Techniques in hybrid seed production of vegetables and flower crops

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techniques in hybrid seed production

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University of Horticultural Sciences, Bagalkot KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI Arunkumar H.S. UHS10PGM38 Dept of CIB 2 nd Seminar Techniques of hybrid seed production in vegetables and flower crops 2

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3 Introduction Male Sterility Tomato Chilli Onion Self Incompatibility Cauliflower Radish Conclusion Future thrust CONTENTS 3 Introduction Emasculation & Pollination Male Sterility Self Incompatibility Conclusion CONTENTS

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Introduction 4

Seed…………………..? : 

Seed…………………..? An embryo, a living organism embedded in the supporting or the food storage tissue. A carrier of new technologies A basic tool for secured food supply The principal means to secure crop yields in less favorable production areas A medium for rapid rehabilitation of agriculture in cases of natural disaster 5

For hybrid seed production one should know: 

For hybrid seed production one should know

Principles of Seed Production: 

Principles of Seed Production 7

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India is the second largest producer of vegetable crops in the world next to China. India produced 146.5 million tons of vegetables from an area of 8.49 million hectares during 2011. India shares about 14% of the world’s output of vegetables from about 2% of cropped area in the country. The diverse climatic condition’s enable to produce different indigenous and exotic vegetables in India . (Anon., 2011) 8 Karnataka produced 9056.4 million tons of Vegetables from an area of 466.3 thousand hectares and productivity of 19.4 MT/Ha during 2011.

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9 India produced 1031 million tons of loose flowers and 69027 million tons of cut flowers in an area of 191 thousand hectares. Karnataka produced 203.9 million tons of loose flowers and 5860.0 million tons of cut flowers from an area of 27.0 thousand hectares during 2011.

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Scope and Importance of Vegetables Vegetables are considered as " Protective Supplementary Food " Different uses of vegetables - - Cooking, salad, pickle, fried, stuffed - High medicinal value - Use as ornaments - Different industrial uses Vegetable make effective use of land and labour resources They play an important role in national economy Increase vegetable production through use of hybrid seeds 14

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Why hybrids are adopted by farmers? Greater productivity Longer harvest duration Better adaptability to variable environments Better tolerance to diseases and pests Uniform produce Better market acceptability Better nutritional quality 15

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SEED MARKET The global commercial seed market is worth $ 37 bn. India is fifth largest and fastest growing seed market in the world. Vegetables seed market in India is about Rs 6 bn. Seed cost as a percentage of revenue per acre for a farmer in India is less than 5% as against 15% in other countries. 16 Onion seeds Chilli seeds Tomato seeds Marigold seeds

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Table 1: The most commonly utilized mechanisms for developing commercial hybrids in vegetables and flowers. Mechanism Commercially exploited in: Hand emasculation + HP Tomato, eggplant, sweet pepper, okra, hot pepper Remove staminate flowers + HP Cucurbits (bitter gourd, bottle gourd, etc. ) Male sterility + HP Tomato, hot pepper, sweet pepper Male sterility + NP Onion, cabbage, cauliflower, carrot, hot pepper, Petunia, Marigold, Dianthus, Zinnia Self incompability + NP Most of cole vegetables like broccolis, cabbage , Petunia, Marigold, Ageratum, Bellis etc. Gynoecism + NP Cucumber, muskmelon. Remove staminate flowers + NP Cucurbits including bitter gourd, summer squash, winter squash, etc. HP = Hand pollination NP = Natural pollination 17 Sanjeet Kumar a & Singh , 2011, Varanasi

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Emasculation and Pollination

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Steps in emasculation and pollination Next day opening flower buds are selected To be done before anther dehiscence To be done with hand/ forceps 2.Pollen Collection 1. Emasculation: Flower collection Anther cones are taken and put them in glassine envelopes Dry anthers at 30℃ for 24 hrs Put the dried anther cones in a cup then tranfer to pollen lid cup 3. Pollination

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Table-2 : Effect of crossing ratio and pollination time on tomato hybrid seed production Kumar et al., 2008 , Dharwad Treatments Fruit set(%) Seed weight/fruit(g) No. of seeds /fruit 1000 seed weight(g) Seed yield/plant(g) Crossing ratio ( C ) C1 (3female : 1 male) 41.51 0.335 69.58 3.8 6.91 C2 (4female : 1 male) 44.32 0.361 73.66 3.97 7.38 C3 (5female : 1 male) 39.24 0.319 65.85 3.78 6.47 C4 (6female : 1 male) 23.77 0.312 63.39 3.45 6.33 S.Em ± 0.78 0.006 0.91 0.09 0.1 CD at 5 % 2.23 0.017 2.6 0.26 0.28 Pollination time(P) P1 (7.00 am) 36.13 0.313 64.59 3.53 6.08 P2 (8.00 am) 36.64 0.333 71.34 3.71 6.85 P3 (9.00 am) 37.92 0.349 76.96 3.93 7.14 P4 (10.00 am) 43.02 0.378 81.41 4.2 7.66 P5 (11.00 am) 37.82 0.34 59.58 3.89 7.12 P6 (12.00 noon) 31.73 0.278 54.58 3.23 5.8 S.Em ± 0.96 0.007 1.12 0.11 0.12 CD at 5 % 2.73 0.021 3.19 0.32 0.35 20

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Continued …………. Treatments Fruit set(%) Seed weight/fruit(g) No. of seeds /fruit 1000 seed weight(g) Seed yield/plant(g) C1P1 41.56 0.316 66.46 3.4 6.09 C1P2 41.34 0.333 73.2 3.67 7.09 C1P3 42.37 0.35 79.14 3.77 7.23 C1P4 45.03 0.367 82.53 4.13 7.98 C1P5 41.94 0.337 60.93 3.95 7.17 C1P6 36.85 0.308 55.26 3.74 5.93 C2P1 41.19 0.321 70.46 3.7 6.36 C2P2 42.48 0.34 79.13 3.77 7.45 C2P3 44.14 0.377 83.93 4.18 8.08 C2P4 57.15 0.442 84.13 4.56 8.39 C2P5 42.24 0.373 65.13 4.06 7.92 C2P6 38.42 0.312 59.2 3.57 6.1 C3P1 28.6 0.301 63.26 3.53 5.88 C3P2 28.02 0.325 69 3.71 6.7 C3P3 40.77 0.355 7.25 3.93 7.11 C3P4 42.84 0.35 81.53 4.14 7.45 C3P5 40.33 0.327 53.53 3.93 7.01 C3P6 34.91 0.279 53.53 3.55 6.48 C4P1 22.89 0.312 58.2 3.5 6 C4P2 24.75 0.335 64.06 3.7 6.16 C4P3 24.4 0.335 70.53 3.83 6.15 C4P4 27.05 0.355 72.46 3.98 6.81 C4P5 26.8 0.324 59.8 3.62 6.39 C4P6 16.76 0.211 50.33 2.04 4.69 Mean 37.21 0.332 68.12 0.375 6.78 S.Em± 1.92 0.015 2.24 2.3 0.24 CD at 5 % 5.75 0.042 6.38 6.5 0.7 21

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Treatments No. of fruits crossed per plant Fruit set (%) Seed weight/fruit ( g ) No. of hybrid seeds/fruit Hybrid seed yield/plant(g) 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean D 1 70.33 70.48 70.41 35.95 35.53 35.74 0.26 0.25 0.25 49.70 48.49 49.10 6.51 6.47 6.48 D 2 70.22 71.85 71.04 49.77 48.96 49.37 0.28 0.28 0.28 53.17 52.36 52.75 6.64 6.79 6.80 D 3 69.67 69.83 69.75 32.88 32.34 32.61 0.26 0.25 0.04 48.50 46.46 46.53 6.48 6.35 6.35 SEm + 0.50 0.86 0.49 0.61 0.62 0.88 0.00 0.01 0.00 1.12 1.14 0.71 0.03 0.03 0.02 CD at 5% NS NS NS 1.83 1.86 2.63 0.01 0.01 0.01 3.36 3.42 2.13 0.08 0.08 0.05 Time of pollination P 1 70.22 70.27 70.25 38.79 38.61 38.78 0.26 0.26 0.26 49.78 48.55 49.16 6.51 6.51 6.51 P 2 70.13 71.73 70.93 40.63 39.87 40.25 0.27 0.27 0.27 51.17 51.05 51.11 6.64 6.63 6.64 P 3 69.87 70.16 70.01 39.19 38.36 38.70 0.25 0.25 0.25 48.50 47.71 48.10 6.48 6.48 6.48 SEm+ 0.50 0.86 0.49 0.38 0.42 0.48 0.01 0.01 0.00 2.36 2.48 1.71 0.03 0.03 0.02 CD at 5% NS NS NS 1.14 1.26 1.44 0.01 0.01 0.01 7.05 7.41 5.11 0.08 0.08 0.05 Table 3: Effect of duration of emasculation and pollination time on chilli hybrid (HCH-9646) seed production Priya et al., 2009, Dharwad

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Treatments No. of fruits crossed per plant Fruit set (%) Seed weight/fruit(g ) No. of hybrid seeds/fruit Hybrid seed yield/plant(g) 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean 2006 2007 Mean D1P1 70.45 70.23 70.35 34.79 35.38 35.08 0.26 0.25 0.25 50.11 48.12 49.11 6.47 6.47 6.47 D1P2 69.83 70.11 69.97 36.52 35.87 36.20 0.26 0.25 0.25 50.48 49.19 49.83 6.48 6.47 6.48 D1P3 70.71 71.10 70.91 36.55 35.35 35.95 0.26 0.25 0.25 48.52 48.17 48.34 6.49 6.48 6.49 D2P1 70.44 70.71 70.57 44.18 43.57 43.87 0.27 0.27 0.27 50.45 50.16 50.30 6.64 6.64 6.64 D2P2 70.78 74.96 72.87 33.15 52.10 53.63 0.30 0.30 0.30 57.49 57.12 57.30 7.00 7.16 7.08 D2P3 69.44 69.90 69.67 52.00 51.21 51.60 0.27 0.26 0.26 51.48 49.71 50.64 6.68 6.67 6.67 D3P1 69.78 69.86 69.82 37.40 36.87 37.10 0.27 0.26 0.26 49.78 47.38 48.08 6.42 6.42 6.42 D3P2 69.78 70.13 69.96 32.21 31.63 31.92 0.27 0.26 0.26 45.54 46.85 46.19 6.36 6.35 6.35 D3P3 69.45 69.50 69.47 29.03 28.53 28.78 0.25 0.24 0.24 45.49 45.16 45.32 6.29 6.28 6.28 Mean 70.07 70.72 70.40 39.54 38.95 39.24 0.26 0.26 0.26 49.81 49.10 35.59 6.55 6.54 6.54 SEm ± 0.87 1.49 0.86 1.95 2.38 1.53 0.01 0.01 0.01 4.09 4.29 2.96 0.05 0.05 0.03 CD at 5% NS NS NS 5.83 7.11 4.57 0.02 0.02 0.02 12.22 12.82 8.85 0.14 0.14 0.08

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Pollination method Mean number per inflorescence Seed yield per 5 inflorescences(g) flowers fruits non-pollinated flowers Osmia rufa 398.4 273.4 b 125.0 b 13 . 99 Free pollination 560.8 411.8 a 149.0 b 13.84 Self-pollination 496.4 44.8 c 451.6 a 1.52 Table 4: Results from the application of different methods to pollinate onion cv. Grabowska Wilkaniec et al., 2004 Czech Means followed by different characters are significantly different ( Tukeya’s test α =0.05)

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Pollination method Weight of 1000 seed (g) Germination capacity(%) Germination energy(%) Osmia rufa 3.14 92a 90a Free pollination 3.05 86a 85a Self pollination 2.74 66b 62b Table 5:Properties of onion seeds cv. Grabowska depending on pollination method Wilkaniec et al., 2004, Czech Means followed by different characters are significantly different ( Tukeya’s test α =0.05)

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Inbred line Air temperature and pollination time Average seed per pod 22 0 c 8.00 hrs 30 0 c 13.00 hrs 20 0 c 18.00 hrs Average VB-4PE-1 10.4 6.0 8.4 8.3 B-21PE-3 13.6 6.9 7.1 9.2 E-34 4.0 2.6 4.0 3.5 DT-46 14.2 6.2 7.0 9.1 Average 10.5 5.4 6.6 7.5 LSD 0.05 Inbred line-1.3 Air temperature and pollination-1.4 Table 6: Seed setting through heterogamic bud pollination as affected by air temperature and time of pollination in cabbage inbred lines Hamid et al ., 2010, Russia

Limitations of Emasculation and Pollination : 

Limitations of Emasculation and Pollination Time consuming Labour intensive (Skill is necessary for commercial seed production) Increased cost of production

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WHY MALE STERILITY AND SELF INCOMAPATABILITY? Production of large scale of F 1 seeds. Reduced cost of hybrid seed production. Speedup the hybridization programme. Commercial exploitation of hybrid vigour. 28

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MALE STERILITY 29

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A) Genetic male sterility( GMS) : - wide occurrence in plants - mostly governed by a single recessive gene , ms - male sterile alleles arise spontaneously or may be artificially induced B) Cytoplasmic male sterility (CMS) : - determined by the cytoplasm - it is the result of mutation in the mitochondrial genome (mtDNA) - CMS transfer easily to a given strain C) Cytoplasmic-genetic male sterility (CGMS) : - nuclear gene restores the fertility in the male sterile line - also known as nucleoplasmic male sterility - fertility restorer gene R is required D) Chemically induced male sterility : - IAA, IBA, Ethrel , etc. Kaul (1988) 30 Types of male sterility

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31 Natural population Artificially induced through mutagenesis Genetic engineering Protoplast fusion SOURCES FOR MALE STERILITY

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How to identify male sterile plant?

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Figure 1 : By microspore appearance under 100x magnification (0.5% aceti -carmine stain) Gniffke et al., 2009, Taiwan

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P 1 P2 Male sterile Male Fertile Male sterile F 1 Male sterile Male Fertile Male sterile Back cross Genetic Male Sterility ♀ ♂ ♂ ♀

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This was proposed by Jones, in 1908 in onion and subsequently reported in carrot, chinese cabbage, Radish, Garlic, Ornamental plants etc. Advantage :- All progenies are male sterile. (F 1 progenies) Longer flowering parent and longer life span of flower can be seen in male sterile plant. That is more advantage in ornamental plants . Cytoplasmic male sterility Useful to produce single cross/double cross hybrids, in crop where the vegetative part is commercial product. Disadvantage:- It is not useful to produce the hybrid seed in crop where the fruit/seed is commercial product.

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Male sterile Male sterile Male fertile F 1 seeds 100% male sterile Cytoplasmic male sterility

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This CGMS was first reported in onion by Jones and Davis- 1944 . It is also seen in Barley, Sorghum, Bajra , Rice, Maize, Brassica , Sugar beat etc. There are two steps are involved: Maintenance or multiplication of ‘A’ line (Male sterlie ) ‘B’ line and ‘R’ line parents are multiplied in separate isolated blocks independently. So this stage is called Foundation seed production stage. Cytoplasmic genetic male sterility ‘A’ line can be maintained by crossing the homozygous male sterile line having sterile cytoplasm with homozygous male fertile pollinator parent having fertile cytoplasm.

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A X B FS seed stage F 1 100% Male sterile.

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2) Hybrid seed production:- ‘A’ line X ‘R’ line CS Seed stage F 1 100% male fertile.

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Table 7: Mechanisms for the development of hybrids in vegetables Mechanism Vegetables Remark Reference Nuclear male Sterility Tomato Monogenic recessive mutant was utilized to develop cost effective experimental crosses. Sawhney ., 1997 Watermelon The utilization of monogenic recessive mutant was proposed. Zhang et al., 1994 Bottle gourd, Okra Male sterile plants were identified and utilized to develop experimental crosses. Dutta ., 1983 Cytoplasmic male sterility Radish The combined use of both MS and SI mechanisms to enhance efficiency of hybrid seed production has been proposed. Cho et al., 1985 Functional male sterility Eggplant , Tomato A monogenic recessive mutant was identified and proposed for commercial utilization. Phatak & Jaworski ., 1989 Transgenic male sterility Tomato, Cauliflower, etc. Many vegetables are at the edge of commercial utilization. Williams et al., 1997 40 SanjeetKumara & Singh, 2011, Varanasi

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41 Mutant Description Inheritance Governing by single recessive gene Stamenless Stamens absent Monogenic recessive sl Positional sterility Stigma exerted Monogenic recessive ps Pollen sterility Pollen abortive Monogenic recessive ms series Functional sterility Anthers do not dehisce Monogenic recessive ps-2 Table 8: Different male sterile mutants in tomato. Opena et al., 2009, Taiwan

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Table 9: Total fruit weight and hybrid seed per plant obtained by different pollination methods in chilli . Gniffke et al., 2009, Taiwan CMS sterile line:- CCA7234, CCA7235 Restorer:-9852-161 ns: No seed to test

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CMS/fertile line No. of grown plants No. of harvested plants Weight of Harvested seeds (g) Mean weight of seeds per plant(g) BR CMS 24 20 15 0.8 BR 19 16 16 90 5.6 FT CMS 24 22 43 2.0 FT 13 16 16 157 9.8 Table 10: Reproduction of CMS lines by means of pollination with fertile analogues in cauliflower Kucera et al., 2006, Czech Republic

0: 

0 Table 11:Time(minutes) required for crossing 50 flower buds on male fertile ‘Ms33 IPA’ (MF) and male sterile ‘ ms 331PA’ (MS) plants in tomato Dhaliwal and Cheema , 2008, Ludhiana Worker Emasculation Pollination on MF Emasculation + pollination on MF Pollination on MS Time saved in MS over MF, % 1 22.0 44.0 66.0 37.7 42.9 2 26.1 34.9 60.5 26.3 56.5 3 38.7 45.1 83.8 33.3 60.3 4 37.3 43.8 75.5 34.2 54.7 5 32.8 41.1 73.9 32.7 55.8 Mean 30.7 41.7 71.9 32.8 54.4 CD at 5% 1.5 1.6 2.0 1.1 -

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SELF INCOMPATIBILITY 45

Self incompatibility:: 

Inability to set seed from application of pollen produced on same plant or it refers to failure of viable pollen of a given plant to fertilise the ovules of the same plant, but it capable of fertilising effectively the ovules of the most other plant of the same variety. Brew baker-1957 . stated that self incompatibility occurs in more than 3000 sp belonging to 250 genera, spread in about 70 families. Self incompatibility :

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Gametophytic. SI reaction of a pollen is determined by its own genotype not by the genotype of the plant on which the pollen is produced Sporophytic. SI reaction of pollen is governed by the genotype of the plant on which the pollen is produced and not by the genotype of the pollen. McCubbin and Dickinson (1997) Types of self incompatibility 47

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Stable self incompatibility. High seed set of self pollination at bud stage. Favorable and uniform economic characters. Desirable combination ability. 48 The superior self incompatible lines for hybrid seed production should possess the following characters

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Line A Line B Parents S 11 S 22 Propagation Bud pollination S 11 Χ S 22 SCH S 12 Two season and two parents are required . Single cross hybrid

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Parents A X B C X D S 11 S 22 S 33 S 44 1 st season propagation through propagation through bud pollination bud pollination 2 nd season S 11 X S 22 S 33 X S 44 SCH S 12 S 34 Double cross hybrid

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3 rd season S 12 X S 34 (SCH) (SCH) DCH - S 13, S 14, S 23, S 24 [S 1234 ] The seeds are given to farmer for commercial cultivation for production maintain 1:1 or 2:4 ratio and here 4 parents and 3 season are required.

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Inbred A B C D E F S 11 S 22 S 33 S 44 S 55 S 66 transplant selfed are produced by inbreed in to small isolation block and cross AXB. Self in bud pollination SCH S 12 (AXB) S 33 (C) SCH S 45 (DXE) S 66 (F) After that sow the seeds in proportion of 3:1 ratio in both cross . Triple cross hybrid seed production

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AB: C DE:F S 12 X S 33 S 45 X S 66 s 13 : s 23 S 46 :S 56 We get the 3 way cross We get the 3 way cross 3 way cross X 3 way cross Seeds of two 3 way crosses are mixed and distributed commercial seed firm for final cross. Raise the crop and left for natural pollination. Then harvest the seeds of TCH for commercial cultivation. Here 4 seasons and 6 parents are required.

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Fig. 4: Male sterile and self incompatibility genotypes available in India Male sterility Self incompatibility TOMATO Ludhiana ms33 IPA ms2 IPA ps2 L 3841 ps2 NS 101 ps2 San Pedro ps2 UC 82-B ms 10 36 ms 45 ms 15 47 CHILLI Anand CCA 4261 CCA 4759 CCA 4758 Ludhiana CCA 4261 ONION Bangalore Ms1 Ms2 ms3 RADISH IARI NEW DELHI Pusa Chetaki Pusa Desi Half Red Acc. No. 30205 Acc. No. 282 Chinese Pink BDJ-689 CAULIFLOWER Varanashi IIVR-1 IIVR-50 kataki Early-29 HAZIPUR-4(BP) Pusi – 4 Agahani JBT -23/60 Late Agahani Aghani-8 Pusi Hazipur Agahani long leaf Agahani small leaf Kuwari-1 Kataki-12 54 Sanjeet Kumar a & Singh , 2011, Varanasi

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Lines Number of pod Number of seed Unopened flower Opened flower Unopened flower Opened flower Seed set 1 (%) Conclusion 2 23 15 15 210 80 38.1 WSI 23-3-4 15 15 236 69 29.24 WSI 27 15 15 193 96 49.74 WSI 27-3-7 15 15 231 51 22.01 SI 40-9 15 15 267 66 24.72 SI 142 15 15 253 88 34.78 WSI 142-5 15 15 245 79 32.24 WSI 1 - 0-25%- High Self incompatibility, 26-50% weak self incompatibility, 51-75% Very weak self incompatibility and 76-100% Highly weak self incompatibility. 2 -SI= Self incompatibility WSI= Week self incompatibility Table 12: Levels of Self incompatibility of Chinese cabbage lines, tested by seed set analysis. Ekapote et al., Thailand , 2003

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SI subline Number of grown plants Number of harvested plants Weight of harvested seeds(g) Mean weight of seeds per plant(g) 3/1 6 6 39 6.5 4/1 6 5 30 6 4/2 6 3 15 5 5/1 6 4 18 4.5 6/1 6 4 16 4 8/1 6 4 19 4.8 Table13:Reproduction of SI MT lines by means of NaCl spraying in cauliflower Kucera et al., 2006, Czech republic MT- Montano

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F1 plant Parents Parental genotypes F1 genotype Mean self seed December July October 416 579-1 x 137-3 S1S1 x S1S1 S1S1 0.0 0.0 0.0 418 579-1 x 582-5 S1S1 x S1S1 S1S1 0.8 0.0 0.0 417 579-1 x 192-3 S1S1 x S3S3 S1S3 174.6 3.3 64.7 419 579-1 x 604-1 S1S1 x S2S2 S1S2 177.6 18.3 93.0 423 604-1 x 192-6 S2S2 x S3S3 S2S3 215.6 28.6 115.2 Table 14:Winter environment increases self seed yield in self incompatible (SI) Petunia hybrida clones. Flaschenreim and Ascher , 1980,

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Cultivars A B C D E F G H ISI 1.1± 0.1 0.07± 0.005 0.19 ± 0.03 0.35 ± 0.03 0.3 ± 0.02 0.25 ± 0.03 0.3 ± 0.15 0.16 ± 0.05 Seed set following self pollination(%) 95±3.2 3±0.8 15 ± 1.5 28 ± 1.8 20 ± 1.8 16 ±1.0 19±1.1 12±0.8 Column headings: A – Bravo cool water mix; B – Bravo purple star; C – Flash red picotee ; D – Flash salmon; E – Bravo pink; F – Bravo purple; G – Bravo apple blossom; H – Bravo peach flare. Table 15: Index of self incompatibility (ISI) and percentage of seed set following self-pollination in different cultivars of Petunia hybrida . Hakimeh and Farkhondeh , 2009, Iran Seed set percent following self-pollination as: 0 – 3% (class 0) =self-incompatible; 3 – 30% (class 1)=slightly self-incompatible; and >30% (class 2)=highly selfcompatible . The index of self incompatibility (ISI) was determined by dividing the percentage of seed set from self-pollination by the percentage of seed set from cross-pollination

Conclusion: 

Conclusion Hand emasculation and pollination is time consuming method but can be practiced if skilled labour available. Eg : Tomato, Chilli, Bhendi , etc., Male sterility and self incompatibility are two best methods of hybrid seed production. GMS: Chilli CMS: Onion, Potato, etc., CGMS: Chilli, etc., SI: Colecrops , Petunia, etc.,

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In the light of rapid advancement of bio-technology, it may be anticipated that transgenic male sterility systems can be increasingly utilized in near future. Future