperature ranged from 27.1 C (August) to 37.1 C (April) mean 0 0 minimum temperature ranged from 12.90 C (January) to 21.50 C (May). The mean monthly maximum relative humidity was 85 per cent (September) while the mean monthly minimum relative humidity was 39 per cent (February).

3.3 SOILS
The experiment was conducted in black clayey soil. The composite soil sample from experimental site was collected from 0 to 30cm depth before the start of the field experiment and was analyzed for physical and chemical characteristics by following the standard procedure. The results are presented in Appendix I.

Table 1. Monthly meteorological data for experimental year 2005-06 of Main Agricultural Research Station, University of Agricultural Sciences, Dharwad

Average of 67 years Month Rainfall (mm) 29.1 82.4 104.5 137.5 92.5 108 123.1 34.0 7.1 2.0 2.0 5.9 46.4 773.5 No. of rainy days 3 8 13 23 21 12 11 3 0.8 0 0 0.7 3 98.6 Rainfall (mm) 75.0 29.4 151.0 290.2 138.8 194.5 89.4 38.0 Trace Trace Trace 5.2 1.5 1013.0

2005-06 No. of rainy days 5 3 10 19 15 14 9 1 Trace 0 0 1 1 78 Relative humidity (%) 53 55 76 83 81 85 70 51 53 52 39 45 49 -

Temperature (0C) Maximum 36.3 37.0 30.9 27.4 27.1 27.5 29.6 29.4 28.9 28.9 32.4 34.1 37.1 Minimum 21.3 21.5 21.5 21.0 20.4 20.3 19.1 14.9 13.1

April May June July August September October November December January February March April

13.1 14.9 18.1 20.3 -

3.4 PREVIOUS CROP IN THE EXPERIMENTAL SITE

Sunflower crop was grown during Rabi season of 2004 in the experimental site

3.5 EXPERIMENTAL DETAILS

3.5.1 EXPERIMENT I: IDENTIFICATION AND GROUPING OF SESAMUM GENOTYPES THROUGH MORPHOLOGICAL CHARACTERISTICS. 3.5.1.1 Treatment details
Genotypes G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 : DS-1 : ORM-17 : Kanak : Chandana : AKT-101 : Usha : Thilarani : Phule Til-1 : VS-9701 : YLM-17 : Thilak G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 : TKG-21 : T-12 : SVPR-1 : T-13 : GT-2 : Uma : E-8 : TC-289 : RT-54 : Paiyar : TKG-55

3.5.1.2 Experimental design and layout

The field experiment was laid out in a randomized block design with three replications.

3.5.1.3 Plot size

Gross plot size - 2.0 X 1.2 m Net plot size - 1.8 X 0.9 m

3.5.1 Cultural operations

3.5.1.1 Land preparation The land was ploughed by mould board plough followed by two harrowings to bring the soil to fine tilth so as to facilitate sowing. The residues of the previous crop and weeds were removed from the experimental area. The land was levelled with the help of plank. 3.5.1.2 Fertilizer application The recommended dose of 40:20:20kg NPK per ha was supplied in the form of urea, single super phosphate and muriate of potash. Fifty per cent of nitrogen (20kg per ha) and entire quantity of phosphorus and potash were applied in the rows five centimeters away from the seed row. Remaining fifty per cent of nitrogen was applied 30 days after sowing as top dressing. 3.5.1.3 Seed source and sowing The seeds of the genotype DS-1 was obtained from the National Seed Project, University of Agricultural Sciences, Dharwad and the seeds of the genotype E-8 from the oilseed scheme, MARS, Dharwad. The seeds of the other genotypes were obtained from the Project Coordinator (Sesame), Jabalpur. Sowing was done in rows of 30cm apart by mixing seeds with sand. Before sowing, the seeds were treated with thiram @ 3g/kg of seeds, to control seed borne diseases. 3.5.1.4 After care

The plants were thinned out 15 days after sowing leaving a single healthy seedling at a distance of 10cm per hill. The crop was kept weed free and three hand weedings were carried out during the crop growth period. Prior to hand weeding, hoeing operation was done at initial crop growth period. Proper soil moisture was maintained throughout the crop growth period through supplementary irrigations. Necessary plant protection measures were taken to control pest and disease. Dimethoate @ 1.7ml per litre of water was sprayed to control thrips and white flies. DM-45 @ 2g per litre of water was sprayed for the control of Cercospora leaf spot.

3.5.1.5 Collection of experimental data

3.5.1.5.1 Sampling procedure Five plants were randomly selected in each genotype and replication and labelled. Observations on plant morphological characteristics were recorded on these plants at different stages of crop growth. 3.5.1.5.2 Observations recorded

i) Plant height (cm)

The plant height at the time of harvest was measured from the base of plant at ground level to the tip of the plant in centimeters and the genotypes were grouped as short, medium and tall types.

ii) Number of primary branches per plant

The number of primary branches were counted and recorded in number. Based on the number of primary branches, the genotypes were grouped as few and high branched types.

iii) Number of nodes per plant

The number of nodes were counted and recorded in number. Based on the number of nodes, the genotypes were grouped as less and more nodal types.

iv) Internodal length (cm)

The internodal length between two nodes was measured in centimeters. Based on internodal length, the genotypes were grouped as short and long internodal types.

v) Stem pigmentation
The stem pigmentation was recorded on visual assessment at peak flowering stage and the genotypes were grouped as weak, medium and strong stem pigmented types.

vi) Number of leaves per plant

The number of leaves were counted at peak flowering stage and recorded in number. Based on the number of leaves, the genotypes were grouped as having few, medium and many leaf types.

vii) Leaf length (cm)

The leaf length of the sixth leaf from the top of the plant was measured in centimeters at peak flowering stage. Based on the leaf length, the genotypes were grouped as short, medium and long leaf types.

viii) Leaf shape

The leaf shape of the sixth leaf from the top of the plant was recorded on visual assessment basis at peak flowering stage and the genotypes were grouped as lobed types.

ix) Leaf colour

The leaf colour of the sixth leaf from the top of the plant was recorded on visual assessment basis at peak flowering stage and the genotypes were grouped as light green, medium green and dark green coloured types.

x) Leaf petiole pigmentation

The leaf petiole pigmentation was recorded on visual assessment basis at peak flowering stage and the genotypes were grouped as light, medium and dark pigment leaf petioled types.

xi) Days to 50 per cent flowering

The number of days taken from sowing to the opening of flowers in 50 per cent of plant population in each genotype was recorded and the genotypes were grouped as early and late flowering types.

xii) Flower petal colour

The petal colour of flowers was recorded on visual assessment basis at peak flowering stage and the genotypes were grouped as light pink, medium pink and dark pink coloured types.

xiii) Flower hairiness

The flower hairiness was recorded on visual assessment basis at peak flowering stage and the genotypes were grouped as low, medium and dense hairiness types.

xiv) Number of pods per axil

The number of pods per axil were counted and recorded as numbers at maturity and the genotypes were grouped as single, alternate and multiple poded types.

xv) Number of pods per plant

The number of pods were counted and recorded in number. Based on the number of pods, the genotypes were grouped as less, moderate and more poded types.

xvi) Pod length (cm)

The pod length of the fifth pod from the top of the plant was measured in centimeters at maturity. Based on the pod length, the genotypes were grouped as short and long poded types.

xvii) Pod shape

The pod shape of the fifth pod from the top of the plant was recorded on visual assessment basis and the genotypes were grouped as oblong and round pod shaped types.

xviii) Pod beak

The pod beakness of the fifth pod from the top of the plant was recorded on visual assessment basis at maturity and the genotypes were grouped as short and long pod beaked types.

xix) Pod pubescence

The pod pubescence of the fifth pod from the top of the plant was recorded on visual assessment basis at maturity and the genotypes were grouped as nil, medium and dense pod pubescence types.

xx) Number of locules per pod

The number of locules per pod were counted and recorded as number at maturity and the genotypes were grouped as four and six loculed types.

xxi) Pod dehiscence

The pod dehiscence was recorded on visual assessment basis at maturity and the genotypes were grouped as dehiscent and indehiscent types.

xxii) Days to maturity

The number of days taken for each genotypes from sowing to physiological maturity (yellowing of pods and leaves) was recorded and the genotypes were grouped as early and late maturity types.

xxiii) Seed colour

The seed coat colour of each genotypes was observed under natural day light condition and the genotypes were grouped as brown, black and white seed coloured types.

xxiv) Thousand seed weight (g)

Thousand seeds each in three replications were counted and weighed separately and The mean weight was expressed in grams. Based on this, the genotypes were grouped as medium and bold types.

xxv) Oil content (%)

The oil content of each genotype was determined with the help of NMR (Nuclear Magnetic resonance spectrometer) and the genotypes were grouped as low, and high oiled types.

xxvi) Seed germination and seedling characters

a. Seed germination (%) The germination test was conducted as per the ISTA procedure (Anon., 1996), using between paper method. The rolled paper towels were placed at slanting position in a cabinet seed germinator at constant temperature of 25 10 C and 95 1 per cent relative humidity. Final count on normal seedlings was recorded on seventh day and per cent germination was computed and expressed in percentage. b. Seedling length (cm) Ten normal seedlings were selected at random from the germination test. The length between the collar region and the tip of the primary shoot was measured as shoot length (cm). The length between the collar region and the tip of primary root was measured as root length (cm). The seedling length was computed by using the following formula, Seedling length = (Shoot length (cm) + Root length (cm) c. Seedling vigour index (SVI) The seedling vigour index was computed by multiplying the germination percentage with total seedling length and expressed as whole number (Abdual Baki and Anderson, 1973).

3.5.1.5.3 Statistical analysis

The mean values of the genotypes in each replication were used for analysis of variance. Result and values were subjected to RBD analysis and, the significant of differences among all the genotypes were tested by F test. Critical differences were calculated at five per cent level (Cochran and Cox, 1965).

3.5.2 EXPERIMENT II: IDENTIFICATION AND GROUPING OF SESAMUM GENOTYPES THROUGH CHEMICAL TESTS
The chemical tests are spot tests and they are useful in identification by representing seed coat colour reaction to chemicals. The differential response of seeds of sesamum genotypes to chemical solution tests was used as a tool as per standard procedures given by various workers to identify different genotypes. The study included the following.

3.5.2.1 Sodium hydroxide (NaOH) test

The seeds (one gram) of sesame genotypes were washed in distilled water and then soaked in 10ml of five per cent NaOH solution in test tube for one hour at an ambient temperature. The solution was decanted and used for observation. Based on the change in colour solution of the genotypes were grouped as light brown, brown and dark brown.

3.5.2.2 Potassium hydroxide (KOH) test

The seeds (one gram) of sesame genotypes were washed in distilled water and then soaked in 10ml of six per cent KOH solution for one hour in test tube at an ambient temperature. The solution was decanted and used for visual observation. Based on the change in colour of the solution the genotypes were grouped as light yellow, yellow, light brown, brown and dark brown.

3.5.2.3 Seedling growth response to GA3

The seeds of sesame genotypes were surface sterilized by washing in distilled water. Fifty seeds each in three replications were placed on two layers of blotter paper moistened 0 with 25ppm GA3 solution and incubated at 25 1 C as per ISTA procedure (Anon., 1996). The water soaked blotter papers were used as the control. On seventh day, the coleoptile length of twenty five randomly selected seedlings was measured and the growth response was recorded as per cent increase in coleoptile length over control. The per cent increase in coleoptiles length over control was calculated using the following formula Coleoptile length in GA3 Per cent increase over control Coleoptile length in control = Coleoptile length in control X 100

The genotypes were grouped based on per cent increase of coleoptiles length over control as follows a) Very low response b) Low response c) Moderate response : < 10 per cent increase : 10-30 per cent increase : > 30 per cent increase

3.5.2.4 Seedling growth response to 2, 4-D

The seeds of sesame genotypes were surface sterilized by washing in distilled water. Fifty seeds each in three replications were placed on two layers of blotter paper moistened with 2ppm of 2, 4-D solution and then incubated at 2510 C as per ISTA procedure (Anon., 1996). The water soaked blotter papers were used as control. On seventh day, coleoptile length of twenty five randomly selected seedlings was measured and the sensitivity response of genotypes was recorded as percent decrease in coleoptile length over control. The decrease in coleoptiles length over control was calculated using the formula such as, Coleoptile length in control Per cent decrease over control Coleoptile length in 2, 4-D = Coleoptile length in control The genotypes were grouped based on per cent decrease of coleoptile length over control as follows a) Susceptible b) Highly susceptible : < 85 per cent : > 85 per cent X 100

3.5.3 EXPERIMENT III: IDENTIFICATION AND GROUPING OF SESAMUM GENOTYPES THROUGH MOLECULAR MARKERS
3.5.1 Plant material
As a part of the development of a molecular tool kit for the study of diversity within the plant germplasm collections, RAPD technology has been applied to the selected sesamum genotypes. The following sesamum genotypes were used for the study.

Kanak Usha VS-9701 YLM-17 T-12

SVPR-1 T-13 E-8 Paiyar TKG-55

3.5.2 Laboratory procedure and techniques

3.5.2.1 Stock solutions prepared a. Extraction buffer 0.5 M EDTA (PH 8.0) 1.0 M Tris 4.0 M Nacl 10% CTAB b. Chloroform: isoamyl alcohol (24:1) c. Isopropanol d. 70% Ethanol e. T10E1 buffer: Tris 10mM Containing 1mM EDTA

3.5.2.2 DNA extraction procedure

Seed samples were germinated in plastic cups until seedlings were grown upto two to three leaves stage. Five to six such seedlings were taken from each genotype. The young seedlings leaf samples (two grams) were ground into fine powder in liquid nitrogen using autoclaved or sterilized pestle and mortar. The ground tissue was transferred to 50ml centrifuge tube containing 10ml extraction buffer, preheated to 650 C. The tubes containing round tissue were placed in water bath (with gentle shaking) for 0 10-15 minutes at 65 C with periodical shaking at an interval of five minutes. Later, the tubes with leaf tissue extract were incubated at room temperature for 15 minutes. Ten mililitre of chloroform: isoamyl alcohol (CIA) mixture (24.:1) was added to tissue extract and the contents were mixed by shaking gently. Then the tubes were centrifuged for 10 minutes at 15,000 rpm at room temperature. The supernatant was transferred to fresh centrifuge tubes carefully and 10ml of chilled isoproponal was added to each tube and mixed by inverting and incubated at 200 C for overnight. The content was centrifuged for 10 minutes at 15,000 rpm at was discarded. 40 C. The supernatant

The DNA pellet obtained was washed with 70 per cent ethanol and the tubes were inverted on blotter paper to dry the pellet. The DNA was dissolved in 200 l T10 E1 buffer and stored at -200 C.

3.5.2.3 DNA quality and quantity estimation

The concentration of DNA was estimated spectrophotometrically and also by gel electrophoresis using 0.8 per cent agarose with known concentrations of DNA. In spectophotometric analysis, 5 l of DNA was diluted to 3000 l of T10 E1. The spectrophotometer readings were recorded at 260 and 280 nm. A good DNA preparation generally exhibits the following spectral properties. A260/A280 = 1.80

DNA concentration was calculated using OD values at 260 nm using the following formula. Concentration of DNA (l/ml) = OD at 260 nm X 50 To test the quality of DNA, samples were run on 0.8 per cent agarose gel in IXTAE buffer and stained with ethidium bromide. DNA was evaluated by comparing it with a standard digested DNA sample.

3.5.2.4 RAPD-PCR amplification

3.5.2.4.1 Requirements a. Random primers: Commercial kits were obtained from operon technologies Inc. Alameda, USA. b. Template DNA: Crude genomic DNA extracts from leaf samples of young seedlings of different genotypes. c. dNTPs: The four individual dNTPs viz., dATP, dGTP, dCTP and dTTP were obtained from M/S Bangalore Genei Pvt. Ltd., Bangalore and were used at a concentration of 2.5 mM each.

d. Taq DNA polymerase: Taq DNA polymerase (3U/l) and 10 X Taq assay buffer were obtained from M/s Bangalore Genei Pvt. Ltd., Bangalore. e. Chemicals: Analytical grade chemicals were obtained locally f. Polymerase chain reaction:

Requirements: DNA, random primers, dNTPs, Taq DNA polymerase, 10 X Taq assay buffer, deioinised distilled water and thermal cycler.

3.5.2.5 Stock solutions

a. 100 M random primer b. 25 ng l template DNA c. 3.0 U l-1 DNA polymerase A total of 12 random primers used for the study are as followers List of primers used in the study Sl. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12 Primers OPP-09 OPP-10 OPP-03 OPA-11 OPA-13 OPP-01 OPP-07 OPA-12 OPA-14 OPA-08 OPA-15 OPP-10
-1

3.5.2.6 Master mix for PCR (25 l tube-1)

Sl.No. 1. 2. 3. 4. 5. 6. 7. Components Taq assay buffer (10X) Mgcl2 (25mM) dNTPs (2.5 mM) Primer (5 pM/l) Template DNA Taq DNA polymerase (3.00 U/1) Deionised distilled water Total reaction volume Quantity (l/reaction) 2.50 l 1.00 l 2.00l 2.0 l 1.0 l 16.001 16.00 l 25.00 l

Master mix required for a set of 10 reactions was prepared fresh, from the original stocks. The master mix was distributed (24 l tube-1) to 10 tubes containing 1.0 l each of the template DNA from different genotypes and mixture was given a short spin to mix the contents.

3.5.2.7 Thermal cycling

Sterile microfuge tubes were numbered from 1 to 10. 1.0 l of template DNA from individual genotypes was added to each tube. 24 l of master mix was added to all the tubes and was given a short spin to mix the contents. The tubes were placed in the thermal cycler for amplification.

The PCR reaction was carried out using master cycler gradient 5331 eppendorf version 2.30-31-09, Germany. This cycler was programmed as under. Sl.No. 1. 2. 3. 4. 5. 6. Steps Initial denaturation Final denaturation Primer annealing Primer extension Final extension Dump Temperature ( C) 94 94 38 72 72 4
0

Duration (Min) 5 1 1 2 8 Until removed

0

Number of cycles 1

42 1

After the completion of PCR, the products were stored at 4 electrophoresis was done.

C until the gel

3.5.3

Separation of electrophoresis

amplification

products

by

agarose

gel

3.5.3.1 Requirements
a. Electrophoretic unit: Gel casting trough, gel preparation comb, power pack, UV transilluminator. b. Agarose c. Bromophenol blue d. Ethidium bromide (0.5g ml ) e. 50 X TAE pH-8.0 f. Working solution (1 X TAE)
-1

3.5.3.2. Procedure
1.8 g of agarose was weighed and added to a conical flask containing 100ml of 1 X TAE buffer. The agarose was melted by heating the solution on an electric heater and the solution was stirred to ensure even mixing and complete dissolution of agarose. The solution was then cooled to about 40-45 C. Two to three drops of ethidium bromide (0.5 g ml ) was added. The solution was poured into the pre levelled gel casting platform after inserting the comb in the trough. While pouring, sufficient care was taken for not allowing the air bubbles to trap in the gel. The gel was allowed to solidify and the comb was removed after placing the solidified gel into the electrophoretic apparatus containing sufficient buffer (1 X TAE) so as to cover the well completely. The amplified products (25l) to be analysed were carefully loaded along with the marker ( DNA EcoRI and Hind III double digest, Bangalore Genei. Bangalore) into the sample wells, after additing 2-3 l of loading dye (Bromophenol blue) with the help of a micropipette. Electrophoresis was carried out at 50-55 volts, until the tracking dye migrated to the end of the gel. Ethidium bromide stained DNA bands were viewed under UV transilluminator and photographed for documentation.
-1 0

3.5.3.3 Analysis of amplified profiles

Amplified fragments were scored as 1 for the presence and o for the absence of band generating the O and 1 matrix and per cent polymorphism was calculated by using the following formula. Number of polymorphic bands Per cent polymorphism = Total number of bands X 100

IV. EXPERIMENTAL RESULTS

The results obtained from the studies on identification and grouping of sesamum genotypes through morphological characters, chemical tests and molecular markers are presented in this chapter.

4.1

IDENTIFICATION AND GROUPING OF SESAMUM GENOTYPES THROUGH MORPHOLOGICAL CHARACTERISTICS

4.1.1 Plant height (cm)

The plant height varied among the different sesamum genotypes. (Table 2). The mean plant height of the genotypes was 91.65cm. Highest plant height was observed in E-8 (119.56cm) and the lowest plant height was recorder in GT-2 (75.45cm). Based on the plant height, the genotypes were grouped into three categories as short (<100cm), medium (100115) and tall (<115cm) types. Among the 22 genotypes, 17 genotypes were short (ORM-17, Kanak, Chandana, AKT-101-1, Usha, Phuile Til-1, Thilak, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, TC-289, RT-54, Paiyar, TKG-55), four genotypes wee medium (DS-1, Thilarni, VS-9701, YLM-17) and one was tall (E-8) in plant height.

4.1.2 Number of primary branches per plant

The number of primary branches per plant varied among the sesamum genotypes. (Table 2). The mean number of primary branches per plant was 3.75. Significant higher number of primary branches were recorded in VS-9701 (5.46) and the lowest was in TKG-55 (2.13). Based on the number of primary branches, the genotypes were grouped into two categories as few branched (<3.0) and high branched (>3.0). Among the 22 genotypes, three were few branched (GT-2, TC-289, TKG-55) and nineteen genotypes were highly branched (DS-1, ORM-17, Kanak, Chandana, AKT-101, Usha, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, TKG-21, T-12, SVPR-1, T-13, Uma, E8, RT-54, Paiyar) types.

4.1.3 Number of nodes per plant

The number of nodes per plant varied significantly among the sesamum genotypes (Table 2). The mean number of nodes per plant were 13.71. Highest number of nodes were recorded in E-8 (18.86) and lowest were recorded in TC-289 (8.80). Based on the number of nodes the genotypes were grouped into two categories as less with less than 15 number of nodes per plant and more with more than 15 number of nodes per plant. Among the 22 genotypes, 13 genotypes were grouped as having less (DS-1, ORM-17, Kanak, AKI-101, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, TC-289, Paiyar, RT-54, TKG-55) and eight genotypes as having more (Chandana, Usha, Thilarani, Phule Til-1, VS-9701, YLM17, Thilak, E-8) number of nodes per plant.

4.1.4 Internodal length (cm)

The internodal length varied significantly among the sesamum genotypes (Table 3). Highest internodal length was recorded in SVPR-1 (4.44cm) and lowest was recorded in E-8 (3.26cm). The mean internodal length of genotypes was 3.79cm. Based on the internodal length the genotypes were classified into two categories as the genotypes with short internodal length (<4.0cm) and long internodal length (> 4.0 cm). Among the 22 genotypes, 18 genotypes were short in internodal length (DS-1, ORM17, Kanak, Chandana, usha, YLM-17, VS-9701, Phule Til-1, Thilarani, Thilak, TKG-21, T-12, GT-2, Uma, E-8, RT-54, Paiyar, TKG-55) and four were long (TC-289, SVPR-1, T-13, AKT101) in internodal length.

Table 2. Identification and grouping of sesamum genotypes based on plant height, number of primary branches per plant and number of nodes per plant

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5% Short Medium Tall

Plant height (cm) 113.47 77.23 78.00 90.40 76.36 80.38 100.20 96.40 107.00 105.30 98.60 88.66 88.97 86.14 94.47 75.45 91.50 119.56 77.15 97.00 96.50 77.29 91.65 5.40 14.92 : <100 cm : 100-115 cm : > 115 cm

Groups

Number of primary branches per plant 4.80 3.53 3.53 3.93 3.93 4.53 5.13 3.13 5.46 4.26 4.66 3.60 3.06 3.33 3.73 2.73 3.60 3.06 2.40 3.06 5.0 2.13 3.75 0.40 1.10 Few high : < 3.0 : > 3.0

Groups

Number of nodes per plant 14.06 11.46 12.60 15.66 14.06 15.26 17.06 18.36 15.86 15.86 16.93 10.93 12.86 12.00 12.60 10.80 13.60 18.86 8.80 11.13 13.80 9.20 13.71 1.30 3.59

Groups

Medium Short Short Short Short Short Medium Short Medium Medium Short Short Short Short Short Short Short Tall Short Short Short Short

High High High High High High High High High High High High High High High Few High High Few High High Few

Less Less Less More Less More More More More More More Less Less Less Less Less Less More Less Less Less Less

Less More

: < 15 : > 15

Table 3. Identification and grouping of sesamum genotypes based on internodal length, stem pigmentation and number of leaves per plant Number of leaves per plant 81.46 75.66 69.33 83.60 75.93 85.66 94.13 75.66 93.86 81.86 97.00 65.66 68.06 60.83 66.44 50.60 71.86 62.66 45.53 54.53 82.33 48.13 72.30 2.21 6.10 Few Medium Many : < 60 : 60-80 : > 80 Many Medium Medium Many Medium Many Many Medium Many Many Many Medium Medium Medium Medium Few Medium Medium Few Few Many Few

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5% Short Long

Internodal length (cm) 3.82 3.70 3.30 3.64 4.33 3.37 3.60 3.98 3.85 3.86 3.93 3.40 3.73 4.44 4.27 3.76 3.35 3.26 4.35 3.61 3.86 3.97 3.79 0.16 0.44 : < 4.0 cm : > 4.0 cm

Groups Short Short Short Short Long Short Short Short Short Short Short Short Short Long Long Short Short Short Long Short Short Short

Stem pigmentation Strong Medium Weak Strong Strong Medium Weak Weak Strong Weak Strong Medium Weak Weak Medium Medium Weak Medium Medium Weak Medium Strong

Groups

4.1.5 Stem pigmentation

The stem pigmentation varied among the sesamum genotypes (Table 3, Plate-1). Based on the stem pigmentation, the genotypes were grouped as weak, medium and strong stem pigmentation types. Among the 22 genotypes, eight genotypes were weak (Kanak, Thilarani, Phule Til-1, YLM-17, T-12, SVPR-1, Uma, RT-54), eight genotypes were medium (ORM-17, Usha, TKG21, T-13, GT-2, E-8, TC-289, Paiyar) and six were strong (Chandana, AKT-101, VS-9701, Thilak, TKG-55, DS-1) in stem pigmentation.

4.1.6 Number of leaves per plant

The number of leaves per plant varied significantly among the genotypes. The mean number of leaves per plant were 72.30. Highest number of leaves were recorded in the genotype Thilak (97.0) and lowest was recorded in TC-289 (45.53). Based on the number of leaves the genotypes were classified into three categories as few (< 60 leaves per plant), medium (60-80 leaves per plant) and many (>80 leaves per plant). Among the 22 genotypes, four genotypes were grouped as having few (GT-2, TC-289, RT-54, TKG-55), ten genotypes as having medium (ORM-17, Kanak, AKT-101, Phule Til-1, TKG-21, T-12, SVPR-1, T-13, Uma, E-8) and eight genotypes as having many (Paiyar, Thilak, YLM-17, VS-9701, Thilarani, Ush, Chandana, DS-1) number of leaves per plant.

4.1.7 Leaf length (cm)

The leaf length varied significantly among the sesamum genotypes (Table 4, Plate-1). The mean leaf length was 8.47cm. The highest leaf length was recorded in E-8 (11.06cm) and the lowest leaf length was observed in T-13 (5.82cm). Based on the leaf length, the genotypes were grouped into three categories viz., short (<8.0cm), medium (8.0-10.0cm) and long (> 10.0cm) types. Among the genotypes, eight had short (ORM-17, Kanak, Chandana, Usha, TKG-21, T-12, T-13, Paiyar), 11 were having medium (DS-1, AKT-101, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, SVPR-1, GT-2, Uma, RT-54) and three had long (TKG-55, E-8, TC289) leaf length.

4.1.8

Leaf shape

The leaf shape did not vary among the sesamum genotypes under study. All the genotypes were having the Lanceolate shape with entire margin (Table 4).

4.1.9 Leaf colour

The leaf colour varied among different sesamum genotypes (Table 4, plate-1). Based on the leaf colour the genotypes were grouped into three categories as dark, medium and light green types. Among the 22 genotypes, seven genotypes had light green leaves (Chandana, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, RT-54). three genotypes medium green leaves (DS-1, E-8, Paiyar) and 12 genotypes dark green leaves (ORM-17, Kanak, AKT-101, Usha, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, TC-289, TKG-55),

4.1.10 Leaf petiole pigmentation

The leaf petiole pigmentation varied among the genotypes. (Table 4, Plate 2). Based on the leaf petiole pigmentation the genotypes were grouped into three categories as dark, medium and light leaf petiole pigmented types. Among the 22 genotypes, nine were light pigmented (DS-1, ORM-17, AKT-101, Usha, VS-9701, TKG-21, T-12, TC-289, TKG-55), nine were medium pigmented (Kanak, Phule Til-1, SVPR-1, T-13, GT-2, Uma, E-8, RT-54, Paiyar) and four were darkly pigmented (Chandana, Thilarani, Thilak, YLM-17),

Table 4. Identification and grouping of sesamum genotypes based on leaf length, leaf shape, leaf colour and leaf petiole pigmentation Leaf length (cm) 9.57 6.20 7.08 7.54 8.24 7.63 8.14 9.95 9.14 8.78 9.15 7.69 7.60 8.06 5.82 9.04 9.25 11.06 10.14 8.50 7.38 10.15 8.47 0.20 0.55 Leaf petiole pigmentation Light Light Medium Dark Light Light Dark Medium Light Dark Dark Light Light Medium Medium Medium Medium Medium Light Medium Medium Light

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5%

Group Medium Short Short Short Medium Short Medium Medium Medium Medium Medium Short Short Medium Short Medium Medium Long Long Medium Short Long

Leaf shape Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate Lanceolate

Leaf colour Medium green Dark green Dark green Light green Dark green Dark green Light green Light green Light green Light green Light green Dark green Dark green Dark green Dark green Dark green Dark green Medium green Dark green Light green Medium green Dark green

Short leaf length Medium leaf length Long leaf length

: < 8.0 cm : 8.0-10.0cm : > 10.0 cm

Plate 1. Stem pigmentation, leaf length, leaf colour of sesamum genotypes

Table 5. Identification and grouping of sesamum genotypes based on days to 50 per cent flowering and days to maturity. Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5% Days to 50 % flowering 42.7 40.3 42.0 43.3 38.3 39.7 44.3 46.0 44.7 42.7 43.7 38.7 39.7 39.7 43.0 39.0 42.0 43.3 41.3 39.3 42.3 41.0 41.7 1.3 3.3 Early maturity Late maturity Groups Late Late Late Late Early Early Late Late Late Late Late Early Early Early Late Early Late Late Late Early Late Late Day to maturity 94.0 94.3 94.7 92.3 81.0 84.7 89.3 96.0 90.0 80.7 90.0 80.3 84.7 83.3 91.0 81.7 92.7 86.0 92.0 81.7 91.3 90.3 88.2 2.9 8.1 : < 85 days : > 85 days Groups Late Late Late Late Early Early Late Late Late Late Late Early Early Early Late Early Late Late Late Early Late Late

Early flowering : < 40 days Late flowering : > 40 days

Table 6. Identification and grouping of sesamum genotypes based on flower petal colour and flower hairiness

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55

Flower petal colour Light Pink Light Pink Light Pink Medium Pink Light Pink Light Pink Light Pink Medium Pink Light Pink Medium Pink Light Pink Dark Pink Dark Pink Dark Pink Dark Pink Light Pink Light Pink Dark Pink Dark Pink Light Pink Light Pink Dark Pink

Flower hairiness Medium Low Low Medium Medium Dense Low Medium Low Medium Low Medium Dense Dense Medium Dense Dense Low Medium Medium Low Low

Plate 2. Leaf petiole pigmentation, flower petal colour, flower hairiness of sesamum genotypes

4.1.11 Days to 50 per cent flowering

The days to 50 per cent flowering varied significantly among the genotypes Table 5). The average days taken by the genotypes for fifty per cent flowering were 41.7. The genotype Phule Til-1 took more days to 50 per cent flowering (46.0), where as the genotype AKT-101 took less days for 50 per cent flowering (38.3). The genotypes were grouped into two categories as early (AKT-101, Usha, TKG-21, T-12, SVPR-1, GT-2, RT-54) and late (DS-1, ORM-17, Kanak, Chandana, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, T-13, Uma, E-8, TC-289, Paiyar, TKG-55) flowering types.

4.1.12 Days to maturity

The days to maturity varied significantly among the genotypes (Table 5). The average days taken by the genotypes for maturity were 88.2. The genotypes Phule Til-1 took more days for maturity (96.0) where as the genotype TKG-21 took less days for maturity (80.3). Based on the days to maturity the genotypes were grouped into two categories as early (AKT101, Usha, TKG-21, T-12, SVPR-1, GT-2, RT-54) and late (DS-1, ORM-17, Kanak, YLM-17, Chandana, Thilrani, Phule Til-1, VS-9701, Thilak, T-13, Uma, E-8, TC-289, Paiyar, TKG-55) maturity types.

4.1.13 Flower petal colour

The flower petal colour varied among the genotypes (Table 6, Plate-2). Based on the flower petal colour the genotypes were grouped into three categories as light pink, medium pink and dark pink. Among the 22 genotypes, 12 genotypes (DS-1, ORM-17, Kanak, AKT-101, Usha, Thilarani, VS-9701, Thilak, GT-2, Uma, RT-54, Paiyar) had light pink, three genotypes (Chandana, Phule Til-1, YLM-17) had medium pink and seven genotypes (TKG-21, T-12, SVPR-1, T-13, E-8, TC-289, TKG-55) had dark pink petal colour.

4.1.14 Flower hairiness

The genotypes varied among themselves for flower hairiness (Table 6, Plate-2). Based on the flower hairiness, the genotypes were grouped into three categories as low, medium and dense hairy types. Among the 22 genotypes, eight genotypes were low (ORM-17, Kanak, Thilarani, VS9701, Thilak, E-8, Paiyar, TKG-55), nine were medium (DS-1, Chandana, AKT-101, Phule til1, YLM-17, TKG-21, T-13, RT-54, TC-289) and five were dense (Usha, T-12, SVPR-1, GT-2, Uma) in flower hairiness.

4.1.15. Number of pods per axil

The number of pods per axil varied among the different sesamum genotypes (Table 7, Plate-3). Based on the number of pods per axil, the genotypes were grouped into two groups as single and alternate pod bearing types and multiple pod bearing types. Among the 22 genotypes, 19 were single and alternate pod bearing (DS-1, ORM-17, Kanak, Chandana, AKT-101, Usha, Thilarani, Phule Til-1, VS-9701, YLm-17, Thilak, T12, SVPR-1, T-13, Uma, TC-289, RT-54, Paiyar, TKG-55) and three were multiple pod bearing (TKG-21, GT-2, E-8) types.

4.1.16 Number of pods per plant

The number of pods varied significantly among the different sesamum genotypes (Table 7). Highest number of pods was recorded in the genotype DS-1, (79.60) and the lowest in Phule Til-1 (34.93). Based on the number of pods the genotypes were grouped into three categories as less (< 40 pods per plant), moderate (40-60 pod per plant) and more (> 60 pods per plant). Among the 22 genotypes, five had less (Kanak, Phule Til-1, TKG-21, GT-2, TC-289), twelve had moderate (ORM-17, Chandana, AKT-101, Usha, YLM-17, T-12, SVPR-1, T-13, Uma, RT-54, Paiyar, TKG-55) and five had more (DS-1, Thilarani, VS-9701, Thilak, E-8) pod numbers per plant.

Table 7. Identification and grouping of sesamum genotypes based on number of pods for axil, number of pods per plant and pod length Number of pods per axil S&A S&A S&A S&A S&A S&A S&A S&A S&A S&A S&A Multiple S&A S&A S&A Multiple S&A S&A S&A S&A S&A S&A Number of pods per plant 79.60 44.33 39.20 46.20 43.60 42.73 65.86 34.93 64.40 50.60 63.20 39.60 42.40 45.80 55.60 36.60 51.60 68.27 39.44 49.60 44.40 46.60 49.75 2.14 5.91 : Single and alternate : < 40 : 40-60 : > 100 Short Long pod length pod length : < 2.0 cm : > 2.0 cm Pod length (cm) 2.23 1.93 2.13 2.19 2.31 2.20 2.02 2.52 2.11 2.30 2.17 2.20 2.26 2.28 2.14 2.13 2.12 3.08 2.20 2.34 1.88 2.35 2.23 0.07 0.19

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em CD at 5% S and A Less Moderate More

Groups More Moderate Less Moderate Moderate Moderate More Less More Moderate More Less Moderate Moderate Moderate Less Moderate More Less Moderate Moderate Moderate

Groups Long Short Long Long Long Long Long Long Long Long Long Long Long Long Long Long Long Long Long Long short Long

Table 8. Identification and grouping of sesamum genotypes based on pod shape, pod beak, pod pubescence, number of locules per pod and pod dehiscence Pod pubescence Dense Nil Nil Medium Dense Medium Nil Medium Nil Nil Nil Dense Nil Medium Dense Medium Medium Nil Medium Dense Medium Medium Number of locules per pod Four Four Four Four Four Four Six Four Four Four Four Four Six Six Six Six Four Four Four Four Four Four

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55

Pod shape Round Round Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Oblong Round Round Oblong

Pod beak Short Long Long Short Short Long Long Long Long Long Long Long Long Long Short Short Long Short Long Short Long Long

Pod dehiscence Indehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent Dehiscent

Plate 3. Number of pods per axil, pod length, pod shape, pod beak pod pubescene of sesamum genotypes

4.1.17 Pod length (cm)

The sesamum genotypes varied significantly among themselves for pod length (Table 7, Plate-3). The mean pod length was 2.23. Significant highest pod length was recorded in E8 (3.08 cm) and lowest pod length was observed in Paiyar (1.88 cm). Based on the pod length, the genotypes were grouped into two categories as having short pod length (< 2.0 cm) and long pod length (> 2.0cm) types. Among the genotypes, two were short (ORM-17, Paiyar) and 20 were long (DS-1, Kanak, Chandana, AKT-101, Usha, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, E-8, TC-289, RT-54, TKG-55) in pod length.

4.1.18 Pod shape

The sesamum genotypes varied for pod shape (Table 8, Plate-3).Based on the pod shape the genotypes were grouped into two categories as round and oblong. Among the 22 genotypes, 18 genotypes had oblong pods (Kanak, Chandana, AKT101, Usha, Thilarani, Phile Til-1, VS-9701, YLM-17, Thilak, TKG-21, T-12, SVPR-1, T-13, GT2, Uma, E-8, TC-289, TKG-55) and four genotypes had round (DS-1, ORM-17, RT-54, Paiyar) pods.

4.1.19 Pod beak

The pod beak varied among the genotypes (Table 8).Based on the pod beak the genotypes were grouped as short and long beaked types. Among the 22 genotypes, seven were with short beaks (DS-1, Chandana, AKT-101, T-13, GT-2, E-8, RT-54) and 15 were with long pod beaks (ORM-17, Kanak, Usha, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, TKG-21, T-12, SVPR-1, Uma, TC-289, Paiyar, TKG55).

4.1.20 Pod pubescence

The genotypes varied for pod pubescence (Table 8, Plate-3). Based on the pod pubescence, the genotypes were grouped into three categories as nil, medium and dense types. Among the 22 genotypes, eight were grouped into nil (ORM-17, Kanak, Thilarani, VS9701, YLM-17, Thilark, T-12, E-8), nine into medium (Chandana, Usha, Phule Til-1, SVPR-1, GT-2, Uma, TC-289, Paiyar, TKG-55) and five into dense (DS-1, AKT-101, TKG-21, T-13, RT-54) pod pubescence types.

4.1.21 Number of locules per pod

The number of locules per pod varied among the sesamum genotypes (Table 8, Plate-4). Based on the number of locules per pod the genotypes were grouped into four loculed (DS-1, ORM-17, Kanak, Chandana, AKT-101, Usha, Phule Til-1, VS-9701, YLM-17, Thilak, TKG-21, Uma, E-8, TC-289, RT-54, Paiyar, TKG-55) and six loculed (Thilarani, T-12, SVPR-1, T-13, GT-2) types.

4.1.22 Pod dehiscence

The pod dehiscence varied among the genotypes (Table 8, Plate-4). Based on the pod dehiscence the genotypes were grouped into two categories as dehiscent (ORM-17, Kanak, Chandana, AKT-101, Usha, Thilarani, Phule Til-1, VS-9701, YLM-17, Thilak, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, E-8, TC-289, RT-54, Paiyar, TKG-55) and indehiscent (DS-1) types.

Table 9. Identification and grouping of sesamum genotypes based on seed colour, thousand seed weight and oil content

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5% Medium size Bold size

Seed colour White Black Brown Brown White Brown Black White Black Black Black White White White White White Brown White White Brown Black White

1000 seed weight (g) 3.07 3.04 3.18 3.13 3.20 3.18 2.67 3.52 2.93 3.38 3.33 3.51 3.16 3.23 3.35 3.45 3.27 3.67 3.57 3.39 3.31 3.60 3.27 0.02 0.05

Groups Medium Medium Medium Medium Medium Medium Medium Bold Medium Medium Medium Bold Medium Medium Medium Medium Medium Bold Bold Medium Medium Bold

Oil content (%) 48.5 42.2 44.0 44.7 49.0 44.2 46.6 48.8 44.3 40.0 49.3 47.9 48.6 48.9 49.0 48.9 45.1 49.2 48.8 42.0 47.8 48.2 46.63 0.30 0.85

Groups High Low Low Low High Low High High Low Low High High High High High High High High High Low High High

: < 3.5 g : > 3.5 g

Low High

: < 45 per cent : > 45 per cent

Plate 4. Number of loules per pod, pod dehiscence, seed colour, NaOH test of sesamum genotypes

Table 10. Identification and grouping of sesamum genotypes based on germination, seedling length and seedling vigour index.

Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-1 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean S.Em C.D. at 5%

Germination (%) 94.66 96.00 86.66 89.33 80.00 92.00 93.33 74.66 90.66 93.33 84.00 73.33 88.00 75.33 81.33 76.66 82.66 90.66 78.66 72.00 81.33 82.66 84.42

Seedling length (cm) 6.40 7.89 4.60 7.46 8.51 6.10 6.33 5.86 5.76 8.16 7.25 5.13 5.63 6.93 4.18 4.49 4.62 6.82 4.57 4.20 4.36 7.50 6.03 0.82 2.26

Seedling vigour index 609.33 762.93 399.86 663.33 676.42 560.00 590.88 519.26 519.26 759.73 615.93 372.13 500.80 520.03 340.09 344.20 317.08 615.14 360.00 302.40 353.94 618.81 514.70 75.63 202.01

Groups High High Low High High High High High High High High Low High High Low Low Low High Low Low Low High

Less vigorous High vigorous

: < 500 : > 500

Table 11. Identification and grouping of sesamum genotypes based on NaOH and KOH tests.

Colour of the solution Genotypes DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 NaOH test Light brown Dark brown Brown Brown Brown Brown Dark brown Brown Brown Dark brown Dark brown Light brown Dark brown Light brown Light brown Light brown Light brown Light brown Light brown Brown Dark brown Light brown KoH test Light yellow Dark brown Light brown Light brown Light brown Light brown Dark brown Light brown Dark brown Dark brown Dark brown Light brown Brown Yellow Yellow Yellow Yellow Light yellow Light yellow Light yellow Brown Light yellow

4.1.23 Seed colour

The seed colour varied among the sesamum genotypes (Table 9, Plate-4). Based on the seed colour the genotypes were grouped into three groups viz., black, brown and white. Among the 22 genotypes, six genotypes had black (ORM-17, Thilarani, VS-9701, YLM-17, Thilak, Paiyar), five genotypes brown (Kanak, Chandana, Usha, Uma, RT-54) and 11 genotypes white, (DS-1, AKT-101, Phule Til-1, TKG-21, T-12, SVPR-1, T-13, GT-2, E-8, TC-289, TKG-55) seed colour.

4.1.24 Thousand seed weight (g)

The thousand seed weight varied significantly among different sesamum genotypes (Table 9). The mean thousand seed weight of the genotypes was 3.27. Significantly highest 1000 seed weight was observed in the genotype E-8 (3.67 g) and lowest was observed in the genotype Thilarani (2.67 g). Based on the thousand seed weight, the genotypes were grouped into two categories as medium sized with the thousand seed weight less than 3.5 g and bold sized with the thousand seed weight more than 3.5 g. Among the 22 genotypes, seventeen genotypes were grouped into medium (DS-1, ORM-17, Kanak, Usha, Chandana, AKT-101, Thilarani, VS-9701, YLM-17, Thilak, T-12, T-13, SVPR-1, GT-2, Uma, RT-54, Paiyar) and five genotypes into bold in size (Phule Til-1, TKG-21, E-8, TC-289, TKG-55) for thousand seed weight.

4.1.25 Oil content (%)

The oil content varied significantly among the sesamum genotypes (Table 9). The mean oil content of the genotypes was 46.63 per cent. The oil content of the genotypes ranged from 49.3 (Thilak) to 40.0 per cent (YLM-17). Based on the percentage of oil content, the genotypes were grouped into two groups as having low (< 45%) and high (> 45%) on content. Among the 22 genotypes, eight genotypes had low (ORM-17, Kanak, Chandana, Usha, VS-9701, YLM-17, RT-54) and 14 genotypes had high oil content (DS-1, AKT-101, Thilarani, Phule Til-1, Thilak, TKG-21, T-12, SVPR-1, T-13, GT-2, Uma, E-8, TC-289, Paiyar, TKG-55).

4.1.26 Germination percentage

The germination percentage varied among the sesamum genotypes (Table 10). The mean seed germination percentage of the genotypes was 84.4 percent. Highest seed germination percentage was observed in ORM-17 (96.0) and the lowest was observed in RT54 (72.0).

4.1.27 Seedling length (cm)

The seedling length varied among the genotypes (Table 10) The seedling length ranged from 4.18 cm (T-13) to 8.51 cm (AKT-101) with a mean seedling length of 6.03cm.

4.1.28 Seedling vigour index

The seedling vigour index varied significantly among the genotypes (Table 10). The mean seedling vigour index of the genotypes was 514.7. Highest seedling vigour index was noticed in ORM-17 (762.9) and the lowest seedling vigour index was noticed in RT-54 (302.4). Based on the variation in the seedling vigour, the genotypes were grouped into two groups as low vigorous (<500) and high vigorous (> 500) types. Among the 22 genotypes, eight genotypes were low vigour and 14 genotypes were high vigour types.

4.2

IDENTIFICATION AND GROUPING OF GENOTYPES THROUGH CHEMICAL TESTS

SESAMUM

The results of various chemical tests are presented for varietal characterization.

Table 12. Identification and grouping of sesamum genotypes based on coleoptile growth response to GA3

Coleoptile growth (cm) Genotypes Control DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean Very low response Low response Moderate response 3.90 4.06 3.03 3.86 4.55 3.73 3.63 3.55 3.60 3.73 3.80 3.13 3.11 4.08 2.33 3.12 3.25 4.18 2.93 2.31 2.87 3.3 3.45 GA3 5.78 5.43 3.60 4.83 6.20 3.90 4.17 4.83 4.03 3.88 4.96 4.18 4.20 4.60 4.75 4.48 3.80 5.56 3.98 3.23 4.00 3.49 4.47 : < 10 per cent : 10-30 per cent : > 30 per cent

Per cent increase over control

Groups

48.20 33.70 18.81 25.12 36.26 4.55 29.75 36.05 11.94 4.02 30.52 33.54 35.04 12.74 103.86 43.58 16.92 33.01 35.83 39.82 39.37 4.80 30.79

Moderate Moderate Low Low Moderate Very low Low Moderate low Very low moderate Moderate Moderate Low Moderate Moderate Low Moderate Moderate Moderate Moderate Very low

Plate 5. KOH test, seedling response to GA3, seedling response to 2,4-D of sesamum genotypes

4.2.1 Sodium hydroxide (NaoH) test

Varied response of sesamum genotypes to sodium hydroxide test was observed (Table 11, Plate-4). Based on the colour development of the decanted solution, the genotypes were grouped into three groups viz., light brown, brown, and dark brown. Among the 22 genotypes the response of nine genotypes was light brown (DS-1, TKG-21, SVPR-1, T-13, GT-2, Uma, E-8, TC-289, TKG-55), seven genotypes was brown (Kanak, Chandana, AKT-101, Usha, Phule Til-1, VS-9701, RT-54) and six genotypes was dark brown (Thilarani, ORM-17, YLM-17, Thilak, T-12, Paiyar) in colour.

4.2.2 Potassium hydroxide (KOH) test

The genotypes exhibited varied response to potassium hydroxide test. (Table 11, plate-5) Based on the change in colour of solution the genotypes were grouped into five groups viz., light yellow, yellow, light brown, brown and dark brown responsive to KOH test. Among the 22 genotypes, five genotypes were light yellow (DS-1, TKG-55, TC-289, RT-54 and E-8), four genotypes were yellow (SVPR-1, T-13, GT-2, Uma), six genotypes were light brown (Kanak, Chandana, AKT-101, Usha, Phule Til-1, TKG-21), two genotypes were brown (T-12, Paiyar) and five genotypes were dark brown (ORM-17, Thilarani, VS-9701, YLM-17, Thilak) in colour.

4.2.3 Coleoptile growth response to gibberllic acid

The coleoptile length of sesamum genotypes showed varied response to GA3 (Table 12, Plate-5). The highest increase in coleoptile length was observed in genotype T-13 (103.86 per cent) and the lowest was observed in YLM-17 (4.02%). Based on the per cent increase in coleoptile length over control the genotypes were grouped into three categories as very low response, low response, and moderate response to gibberllic acid. Among the 22 genotypes the coleoptile growth of three genotypes had very low response (Usha, YLM-17, TKG-55), six genotypes had low response (Kanak, Chandana, Thilarani, VS-9701, SVPR-1, Uma), and 13 genotypes had moderate response (DS-1, ORM17, AKT-101, Phule Til-1, Thilark, TKG-21, T-12, T-13, GT-2, E-8, TC-289, RT-54, Paiyar) to gibberllic acid.

4.2.4 Coleoptile growth response to 2, 4-D

The coleoptile of sesamum genotypes showed varied response to 2,4-D (Table13, Plate-5). The mean length of coleoptile under control and at 2, 4-D was 3.45cm and 0.54cm, respectively. The highly affected genotype` was ORM-17 (92.36%) and the least affected genotype was T-13 (75.96%). Based on the percent decrease in coleoptiles length over control, the genotypes were grouped into two categories viz., susceptible (< 85%) and highly susceptible (> 85%). Among the 22 genotypes,14 genotypes were susceptible (Chandana, Phule Til-1, YLM-17, Thilak, TKG-21, E-8, TC-289, Paiyar, TKG-55, RT-54, GT-2, Uma, T-13, T-12) and eight genotypes were highly susceptible (DS-1, ORM-17, Kanak, AKT-101, Usha VS-9701, SVPR-1, Thilarani) to 2, 4-D.

Table 13. Identification and grouping of sesamum genotypes based on coleoptile growth response to 2, 4-D

Coleoptile length (cm) Genotypes Control DS-1 ORM-17 Kanak Chandana AKT-101 Usha Thilarani Phule Til-1 VS-9701 YLM-17 Thilak TKG-21 T-12 SVPR-1 T-13 GT-2 Uma E-8 TC-289 RT-54 Paiyar TKG-55 Mean Susceptible Highly susceptible 3.9 4.06 3.03 3.86 4.55 3.73 3.63 3.55 3.60 3.73 3.80 3.13 3.11 4.08 2.33 3.12 2.25 4.18 2.93 2.31 2.87 3.33 3.45 2, 4-D 0.47 0.31 0.33 0.62 0.59 0.50 0.53 0.55 0.48 0.68 0.75 0.61 0.63 0.60 0.56 0.71 0.61 0.79 0.45 0.49 0.46 0.57 0.54 : < 85 per cent : > 85 per cent

Per cent decrease over control 87.94 92.36 89.10 83.93 87.03 86.59 85.39 84.50 86.66 81.76 80.26 80.51 79.74 85.29 75.96 77.24 81.23 81.10 84.64 78.78 83.97 82.88 83.49 Groups

Highly susceptible Highly susceptible Highly susceptible susceptible Highly susceptible Highly susceptible Highly susceptible Susceptible Highly susceptible Susceptible Susceptible Susceptible Susceptible Highly susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible

Table 14. Analysis of RAPD banding pattern for sesamum genotypes

Total number of Primers bands OPP 07 OPP-09 OPP-03 OPA-10 OPA-13 OPP-01 OPA-14 OPA-15 OPA-08 OPA-12 Total Average 6 7 7 10 10 8 4 2 4 4 62 6.2 4 5 7 10 6 7 4 2 4 3 52 5.2 Polymorphic bands

Per cent polymorphism (%) 66.66 71.22 100 100 60 87.5 100 100 100 75 86.05 -

Fig. 1. Dendrogram obtained from pooled data of RAPD profile of sesamum genotypes

LEGEND

1: Kanak 2: Usha 3: VS-9701 4: YLM-17 5: T-12 6: SVPR-1 7: T-13 8: E-8 9: Paiyar 10: TKG-55 M : Marker

Plate 6. RAPD profile of ten sesamum genotypes

Table 15. Genetic similarity coefficient based on RAPD data among the ten sesamum genotypes Kanak Kanak Usha VS-9701 YLM-17 T-12 SVPR-1 T-13 E-8 Paiyar TKG-55 1.00 0.70 0.72 0.75 0.71 0.67 0.71 0.64 0.62 0.64 1.00 0.94 0.86 0.84 0.71 0.83 0.80 0.83 0.85 1.00 0.90 0.84 0.70 0.82 0.82 0.85 0.83 1.00 0.86 0.76 0.84 0.72 0.78 0.80 1.00 0.69 0.78 0.73 0.71 0.76 1.00 0.75 0.65 0.63 0.76 1.00 0.76 0.67 0.76 1.00 0.83 0.86 1.00 0.85 1.00 Usha VS-9701 YLM-17 T-12 SVPR-1 T-13 E-8 Paiyar TKG-55

4.3

IDENTIFICATION AND GROUPING OF SESAMUM GENOTYPES THROUGH MOLECULAR MARKERS

Ten sesamum genotypes were subjected to RAPD assay to study the genetic diversity. Ten random decamer primers revealed a high DNA polymorphism among the genotypes (Table 14, Plate-6).Ten primers produced a total of 62 amplified products. Among these 52 were polymorphic with an average of 86.05 per cent polymorphism out of the ten primers used for amplification. Only five primers (OPP-03, OPA-10, OPA-14, OPA-15, OPP08) showed highest (100%) polymorphism. The primer OPA-13 gave the lowest polymorphism (60%). The number of bands ranged from 2 (OPA-15) to 10 (OPA-10 and OPA-13) with an average of 6.2 bands per primer. Genetic similarity coefficient ranged from 0.62 to 0.94 (Table 15). Highest genetic coefficient similarity (0.94) was observed between Usha and VS-9701; whereas least similarity (0.62) was observed between Kanak and Paiyar. Further, the dendrogram constructed from the pooled data (Fig.1) clearly showed, two major clusters namely cluster A and B. Only one genotype (Kanak) was found on cluster B. Cluster A was again divided into sub clusters (A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14,). Two main clusters had 68 per cent similarity.

V. DISCUSSION
The varietal description for identification of crop varieties has assumed critical importance in national and international seed programmes and there is a considerable need for the development of reliable methods and identifiable characters for the purpose. The characters for which a variety is distinct from other could be morphological, chemical and biochemical in nature which aids in varietal identification. With the introduction of New Seed Policy and globalization of agriculture, a large number of new varieties of all crops including sesamum are being released by state and central government, local and foreign private seed companies. Due to the mushrooming growth of seed industry, it is essential to ensure the quality of seed materials in the market. To ensure the quality seeds, one should be equipped to identify varietal purity both at seed and seedling stages. Varietal description provided by the concerned plant breeders are inadequate to distinguish the variety at seed level or in the laboratory. Therefore, there is a great need to develop keys to identify varietal purity based on the seed characteristics in the laboratory on the routine basis. This is very much applicable to have a quick and effective check on the quality of seed moving in commercial channels. In the present investigation, attempts were made to characterize 22 sesamum genotypes based on the morphological, chemical and biochemical methods. The results obtained from the above study have been discussed in this chapter.

5.1 PLANT MORPHOLOGICAL CHARACTERISTICS

Use of plant diagnostic characteristics to identify a variety has been classical taxonomic approach for both varietal purity and varietal identification. The plant morphological characteristics such as plant height, number of primary branches per plant, number of nodes per plant, internodal length, and stem pigmentation were observed and classified into different groups. Based on the grouping seed keys were prepared (Fig. 2). The plant height is one of the important characteristics, which help in differentiating the genotypes as short, medium and tall. The sesamum genotypes exhibited significant variability in plant height ranging from 75.45 cm (GT-2) to 119.56 (E-8) with a mean plant height of 91.65 cm. Based on this variation in plant height, the 22 genotypes under study were grouped into short (<100 cm) with 17 genotypes, medium (100-115c cm) with four and tall (>115 cm) with one genotype. This grouping is based on the classification of sesamum varieties earlier by Rhind and Thein (1933). Similar classification was reported by Surendra Prakash and Singhal (1997) in peas, Ravikumar (1999) in soybean, Jain (2001) in mungbean, Mate and Shelar (2006) in sorghum varieties. The wide variation in plant height might be due to variation in genetic constitution of the genotypes as the plant height is controlled by three to ten pairs of genes with heritability value of 40 to 50 per cent in sesamum (Weiss, 1971). The plant height might have also been influenced by agronomic and environmental conditions. The number of primary branches determines ultimately the pod bearing ability of plant which will inturn contributes to the yield, hence identification and selection of genotypes with more branching ability is necessary. In the present study, the genotypes exhibited varied branching ability with a mean primary branches of 3.75. The genotype VS-9701 recorded the higher primary branches of 5.46, whereas it was lowest in TKG-55 (2.13). Based on the number of primary branches, 22 genotypes were classified as plants with less branches (<3.0) and more branches (>3.0). Similar results of varied branch numbers and grouping of sesamum varieties were reported by Rhind and Thein (1932) and Choudhary et al. (1977). The branching habit has also been affected by environmental conditions, sowing seasons, seed rate and spacing (Weiss, 1971). Number of primary braches in sesame is highly heritable and is influenced by the genetic content of the genotype (Shadakshari et al., 1995). The number of nodes on main stem and the internodal length influences the plant height and the number of branches (Weiss, 1971). In the present study the genotypes exhibited variability with respect to number of nodes per plant and internodal length. The number of nodes per plant ranged from 8.8 (TC-289) to 18.86 (E-8) with a mean of 13.71 cm. Among the 22 genotypes, 14 genotypes were with less number of nodes (<15.0). Similarly, 18 genotypes were with short internodal length (<4.0 cm). Similar variation in these characters

were earlier reported by Jayaramaiah et al. (1993) in sunflower germplasms, Muralimohan Reddy et al. (2004) in castor varieties and hybrids. The variation is genetically controlled and further influenced by environmental conditions and agronomic practices. The stem pigmentation is one of the conspicuous characteristics in varietal identification. Based on the stem pigmentation, the genotypes were grouped as weak (eight genotypes), medium (eight genotypes) and strong (six genotypes) pigmented. Similar results of varied stem pigmentation were reported earlier by Muralikrishna et al. (1990) in cotton, Jain (2001) in mungbean and Yadav and Srivastava (2002) in chickpea varieties. Stem pigmentation is under genetic control and it may also be affected by light intensity and temperature prevailing during the crop growth, thus resulting in variation in the degree of pigmentation in black gram varieties. (Chakrabarthy and Agrawal, 1989b).

5.2 LEAF CHARACTERS

The number of leaves per plant, leaf area or size of leaf and chlorophyll pigmentation plays an important role in the yielding ability of the genotype as the leaves are the food synthesis site of the plants. In the present study, the characteristics related with have been recorded and based on these characteristics the genotypes were categorized into different groups and seed key was prepared (Fig. 2) The genotypes varied significantly for leaf number with a maximum leaf number of 97.0 in Thilak and minimum of 45.53 in TC-289. Based on this variation in leaf number, the genotypes were grouped into three groups namely few (<60 leaves per plant), medium (60-80 leaves per plant) and many (> 80 leaves per plant). Similar grouping was earlier reported by Ranjendra Prasad et al. (2003) in sunflower hybrids and their parental lines. Similarly the genotypes exhibited significant variability in leaf length. The highest leaf length was noticed in E-8 (11.06 cm) whereas it was lowest in the genotype T-13 (5.82 cm). The variability helped in grouping the genotypes into three categories namely short (<8.0 cm), medium (8.0-10.0 cm) and long (> 10.0 cm) types. Similar variations and grouping of genotypes in these leaf characters were reported earlier by Rosta (1975) in rice varieties and Surendra Prakash and Singhal (1997) in pea varieties. The genetic constituent of the genotype coupled with environmental and agronomic factors might have led to the variations in these characters. Though, Kashiram (1930) and Weiss (1971) observed varied leaf shapes such as lobed, lanceolate and linear in sesamum genotypes, the genotypes under the present study did not vary in their leaf shape. All the genotypes had lanceolate leaf shape. The leaf colour is one of the important characteristics used for grouping of the genotypes. The leaf colour depends upon the intensity of chlorophyll pigmentation which inturn varies with genotypes according to their genetic constitution. The genotypic variation in colour of the leaf is also governed to some extent by the varied response to environmental conditions such as light intensity and nutrition. Thus in the present investigation, leaf colour of the sesamum genotypes varied from light green to medium green to dark green. This variation helped in grouping the genotypes into three categories. Among the 22 genotypes, seven genotypes were light green, three genotypes were medium green and 12 genotypes were dark green in colour. Similar results were reported earlier by Rosta (1975) in rice, Surendra Prakash and Singhal (1997) in pea, Jawaharlal (1994) and Ezilkumar (1999) in cotton genotypes. The leaf petiole pigmentation at peak flowering stage varied among the 22 sesamum genotypes. These genotypes were grouped into three groups as light (nine), medium (nine) and dark (four) pigmented based on the intensity of anthocyanin pigmentation. Similar results were also reported by Ezilkumar (1999) in cotton, and Jain (2002) in mung bean varieties. Rajendra Prasad et al. (2003) grouped the sunflower hybrids and their parental lines based on leaf petiole pigmentation as present or absent. The variation in leaf petiole pigmentation among the genotypes may be due to varied intensity of anthocyanin pigmentation, which is governed genetically and also influenced by the light intensity and nutritional status of the soil under which the crop is raised.

5.3 FLOWER CHARACTERISTICS

Based on the flower characteristics such as days to 50 per cent flowering, flower petal colour and flower hairiness the genotypes could be grouped into different categories. These characters are also useful in genetic purity testing of the seed lots. Based on this seed keys were prepared (Fig.3). The days to 50 per cent flowering varied with the genotypes, ranging from 38.3 days (AKT-101) to 46.0 days (Phule Til-1) with an average of 41.68 day. The sesamum genotypes were grouped as early (<40 days) and late (>40 days) flowering types. Among the 22 genotypes, seven genotypes were early in flowering and 15 genotypes were late in flowering. Similar results were reported by Srivastav and Kaushal (1972) in sesame, Stahi and Pandey (1981) in soybean and Mudzana et al. (1995) in fababean varieties. Reasons attributed for difference in days to 50 per cent flowering among the genotypes is that the character is dependent on a minor gene complex (Weiss, 1971). The days required from sowing to fifty per cent flowering is a genetical character in peas as reported by Surendra Prakash and Singhal (1997). The environmental conditions also have selective influence on flowering. The petal colour of the flower is one of the important characters for characterization. Based on the variation in the flower petal colour, the genotypes were grouped into three categories namely light pink (12 genotypes), medium pink (three genotypes) and dark pink (seven genotypes) types. Similar results were observed by Kashiram (1930) in sesamum, Edgar et al. (1970) in soybean, Ezilkumar (1999) in cotton, Tarasatyavathi et al. (2004) and Kumar et al. (2005) in jute varieties. Langham (1947a) reported that the actions of the genes were responsible for variations in the petal colour of the genotypes. The genes determine the colour of the petal by developing or blocking of anthocyanin pigmentation. The flower hairiness also varied with the genotypes. Based on the the flower hairiness the genotypes were grouped into three categories as low (eight), medium (nine) and dense (five) haired types. Similar results of varied flower hairiness were reported by Kashiram (1930), Mohammed and Alam (1933), Rhind and Thein (1933) in sesamum and Bahrenfus and Fehr (1984) in soybean cultivars. Langham (1947a) opined that the flower hairiness is governed by specific genes.

5.4 POD CHARACTERISTICS

The pod characteristics influence the yielding ability of the plant. The genotypic variation was observed for various characteristics such as number of pods per axil, number of pods per plant, pod length, pod shape, pod beak, pod pubescence, number of locules per pod, pod dehiscence which help for classifying the genotypes into different groups. Based these groupings, seed keys were prepared and presented (Fig. 4 and 5). The number of pods per axil and arrangement of these pods on the stem varied with the genotypes, thus leading to grouping them as single podded with alternate arrangement and multiple podded types. Among the genotypes, 19 were single and alternate type and three were multiple types. Similar results were reported by Rhind and Thein (1932) in sesamum, Yadav and Srivastava (2002) in chickpea varieties. The single podedness or multipodedness per axil depends on the gene actions as single podedness is governed by dominant genes and the multipodedness is governed by its recessive counterpart (Langham, 1947a). The number of pods per plant varied among the genotypes with highest pods per plant noticed in the genotype DS-1 (79.67) and least was observed in genotype Phule Til-1 (34.93). Based on this character the genotypes were grouped as less (<40), moderate (40-60) and more (>60) pod bearing types. Similar results were observed by Gupta and Gupta (1977) in sesamum, Rasaily et al. (1986) in soybean, Yadav and Srivastava (2002) in chick pea varieties. The variation in pod number may be due to pod bearing ability of the genotype itself and varied response to environmental conditions and nutritional status of the soil to some extent. The pod length varied among the genotypes. Based on this variation in pod length, the genotypes were grouped as short (<2.0 cm) and long (72.0 cm). The highest pod length was observed in the E-8 genotype (3.08 cm) and the lowest was observed in the genotype Paiyar (1.88 cm) with a mean pod length of 2.23 cm. Among the 22 genotypes, two genotypes were short and 20 genotypes were long in pod length. Similar classification of varieties was reported by Gupta and Gupta (1977) in sesamum, Mudzana et al. (1995) in faba bean, Bonetti et al. (1995) in bean, Surendra Prakash and Singhal (1997) in pea cultivars. Weiss (1973) reported that the inheritance of capsule length was found to be conditioned by two to five pairs of factors with a heritability value of 50 to 70 per cent indicating the genetic influence in determining the pod length. The pod shape varied with the genotypes. Based on the shape of pod, the genotypes were grouped as oblong (eighteen genotypes) and round (four genotypes). Similar results were reported by Kooistra (1964) in pea and Sankarapandian (2002) in cowpea varieties. Weiss (1971) reported that each pod shape tends to be a varietal characteristic, but environment is a major modifying factor and several forms may occur in one plant in sesame. The pod beakness also varied among the genotypes under study. Based on the pod beakness variation, the genotypes were grouped into two categories as short beaked (seven genotypes) and long beaked (fifteen) types. Similar type of categorization was reported earlier by Surendra Praksh and Singhal (1973) in pea varieties and expression of pod beak character is controlled by the gene bt in pea. The pod pubescence varied among the sesamum genotypes. Based on the intensity of pubescence on pod, the genotypes were catgorised as nil (eight genotypes), medium (nine genotypes) and dense (five genotypes) pod pubescence types. Similar type of grouping were reported earlier by Kashiram (1930), Mohammed and Alam (1933) in sesame, Edgar et al. (1970) and Tarasatyavathi et al. (2004) in soybean varieties. Rhind and Thein (1932) reported that the genetic and environmental factors influences pod hairiness. The number of locules per pod varied with the variety. Based on the variation in the number of locules per pod the genotypes were grouped as four (seventeen genotypes) and six (five genotypes) loculed type. Similar type of results were reported by Rhind and Thein (1932) in sesamum and Yadav and Srivastava (2002) in chickpea varieties. The genotypic expression of four and six loculed pods in mainly controlled by the gene actions as four locules expression is governed by dominant gene and six loculed by recessive gene in sesamum (John, 1934).

The pod dehiscence varied with the genotypes. Based on the dehiscence characters of pod, the genotypes were grouped as dehiscent (21 genotypes) and indehiscent (one genotype) types. Similar results were reported by Muralimohan Reddy et al. (2004) in castor varieties and Kumar et al. (2004) in jute varieties. Weiss (1971) reported that two alleles control the dehiscence and indehiscence act by influencing the monocarp development in sesamum. The pod dehiscence is also influenced by the environmental conditions especially the temperature at the time of maturity The days to maturity varied with the genotypes ranging from 80.26 days (TKG-21) to 96.0 days (Phule Til-1) with an average days to maturity of 88.23. Seven genotypes were grouped as early (85 days) and fifteen genotypes was grouped as late (> 85 days) flowering types. Similar classification was reported earlier by Kashiram (1930), Mohammed and Alam (1933) in sesamum, Bonetti, et al. (1995) in bean, Yadav and Srivastav (2002) in chickpea and Tarasatyvathi et al.(2004) in soybean varieties. Though the duration of the crop growth is a genetically controlled character it is also influenced by the environment and crop growth conditions such as soil moisture etc.

5.5 SEED CHARACTERISTICS

Seed morphological characteristics are useful in development of varietal identification keys and also in genetic purity testing. The seed characteristics such as seed colour, thousand seed weight have been recorded and based on the variations, in these characteristics, the genotypes were grouped into different categories and varietal identification keys were prepared (Fig.6). The seed colour is one of the important parameters useful in varietal identification. Based on the variations in the seed colour, the genotypes were grouped as black (six genotypes), brown (five genotypes) and white (11 genotypes). Similar pattern of grouping was reported earlier by Mohammed and Alam (1933), Rhind and Thein (1933) in sesame, and Arunkumar et al. (2004) in pearl millet hybrids and their parental lines. The seed colour difference was mainly due to heritable characters of the genotypes. Nohara (1933) attributed the difference between brown and black seed colour due to two genes namely B1 and B2. Seed colour is also selectively influenced by environmental conditions such as rainfall during harvest and incidence of the diseases. Thousand seed weight varied significantly with the genotypes. Thousand seed weight ranged from 2.67 g (Thilarani) to 3.67 g (E-8) with a mean weight of 3.27 g. Based on this, the genotypes were grouped as medium (< 3.5 g) and bold (< 3.5 g). Among the genotypes, 17 genotypes were medium and five genotypes were bold in seed size. The same pattern of classification was earlier reported by Elsaeed (1967) in broad bean, Tiwari (1978), Paramesh (1983) in soybean, Chakrabrathy and Agrawal (1990) in blackgram, Muralimohan Reddy et al. (2004) in castor and Rajendraprasad et al. (2004) in sunflower hybrids and their parental lines. The variation among the genotypes may be due to inherent genotypic differential conditions that had existed during the crop growth, seed development and maturation. The genotypic variation in test weight may also be due to the varied capacity of the genotype to utilize the reserved food material.

5.5.1 Oil content

The genotypes exhibited significant variation for the oil content. The highest oil content was noticed in the genotype Thilak (49.3%) and the lowest was noticed in YLM-17 (40.0%) with a mean of 46.63 per cent. Based on this, the genotypes were grouped as low (< 45 %) with 15 genotypes and high oil (> 45%) with seven genotypes. Similar results were earlier reported by Luan and Han (1990) in groundnut, Shadakshri et al. (1995) in sesamum. The variation in oil content might be a genetic factor. Weiss (1971) reported that two to seven polygenes were involved in the inheritance of oil content.

5.6 SEEDLING CHARACTERS

The seed germination percentage varied among the genotypes due to the quality parameters and could be attributed to better development of seeds The seed germination percentage ranged from 72.0 (RT-54) to 96.0 per cent (ORM-17) with a mean seed germination percentage of 84.4 per cent. The seedling length varied among the genotypes. The highest seedling length was noticed in the genotype T-13 (4.18 cm) and the lowest in the genotype AKT-101 (8.41 cm) with mean seedling length of 6.03 cm. The variation in seedling length was due to it better quality of seeds of the genotypes (Fehr. 1973). The seedling length was found to be important characteristic in the blackgram varieties identification as reported by Cakrabarthy and Agrawal (1989b). Among the genotypes studied, the seedling vigour index of the genotypes ranged from 762.93 (ORM-17) to 302.4 (RT-54). The variation in seed vigour may be due to varied germination and seedling length recorded with the genotypes. It is capability of the genotype to produce the quality seeds based on the utilization of the resources such a light, moisture and nutrients. The translocation of food reserves from plant to seed, leading to better development of seed is the genotypic character, thus, resulting into low and high vigour types.

5.7 SEED AND SEEDLING RESPONSE TO CHEMICAL TESTS

Varietal identification by morphological characters is laborious, time consuming, tedious, cumbersome and costly affair. A number of chemical tests have been developed for varietal identification such as sodium hydroxide test, potassium hydroxide test, gibberllic acid response test and 2, 4-D soak test. These chemical tests are very quick, easy and reproducible (Agrawal and Sharma, 1989. Ashwani Kumjar et al. 1995. Very often these tests provide supportive evidence for the morphological evaluation of the seeds (Vanderburg and Vanzal, 1991). On the basis of colour reaction with sodium hydroxide solution, the genotypes were grouped into three categories as light brown (nine genotypes), brown (seven genotypes) and dark brown (six genotypes). Similar classification by NaoH test reported earlier by Sambasivarao et al. (2002) in rice, Ponnuswamy et al. (2003) in cotton and Biradarpatil (2006) in safflower genotypes. The colour reaction to sodium hydroxide solution was obtained in sesamum due to reaction of seeds to secondary metabolites (Vanderburg and Vanzwol, 1991). The difference in colour reaction of seeds seems to be due to differences in genetic back ground concerning the enzyme system (Chakrabarthy and Agrawal, 1990). The potassium hydroxide test is useful in determining the varietal difference based on the chemical reaction. On the basis of colour reaction with KOH solution, the genotypes were into five groups as light yellow (five genotypes), yellow (four genotypes), light brown (six genotypes), brown (two genotypes) and dark brown (five genotypes). Similar groupings were reported by Mckee (1973) in wheat, Rosta (1975) and Ranagmude et al. (1988) in rice, Sambasiva Rao. et al.(2002) in groundnut and Biradarpatil et al. (2006) in safflower genotypes. Varied colour reaction may be due to the chemical composition of seed or selective action of enzymes present which may be governed genetically. The findings of the present investigation (KoH and NaoH) which are simple, quick, and cheap for determining the varietal differences in sesamum genotypes could be used as routine genetic purity test. The varied coleoptile growth response of sesamum genotypes to gibberllic acid (25ppm) has been observed in the present study. Based on the differential growth response of coleoptile length to GA3, the genotypes were grouped into three categories as very low response (<10 per cent), low response (10-30 per cent) and moderate response (< 30 percent). The per cent increase in coleoptile length over control ranged from 4.02 per cent (YLM-17) to 103.86 per cent (T-13). Among the genotypes, three genotypes showed very low response, six genotypes showed low response and 13 genotypes showed moderate response. Similar groupings were reported by Gupta and Agrawal (1988) in rice, Agrawal and Pawar (1990) in soybean Chakrabarty and Agrawal (1990) in blackgram, Sambasivarao et al. (2002) in groundnut genotypes. The difference in seedling length may be due to differences in genetic make up of genotypes and selective response to the growth regulator.

The genotypes showed varied response to 2,4-D application (2ppm). The per cent decrease in coleoptile length over control ranged from 75.96 per cent (T-13) to 92.36 per cent (ORM-17). Based on this, the genotypes were grouped into two groups as susceptible (<85 per cent) and highly susceptible (> 85 per cent). Among the genotypes, 13 genotypes were susceptible and nine were highly susceptible. Similar grouping was reported by Chakrabarty and Agrawal (1990) in blackgram, Shivakumarrao et al. (2000) in rape seed and mustard and Sambasivarao et al. (2002) in groundnut. The difference in decrease in coleoptile growth among the genotypes may be due to differential ethylene production upon application of 2, 4D (Sundaru et al., 1983)

5.8 MOLECULAR MARKERS

Genetic variation is a pre requisite for any crop improvement programme to be successful. DNA based molecular markers acted as versatile tools to study variability and diversity in different plant species. Though a range of plant characters are currently available for distinguishing between closely related individuals, their sensitivity to environment and less genome coverage hinders their usage. DNA based molecular markers clearly allow the comparison of genetic material of two individual plants avoiding any environmental influence on gene expression. Presently, many kinds of DNA based molecular markers such as RFLP, RAPD and AFLP etc., are available which detect polymorphism at the DNA level. The present study employed RAPD technique to assess genetic polymorphism. The major advantages of the RAPD technique is that, it does not need sequence information to start with. The polymorphism among genotypes can be detected by using random primers variation in the banding pattern of the amplification products which occur because of variation in the length of DNA sequences flanked by the primers. The present study utilized ten sesamum genotypes for RAPD analysis with twelve random decamer primers. The primers produced high degree of polymorphism with an average of 86.05 per cent. Among the primers used, five primer (OPP-03, OPA-10, OPA-14, OPA-15, OPP-08) gave highest polymorphism (100%). On an average 6.2 bands per primer were amplified. High level of polymorphism (78%) based on RAPD has been reported among 38 sesame populations by Gulhanercan et al. (2004), whereas Bhat and Suman Lakhanpaul (2000), reported low level of polymorphism among 48 sesame cultivars because of narrow genetic diversity among the sesame cultivars. Similar varied levels of polymorphism have been reported by Multani and Lyon (1995) in cotton, Encheva et al. (2005) in sunflower. Silvanacreste et al. (2005) in groundnut and Subramanian et al. (2000) in groundnut genotypes. The dendrogram constructed from the pooled data revealed two distinct clusters. The clustering pattern was not influenced by any definite morphological character, geographic area of adoption or ploidy level. The set of primers used were not able to group the genotypes into phenotypically intended categories. It was clear from the study that, the genotype Kanak which was present in cluster B is genetically distinct from other genotypes studied. The genotypes which exhibited low diversity morphological studies, exhibited higher diversity at molecular level. For instance, higher diversity was found between Kanak and Paiyar at the molecular level but both the genotypes showed near similarity in morphological studies. In cluster A, Usha and VS-9701 showed 94 per cent similarity at molecular level, but showed dissimilarity in morphological studies. The difference between morphological and molecular diversity may be due to the screening or use of limited number of RAPD markers. The grouping of genotypes or diversity is independent of geographical location and ploidy level or even phenotypic markers.

PRACTICAL UTILITY
1. The morphological characters studied can be utilized for identification and characterization of sesamum genotypes in DUS testing. 2. These characters are also helpful in genetic purity testing.

3. The chemical test results are useful in identifying and the grouping varieties and also in genetic purity testing. 4. The RAPD markers are useful in identifying the varieties at DNA level, which is not influenced by the environment. These molecular markers are helpful in assessing genetic purity at a shortest time.

FUTURE LINE OF WORK

1. To develop and standardize a biographic characteristics descriptor for identification of crop varieties specially for seed industry based on morphological traits, chemical tests and electrophoretic banding pattern. 2. Seed keys have to be developed for the genetic purity test on routine basis in the seed testing laboratories. 3. As an expansion of present RAPD investigations, further research in this crop is to be carried out on the levels of genetic variation among sesame cultivars using, other molecular markers.

VI. SUMMARY
Varietal identification and cultivar purity testing are very important aspects in seed production. All sectors of seed industry benefit from the ability to assess cultivar purity and identity. Therefore, information on well expressed and distinct characters of sesamum variety should be made available to the seed producing and certificating agencies in order to monitor the genetic purity of seeds. Therefore, a study was undertaken at the National Seed Project (NSP), University of Agricultural Sciences, Dharwad, to reveal the objectives of varietal differences in seed and seedling attributes, plant morphological characteristics, seedling growth response to various chemicals and also through molecular markers. The results of the present study are summarized in this chapter.

6.1 PLANT MORPHOLOGICAL CHARACTERISTICS

The 22 sesamum genotypes were studied for various plant morphological characteristics such as plant height, number of primary branches per plant, number of nodes per plant, internodal length, stem pigmentation. The plant height of the genotypes ranged from 75.45 cm to 119.56 cm. Based on the plant height, genotypes under study were grouped into three categories as short (<100cm), medium (100-115cm) and tall (>115) cm). Seventeen genotypes were short, four genotypes were medium and one genotype was tall in plant height. Based on number of primary branches per plant, genotypes were grouped as less branched (<3.0) and more branched (>3.0) types. The sesamum genotypes were grouped as less noded (14 genotypes) and more noded (8 genotypes) types based on the number of nodes per plant which ranged from 8.80 (TC-289) to 18.86 (E-8) with average of 13.71 nodes per plant. The internodal length of genotypes were grouped as short (<4.0cm) with 18 genotypes and long (>4.0 cm) with four genotypes. The internodal length ranged from 3.26 (E-8) to SVPR-1 (4.44 cm) with a mean of 3.79 cm. The stem pigmentation was used to group the genotypes as weak (eight genotypes), medium (eight genotypes) and strong (six genotypes).

6.2 LEAF CHARACTERISTICS

The genotypes were grouped based on the leaf characteristics such as number of leaves per plant, leaf length, leaf shape, leaf colour, leaf petiole pigmentation. Based on the variation in the number of leaves per plant, four genotypes were grouped as few (<60 leaves per plant), ten genotypes were grouped as medium (60-80 leaves per plant) and eight genotypes were many (>80 leaves per plant) leaved types. The genotypes were grouped based on the leaf length into three groups as short (<8.0 cm) with eight genotypes, medium (8.0-10.0 cm) with 11 genotypes and long (>10.0 cm) with three genotypes. The shape of leaf did not vary among the genotypes studied. All the genotypes were Lanceolate with margin entire leaf shape. The sesamum genotypes under study were grouped based on leaf colour into three categories namely, light green (seven genotypes), medium green (three genotypes) and dark green (12 genotypes). The pigmentation intensity of the petiole varied among the genotypes. Based on this the genotypes were grouped as light (nine genotypes), medium (nine genotypes) and dark (four genotypes) leaf petiole pigmented.

6.3 FLOWER CHARACTERISTICS

The genotypes were grouped based on the flower characteristics like 50 per cent flowering, flower petal colour and flower hairiness.

The genotypes were grouped as early flowering (seven genotypes with less than forty days) and late flowering (15 genotypes with more than forty days) types based on 50 per cent flowering. The genotypes were classified into light pink (12 genotypes), medium pink (three genotypes) and dark pink (seven genotypes) based on flower petal colour. The genotypes were further divided into three groups based on the flower hairiness. Among the genotypes, eight genotypes were low, nine genotypes were medium and five genotypes were dense pubescence types.

6.4 POD CHARACACTERISTICS

The pod characteristics such as number of pods per axil, number of pods per plant, pod length, pod shape, pod beak, pod dehiscence, number of locules per pod were observed and the genotypes were grouped based on these pod characteristics. The genotypes could be classified into two groups viz., single and alternate (19 genotypes) and multiple types (three genotypes) based on the number of pods per axil. The genotypes were grouped based on the variation in the number of pods per plant into three categories. Out of 22 genotypes, five genotypes were less (<40 pods per plant), 12 were moderate (40-60 pods per plant) and five were more (< 40 pods per plant) podded types. The pod length of the geotyeps was observed and the genotypes were grouped as short (<2.0 cm) with two genotypes and long (>2.0 cm) with 20 genotypes. Based on shape of pod genotypes were grouped as oblong (18 genotypes) and round (four genotypes) types, whereas, based on the pod beak, the genotypes were grouped as short (seven genotypes) and long (15 genotypes) beaked types. The genotypes under study were also grouped based on the pod pubescence into three groups viz., nil (eight genotypes) medium (nine genotypes) and dense (five genotypes) pod pubescence types. The genotypes were grouped based on variation in the number of locules per pod. Among the genotypes, 17 genotypes were four loculed types and five genotypes were six loculed types. Based on the pod dehiscence, the genotypes were grouped as dehiscent type (21 genotypes) and indehiscent type (one genotype). The genotypes could be classified into two groups based on the days to maturity. Among the genotypes, seven genotypes were early in maturity (<85 days) and fifteen genotypes were late in maturity (> 85 days).

6.5 SEED CHARACTERISTICS

Among the sesamum genotypes studied, three groups were observed on the basis of seed colour such as black (six genotypes), brown (five genotypes) and white (eleven genotypes) seeded types. While, on the basis of thousand seed weight, the genotypes were grouped into medium sized (< 3.5 g) with 17 genotypes and bold sized (> 3.5 g) with five genotypes. Based on the variation in the oil content percentage, the genotypes were grouped into two groups as low (seven genotypes) with less than 45 per cent oil content and high (15 genotypes) with more than 45 per cent oil content.

6.6 SEEDLING CHARACTERISTICS

The seed germination, seedling length, and vigour index varied among the genotypes. Based on the variation in the seedling vigour index, the genotypes were grouped as low vigorous (eight genotypes) and high vigorous (14 genotypes) types.

6.7 CHEMICAL TESTS

The seeds were subjected to NaOH and KOH test for differentiating the genotypes. Based on the colour of the solution, the genotypes were grouped as light brown (nine genotypes), brown (seven genotypes) and dark brown (six genotypes) to sodium hydroxide

test and light yellow (five genotypes), yellow (four genotypes), light brown (six genotypes), brown (two genotypes) and dark brown (five genotypes) to potassium hydroxide test. Based on the coleoptile growth response to gibberllic acid, the genotypes were grouped as very low response (three genotypes), low response (six genotypes) and moderate response (thirteen genotypes). Based on the coleoptile growth response to 2, 4-D, genotypes were grouped as susceptible (14 genotypes) and highly susceptible (eight genotypes) types.

6.8 MOLECULAR MARKERS

Genetic diversity at molecular level was estimated by using RAPD primers. RAPD profiles for all the 10 genotypes were generated with 10 random decamer primers. The level of polymorphism generated (86.05%) among the genotypes was high. On an average 6.2 bands per primer were produced. Between Paiyar and Kanak genotypes, minimum similarity (62%) was noticed whereas maximum similarity was noticed in between Usha and VS-9701 (94%) at molecular level. The dendrogram constructed from the pooled data revealed two distinct clusters.

VII. REFERENCES
ABDUL BAKI, A.A. AND ANDERSON, J.D., 1973, Vigour determination in cotton by multiple criteria. Crop Sciences, 13: 630-633. AGRAWAL P.K. 1987, Cultivar purity test In. Techniques in Seed Science and Technology. South Asian Publishers, New Delhi, p.160. AGRAWAL, R.L. AND SHARMA, B.L., 1989, Identification of mungbean varieties on the basis of seedling growth response to GA3 and DDT treatment. Seed Research, 17(1): 84-87. AGRAWAL, R.L. ANIL PAWAR A., 1990, Identification of soybean varieties based on seed and seedling characteristics. Seed Research, 18(1): 77-81. AGRAWAL., R.L., 1984, Identification of crop varieties Oxford and IBH Publishing Company, New Delhi, India, p. 227. ANNONYMOUS, 1992, NBPGR Annual Report, New Delhi, p. 125. ANONYMOUS, 1985, NBPGR Annual Report, New Delhi, p. 133. ANONYMOUS, 1990, NBPGR Annual Report, New Delhi, p. 197. ANONYMOUS, 1996, International rules for seed testing. Seed Science and Technology, 29: 1-335. ANONYMOUS, 1998, Annual Report, All India Coordinated National Seed Project (Crops). IARI, New Delhi, pp. 122-143. ANONYMOUS, 2002, Annual Report, All India Coordinated National Seed Project (Crops), IARI, New Delhi, p. 257. ANONYMOUS, 2004, Annual Report, All India Coordinated National Seed Project (Crops), IARI, New Delhi, p 258. ANONYMOUS, 2004, Annual Report, http:/www.icar.org.in/ an. ANONYMOUS, 2005, Annual Report, of All India Coordinated National Seed Project (Crops), IARI, New Delhi, p. 201. ARUNKUMAR, M.B., BHERRY, R.J., MALAVIKA DADLANI, ANURADHA VARIER AND SHARMA, S.P., 2004, Characterization of pearl millet hybrids and parental lines using morphological characters. Seed Research, 32(1): 15-19. ARUNKUMAR, M.B., VARIER, A., SHERRY, R.J., ARUNKUMARI, R., DADLANI, M. AND SHARMA, S.P., 2005, Characterization of pearlmillet (Pennisetum glacum (L.) R.Br.) genotypes by seedling anthocyanin pigmentation and seed characters. Seed Science and Technology, 33: 215-226. ASHWANIKUMAR, CHOWDHURY, R.K. AND KAPOOR, R., 1993, Varietal identification in pearlmillet through morphological characters. Seed Research, 25(1): 53-58. ASHWANIKUMAR, K., CHOWDHURY, R.K., KAPOOR, R. L. AND DAHIYA, O.S., 1995, Identification of pearlmillet hybrids and their parental lines using seeds and seedling characters, chemical tests and gel electrophoresis. Seed Science and Technology, 23: 21-32. BAHRENFUS, J.B. AND FEHR, W.R., 1984, Registration of Harper and Lokata soybean. Crop Science, 24: 385. BANSAL, R., MALIK. C.P. AND THIND, S.K., 1992, Effect of GA3 in some early cultivars at early seedling stage. Oryza, 29: 51-55. BEDIGIAN, D. AND HARLAN, J.R., 1986, Evidence for cultivation of sesame in the ancient world. Economic Botany, 40: 137-154. BHAGAT, N.R., TASLIM AHMD, H.B., LALWANI AND HARENDER SINGH, 1985, Status of National resources of cultivated groundnuts. Indian Journal of Genetics and Plant Breeding, 45: 171-177.

BHAT, K.V. AND SUMAN LAKHANPAUL, 2000, Development of molecular markers for the study of genetic diversity in sesame. NBPGR. Annual Report, New Delhi, pp. 57-58. BIRADARPATIL, N.K., SANGEETA MACHA, MOTAGI, B.N., VIJAYKUMAR, A.G. AND HANCHINAL, R.R., 2006, Characterization of safflower varieties through chemical tests. Abstract XII National Seed Seminar, 24-26 February, ANGRAU Hyderabad, p 168. BONETTI, A., MIGGIANO, A., DINELLI, G. AND LOVATO, A., 1995, Identification of bean (Phaseolus vulgaris L.) cultivars grown in Italy by field and electrophoresis tests: a comparative study. Seed Science and Technology, 23: 69-84. CHAKRABARTHY, S.K. AND AGRAWAL, R.L., 1989a, Identification of black gram varieties-I, utilization of seed characteristics. Seed Research, 17: 23-28. CHAKRABARTHY, S.K. AND AGRAWAL, R.L., 1989b, Identification of black gram varieties II, utilization of morphological characteristics of seedling. Seed Research, 17 (1): 139-142. CHAKRABARTHY, S.K., AND AGRAWAL, R.L., 1990, Identification of black gram varietiesIII: utilization of seedling growth response to added chemicals. Seed Research, 18 (1): 34-39. CHAUDHARY, P.M., PATIL, G.D. AND ZOPE, R.E., 1977, Genetic variability and correlation studies in sesame (Sesamum indicum L.). Journal of Maharashtra Agricultural Universities, 2: 30-33. COCHRAN, W.G. AND COX, G.M., 1965, Experimental Design, Asia Publishing House, Bombay, pp. 293-305. DEEPAMALA, SEHGAL. AND SOOMNATH RAINA, 2005, Genotyping safflower (Carthamus tinctorius) cultivars by DNA fingerprints. Emphatic, 146: 67-76. DOLDI, M.L., VOLLMANN, J. AND LELLY, T., 1997, Genetic diversity in soybean as determined by RAPD and micro satellite analyses. Plant Breeding, 116 (4): 331-335. EDGAR, E., HARIWING. AND CALTION, J., EDWARD, J.R., 1970, Effects of morphological characteristics upon seed yield in soybean. Agronomy Journal, 62: 64-65. ELSAEED, E.A.K., 1967, Seed size as a varietal differences in broad beans (Vicia faba L.). Journal of Agricultural Sciences, Coimbatore. pp. 68-73. ENCHEVA, J., KOHLER, H., CHRISTOV, M. AND FRIEDT, W., 2005, Intergeneric hybrids between cultivated sunflower (Helianthus annuus L.) and Verbesina helianthoides (Genus verbesina), RAPD analysis. Helia, 28: 37-44. EZILKUMAR, S., 1999, Studies on varietal identification in hybrids, parents and varieties in cotton (Gossypium spp.). M.Sc. (Agri.) Thesis, Tamil Nadu Agricultural University, Coimbatore FEHR, W.R., 1973., Breeding for soybean hypocotyle length at 250 C. Crop Science, 13: 600-603. GULHANERCAN, A., MELIH TASKIN AND KENAN TURGUT, 2004, Analysis of genetic diversity in Turkish sesame (Sesamum indicum L.) populations using RAPD markers. Genetic Resources and Crop Evaluation, 51: 599-607. GUPTA, P.K. AND AGRAWAL, R.L., 1988, Determination of varietal purity of paddy varieties by laboratory evaluation. Oryza, 25: 310-314. GUPTA, V.K. AND GUPTA, Y.K., 1977, Variability, interrelationship and path coefficient analysis for some qualitative characters in sesame (Sesamum indicum L.) cultivars. Indian Journal of Heredity, 9: 31-37. JAIN, S.K., KHARE, D., BHALE, M.S. AND RAUT, N.D., 2002, Characterization of mung bean varieties for verification of genetic purity. Seed Tech News, 32 (1): 200201.

JAWAHARLAL., 1994, Studies on varietal characterization in inbreds, hybrids and varieties of cotton (Gossypium spp.) through physical, physiological and bio-chemical methods. M.Sc. (Agri.) Thesis, Tamil Nadu Agricultural University, Coimbatore. JAYARAMIAH, H., VIRUPAKSHAPPA, K., JAYARAMEGOWDA AND NAGARAJU, 1993, Germplasm utilization and enhancement in sunflower. In Sustainability in oilseed, (Eds. M.V. R Prasad et al.). Indian Society of oilseed Research, Hyderabad, 113-115. JOHN, C.M., 1934, Inheritance studies in gingelly (Sesamum indicum L.) Proceeding, Association Economic Biology. Coimbatore, 2: 33-40. KANDASWAMY, M., 1985, Genetic variation and genotypic environment interaction in sesamum ((Sesamum indicum L.). Madras Agricultural Journal, 72: 156-161. KARIVARTHARAJU, T.V., 2005, Physiological and biochemical techniques for crop variety identification. Training Manual, DUS test in cotton with reference to PPV and FR legislation, 2001, pp. 101-115. KASHIRAM, 1930, Studies in Indian oil seed (4) The types of Sesamum indicum D.C. Mem. Dep.Agric. India. Bot. 18: 127-147. KIRANKUMAR REDDY, C., 2004, Studies on laboratory techniques for identification of cotton (Gossypium spp.) genotypes. M.Sc (Agri.) Thesis, Acharya N.G. Ranga Agricultural University, Hydrabad. KOOISTRA, E., 1964, Identification research on pulses. Proceedings of International Seed Testing Association, 29(4): 937-947. KUMAR, D., ARPITHA DAS, MAHATO, P., LAKSHMAN, S.S. AND MANDI, S., 2005, Identification key for notified varieties and varieties of common knowledge of jute (Corchorus olitorius (L.) and C. capsularis (L.)). Indian Journal of Genetics, 65(3): 236-238. KURDIKERI, M.B. AND KURDIKERI, C.B., 1988, Seedling vigour in cotton as influenced by seed soaking treatment. Seed Research, 16(1) : 224-227. LANGHAM, D.G. (1947a), Genetics of sesame IV. Some Genetic variations in the colour of the sesame flower. Journal of Hereditary, 38: 221-224. LANGHAM, D.G., (1947b), Genetics of sesame V. Some morphological differences of the sesame flower (Sesamum indicum L.) Journal of Hereditary, 38: 347-350. LAWSON, W.R., HENRY, R.J., KOCHMAN, J.K., KONG, G.A., 1994, Genetic diversity in sunflower (Helianthus annuus L.) as revealed by random amplified polymorphic DNA analysis. Australian Journal of Agricultural Research, 45:1319-1327. LEE, S.C., SEO, H.I., KIM, J.H. AND CHOI, K.G., 1992, Effect of seed size, temperature and GA3 treatment on hypocotyls elongation in soybean. Korean Journal of Crop Science, 37(1): 68-77. LIRINDE, M.A., 1986, Laboratory methodology for cultivar purity testing in US rices. Dissertation Abstracts International B (Sciences and Engg.), 47 (4): 1336B1337B. LUAN, W.Q. AND HAN, S.P., 1990, Preliminary analysis of nutritive value of groundnut germplasm resources in shangadong province. Crop Genetic Research, 2: 2225. MATE, S.N. AND SHELAR, V.R., 2006, Morphological characterization of sorghum hybrids and their parental lines. Abstract XII National Seed Seminar, 24-26 February, ANGRAU, Hyderabad, pp. 175-176. McKEE, 1973, Chemicals and biochemical techniques for varietal identification. Seed Science and Technology, 1: 181-199. MIKHAILOV., U.G. AND TRAVYANKO, D.A., 1987, Inheritance of seed coat pigmentation in soybean hybrids. Tristologiya. T. gentika, 21(6): 468-472.

MOHAMMED, A. AND ALAM, Z., 1933, Types of Sesamum indicum in Punjab. Indian Journal of Agricultural Sciences, 3: 897-911. MUDZANA, G., PICKETT, A.A., JARMAN, R.J. AND COOKE, R.J., 1995, Variety discrimination in faba beans: an integrated approach. Plant Varieties and Seed, 8: 135-145. MULTAN, D.S. AND LYON, B.R., 1995, Genetic fingerprinting of Australian cotton cultivars with RAPD markers. Genome, 38: 1005-1008. MURALIKRISHNA, S., SAXENA, O.D. AND DESAI, D. B., 1990. Comparative evaluation of techniques for identifying parents and hybrids of cotton. Institution Conference of Science and Technology., New Delhi, pp. 21-25. MURALIMOHANREDDY, B., KESHAVULU, K., ANKAIAH, R., MANOHAR REDDY, N., SAMBASIVA RAO, P., BAYYAPU REDDY, K., ANURADHA VARIER, ARUNA KUMARI, K., 2004, Morphological, chemical and electrophoretic descriptors for castor (Ricinus communis L.). Manual, National Seed Project (Crops), IARI, New Delhi, pp. 1-22. NAGAPADMA, K., MURALIMOHANREDDY, B., ANKAIAH, R. AND SARADA, P., 1996, Characterization of twenty three maize inbred lines through seed morphology, leaf stomatal studies and response of seedling to added chemicals. Seed Research, 24 (1): 15-19. NAKAGAWA, J., BANZATTO, N.V. AND SAVY FILHO, A., 1978, Characterization of sesame (Sesamum indicum L.) cultivars. Review de Agricultura Paracicab, Brazil, 53: 179-181. NOHARA, S., 1932, Genetical studies on Sesamum indicum L. Journal College of Agriculture Tokyo, Imperial University, 12: 227-386. PAIN, S.K. AND BASU, M.K., 1985, Growth and metabolism of rice seedling the effect of GA. Indian Journal of Agricultural Research, 19 (2): 59 -68. PALANISAWAMY, V., NATESAN, P., SUNDARALINGAM, P., BALAMURAGAN, P. AND KRISHNASAMY, V., 1998, Identification of red rice cultivars. Tamil Nadu Agricultural University, News letter, 28: 7. PARAMESH, R., 1983, Screening of soybean cultivars for seed size, seed vigour and establishment of the relationship with yield and yield components. M.Sc. (Agri.) Thesis, University of Agricultural Sciences, Bangalore. PARANI, M., SINGH, K.N., SREE RANGASAMY AND RAMALINGAM, R.S., 1997, Identification of Sesamum alatum x Sesamum indicum hybrid using protein, isozyme and RAPD markers. Indian Journal of Genetics. 57 (4): 381-388. PATIL, K.B., SURYA WANSHI, B. AND DESHMUKH, 1996, Morphological key characters for identification of cotton hybrids. Technical bulletin NSP (Crops), Published by National Seed Project (Crops), IARI, New Delhi. PAUKENS, J., 1975, Methods for determining cultivar trueness and purity in maize (Zea mays L.). Seed Science and Technology, 3: 176-181. PONNUSWAMY, A.S., BHASKARAN, M. AND SASTRI, G., 2003, Variety characterization in cotton by physical, chemical and bio-chemical methods. Training Manual, Variety characterization by image analysis and electrophoresis, pp. 106-120. PRAKASH, V. AND LAL, R.K., 1968, Effect of seed treatment with CCC, B-nine, gibberllic acid and kinetin on morphological and biochemical changes in cotton. Indian Journal of Experimental Biology, 6: 44-46. PRASAD, R., 2002, Text Book of field Crops Production, Indian Council of Agricultural Research, New Delhi, p 821. RAJENDRA PRASAD, S., SHANKAR, A.N., RAUT, N.D., KHARE, D., SRIVASTAVA, R., DADLANI, M. AND VEENA VASHISHT, 2003, Morphological, chemical and

electrophoretic descriptors for sunflower varieties. Manual National Seed Project (Crops), IARI, New Delhi, pp. 1-33. RAMACHANDRA, M., RAMANATHAN, T. AND SRIDHARAN, C.S., 1972, Association of certain morphological characters with yield in Sesamum indicum L. Madras Agricultural Journal, 59: 9-10. RASAILY, S.K., DESAI, N.D. AND KUKUDIA, M.U., 1986, Genetic variability in soybean (Glycine max (L.) Herill). Gujarat Agricultural University Research Journal, 11(2): 57-60. RAVIKUMAR, 1999, Identification of soybean varieties based on seed, seedling and plant characters. M.Sc. (Agri.) Thesis, University of Agricultural Sciences, Bangalore. REDDY, P.N., REDDY, K.N. AND SINGH, S.P., 1989, Effect of seed size on qualitative and quantitative traits in soybean. Seed Science and Technology, 17: 289-295. RHIND, D. AND THEIN, B., 1933, The classification of Burmese sesame, Sesamum oriental Linn. Indian Journal of Agricultural Sciences, 3: 478-495. ROBERT, E. ALLAN, VOGEL, O.A. AND CRADDOCK, J.C., 1980, Effect of gibberllic acid upon seedling emergence of slow and fast emerging wheat varieties. Agronomy Journal, 46: 30-32. ROSTA, K., 1975, Variety determination in rice. Seed Science and Technology, 3: 161-168. SAMBASIVA RAO, P., MURALI MOHAN REDDY, B., BAYYAPU REDDY, K. AND BHARATHI, M., 2002, Varietal identification of rice (Oryza sativa L.) by chemical tests and electrophoresis of total soluble seed proteins. Seed Tech News, 32 (1): 93-94. SAMBASIVARAO, P. MURALIMOHANREDDY, B., BHARATHI, M. AND BAYYAPU REDDY, K., 2002, Varietal identification in groundnut (Arachis hypogaea L.) by chemical tests and electrophoresis of total soluble seed proteins. Seed Tech News, 32(1): 93. SANJEEVAIAH, B.S. AND JOSHI, M.S., 1974, Correlation and genetic variability in sesamum. Current Science, 3: 144-146. SANKARAPANDIAN, R., 2002, Identifying some pulse cultivars based on key characters alone among popular varieties of Tamil Nadu. Seed Tech News, 32 (1): 146 SATISHA, 1995, Evaluation of sunflower (Helianthus annuus L.) germplasm for yield components. M.Sc. (Agri.). Thesis, University of Agricultural Sciences, Bangalore. SHADAKSHARI, Y.G., VIRUPAKSHAPPA, K. AND SHIVASHANKAR, G., 1995, Genetic variability studies in germplasm collection of sesamum (Sesamum indicum L.). Mysore Journal of Agricultural Sciences, 29: 133-137. SHIVAKUMAR, 2000, Characterization of rapeseed and mustard (Brassica spp.) cultivars using field and laboratory techniques. Seed Tech News, 31 (1): 31. SHIVASUBRAMANIAN, S. AND RAMAKRISHNAN, V., 1978, Identification of rice varieties by laboratory techniques. Seed Research, 6(1) : 71-76. SHRIVASTAV, S.R. AND KAUSHAL, P.K., 1972, Phenotypic variability in sesamum (Sesamum indicum L.) JNKVV Research Journal, 6: 140-144. SILVANACRIESTE, SIN, MOISTSAI, JOSE, F.M, VALLS, MARCOS, A,, GIMENES AND CATALIN ROHERO LOPES, 2005, Genetic characterization of Brazilian annual Arachis species from sections Arachis and heteranthae using RAPD markers. Genetic Research and Crop Evaluation, 52: 1079-1086. SINGH, K. AND AFRIA, B.S., 1990, Seed germination, seedling growth and establishment responses of cotton cultivars as regulated by growth substantance. Seed Research, 18(1): 25-30.

STAHI, J.P. AND PANDEY, M.P., 1981, Association of seed impermeability with flowering and maturity duration in soybean. Tropical grain legume Bulletin, 22: 119-21. SUBRAMANIAN, V., GURTY, S., NAGESWARA, R.C. AND NIGAM, S.N., 2000, Identification of DNA polymorphism in cultivated groundnut using random amplified polymorphic DNA (RAPD) assay. Genome, 43: 656-660. SUNDARU, M., BABA, I., TANABES, T., TAMAI, F. AND MATODA, Y., 1983, Varietal differences of Indonesian rice plants in their susceptibility to 2,4-D injury and inter-relationship with ethylene. Japenese Journal of Crop Sciences 52(3): 323330. SURENDRA PRAKASH AND SINGHAL, N.C., 1997, Characterization of some Indian pea (Pisum sativum L.) cultivars. Seed Research, 25 (1): 53-58. TARASATYAVATHI, C., BHARADWAJ, C.H., HUSAIN, S.M., KARMAKAR, P.G., TIWARI, S.P., JOSHI, O.P. AND YOGENDRA MOHAN, 2004, Identification key for soybean (Glycine max) varieties released or notified in India. Indian Journal of Agricultural Sciences, 74 (4): 215-218. TERAO, H., 1986, Studies on the mesocotyl elongation of seedling in Japanese paddy rice cultivars. Oryza sativa L. Bulletin Institutional Tropical Agriculture, 9 : 77-87. THANGAVEL, P., BHARATHI, A., NATARAJAN, N. AND EVERA, T., 2005, Varietal grouping in sorghum by seed and seedling morphology and response to chemical testing. Karnataka Journal of Agricultural Sciences, 18 (3): 664-672. TIWARI, D.K., TIWARI, J.P. AND AGARWAL, V.K., 1978, Evaluation of soybeans for high germinability and field emergence. Seed Research, 6(2); 125-128. UPADHYAYA, M.D., ROOMIROORTIZ, DAULA, I. BRAMEL AND SUBE SINGH, 2002, Phenotypic diversity for morphological and agronomic characteristics in chickpea core collection. Euphytica, 123: 333-342. VANAGAMUDI, K., PALANISAMY, V. AND NATESAN, P., 1988a, Variety determination in rice-phenol and potassium hydroxide tests. Seed Science and Technology, 16: 465-479. VANAGAMUDI, K., PALANISAMY, V., NATESAN, P. AND KARIVARTHARAJU, T.V., 1988b, Variety determination in rice-Examination of the hulled grain. Seed Science and Technology, 16: 457-464. VANDERBURG, N.J. AND VANZWOL, R.A., 1991, Rapid identification techniques used in laboratories of the International Seed Testing Association: a survey. Seed Science and Technology, 19: 687-700. VENKATAREDDY, D.M., 1991, Investigations on seed technology of soybean, Ph.D. Thesis, University of Agricultural Sciences, Bangalore. VIRUPAKSHAPPA, K. AND SINDAGI, S.S. 1987, Characterization, evaluation and utilization of sunflower germplasm accessions, a catalogue. Project Coordinating Unit Sunflower, UAS, GKVK, Bangalore, pp. 33. WAX, L.M., BERANARD, R.L. AND HAYERS, R.M., 1974, Response of soybean cultivars to bentazon, bromoxynil, chloroxuron and 2,4-D. Weed Science, 22(1): 35-41. WEISS, E.A., 1971, Castor, sesame and safflower, Leonard Hill Books, London, 311-355. YADAV, R.D.S. AND SRIVASTAVA, J.P., 2002, DUS characteristics of chickpea varieties. Seed Tech News, 32(1): 29-30.`

APPENDIX- I
Physical and chemical properties of the soil of the experimental site

Sl. No. A.

Particulars Particle size analysis

Values

Method employed International Pipette Method (Piper, 1966)

1 2 3 4 B. 1

Coarse sand (%) Fine sand (%) Silt (%) Clay (%) Chemical properties Available nitrogen (kg Nha-1)

5.9 14.32 26.55 53.23

295.00

Alkaline permanganate method (Jackson, 1967)

2.

Available phosphorus (kg P2O5 ha1

24.82

Olsens method (Jackson, 1967)

) 307.64 Flame photometer method (Jackson, 1967)

3.

Available potassium (kg K2O ha-1)

Soil pH

7.7

pH meter (Piper, 1996)

CHARACTERIZATION OF SESAME GENOTYPES THROUGH MORPHOLOGICAL, CHEMICAL AND RAPD MARKERS

SUHASINI K.S. 2006 DR. N.K. BIRADAR PATIL MAJOR ADVISOR

ABSTRACT
The experiment was conducted at the Main Agricultural Research Station, UAS, Dharwad during kharif, 2005 for identification of sesamum genotypes through morphological characters, chemical and moleculer tests were carried out at Seed Research Laboratory of National Seed Project. The 22-sesamum genotypes were grouped in to different groups based on morphological characteristics such as plant height, number of primary branches per plant, number of nodes per plant, internodal length and stem pigmentation, number of leaves per plant, leaf length and leaf shape, leaf colour, and leaf petiole pigmentation. The flower characteristics such as 50 per cent flowering, flower petal colour, flower hairyness and pod characteristics viz., number of pods per axil, number of pods per plant, pod length, pod shape, pod beak, pod dehiscence, number of locules per pod were used for grouping the genotypes. Based on the seed characteristic such as seed colour thousand seed weight, oil content percentage, and the seedling characteristics the genotypes were classified into different groups. The seeds were subjected to NaOH and KOH test for differentiating the genotypes. Based on the colour of the solution, the genotypes were grouped as light brown, brown and dark brown in NaOH test and light yellow, yellow, light brown, brown and dark brown in KOH test. Based on the coleoptile growth response to GA3, the genotypes were grouped as very low response, low response and moderate response and based on the coleoptile growth response to 2,4D genotypes were grouped as susceptible and highly susceptible. RAPD profile for all the 10 genotypes were generated with 10 random decamer primers. Between Paiyar and Kanak genotypes minimum similarity and maximum similarity between Usha and VS-9701 was noticed at molecular level.