Male sterility in plants

Male sterility-classification, mechanism, site of action, utilization in plant breeding,advantages and disadvantages

1. STERILITY IN CROP PLANTS ARUN CHACKO PhD SCHOLAR PLANT BREEDING AND GENETICS

2. Sterility : An inability of a living organism to effect sexual reproduction ie ., both anthers and ovules are non-functional. Male sterility : It is the failure of plants to produce functional anthers, pollen, or male gametes. Characterised by non-functional pollen grains.

3. Male Sterility Absence of male individuals in dioecious strain, Absence of male organ in a normal bisexual plant, Failure to produce normal sporogenous tissue in stamens, Inhibition at various stages of pollen development, Failure to mature, dehisce or function on a compatible stigma, Pollen Sterility: Pollen abortion and Pollen failure.

4. 1694: Detection of sex in higher plants by Camerarius . 1763: Anther abortion within species and species hybrids ( Koelreuter ). ‘Male contabescens ’ and Hybrid contabescens . Bateson et al .(1908) : ‘male sterility is not simply a random sporadic event but a genetically conditioned system in Sweet pea, where sterility is controlled by one recessive gene’.

5. Correns ( 1908 ): maternal control of gynodioecy . Evidence for non- mendalian inheritance and studies came with gene-cytoplasm interactions and led to discovery of sex types, sex reversals, male sterility in higher plants.

6. Occurrence of male sterility Mutation of either nuclear or cytoplasmic genes or both. Male sterility > Female sterility Male sporophyte and gametophyte are more vulnerable to intrinsic and extrinsic forces than the protected ovule and embryo sac. Pollen : ease of availability and detection Pollen: Carmine, lactophenol or iodine test Female sterility : artificial crossing and developmental studies of seed formation.

7. Mechanism of Male Sterility Abnormal microsporogenesis leads to non-viable microspores. Factors: Mitochondrial mutation Barrier of Tapetal layer Improper timing of Callase activity Operon concept of Gene Action

8. Callase enzyme: for normal pollen development. Early or delayed callase activity leads to male sterility. Heslop -Harrison (1967) : CMS results from operon type control rather than due to cytoplasmic factors. Demonstrated that a specific condition guided by the nuclear gene is propagated by in the cytoplasm thus a permanent repression upon genetic system that determines the pollen producing capacity of the plant. But failed to explain fertility restoration in male sterility system.

9. Male Sterility Gableman (1956) All classifications is either based on phenotypic or on genotypic basis. Pollen Staminal Functional

10. General classification of Male Sterility ( Kaul , 1988)

11. Sterility Type Phenotypic Alteration Effect on male sex organs Action of the male sterility gene Env . Influence on MS gene action Influence of other genes on MS gene action 1. Structural No anthers Transformed anthers Faulty development Form and function impaired During anther development and differentiation before the onset of microsporogenesis High High 2.Sporogeneous No meiosis Abnormal microsporogenesis Function impaired Just before or during or after microsporogenesis High Medium 3.Functional Viable pollen but not liberated Mechanical barrier Functional ability inhibited After microporogenesis , before anther dehiscence Low Low

12. Genic Male Sterility (g- mst ) Occurrence: Spontaneous or artificial mutation In Self-pollinated sp., loss of chromosome carrying mft (male fertile) gene results in male sterility and the plants become weak, slow growing and inviable . Observed in O. lamarckiana , Pisum sativum .

13. Origin of ms alleles Spontaneous mutation Chemical and physical mutagens X-rays, EMS, Et-Br, Acetone, Colchicine

14. Gene Control : Mostly by single recessive gene (ms)

15. Dominant gene governing: eg . Safflower Both recessive and polygene: Medicago sativa Recessive, dominant and Polygene: T.aestivum Several different ms genes act monogenically to produce male sterility.

16. Sterility maintenance: Sterile Fertile 1: 1 Male fertile Male fertile: Male sterile r r R R R r r r Fertility Restoration r r R r R r

17. Site of action of ms allele Time, site, stage and sex specific.

18. STAGE MODE OF ACTION Staminal initiation Rudimentary stamen, undifferentiated, forming minute cellular protuberence . Staminal development and differentiation Stamens malformed, anther sacs rudimentary and misformed , lump of unorganised cells are formed. Anther sac development and archesporial differentiation misdifferentiation , non-functional cells Microsporangial differentiation and development Anther sacs formed, microsporangia misdifferentiated .

19. STAGE MODE OF ACTION PMC formation Anther sacs developed & differentiated but PMC fail to separate out from each other and doesn’t acquire the required genetic autonomy to undergo meiosis. -Their chromatin & nucleoli contract, fragment and degenerate with cytoplasmic content. Premeiosis PMC separate out, no chromosome despiralization occurs, DNA synthesis drastically reduced, unique meiotic histones are not produced. Meiotic entry Chromatin compacts and fragments, PMC degenerate. Tetrad formation Unable to develop, separate and produce callose or to be released from PMCs and finally degenerate.

20. STAGE MODE OF ACTION Microspore liberation Enzyme callase either absent/insufficiently produced, microspore remain enlarged with the PMCs and finally degenerate. Primexine and Sporopollenin deposition Either absence/ misfunction of primexine or abnormal deposition. Pollen development Failure of nucleus to undergo meiosis, followed by cytoplasmic disintegration, low enzyme activity, rRNA and ascorbic acid content. Pollen maturation Generative cells follows vegetative cells degeneration, pollen remains sticky and degenerates Pollen liberation Exine sporopollenin becomes hydrophilic & sticky, pollen release prevented. Anther sac dehisence Anther sac becomes hard and non-dehisce.

21. Molecular mechanism of ms action Changes in content & proportion of amino acids. High deficiency in DNA & RNA Reduced carbohydrate and protein Reduction in activities of callase , cytochrome oxidase . Altered proportion of growth regulators. Delay tapetum degeneration, so that no nucleotide is provided to PMC causing degeneration and male sterility.

22. Allelic relationship between genes causing GMS Identified by crossing the male steriles from different sources with their fertile counter parts ( MsMs )and intercrossing F1s. Intercross progenies ( sterile:fertile ) 3:1  Allelic to each other. 4:0  Non-allelic, all fertile.

23. Advantages of GMS 1.Large number of parents can be used in crosses, because all the genotypes have dominant genes for male sterility. 2.Only female parents of a good hybrid has to be converted. 3.GMS generally does not have undesirable agronomic characters. 4.It is possible to breed the varieties from segregating population of GMS.

24. Disadvantages 1.GMS is less stable. Sometimes, sterile plants become fertile under low temperature conditions. 2.In GMS, the lines segregate into male sterile and fertile plants in 1: 1 ratio. 3.Conversion of a genotype into GMS needs selfing after each backcross to isolate recessive genes and hence more number of generations are required. 4.It requires more area as 50% of the population is fertile. 5.The quantity of seed produced is less. 6.There is possibility of admixture if fertile plants are not properly rogued out.

25. Types of GMS Environment insensitive GMS : ms gene expression is much less a ffected by the environment. Environment sensitive GMS : ms gene expression occurs within a specified range of temperature and /or photoperiod regimes (Rice, Tomato, Wheat etc.). Temperature-sensitive GMS Photoperiod-sensitive GMS Transgenic GMS

26. Temperature-sensitive Genetic Male Sterility (TGMS) Controlled by recessive genes tms1 and tms2 . Complete male sterility by the ms gene at higher temperatures. Temp. below critical temperature Normal fertility. 1 st TGMS line of indica rice: Annong S-1 (Spontaneous mutant in China) Critical Sterility Point in rice: 27-28 C Temp. 24 C or below can cause male fertile lines.

27. TGMS lines in rice: IR 68945, Pei-Ai 645, Hennong -S, Norin PL 12 UPRI 95-140 and UPRI 95-167 : Spontaneous mutant. Pei-Ai 645 : 23.3 C or above can cause male sterile lines. PMC stage to meiosis : Temperature sensitive.

28. Photoperiod-Sensitive GMS ms gene expressed at prevailing photoperiod and provided the temp. is within a critical range. Governed by two recessive genes. For Rice, Sensitive stage: 2 rachis differentiation to PMC formation. 23-29 C. Long day condition (> 13hr 45min.)

29. An ideal PGMS line A low critical temperature for fertility induction. A high critical temperature for strerility induction. Wide temp. range for photoperiod sensitivity. Strong interaction between photoperiod and temperature.

30. Advantages of EGMS No need for a maintainer line Any genotype can be utilized as pollinator parent. Seed production programme is simple and more efficient. Negative influence of male sterility inducing cytoplasm on the F1 plants can be avoided.

31. Photo-thermo sensitive GMS Adaptability of PGMS line: Critical day length and intensity of interaction between photoperiod and temperature. Adapted PGMS line: strong interaction Higher temp. complement with short photoperiod in low altitudes. Low temp. complement with higher photoperiod in high altitudes.

32. Transgenic Genetic Male Sterility 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

33. The first transgene designed to confer GMS was reported and were used to transform Tobacco and oilseed rape plants. Tapetal -specific transcriptional activity of the tobacco TA29 gene. Upstream regulatory elements of TA29 gene used to drive the expression of transgenes (extracellular RNAses from bacteria). Two genes were used: barnase from Bacillus amyloliquefaciens RNAse-T1 from Aspergillus oryzae RNase genes selectively destroyed the tapetal cells during anther development and prevented pollen formation Herbicide ( bialophos ) resistant gene (bar) used as selectable marker

34. Utilization of GMS in Plant Breeding Male Sterile (A): ms ms Maintainer line (B): Ms ms Progeny of [ ms ms ] x [ Ms ms ] used as female. Rouging of male fertile progeny required . In India, Pigeonpea hybrid: ICPH-8,-4, CoH1,2, AKPH4104, AKPH2022.

35. Cytoplasmic Male Sterility In many gynodioecious populations, the male-sterile trait is transmitted matrilineally , and the genetic determinants are therefore believed to reside in the cytoplasm. These cytoplasmic determinants usually coexist in a population with autosomal genes that repress their action and cause the restoration of pollen fertility. ( Kheyr -Pour 1980, 1981; van Damme 1983)

36. Cytoplasmic male sterility (CMS) was first discovered by Correns (1906). Cytoplasmic male sterility (CMS) is the maternal transmission of failed pollen production in hermaphroditic plants leading to a mixture of male-sterile and hermaphroditic individuals in the population ( gynodioecy ).

37. CMS is the result of mutation in mtDNA leading to nuclear-mitochondrial interaction or incompatability . Nuclear genes are not involved. CMS is not influenced by environmental factors (temperature) so is stable. Sterility Maintenance: Sterile A line x Fertile B line  A line sterile progenies Fertility restoration is not feasible., useful where seed is not the desired end product.

38. TEXAS, OR T, CYTOPLASM ( cms -T) of maize was first described in Texas in the Golden June line of maize. Carries two cytoplasmically inherited traits, male sterility and disease susceptibility, The two traits are inseparable and are associated with an unusual mitochondrial gene, T-urf13, which encodes a 13-kilodalton polypeptide (URF13). An interaction between fungal toxins and URF13, which results in permeabilization of the inner mitochondrial membrane, accounts for the specific susceptibility to the fungal pathogens.

39. Male sterility is characterized by the failure of anther exertion and pollen abortion. Female fertility is not affected by CMS, so male-sterile plants can set seed if viable pollen is provided. cms -T had replaced detasseling as the chief method of pollen control in hybrid corn production

40. After it was determined that cms -T was specifically susceptible to Bipolaris maydis race T, the organism responsible for the blight, its use by the hybrid seed corn industry was largely terminated. Phyllosricra maydis , another fungal pathogen, is also specifically virulent on cms -T. Susceptibility of cms -T maize to the fungal pathogens is due to mitochondrial sensitivity to the pathotoxins , whereas disease-resistant maize types have mitochondria that are insensitive to the pathotoxins .

41. Ogura-CMS turnip: reduction in the size of the fleshy root, distinct defects in microspore development and tapetum degeneration during the transition from microspore mothercells to tetrads. Defective microspore production and premature tapetum degeneration during microgametogenesis resulted in short filaments and withered white anthers

42. The mechanism regulating Ogura-CMS in turnip was investigated using inflorescence transcriptome analyses of the Ogura-CMS and MF lines. 5,117 differentially expressed genes (DEGs) were identified including 1,339 up- and 3,778 down-regulated genes in the Ogura-CMS line compared to the MF line. A number of functionally known members involved in anther development and microspore formation were addressed in the DEG pool, particularly genes regulating tapetum programmed cell death (PCD), and associated with pollen wall formation. Additionally, 185 novel genes were proposed to function in male organ development based on Gene Ontology analyses, of which 26 DEGs were genotype-specifically expressed.

43. Genetics of fertility restoration &Allelic relationship of Rf genes Fertility restoration can be monogenic and digenic . In digenic restoration, non-allelic interaction of different types has been reported. Eg : Rice The fertility restoration controlled by a single dominant gene have more practical value where Rf gene is to be transferred to a different genetic background.

44. The allelic relationship b/w restorer genes can be tested by crossing two restorers and using F1 as pollinator to pollinate CMS line. 3:1  Non-allelic All progenies are fertile, if two restorer lines carried genes are allelic.

45. Allelic condition Restorer 1 x Restorer 2 R 1 R 1 R 1 R 1 F 1 (R 1 R 1 ) x r 1 r 1 CMS line R 1 r 1 All fertile

46. Non-allelic condition Restorer 1 x Restorer 2 R 1 R 1 r 2 r 2 r 1 r 1 R 2 R 2 F 1 (R 1 r 1 R 2 r 2 ) x r 1 r 1 r 1 r 1 CMS line R 1 r 1 R 2 r 2 R 1 r 1 r 2 r 2 r 1 r 1 R 2 r 2 r 1 r 1 r 2 r 2 Fertile Fertile Fertile Sterile Fertile: Sterile  3:1

47. Molecular tagging and transfer of Rf gene Generate an appropriate mapping population.F 2 population from crossing CMS and a restorer line. 150-200 F 2 plants are selected randomly, DNA isolated. Plants are individually tagged and screened for pollen fertility as well as bagged to score spikelet fertility.

48. Based on pollen and spikelet fertility, 10 fully sterile and fully fertile F2 plants are selected. DNA from these plants in each group is used to create fertile and sterile bulks. These bulks are used for Bulk Segregant Analysis (BSA). This helps to find molecular markers which are polymorphic b/w bulks as well as b/w the parents.

49. The polymorphic markers can be used on the individual plants to identify markers closely linked to the Rf gene, which may be used for marker-aided transfer of the Rf gene to a desirable background. Eg : Rice, Sorghum, Brassica

50. Cytoplasmic -Genetic Male Sterility The male sterility which  is governed by both nuclear and cytoplasmic genes. Also called as Nucleoplasmic Male Sterility. When nuclear restoration of fertility genes (“ Rf ”) is available for a CMS system in any crop, it is cytoplasmic -genetic male sterility. There are commonly two types of cytoplasm N (normal) and S (sterile). The genes for these are found in mitochondrion. There are also restores of fertility ( Rf ) genes.

51. Rf genes do not have any expression of their own, unless the sterile cytoplasm is present. Rf genes are required to restore fertility in S cytoplasm which is responsible for sterility. So the combination of N cytoplasm with rfrf and S cytoplasm with RfRf produces plants with fertile pollens, while S cytoplasm with rfrf produces only male sterile plants. R (restorer gene) is generally dominant can be transferred from related strains or species.

52. Hybrid Seed Production using CGMS

53. Transfer of Restorer gene ‘R’ to non-restorer starin

54. Production Double cross maize hybrids using CGMS

55. Sources of CMS & Restorers

56. Hybrids released using CGMS Sl. No. Crop Hybrid CHILLI Arka Megha , MSH-149 MSH-96 2. CARROT Pusa Nayanjyoti 3. ONION Arka Kirtiman , Arka Lalit , Hybrid-63, Hybrid-35

57. Merits of CGMS 1.In CGMS system, CMS is highly stable and is not affected by environmental factors. 2.In CGMS system, CMS 'A' line gives only male sterile plants. 3.Conversion of a genotype in CGMS system 'A' line is quicker and direct. 4.CMS requires less area for maintenance. 5.The quantity of seed produced is more. 6.There is no chance of admixture.

58. Demerits of Cytoplasmic -Genetic Male Sterility 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

59. CHEMICALLY INDUCED MALE STERILITY 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).

60. 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

61. 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. CH A b ased F 2 h y b ri d s a r e f u lly f e r t ile as c o m p a r ed t o f ew sterile hybrids in case of CMS or GMS.

62. 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.

63. 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.

64. PISTILLATE CONDITION In some mutants, only pistillate flowers are produced in place of both male and female flowers. eg : Castor, Cucurbits In castor : it is governed by a single recessive gene. Types: N Type S Type NES Type

65. N Type Pistillate Lines Governed by single recessive gene (n). Produce only pistillate flowers. Maintaining pistillate lines : crossing them with heterozygous monoecious ( Nn ) lines that produce both male and female flowers. Progeny: 50% pistillate , 50% monoecious . Similar to GMS.

66. S Type Pistillate Lines Sex reversal variants: pistillate to begin with and changes to monoecy . In S type, continued selection for increased expression of pistillate condition within sex reversal variants. 50-70% plants are pistillate . Pistillate plants revert back to monoecious in different stages of development. Use of pistillate lines in hybrid seed production requires removal of monoecious plants and early revertants . Eg . Castor VP1 (female parent of GAUCH1) Geeta (female parent of GCH1)

67. NES Type Temperature sensitive N lines. 100% pistillate when temp. during flowering < 35 C., produce male flowers @ temp. > 35 C Plants are multiplied during hot season. Comparable to TGMS and PGMS. Most suited for hybrid seed production. Eg . JP65 (female parent of GCH6)

68. Significance of male Sterility in Plant Breeding M ale st e r ili t y a p r i m ary t o o l t o a v o i d e mascul a t i o n 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 .

69. Limitations in using Male Sterile line 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 F 1 seed will be sterile Isolation is needed for maintenance of parental lines and for producing hybrid seed.