Male sterility, types and utilization in hybrid seed production

Hirdayesh Anuragi Ph.D. Scholar Genetics and Plant Breeding, CCS HAU, Hisar, Haryana

1. MALE STERILITY, TYPES & USES IN HYBRID SEED PRODUCTION PRESENTED BY- HIRDAYESH ANURAGI ADM. NO. 2015A29D PhD GENETICS & PLANT BREEDING CCS HAU HISAR, HARYANA PRESENTED TO- Dr. M. S. PUNIA PROFESSOR GENETICS & PLANT BREEDING CCS HAU HISAR, HARYANA Course: Heterosis Breeding (GP-507)

2. Male sterility Manifestation of Male sterility History of Male sterility Need of Male sterility Detection of Male sterility Creation of Male sterility Classification of Male sterility Cytoplasmic Male sterility (CMS) Genetic Male sterility (GMS) Cytoplasmic genetic Male sterility (CGMS) Transgenic Male sterility Chemical hybridizing agents (CHAs) Applications of Male sterility in Hybrid seed production Contents

3. Male Sterility Male sterility is characterized by nonfunctional pollen grains, while female gametes function normally. I nability to produce or to release viable or functional pollen as a result of failure of formation or development of functional stamens, microspores or gametes. Main reason is mutation. Sterile Sterile Fertile Fertile

4. Manifestations of Male Sterility Absence or malformation of male organs. Failure to develop normal microsporogenous tissue- anther Abnormal microsporogenesis (deformed or inviable pollen) Abnormal pollen maturation Non dehiscent anthers but viable pollen, sporophytic control Barriers other than incompatibility preventing pollen from reaching ovule

5. History of Male Sterility J.K. Koelreuter (1763) observed anther abortion within species & species hybrids . Genic male sterility has been reported in cabbage ( Rundfeldt 1960) , cauliflower ( Nieuwhof 1961 ) Male sterility systems have been also developed through genetic engineering (Williams et al. 1997) and protoplast fusion (Pelletier et al. 1995 ) Male sterility were artificially induced through mutagenesis ( Kaul 1988)

6. Manual emasculation Use of male sterility Use of self-incompatibility alleles Use of male gametocides Use of genetically engineered “pollen killer” genetic system Several forms of pollination control

7. Why Male Sterility ??? Reduced the cost of hybrid seed production. Production of large scale of F1 seeds. A voids enormous manual work of emasculation and pollination. Speed up the hybridization programme. Commercial exploitation of hybrid vigour .

8. Creation of Male Sterility Spontaneous mutations Interspecific hybridization Mutation induction ( EtBr ) Genetic Engineering Chemically induced male sterility (CHAs)

9. Detection of Male S terility system Whether a particular sterile genotype belongs to which MS system can be detected by its progeny performance on crossing with a few normal genotypes . Trend-I - All progenies in all the rows may be sterile- CMS Trend-II - Some rows may consist all fertile Some rows sterile and fertile in 1:1 ratio- GMS Trend-III- Some rows fertile . Some rows sterile and some rows sterile and fertile in 1:1 ratio - CGMS

10. Classification of M ale Sterility Kaul (1988) Classified Male Sterility in three major groups 1. Phenotypic Male Sterility (Morphological ) Structural or Staminal Male Sterility Pollen Male Sterility Functional Male Sterility 2. Genotypic Male Sterility Genetic Male Sterility (GMS) Environmental Sensitive (EGMS) Thermo sensitive genetic male sterility (TGMS) Photoperiod sensitive genetic male sterility (PGMS) Environmental non-sensitive Cytoplasmic Male Sterility (CMS) Cytoplasmic Genetic Male Sterility (CGMS) Transgenic Male Sterility (TMS) 3. Chemically Induced Male Sterility (CHA )

11. Pollen sterility : in which male sterile individuals differ from normal only in the absence or extreme scarcity of functional pollen grains ( the most common and the only one that has played a major role in plant breeding ). Structural or staminal male sterility : in which male flowers or stamen are malformed and non functional or completely absent. Functional male sterility : in which perfectly good and viable pollen is trapped in indehiscent anther and thus prevented from functioning Phenotypic Male Sterility

12. Cytoplasmic Male Sterility (CMS) D etermined by the cytoplasm (mitochondrial or chloroplast genes). Result of mutation in mitochondrial genome ( mtDNA )- Mitochondrial dysfunction. Progenies would always be male sterile s ince the cytoplasm comes primarily from female gamete only. Nuclear genotype of male sterile line is almost identical to that of the recurrent pollinator strain. Male fertile line (maintainer line or B line) is used to maintain the male sterile line ( A line). CMS is not influenced by environmental factors (temperature) so is stable .

13. CMS can used in hybrid seed production of certain ornamental species or in species where a vegetative part is of economic value . But not for crop plants where seed is the economic part because the hybrid progeny would be male sterile . This type of male sterility found in onion, fodder jowar , cabbage etc . Utilization of CMS in P lant Breeding

14. Use of CMS lines

15. Transfer of CMS to new strains (Diversification)

16. Genetic Male Sterility (GMS) Also called as nuclear male sterility . Mostly governed by single recessive gene ( ms ) but dominant gene governing male sterility (safflower). Origin: Spontaneous mutation or artificial mutations (Gamma rays, EMS) are common. ‘ ms ’ alleles may affect staminal initiation, stamen or anther sac development, PMC formation, meiosis, pollen formation, maturation and dehiscence. S.No. Mutagens Crops 1 Colchicine Jowar 2 Ethidium Bromide Groundnut, Maize, wheat 3 Acetone Barley

17. Types of GMS Environment insensitive GMS : ms gene expression is much less affected by the environment. Environment sensitive GMS : ms gene expression occurs within a specified range of temperature and /or photoperiod regimes (Rice, Tomato, Wheat etc.). TGMS : sterility is at particular temperature e.g. In rice TGMS line (Pei- Ai645) at 23.3 C (China). TGMS at high temperature is due to failure of pairing of two chromosomes at metaphase was evident. This abnormality led to abnormal meiosis, abnormal or sterile pollens. Anthers were shriveled and non-dehiscence-Male sterile. However , these lines produced normal fertile pollen at low temp. Sensitive period : PMC formation to Meiosis

18. PGMS : Governed by 2 recessive genes. Sterility is obtained in long day conditions while in short days, normal fertile plant. Rice: - Sterile under Long day conditions (13 hr . 45 min + Temp. 23-29 C) but fertile under short day conditions . Sensitive period : Differentiation of secondary rachis branches to PMC formation

19. Inheritance & Maintenance Of male sterile line

20. Nuclear male sterility and hybrid seed production msms Msms P 1 P 2 X Msms Male fertile Male sterile Male fertile msms Male sterile MsMs Male fertile X F1 Msms Male fertile

21. Cytoplasmic Genetic Male Sterility (CGMS) CGMS is also known as nucleoplasmic male sterility . Case of CMS, where a nuclear gene (R) for restoring fertility in male sterile line is known. R ( restorer gene ) is generally dominant can be transferred from related strains or species . This system is known in cotton , maize , jowar , bajra , sunflower, cotton, rice and wheat etc.

22. Hybrid seed production using CGMS system

23. rr S RR F R r S rr F rr S R r S rr S R r S rr S RR S rr S R r S ♂ ♀ ♂ ♀ Strain A Strain B × × ♀ × rr F ♂ rr F ♂ × 6-7 Back crosses × RR S 1 2 1 : : CMS Restorer Male fertile Non restorer (Strain-C) Male fertile × Male fertile Male sterile Discarded Discarded Discarded Male sterile Male sterile Discarded Male sterile × Self pollinated Male fertile Male sterile Self pollinated Male fertile (Strain-C) Male fertile (Strain-C) ♀ Male fertile Restorer line R is crossed to Male sterile A Male fertile F1 is crossed to Strain C in which R gene is to be transferred Male fertility progeny is back crossed to strain C × Male fertility progeny is back crossed to strain C Male fertile progeny is self pollinated Male fertile progeny is self pollinated. Individual plant progenies grown in next generation and non segregating progenies are selected Transfer of Restorer gene ‘R’ to non restorer strain

24. Inbred A (Cytoplasmic Male Sterile) Inbred B (Non restorer male fertile) Inbred C (Cytoplasmic Male Sterile) Inbred D (Non restorer male fertile) ♂ ♂ ♀ ♀ rr S rr f rr S RR S Single Cross –I A×B (Male Sterile) Single Cross-II C ×D (Male Fertile) rr S R r S ♀ ♂ Double Cross (A×B) × (C×D) rr S R r S 50% 50% Production of Double cross maize hybrids using CGMS (1:1 Segregation for Male Fertility & Sterility)

25. Sources of CMS & Restorer genes in some Crops Crop species Cytoplasm Restorer Genes Rice CMS-CW O. spontanea CMS- bo O. Sativa boroII (single dominant) CMS-WA O. Spontanea (WA, four genes) CMS-W18 O. rufipogon Wheat ( T.aestivum ) T. timopheevi Rf1 and rf2 A. caudata - T. Durum Aegilops ovata - Maize CMS-C Rf4 CMS-S Rf3 CMS-T Rf1 and Rf2

26. Crop species Cytoplasm Restorer Genes Tobaco N. Debneyi - N. Megalosiphon - N. bigelovii - Cotton G. Anomalum - G. Arboreaum - G. harknesii - Sunflower PET-1 (H. petalaris ) 2 polymorphic genes ( Rf1, Rf2 ) Jowar Milo or A1 Msc from kafir race Bajra Tift-23A -

27. S.No . Crop Hybrid Variety Seed Production 1. Maize Ganga 101, Ganga 1, Deccan, Ranjit , Trishulatha , DHM-107, DHM-109 CMS 2. Sorgum CSH1 CMS 3. Bajra HB1 CMS 4. Sunflower BSH1 CMS 5. Rapeseed PGSH51 CMS 6. Red Gram ICPH-8 GMS 7. Rice PRH1 CMS Male Sterility based Hybrids in Important Crops

28. Recombinant DNA techniques for disturbing any or number of developmental steps required for the production of functional pollen within the microspore or for the development of any somatic tissues supporting the microspores . Transgenes for male sterility are dominant to fertility. Also to develop effective fertility restoration system for hybrid seed production. Example: Barnase / Barstar system Transgenic Male Sterility

29. Undesirable effects of the cytoplasm Unsatisfactory fertility restoration Unsatisfactory pollination Spontaneous reversion Modifying genes Contribution of cytoplasm by male gamete Environmental effects Non availability of a suitable restorer line Limitations of Cytoplasmic-Genetic Male Sterility

30. Barnase is extracellular RNase ; barstar is inhibitor of barnase ( Bacillus amyloliquefaciens ) Plants with TA29 promoter- Barnase construct are male sterile Those with TA29-Barstar are not affected by the transgene barnase . Barstar is dominant over the Barnase Fuse the barnase and barstar genes to TA29 promoter–TA29 is a plant gene that has tapetum specific expression . Cross male sterile ( barnase ) with male fertile ( barstar ) to get hybrid seed, which now has both barnase and barstar expressed in tapetum and, hence, is fully fertile Barnase / Barstar system

31. Hybrid seed production using Barnase / Barstar system

32. CHA is a chemical that induces artificial, non-genetic male sterility in plants so that they can be effectively used as female parent in hybrid seed production. Also called as Male gametocides, male sterilants , selective male sterilants , pollen suppressants, pollenocide , androcide etc. The first report was given by Moore and Naylor (1950), they induced male sterility in Maize using maleic hydrazide (MH). Chemical Induced Male Sterility

33. Properties of a n Ideal CHA Must be highly male or female selective. Should be easily applicable and economic in use. Time of application should be flexible. Must not be mutagenic. Must not be carried over in F1 seeds. Must consistently produce >95% male sterility. Must cause minimum reduction in seed set. Should not affect out crossing. Should not be hazardous to the environment.

34. S.No. CHAs Critical stage Crop species 1. Zink Methyl Arsenate Sodium Methyl Arsenate 5 days before heading Rice 2. Ethephon / Ethrel Depends on crop Barley , oat, bajra , rice 3. Mendok Depends on crop Cotton, sugarbeet 4. Gibberellic acid 1-3 days before meiosis Maize, Barley, Wheat, Rice, Sunflower 5. Maleic Hydrazide Early microsporogenesis Maize, wheat, cotton, onion Some important CHAs

35. Hybrid S eed P roduction based on CHAs Proper e nvironmental conditions (Rain, Sunshine, temp, RH etc.) Synchronisation of flowering of Male & Female parents. Effective chemical emasculation and cross pollination CHA at precise stage and with recommended dose GA3 spray to promote stigma exertion. Supplementary pollination to maximise seed set Avoid CHA spray on pollinator row. Conditions required:-

36. Advantages of CHAs Any line can be used as female parent. Choice of parents is flexible. Rapid method of developing male sterile line. No need of maintaining A,B&R lines . Hybrid seed production is based on only 2 line system. Maintenance of parental line is possible by self pollination. CHA based F2 hybrids are fully fertile as compared to few sterile hybrids in case of CMS or GMS.

37. Limitations of CHAs Expression and duration of CHA is stage specific. Sensitive to environmental conditions. Incomplete male sterility produce selfed seeds. Many CHAs are toxic to plants and animals. Possess carryover residual effects in F1 seeds. Interfere with cell division. Affect human health. Genotype, dose application stage specific.

38. Male sterility a primary tool to avoid emasculation in hybridization. Hybrid production requires a female plant in which no viable pollens are borne. Inefficient emasculation may produce some self fertile progenies. GMS is being exploited ( Eg.USA -Castor, India- Arhar ). CMS/ CGMS are routinely used in Hybrid seed production in corn, sorghum, sunflower and sugarbeet , ornamental plants. Saves lot of time, money and labour . Significance of male Sterility in Plant Breeding

39. Existence and maintenance of A, B & R Lines is laborious and difficult. If exotic lines are not suitable to our conditions, the native/adaptive lines have to be converted into MS lines. Adequate cross pollination should be there between A and R lines for good seed set. Synchronization of flowering should be there between A & R lines. Fertility restoration should be complete otherwise the F1 seed will be sterile Isolation is needed for maintenance of parental lines and for producing hybrid seed. Limitations in using Male Sterile line

40. Applications of Male Sterility in Hybrid Seed Production

41. Male sterility system in Rice hybrid seed production Male sterility: a condition in which the pollen grain is unviable or cannot germinate and fertilize normally to set seeds. Male Sterility Systems (genetic and non-genetic): Cytoplasmic genetic male sterility (CMS) Male sterility is controlled by the interaction of a genetic factor (S) present in the cytoplasm and nuclear gene (s). Environment-sensitive genic male sterility (EGMS) Male sterility system is controlled by nuclear gene expression, which is influenced by environmental factors such as temperature (TGMS), daylength (PGMS), or both (TPGMS). Chemically induced male sterility Male sterility is induced by some chemicals (gametocides)

42.     Two Commercial MS Systems for Hybrid Rice

43. TGMS and two-line hybrid Based on the discovery of P(T)GMS mutant Male sterility controlled by 1 or 2 pairs of recessive gene(s) Fertile S-line Multiplication Critical Fertility Point Critical Sterility Point Reproductive Upper Limit Reproductive Lower Limit Sterile F1 Seed Production Partial Sterility Model of Sterility / Fertility Expression for TGMS Rice Temperature low high

44. Advantage & Disadvantage of 2-line hybrid rice system Advantages Simplified procedure of hybrid seed production Multiple and diverse germplasm available as parents Any line could be bred as female 97% (2-line) vs 5% (3-line) of germplasm as male Increased chance of developing desirable & heterotic hybrids Multiple cytoplasm courses as female parents Disadvantages Environmental effect on sterility could cause seed purity problem

45. Requirements for 3 Lines in CMS System A-line Stable Sterility Well developed floral traits for outcrossing Easily, wide- spectum , & strongly to be restored B-line Well developed floral traits with large pollen load Good combining ability R-line Strong restore ability Good combining ability Taller than A-line Large pollen load, normal flowering traits and timing

46. Advantage & Disadvantage of 3-line hybrid rice system Advantages Stable male sterility. Disadvantages Limit germplasm source (CMS, Restorer) Dominant CMS cytoplasm in large area (WA) One more step for parental seed production Time consuming of CMS breeding

47. Male sterility system in Maize hybrid seed production Different ways of inducing male sterility in maize Manual/mechanical emasculation (detasselling) Genic male sterility Cytoplasmic genetic male sterility Gametocides 1. Genetic Male sterility Male sterility determined by single recessive gene 40 loci involved have been identified ( ms1 to ms52 ) ms5 –cloned Problem : impossible to maintain male sterile inbred detasselling required

48. 2 . Cytoplasmic Male sterility 1. CMS-T ( T exas) (Rogers and Edwardson , 1952 ) Highly stable under all environmental conditions Characterized by failure of anther exertion and pollen abortion Susceptible to race T of the southern corn leaf blight - ( Cochliobolus heterostrophus = Bipolaris maydis ) Widespread use of T-cytoplasm for hybrid corn production led to epidemic in 1970 with the widespread rise of Race T. Toxin produced by C. heterostrophus = T-toxin . Fertility restoration is sporophytic Rf1 ( chr. 3) & Rf2 ( chr.9) are responsible for fertility restoration

49. 2. CMS-C ( C harrua ) (Beckett, 1971) Mutations in three genes viz atp 6, atp 9 and cos II - confer CMS phenotype Fertility restoration is Sporophytic Rf4, Rf5, Rf6 are responsible for fertility restoration 3. CMS-S (U S DA) (Jones,1957) Sterility associated with orf355-orf77 chimeric mt gene Fertility restoration is Gametophytic Rf3 ( chr. 2) are responsible for fertility restoration Plasmid like element S1 & S2 T-urf13 gene in T cytoplasm maize Mitochondrial gene T-urf13 is a unique chimeric sequence Effect of URF13 protein- Degeneration of the tapetum during microsporogenesis Disruption of pollen development leading to male cell abortion

50. Reversion to fertility The reversion of CMS strain to male fertility is based on genetic change Reversion can be spontaneous or mutagen induced S-cytoplasm revert rather frequently to male fertility (than T & C). Maize-CMS Restoration of fertility system: different classes of pollen grains are produced, but not all of them are viable

51. A X B ( frfr ) ( frfr ) ms mf AB ( frfr ) ms X C ( FrFr ) mf ABC ( Frfr ) mf Triple Cross Hybrid C X D ( frfr ) ( FrFr ) ms mf CD ( Frfr ) mf A X B ( frfr ) ( frfr ) ms mf AB ( frfr ) ms X ABCD 1 ( Frfr ) mf 1 ( frfr ) ms : : : Double Cross Hybrid

52. Types of Hybrids Single cross hybrid (A×B) Double cross hybrid ( A×B)×(C×D) Three way cross Hybrid (A×B)×C Top cross (C×OPV) Hybrid blends Inter-population hybrids Chance hybrids Male sterility system in Bajra hybrid seed production

53. Hybrid seed production using CGMS Depends on the cytoplasm that produce male sterility and gene that restores the fertility. Steps: Multiplication of CMS (A) line Multiplication of Maintainer (B) line and Restorer (R) line Production of Hybrid seed (A×R) Maintenace of A & B lines: Grow A line and its corresponding B line together in an isolated plots. Isolation distance for A×B production fields is at least 1000m. A ratio of 1A:1B row is maintained. Pollens produced by the B line fertilize the male sterile plant (A) and seeds produced thus Give rise to A line again.

54. Maintenance of R line: Pearl millet R line could be either an inbred line or an Open pollinated variety which can be multiplied as variety. Seeds of R lines are produced by multiplying seeds in isolated plots having distance 1000m. Any plant in the R line plot appearing different from true R type should be uprooted or rogued out before anthesis. Purity of the parental seed is very important because it affects the quality of the hybrid seeds that is generated.

55. Scheme of hybrid seed production in pearl millet Layout of hybrid seed production plot

56. Identification of potential hybrid parents (A,B and R lines) Potential male and female parents for hybrid seed production are identified by crossing male fertile parent (Inbreds, variety, germplasm, breeding stocks in advanced generations) to a male sterile line (A line) and observing their corresponding hybrids in small plots of an observation nursery. A few plants of each cross are subjected to the bagging test i.e. covering the few panicles with the paper bags before anthesis and observing the seed set under the bag after few weeks.

57. CGMS A 1 Tift 23 A (Most of the world hybrids contains A1 Blood), Burton,1958 A 2 , A 3 Not stable cytoplasm A 4 Derived from P. glacum subspecies monodii Does not have effective restorer Used in forage hybrid production

58. Male sterility system in Brassica hybrid seed production Cytoplasmic male-sterile Stamen (anther and filament) and pollen grains are affected It is divided into: a. Autoplasmic A risen within a species as a result of spontaneous mutational changes in the cytoplasm, most likely in the mitochondrial genome b. Alloplasmic A risen from intergeneric , interpecific or occasionally intraspecific crosses and where the male sterility can be interpreted as being due to incompatibility or poor co-operation between nuclear genome of one species and the organellar genome. Another CMS can be a result of interspecific protoplast fusion

59. Raphanus or ogu system Polima or pol system Shiga-Thompson or nap system Diplotaxis muralis or mur system Tournefortii (tour) system Moricandia arvensis or mori system Chinese juncea or jun system 17 systems are available, only difference is the use of male sterile cytoplasmic sources differs for each system Nap system – B.napuus cross b/w winter & spring var. pol system – B.napus var polima mur system -- Diplotaxis muralis x B.campestris cv Yukina tour system – B.juncea collections Various CMS systems

60. Ogu system :- First discovered in Japanese radish ( Raphanus sativus ) by Ogura, 1968 B.napus genome was transferred into the back round of R.sativus ( mst ) through intergeneric crosses followed by back crossing with B.napus . CMS seedling under low temperature showed chlorosis , because chloroplast of R.sativus is sensitive to cold, it is governed by cp -DNA , but mst is governed by mt DNA. Protoplast fusion of R.sativu s with B.napus carried out to have normal green plants with ogu CMS characterisitics This system now has been used for developing alloplasmic male sterile line in B.juncea and B.campestris .

61. Genetic Male Sterility GMS is governed by two genes either recessive or dominant genes(Kaul,1988) One more dominant gene is associated with development of male sterility in B.napus type by means of transgenic male sterility Chemical Male sterility Enthrel – Brassica juncea Zinc methy arsenate- B.napus GA - B.oleracea var capitata

62. B.napaus F 1 interspecific cross x Rhapanus sativus F1 Sterile G-Rs C-Rs G-Bn N-Bn 1/2G-Rs 1/2G-Bn C-Rs mft mst Doubling by colchince Fertile amphidiploid 1/2G-Rs 1/2G-Bn C-Rs mst Development of Male sterile B. napus from R. sativus

63. 1/2G-Rs 1/2G-Bn C-Rs x G- Bn N-Bn G-Bn C-Rs B.napus mst BC 3 Male sterile B.napus mft

64. Development of Alloplasmic Male sterile Brassica campestris x N-Bc B.campestris F 1 interspecific cross x G-Bn S-Rs G-Bct N-Bc 1/2G-Bn 1/2G-Bc S-Rs mft mst G-BC S-Rs BC 4 G- Bc G-Bc Male sterile B.napus

65. Presently genetic male sterility (GMS), cytoplasmic male sterility (CMS) and thermo sensitive genetic male sterility (TGMS) lines are available in India. Development of agronomically superior genetic male-sterile lines in safflower in India have resulted in the development and release of spiny safflower hybrids DSH-129 and MKH-11 in 1997 and NARI-H-15 in 2005, the first non-spiny hybrid safflower NARI-NH-1 in 2001. Male sterility system in Safflower hybrid seed production

66. Genetic Male sterility (GMS) Complete male sterility ms 1 -ms 5 = male sterility in sunflower recessive gene Two types of g- mst Type 1-gmst-Bloomington type Type 2-gmst-Modern type Cultivated Sunflower variety Karlik-68(Dwarf 68)- two recessive genes msi 1 ,msi 2 ( Stable and complete male sterile) Partial male sterility –p mst Male sterility system in Sunflower hybrid seed production

67. CGMS H.petiolaris × H.annuus Repeated backcross of H.annuus results in cms 1 which is extensively used mst in hybrid seed production of sunflower all over the world H.giganteus × H.annuus Cms 3 ( S cytoplasm source) H. annuus subspp lenticularis × H. annuus CV commander Indiana 1

68. Genetic Male Sterility (GMS): R eported in upland, Egyptian and arboreum cottons. In tetraploid cotton, male sterility is governed by both recessive and dominant genes. However , male sterility governed by recessive genes is used in practical plant breeding All three types of male sterility occurs (g mst,c mst,gc mst ) in cotton Sixteen different genes in tetraploid cottons (13 in G. hirsutum and 3 in G. barbadense ) and two in G. arboreum have been identified for genetic male sterility. Sterility is conditioned by dominant alleles at five loci viz , MS4, MS7, MS10, MS11 and MS12 by recessive allele at other loci viz. ms l , ms 2 , ms 3 , ms 13 , ms 14 (Dong A), ms 15 (Lang A) and ms 16 (81 A ). Male sterility system in Cotton hybrid seed production G. hirsutum line Gregg (MS 399) from USA is the basic source of GMS possessing ms 5 ms 6 gene for male sterility.

69. Genetic Male Sterility

70. CMS System In case of CMS, the originally discovered CMS sources involving G. arboreum and G. anomalum cytoplasmic systems having interaction with ms 3 locus were not found effective or stable under different environments. The only stable and dependable CMS source under varied environment was developed through the utilization of G. harknessii . The complete genome of G.hirsutum was transferred into the G. harknessii cytoplasm. A single dominant gene ‘ Rf ’ from G.harknessii is essential for fertility restoration. Fertility enhancer factor 'E' for this CMS restorer system was obtained from a G.barbadense stock. The harknessii system is reported to contribute to good agronomic properties and attraction to honey bees.

71. Sources of Male sterility in Cotton Source of ms cytoplasm Nuclear genome G. anomalum , G . arboreum , G. harknessii G. hirsutum G. anomalum , G. arboreum Heat sensitive , less stable G. harknessii × G. hirsutum Stable cms all over the environment New sources of CMS G. aridum Skovt. × G. hirsutum (D4) G. trilobum × G. hirsutum CMS 8 (D-8) G. sturtianum × G. hirsutum CMS-C1 New sources of CGMS G. anomalum x G. thurberi Cg- mst

72. Mutation G. arboreum , the first spontaneous male sterility mutant was identified in variety DS-5 Chemical based male sterility FW 450(Sodium B- Dichloro - iso -butyrate) MH-30 (Maleic hydrazide ) Ethidium bromide Male sterility based hybrid Production GMS system. CPH2 ( Suguna ), First hybrid based on GMS released at CICR, RS, Coimbatore G. harknessii based cms with fertility restoration gene sources were used in developing the hybrid CAHH 468 (PKV Hy-3).

73. Cytoplasm Nuclear genome Reference S.acaule (4X) S.tuberosum Lamm,1953 S.chacoense (4X) S.tuberosum Rammanna and Hersmen (1974) S.phureja (2x) S.tuberosum Magoon et al.,1958b S.stoloniferum (4x) S.tuberosum Ross (1961) S.Verrucosum (2X) S.tuberosum Abdalla (1970) Inter-specific Hybridization Male sterility system in Potato hybrid seed production

74. FW 450(Sodium B- Dichloro - iso -butyrate) MH-30 (Maleic hydrazide ) Ethidium bromide Chemical mutagens Development of Male sterility Genome transfer S cytoplasm is in the genome of fr genes Unreduced Gamete Production S.tuberosum (2x) × S.tuberosum (4x) Protoplast Fusion S cytoplasm is retained

75. Di haploid S.tuberosum (4x) × S.phureja (4x) (2x) (2x) F1 (4x) Anther culture DiHaploid (2x)