self incompatibility and male sterility


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self incompatibility and male sterility

  1. 1. Speaker:-M.Sc. 2nd year 1A-2010-30-54
  2. 2. • India is the leading vegetable producer in the world occupying an area of 7.98 mha with the production of 133.74 mt ( NHB 2010) and ranks second in vegetable production after China• In H.P. area under vegetables is 79.8 (in000‟HA) with the production of 1390.7 (in 000MT) ( NHB 2010)• Limited cultivated area, increasing population, and ever increasing pressure on land due to urbanization & industrialization has left us only with one solution i.e. to increase the productivity 2
  3. 3. • The productivity of vegetables can be increased by using F1 hybrids along with improved qualities & standardization of agro-techniques• Area, production and productivity of major Cole crops in India Crops Area Production Productivity (IN 000 HA) (IN 000 MT) (IN MT/HA) Cabbage 331.0 7281.4 22.0Cauliflower 347.9 6569.0 18.9 (NHB, 2010) 3
  4. 4. • The cost of hybrid seeds is comparatively higher, which is one of the major constraints in achieving more rapid adoption of the hybrid vegetable technology• With the use of genetic mechanisms i.e. male sterility, self incompatibility etc., we can reduce the cost of hybrid seed production• And these mechanisms will be helpful in economic hybrid seed production and availability of hybrid seed within the reach of poor farmers 4
  5. 5. Self incompatibility:- SI refers to the inability of a plant to set seed upon self pollination despite male and female gamete is viable, there is no seed set in self pollination Lewis (1954) has suggested various classifications of self incompatibility Heteromorphic system• Pin type• Thrum type Homomorphic system• Sporophytic self incompatibility (SSI) i.e. (Cole crops)• Gametophytic self incompatibility (GSI) 5
  6. 6. Gametophytic SI Sporophytic SIThe stigma is smooth and wet The stigma is papilate ( hairy) and dryPollen tube inhibition in style Pollen tube inhibition take place on the stigmatic surface itselfThe pollen-pistil interaction govern The pollen-pistil interaction govern byby haploid genome of each male genome of the plant on which the malegametes and diploid genome of pistil and female gamete produced ( Diplo-tissue ( Haplo-Diplo) Diplo) 6
  7. 7. Sporophytic self incompatibility Gametophytic self incompatibility 7
  8. 8. Male sterility:- Male sterility is defined as the deviant condition in normally bisexual plants (monoecious as well as hermaphrodite) when no viable pollen is formed Kaul (1988) classified male sterility in three major groups• Genetic male sterility• Morphological male sterility:- Not found in Brassicaceae• Environmental genic male sterility (EGMS) TGMS- Thermo sensitive genic male sterility PGMS- Photoperiod sensitive male sterility 8
  9. 9. Phenotypic• Structural male sterility: Abnormalities in male sex organs Tomato (positional sterility (Atanassova 1999) etc.• Sporogenous male sterility: Stamens form, but pollen absent• Functional male sterility: Viable pollen form, but barrier prevents fertilization Eggplant (functional male sterility in eggplant (Phatak and Jaworski l989) )Tomato, cucumber etc. 9
  10. 10.  Genotypic• Genic male sterility: Mendelian inheritance due to nuclear gene (Tomato, Capsicum, cabbage, cauliflower, Watermelon, Lima bean, Lettuce, Muskmelon etc.)• Cytoplasmic male sterility: Non-Mendelian inheritance – cytoplasmic (Tomato, Pepper, Cole vegetables, Onion, Carrot, Radish)• Gene-cytoplasmic male sterility: Both nuclear and cytoplasmic genes (Onion, Carrot, Sugarbeet, Capsicum, Radish etc. ) 10
  11. 11. • Reduced the cost of hybrid seed production• Production of large scale of F1 seeds• It avoids enormous manual work of emasculation and pollination• Speedup the hybridization programme• Commercial exploitation of hybrid vigour 11
  12. 12. • Earliness• Greater productivity• Better adoptability to variable environments• Better tolerance against diseases and pests• Uniform produce• Better market acceptability• Better nutritional quality 12
  13. 13. 13
  14. 14. • First discussion on self-incompatibility by Darwin (1877)• The term self incompatibility was given by Stout (1917)• Bateman (1952, 1954, 1955) gave explanation on incompatibility in three Brassicas plants namely, Iberis amara L., Raphanus sativus L. and Brassica campestris L. 14
  15. 15. • SI phenotype of the pollen is determined by the genotype of the sporophyte (pollen producing parent)• It is controlled by single s-locus with multiple allele but also reported more than one loci, more than 80 s-alleles reported in Brassica family ( On the basis of population) 15
  16. 16. This system was confirmed :- Kale ( Thompson 1957) Radish ( Sampson 1957) Broccoli ( Sampson 1957 and Odland 1962) Cabbage ( Adamson 1965) Cauliflower ( Hoser-krauze 1979) 16
  17. 17. • Bateman (1955) described the control of SI in family Brassicaceae by a single Mendelian locus, the S (Sterility) locus, which exists as multiple alleles The number of S-locus alleles is usually large:- 22 in Iberis (Bateman 1955) 34 in Raphanus (Sampson 1957) 30 in B. rapa (Nou et al. 1993) 50 in Brassica oleracea (Brace et al. 1994) 17
  18. 18. • A classical genetic analysis has grouped the Brassica S-alleles into two categories based on their phenotypic effect on self-incompatibility characteristics First group of alleles Second group of alleles (high-activity) (low-activity)The first group of alleles (high- The second group of allelesactivity) are placed relatively (low-activity) demonstrate ahigh on the dominance scale weak or leaky self incompatibleand exhibit a strong self- phenotypic effect in which 10 toincompatible phenotype in 30 pollen tubes develop perwhich an average of 0 to 10 self-pollinated stigma and theypollen tubes develop per self- are considered to be recessivepollinated stigma 18 (Nasrallah et al. 1991)
  19. 19. • Genotypes (homozygous/heterozygous) of male and female plant at S locus Interactions between the two S alleles Dominance (S1 > S2) Co-dominance (S1 = S2) Mutual wakening (no action by either allele) Intermediate gradation (1-100% activity by each allele) 19
  20. 20.  SI recognition is controlled by a multiallelic gene complex at a single locus, termed the S-locus The S-locus consists of three genes SRK (S-locus receptor kinase); (Stein et al.1991) SP11(S-locus protein 11)/SCR (S-locus cysteine rich); (Suzuki et al.1999; Takayama et al. 2000) SLG (S-locus glycoproteins ) 20
  21. 21. SLG and SRK exhibit a number of characteristics that would be expected for the female determinant of SI They are predominantly produced in the stigma papilla cells, which come into direct contact with pollen Their expression occurs just prior to flower opening They exhibit allelic sequence diversity among all of the S-haplotypes 21
  22. 22.  SP11/ SCR is the male determinant and is predominantly expressed in the anther tapetum Immunohistochemical studies suggest that the SP11/SCR protein is secreted in a cluster from the tapetal cells into the anther locule and translocated to the pollen surface The cloning and sequencing of the S-locus region using fluorescent differential display, succeeded in identifying the male determinant genes, which were named SP11/ SCR 22
  23. 23. 23(Takayama and Isogai 2005)
  24. 24.  Upon pollination, SP11 penetrates the papilla cell wall and binds SRK in an S-haplotype-specific manner This binding induces the autophosphorylation of SRK, triggering a signaling cascade that results in the rejection of self-pollen MLPK (M-locus protein kinase) localizes papilla cell membrane and may form a signaling complex with SRK ARC1(Armadillo repeat-containing 1) identified through protein interaction with SRK The proteasomal degradation of these substrates could result in pollen rejection 24
  25. 25.  Number of seed set after each specific self- or cross- pollination The fluorescent microscopic observations on pollen ability to penetrate style (within 12-15 hr) (Dyki 1978) 25
  26. 26.  Sib-incompatibility is weak in certain inbreds Continuous inbreeding may lead to complete loss of the inbred lines Pseudo-incompatibility Hybrid seeds would be expensive if the self- incompatible lines are difficult to maintain 26
  27. 27.  Stable self incompatibility High seed set of self pollination at bud stage Favorable and uniform economic characters Desirable combination ability 27
  28. 28. Basic steps in the use of SSI• 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 relationships among the S-alleles• Identifying the best combining lines• Maintenance of parental self-incompatible lines• Commercial hybrid seed production 28
  29. 29. • Bud pollination / Sibmating• Treatment with CO2 gas (CO2 enrichment) (Jirik 1985) or sodium chloride ( Kucera 1990) Other methods:-• Electronic aided pollination (EAP); (Roggen et al. 1972)• Steel brush method ( Roggen and Dijik 1972 )• The pollen washing ( Roggen 1974)• Thermally aided pollination (TAP); (Roggen and Dijik 1976) 29
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  31. 31. 31
  32. 32. Crop Name of Hybrid Type of Genetic Developing Mechanism Institution (Parentage) Cabbage KGMR-1, BRH-5 Self-Incompatibility IARI regional station, (KGMR-1=83-1-621 x GA- Katrain 111) Cabbage H-43, H-44 Self-Incompatibility IARI regional station, (H-43=S2S2 x Pusa Katrain Mukta) (H-44=S2S2 x Cornell 83-6Cauliflower Pusa Hybrid-2 (Nov Self-Incompatibility IARI, New Delhi maturing, Group-II), Pusa (Pusa Hybrid-2=CC x 18- Kartik Sankar (group-I) 19) (Pusa Kartik Sankar= CC 14 x 41-5)Cauliflower Xiahua 6 ( heat-resistant ) self-incompatibility X iamen Agricultural Research Institute of Sciences, China, 2006 32
  33. 33. 33
  34. 34.  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) 34
  35. 35. • 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 35 Mehdi and Anwar 2009
  36. 36. • Male sporophyte and gametophyte less protected from environment than ovule and embryo sac• Easy to detect male sterility, because a large number of pollen for study available• Easy to assay male sterility:- Staining technique (caramine, lactophenol or iodine) Female sterility requires crossing• Male sterility has propagation potential in nature 36
  37. 37.  Genic male sterility Wide occurrence in plants Mostly governed by a single recessive nuclear gene , ms Male sterile alleles arise spontaneously or may be artificially induced 37
  38. 38. Cytoplasmic male sterility (CMS) systems Determined by the cytoplasm It is the result of mutation in the mitochondrial genome (mt-DNA) CMS transfer easily to a given strain Most CMS associated genes are chimeric mitochondrial sequences (Schnable and Wise 1998)Chemically induced male sterilityEngineering male sterility 38
  39. 39.  Ogura CMS system (Intergeneric crosses) Ogura male sterility was reported in Japanese radish, (Ogura 1968) It was introduced in cabbage nucleus into the cytoplasm of Ogura male sterile radish (R-cytoplasm) by repeated back crossing (Bannerot et al. 1974) This CMS in cabbage had the problem of chlorosis at low temperature (below 120C), it replace by protoplast fusion (Radish chloroplasts with Brassica oleracea) ; (Robertson et al. 1987) Ogura-CMS is stated to be a mitochondrial DNA encoded male sterility 39
  40. 40.  CMS induced by interspecific cross B. oleracea with rutabaga – B. napus (Chiang, Crete 1985, 1987) transfer resistance against Plasmodiophora brassicae Radish and B.oleracea L. var. italic (broccoli) and later into Cabbage (Pearson 1972) Other sources of CMS:- B. nigra sterile cytoplasm and sterile „Anand‟ cytoplasm from B. rapa in which it was originally derived from the wild species, B. tournefortii (Swarup 2006) One such CMS system tour which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp.(Arumugam et al. 1996) 40
  41. 41. Origins of CMS Intergeneric crosses Interspecific crosses Intraspecific crosses Mutagens (EMS, EtBr) Antibiotic (streptomycin and Mitomycin) Spontaneus 41
  42. 42.  Advantages of CMS• Highly stable and not influenced by environmental conditions Limitations of CMS• Not use where seed is the economic product• CMS line has inferior agronomic performance 42
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  44. 44. Basic steps in the use of CMS• A-line (female parent) of desired genetic background (S- msms)• B-line (maintainer): This is an isogenic line (genetically N- msms)• C-line (male parent): This is a male parental line and also the best specific combiner with A line.• Maintenance of A, B and C lines• Hybrid seed production 44
  45. 45. 45
  46. 46. Crop Name of Hybrid Type of Genetic Developing Mechanism Institution (Parentage)Cabbage KCH-5 Ogura CMS IARI regional station, KatrainCabbage H-11, H-46 CMS IARI regional (H-11=Cornell 83- station, 23 x golden acre), Katrain (H-46=Cornell 83- 23 x golden acre) 46
  47. 47. • GMS has been reported in about 175 plant species (Kaul 1988) including important vegetable crops Salient points• Usually recessive & monogenic• GMS does not have any undesirable agronomic characters Limitations of GMS• Less Stable to temperature and photoperiod• 50% of the fertile plants to be removed from the field• Availability of marker gene- closely linked with ms gene 47
  48. 48. Origin of GMS Spontaneous mutation Mutation by ionizing radiation Chemical mutagens such as ethyl methane sulphonate (EMS) and ethyl imine (EI) Genetic engineering Protoplast fusion 48
  49. 49. 49
  50. 50. LINE A X LINE CMale sterile Male fertile msms MsMs COMMERCIAL HYBRID Msms (AXC) (All male fertile) 50
  51. 51. (Goldberg et al. 1993) 51
  52. 52.  Barnase is extracellular RNase ( from bacterium Bacillus amyloliquefaciens) Barstar is inhibitor of barnase (B. amyloliquefaciens; barstar to protect itself from barnase) Fuse the barnase and barstar genes to TA29 promoter (TA29 is a plant gene that has tapetum specific expression) Plants containing the TA29–barnase construct are male sterile Those with TA29–barstar are not affected by the transgene Barstar is dominant over barnase 52 Mehdi and Anwar 2009
  53. 53. regeneration Agrobacterium- mediated transformation male-sterile plant Promoter which Gene which disruptsinduces transcription normal function of cellin male reproductive specifically 53
  54. 54. Engineering genic male sterility using barnase(A) and restoring fertility to F1 plants using barstar (B) 54 Vinod 2005
  55. 55. The barnase-barstar male sterility-fertility restoration system wasidentified in Cauliflower and Tomato ( Banga and Raman 1998) 55
  56. 56. A (SH/-) X B (-/-) SH/- -/- -/- -/- -/- SH/- SH/-glufosinate -/- -/- SH/- SH/- SH/- -/- -/- X C (R/R) -/- SH/- -/- -/--/- SH/- -/- -/- SH/- -/- -/- SH/- -/- -/- SH/- pTA29-barnase : S (sterility) p35S-PAT : H (herbicide resistance) pTA29-barstar : R (restorer) Fertile F1 (SH/-, R/-) Fertile F1 (-/-, R/-) 56
  57. 57.  Various terms have been used to describe chemicals that induce male sterility in plants Gametocide Selective gametocide Pollen suppression Male sterilant Selective male sterilant Pollen suppressant Pollenocide Androcide 57
  58. 58. • First demonstrated in maize (MH) (Moore 1950; Naylor 1950)Following CHA which are using for inducing male sterility GA, Gibberellins synthesis, CCC inhibitors, ABA (Abscisic acid), FW-450 (Mendok), Dalapon, TIBA (Triiodobenzoic acid), NAA, Ethephon, Dalapon and -chloropropionate Vegetable Applied Remark(s) Reference, chemicals Cole GA Reported Van Der Meer vegetables promising for and Van Dam utilization 1979 58
  59. 59. • It must be highly male or female selective• It should be easily applied and economical in use• The time of its application should be as flexible as possible• It must not be mutagenic• It must not be carried over in F1 seeds• It must consistently produce complete (95% or more) male sterility• It must cause a minimum, preferably zero, reduction in seed set• It should not be hazardous to the environment 59
  60. 60. • EGMS is more popularly termed as “Two line Hybrid Breeding” as against “Three Line Hybrid Breeding” in case of CMS system• Determination of critical environment for sterility and fertility expression (Usually temperature or photoperiod) Vegetable Mutant Temperature Reference Cabbage TGMS, PGMS <100C Rundfeldt 1961Brussels sprouts TGMS <100C Nieuwhof 1968 Broccoli TGMS, PGMS 100-110C Rick 1948; Sawhney 1983 60
  61. 61.  Expression of male sterility trait is associated with Morphological changes Histological changes Cytological changes Biochemical changes and Molecular changes 61
  62. 62. • Variable (complete absence of male reproductive organs)• Male sterile flowers are commonly smaller in size in comparison to the fertile• The size of stamens is generally reduced 62
  63. 63.  Role of tapetum• Premature breakdown of tapetum• Abnormal development of tapetum in male sterile plant first report by (Monosmith 1926) Role of callase• Early or delayed callase activities have been found to be associated with male sterility Role of esterase• Decreased activity of esterase in male sterile plants has been observed in tomato (Bhadula and Sawhney 1987) & in radish (Zhou and Zhang 1994) Role of PGR’s• Reduced level of cytokinins and increased level of abscisic acid associated with GMS and CMS plants 63 Mehdi and Anwar 2009
  64. 64.  In both GMS and CMS systems, male sterility is the consequence of breakdown of tightly regulated pollen development and fertilization processes at any of the pre- or post-meiotic stages i.e. During the formation of tetrad During the release of the tetrad At the vacuolated microspore stage or at pollen dehiscence stage 64 Wu and Yang 2008
  65. 65.  Amino acids• Reduced the level of proline, leucine, isoleucine, phenylalanine and valine• Increased the level of asparagine, glycine, arginine, aspartic acids Soluble proteins• Male sterile anthers contain lower protein content and fewer polypeptide bands Enzymes• Mistiming of callase activity• Decreased the activity of esterase and amylases 65
  66. 66.  CMS resulted from an inability of the mitochondria to meet the energy demands of male gametogenesis Male organs shown lower levels of ATP production and high levels of alternative oxidase activity Mitochondria play a central role in premature programmed cell death (PCD) Pelletier and Budar 200766
  67. 67.  Lack or steady supply of seed Lack of extension work Exorbitant cost of hybrid seed Poor marketing facilities of vegetables Improper quality attributes 67
  68. 68.  Hybrid technology offers tremendous potential for the much needed second Green Revolution Area expansion not possible thus need is to increase production/unit areas Cost effective hybrid seed production (SI, MS etc) 68
  69. 69. To fulfill the demand of vegetables in our country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 69
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