Male sterlity and Self incompatibility.

This is most important PPT on male sterility and self incompatibility.

1. Mr. Devkumar Arya (Lecturer) Department of Genetics and Plant Breeding O.P. Agriculture College Budhwal , Behror , Alwar (Raj..),India Affiliated to Sri Karan Narendra Agriculture University, Jobner ,( Rajsthan ),India Email: [email protected] Male Sterility & Self-incompatibility and their use in hybrid seed production

2. Male sterility Manifestation of Male sterility History of Male sterility Need 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) Use of Male sterility Reference 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. Steri l e Steri l e F e rt i le F e rt i le

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 m ale ster ili ty h a s been r ep o rted in cab b age (R u nd fe l d t 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. Use of genetically engineered “pollen killer” genetic system Several forms of pollination control Manual emasculation Use of male sterility Use of self-incompatibility alleles Use of male gametocides

7. Why Male Sterility ??? Reduced the cost of hybrid seed production. Production of large scale of F1 seeds. Avoids 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. Classification of Male Sterility Kaul (1988) Classified Male Sterility in three major groups Phenotypic Male Sterility (Morphological) Structural or Staminal Male Sterility Pollen Male Sterility Functional Male Sterility 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) Chemically Induced Male Sterility (CHA)

10. Pollen sterility : I n 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 : I n which male flowers or stamen are malformed and non functional or completely absent. Functional male sterility : I n which perfectly good and viable pollen is trapped in indehiscent anther and thus prevented from functioning Phenotypic Male Sterility

11. 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

12. GENETIC MALE STERILITY ENVIROMENT SENSITIVE MALE STERILITY ENVIROMENT INSENSITIVE MALE STERILITY TEMPERATURE SENSITIVE GENETIC MALE STERILITY PHOTOPERIOD SENSITIVE GENETIC MALE STERILITY

13. Temperature sensitive male sterility(TGMS) In this Type of GMS complete male sterility is produced by the ms gene at higher temperature. ( e.g - male sterility at 23.3º C or higher temperature for rice.) Two lines (UPRI 95-140 TGMS and UPRI 95-167 TGMS) have been isolated as spontaneous mutants and registered as germplasm.

14. Photoperiod senstitive genetic male sterility (PGMS) Expression of ms gene is drastically affected by the prevailing photoperiod, provided the temperature is within a critical range (e.g., 23-29 C for rice PGMS) within this temperature range complete sterility obtained in rice plant grown under long day condition (Day lengh more than 13 hr 45 min).

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

16. Cytoplasmic Male Sterility (CMS) Determined by the cytoplasm (mitochondrial or chloroplast genes). Result of mutation in mitochondrial genome (mtDNA)- Mitochondrial dysfunction. Progenies would always be male sterile since 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.

17. It is divided into types :- a. Autoplasmic - CMS has arisen within a species as a result of spontaneous mutational changes in the cytoplasm, most likely in the mitochondrial genome. b. Alloplasmic - CMS has arisen from intergeneric, interspecific 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 .

18. Various CMS systems 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. mur system --Diplotaxis muralis x B.campestris cv Yukina tour system – B.juncea collections

19. 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 Plant Breeding

20. Use of CMS lines

21. Transfer of CMS to new strains (Diversification)

22. Cytoplasmic Genetic Male Sterility (CGMS) Case of CGMS, 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.

23. Hybrid seed production using CGMS system

24. rr S R r S rr 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 rr F × 6-7 Back crosses × RR S 1 1 : 2 : CMS Restorer F ♂ Male fertile Non restorer (Strain-C) Male fertile ♀ × Male fertile Male sterile Discarded Male sterile Discarded Male sterile Discarded Male sterile Discarded × Male fertile Self pollinated Male sterile Self pollinated ♂ Male fertile (Strain-C) ♂ Male fertile (Stra in - 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

25. Inbred A (Cyto p la s m i c Male Sterile) Inbred B (Non restorer male fertile) Inbred C (Cyto p la s m i c Male Sterile) Inbred D (Non restorer male fertile) ♂ ♂ ♀ ♀ rr S rr f rr S RR S Single Cross-II C×D (Male Fertile) rr S R r S ♀ Single Cross –I A×B (Male Sterile) ♂ 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)

26. Limitations of Cytoplasmic -Genetic Male Sterility Unsatisfactory fertility restoration Unsatisfactory pollination Spontaneous reversion Modifying genes Environmental effects Non availability of a suitable restorer line

27. 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

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 t o deve l op e f fec t i v e fer t il i ty re st o ra t ion sys tem for hybrid seed production. Example: Barnase/Barstar system Transgenic Male Sterility

29. Barnase is extracellul a r RNa s e ; barstar is inhibitor o f bar n ase ( 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

30. Hybrid seed production using Barnase/Barstar system

31. 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). 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. Chemical Induced Male Sterility

32. Properties of an 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.

33. 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

34. Hybrid Seed Production based on CHAs Conditions required:- Proper environmental 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.

35. 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.

36. 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.

37. 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

38. 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

39. Content of self incompatibility in plant Introduction History of self incompatibility General features of self incompatibility Classification of self incompatibility Use of SI for hybrid seed production References

40. Self-incompatibility  ( SI ) It refers to the inability of a plant with functional pollen to set seeds when self pollinated. It is the failure of pollen from a flower to fertilize the same flower or other flowers of the same plant. 43

41. General features of Self-incompatibility Prevents selfing and promotes out-breeding so increases the probability of new gene combinations. Causes may be morphological, physiological, genetical or biochemical. Normal seed set on cross pollination. May operate at any stage between pollination and fertilization. Reduces homozygosity . In plants, self-incompatibility is often inherited by a single gene (S) with different alleles (e.g. S 1 , S 2 , S 3  etc.) in the species population

42. Complimentary hypothesis Proposed by bateman in 1952. S.I due to absence of stimulation by the pistil on pollen growth in the like genotype(S1S2xS3S4) Means S.I is due to absence of substance in the pistil or pollen which are essential for pollen tube penetration.

43. Oppositional hypothesis It states that interaction between like alleles(S1S2xS1S2) leads to production of inhibitor which inhibit the growth of pollen tube in the pistil. Interaction between like alleles, a substance is produced in pollen and pistil which property to interfere with normal metabolism of pollen grain and tube(inhibit enzyme or auxin, block pollen tube membrane, inhibit enzyme necessary for penetration.)

44. Criteria Types Flower morphology Heteromorphic self incompatibility Distyly Tristyly Homomorphic self incompatibility Sporophytic self incompatibility Gametophytic self incompatibility Classification of Self-incompatibility 48

45. Criteria Types Genes involved (number) Monoallelic (governed by single gene) Diallelic (governed by two genes) Polyallelic (governed by many genes) Cytology of pollen Binucleate (pollens with two nuclei) Trinucleate (pollens with three nuclei) Expression site Ovarian (expression site is ovary) Stylar (expression site is style) Stigmatic (expression site is stigma) 49

46. The genes responsible for self-incompatibility in heterostylous flowers are strongly linked to the genes responsible for flower polymorphism, so these traits are inherited together. The associated concepts are distyly and tristyly. Heteromorphic self-incompatibility 50

47. What is Distyly ? Here, both stamens and styles are of two types. Stamens may be low and high styles short and long . It is determined by a single gene , with two alleles. The flower with short style and high stamen is called as thrum type and flower with long style and low stamen is called as pin type. Both thrum and pin flowers differ for six characters in addition to stamen and style length. 51

48. Stigma Anther Cross Result Ss (thrum) X Ss (thrum) Incompatible ss (pin) X ss (pin) Incompatible Ss(thrum) X ss (pin) 1:1 ss (pin) X Ss (thrum) 1:1 Distyly Thrum Pin 52

49. What is Tristyly? In tristyly, styles and stamens have three different positions. It is determined by two genes S and M , each with two  alleles. S gives rise to short style, S and M to medium style and s and m to long style. The number of possible genotypes is greater, but a 1:1 ratio exists between individuals of each SI type. 53

50. Short Style Medium Style Long Style 54

51. HOMOMORPHIC SELF INCOMPATITBILITY The homomorphic system is found in the majority of Self incompatibility. The incompatibility is not associated With morphology differences among flowers. SI reaction is Controlled by a single complex locus, the S locus, but in some Cases. More than one gene is involved. It is divided into two group Gametophytic self incompatibility Sporophytic self incompatibility

52. Gametophytic self incompatibility Gametophytic incompatibility was first described by East and Mangelsdorf in 1925 in Nicotiana sanderae . In this system, the Incompatibility reaction of pollen is determined by its own Genotype, and not by the genotype of the plant on which it Is produced. The incompatibility reaction may be controlled by one or two Gene. It is two type Monofactoreal gametophytic self incompatibility Bifactorial gametophytic self incompatibility

53. MONOFACTORIAL GAMETOPHYTIC SYSTEM SI reaction is controlled by single gene designated by S Which usually has 50 or more gene This system is very efficient at preventing fertilization on Self pollination but it is not very efficient at prevnting fertilizationOn sib pollination This system is found in Trifolium , Nicotiana, Solanum etc.

54. BIFACTORIAL GAMETOPHYTIC SYSTEM SI reaction is controlled by two genes designated as S and Z Which are unlinked and exhibit multiple alleles Homozygosity at one of the two loci is common Homozgyosity at both the loci are not found Heterozygotes at both the loci will show partial compatibility When used as males in matings with females heterozygus at Only one locus, but having the same alleles at the two loci as Present in the males; however, when they are used as females, There is complete incompatibility. Polyploidy has no effect on SI reaction

55. Sporophytic self incompatibility This type of SI was first reported by Hughes and Babcock In 1950 in Crepis foetida , and by Gerstel in Parthenium Argentatum (in the same year) In sporophytic system, self incompatibility is governed By a single gene, S, with multiple alleles; The incompatibility reaction of pollen is governed by the Genotype of plant on which the pollen is produced, and Not by the genotype of pollen.

56. Gametophytic self-incompatibility ( GSI) Sporophytic self-incompatibility ( SSI) ( Buchanan, et al ., 2005 )

57. Use of SI for hybrid seed production

58. Basic steps in the use of SI Identification of self-incompatible plants in diverse population/genotypes Development of homozygous self-incompatible lines Identification of S-alleles in the homozygous self-incompatible lines Establishment of inter-allelic relationship among the S-alleles Identifying the best combining lines Maintenance of parental self-incompatible lines Commercial hybrid seed production

59. To fulfill the demand of food to the growing world population. To identify the stable SI and CMS lines. There is need to acquire deeper knowledge about these two important mechanisms for developing location/ environmental specific SI and MS lines. 64 Future Prospects

60. References Singh B.D.2009,Plant Breeding, Kalyani Publications, Ludhiana. Prasad B.K. 2006,Plant Breeding, Kalyani Publications, Ludhiana. Singhal N.C.2003,Hybrid seed production in field crops, Kalyani Publications, Ludhiana. Mukharjee B.K.1995,Heterosis Phenomenon, Kalyani Publications, Ludhiana. Singh R.K. 2006,Plant Breeding, Kalyani Publications, Ludhiana. Singh Phundan.2005,Plant Breeding,Kalyani Publications, Ludhiana. By Google.

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