Transgenic male sterility

Transgenic male sterility usefulness in hybrid seed production

1. welcome

2. What is Male Sterility? Definition : Inability of flowering plants to produce functional pollen. Male sterility is A gronomically important for the hybrid seed production. Flower of male-fertile pepper Flower of male-sterile pepper

3. TRANSGENIC MALE-STERILITY

4. TRANSGENIC MALE-STERILITY Transgenes may be used to produce GMS which is dominant to fertility. In these cases it is essential to develop effective fertility restoration systems for hybrid seed production. An effective restoration system is available in at least one case, Barnase / Barstar system

5. Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues supporting the microspores

6. I. Dominant Male-Sterility Genes Targetting the expression of a gene encoding a cytotoxin by placing it under the control of an ather specific promoter (Promoter of TA29 gene) Expression of gene encoding ribonuclease (chemical synthesized RNAse-T1 from Aspergillus oryzae and natural gene barnase from Bacillus amyloliquefaciens ) RNAse production leads to precocious degeneration of tapetum cells, the arrest of microspore development and male sterility. It is a dominant nuclear encoded or genetic male sterile (GMS), although the majority of endogenous GMS is recessive Success in oilseed rape, maize and several vegetative species

7. Used antisense or co-suppression of endogenous gene that are essential for pollen formation or function Reproducing a specific phenotype-premature callose wall dissolution around the microsporogenous cells Reproducing mitochondrial disfunction , a general phenotype observed in many CMS

8. Barstar Barnase system Barnase (110 amino acids) is a secreted ribonuclease from Bacillu s amyloliquefaciens . Barstar (89 amino acids) is a cytoplasmic barnase inhibitor with which the host protects itself. RNase is linked with bar gene ( glufosinate tolerant), so glufosinate tolerant plant will be male sterile. GM canola containing barstar / barnase system composes about 10% of commercially cultivated crops in Canada and is one of the few GMO cleared for agricultural use in Europe.

9. Dominant nuclear male sterility ( Barnase / Barstar system (Goldberg et al.1993) ) Barnase is extracellular Rnase Barstar is inhibitor of 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 withTA29-Barstar are not affected by the transgene barnase . Barstar is dominant over the Barnase

10. Induced GMS Promoter which induces transcription in male reproductive specifically Gene which disrupts normal function of cell Agrobacterium-mediated transformation regeneration male-sterile plant

11. Cell Ablation Strategy Using Chimeric Barnase and Barstar Genes with Overlapping Cell Specificities. Blocks represent cross-sections through a hypothetical organ system that has four different cell types. The circular cells in the lower right quadrants are the targets of the ablation experiment. (A) Blue represents transcriptional activity of the promoter (lectin gene) fused with the anti-cytotoxic barstar gene. (B) Red represents transcriptional activity of the promoter (TA56 gene) fused with the cytotoxic barnase gene. (C) Combined transcriptional activities of the chimeric barnase and barstar genes. Both chimeric genes are active within the dark gray cells in the upper right quadrant. Only the chimeric barnase gene is active in the target cells present in the lower right quadrant. (D) Selective ablation of the target cells. Barnase/barstar complexes are formed within the dark gray cells in the upper right quadrant protecting them from the cytotoxic effects of barnase. The target cells in the lower right quadrant have been ablated selectively due to the cell-specific activity A Nove1 Cell Ablation Strategy Blocks Tobacco Anther Dehiscence

12. Sterile and fertile anthers

13. Strategies to Propagate Male-Sterile Plant Selection by herbicide application Inducible sterility Inducible fertility Two-component system

14. Selection by Herbicide Application TA29 Barnase NOS-T TA29 Barstar NOS-T Gene for a RNase from B. amyloliqefaciens Tapetum-specitic promoter 35S PAT NOS-T Gene for glufosinate resistance from S. hygroscopicus Gene for inhibitor of barnase from B. amyloliqefaciens fertile

15. Selection by Herbicide Application pTA29-barnase : S (sterility) p35S-PAT : H (herbicide resistance) pTA29-barstar : R (restorer) SH/- SH/- -/- SH/- SH/- -/- SH/- -/- SH/- -/- -/- SH/- -/- SH/- SH/- -/- -/- -/- SH/- SH/- -/- -/- -/- -/- -/- -/- -/- -/- -/- A (SH/-) X B (-/-) glufosinate X C (R/R) Fertile F1 (SH/-, R/-) Fertile F1 (-/-, R/-)

16. Fertility restoration Restorer gene (RF) must be devised that can suppress the action of the male sterility gene ( Barstar ) a specific inhibitor of barnase Also derived from B . amyloliquefaciens The use of similar promoter to ensure that it would be activated in Tapetal cells at the same time and to maximize the chance that Barstar molecule would accumulate in amounts at least equal to Barnase Inhibiting the male sterility gene by antisense. But in the cases where the male sterility gene is itself antisense, designing a restorer counterpart is more problematic

17. Production of 100% male sterile population When using a dominant GMS gene, a means to produce 100% male sterile population is required in order to produce a practical pollination control system Linkage to a selectable marker Use of a dominant selectable marker gene (bar) that confers tolerance to glufosinate herbicide Treatment at an early stage with glufosinate during female parent increase and hybrid seed production phases eliminates 50% sensitive plants Pollen lethality add a second locus to female parent lines consisting of an RF gene linked to a pollen lethality gene (expressing with a pollen specific promoter)

18. Features of commercial value dominant genetic male sterility system Efficient fertility restorer system Easy maintenance of male sterile lines Easy elimination of a male fertile plants from male sterile lines Lack of adverse affects on other traits Stable male sterile phenotype over different environments Satisfactory performance of f1 hybrids

19. Inducible Sterility Male sterility is induced only when inducible chemical is applied. Glutamate Glutamine NH 4 + N-acetyl- L-phosphinothricin (non-toxic) Glufosinate (toxic) N-acetyl-L-ornithine deacetylase (coded by argE ) Male sterility accumulation in tapetal cell Plants of male sterile line were transformed by a gene, argE , which codes for N-acetyl-L-ornithine deacetylase, fused to TA29 promoter. Induction of male sterility can occur only when non-toxic compound N-acetyl-L-phosphinothricin is applied.

20. Inducible Sterility Sterile parent X Fertile parent fertile selfing Plants transformed by TA29- argE fertile Fertile F1 plant N-acetyl-L- phosphinothricin Plants transformed by TA29- argE

21. Inducible Fertility Sterile parent X Restorer selfing If sterility was induced by inhibition of metabolite (amino acids, biotin, flavonols , jasmonic acid) supply, fertility can be restored by application of restricted metabolite and male sterile plant can be propagate by selfing . addition of restricted metabolite Fertile parent Sterile parent Fertile parent Fertile F1 plant

22. Two-Component System Male sterility is generated by the combined action of two genes brought together into the same plant by crossing two different grandparental lines each expressing one of the genes. Each grandparent has each part of barnase. Two proteins which are parts of barnase Two proteins can form stable barnase

23. Two-Component System X F1 (Bn3/-) A (Bn5/Bn3) A2 (Bn3/Bn3) fertile A1 (B5/B5) fertile fertile fertile sterile X A2 (Bn3/Bn3) fertile A1 (B5/B5) fertile B (- -) A1 (Bn5/Bn5) A1 (Bn5/Bn5) X F1 (Bn5/-) fertile A (Bn5/Bn3) sterile selfing selfing Bn3 : 3’ portion of barnase gene Bn5 : 5’ portion of barnase gene

24. Harmone induciblemale sterility based on BCP1 Most attractive system of transgenic male sterility based on antisense construct of B. campestris gene BCP1 drawn by BCP1 promoter linked with a harmone inducible enhances sequence. This system is ready for use in hybrid seed production in Brassica oleracea

25. Sporophytic genes controlling male sterility. Such genes cause male sterile expression by disrupting functioning in Sporophytic tissues. Expected site of transcription of Gametophytic genes is in haploid microspores following meiotic division. Ex: pollen specific gene LAT52 in tomato. Anther specific gene Bcp1 shows different pattern of expression in both diploid Tapetum and haploid microspores. The Gametophytic and Sporophytic activity of this gene was observed. Bcp1 m RNA in both diploid Tapetum and haploid microspores. Bcp1 expression in Tapetal cells initiated shortly after microspore formation and continued until Tapetum degeneration. In unicellular microspores the activity was first detected in the late Vacuolate stage.

26. To confirm the role of Bcp1 in fertility control, LAT52 promoter from pollen specific tomato gene was fused to 0.5 kb of a Bcp1-c DNA insert in a reverse orientation and cloned into p B 1101. The second antisense construct was devoloped by fusion of 0.77 kb of a Bcp1 gene regulatory region with 0.5 kb of an antisense Bcp1 construct. Resulting antisense constructs was subsequently introduced into A. thaliana via 2 separate A . tumefaciens transformation events. The anthers of transformants carrying LAT52 (pollen specific) promoter fused to a reverse Bcp1 c DNA construct had 1: 1 segregation of viable or aborted pollen, indicating Gametophytic male fertility control.

27. Complete male sterility and the absence of typical 1: 1 segregation of fertile and sterile pollens in the anthers of transgenic plants carrying Bcp1 promoter suggested that sterility was the result of down regulating Bcp1 gene expression in the diploid tapetum . Co segregation of male sterile phenotype and presence of antisense insert. In Bcp1 promoter antisense plants low level of male fertility reduction appeared to correlate with dosage levels of antisense gene. 2 copies of antisense gene were needed to ensure complete male sterility expression, while presence of a single copy resulted in leaky male sterile phenotype.

28. Male sterility system based on Bcp1 promoter antisense technology and linked to a harmone inducible promoter is now ready for use in hybrid seed production in B. oleraceae . This trasgenic male sterile line is self-maintainable and remains male fertile so long as it is not sprayed with the concerned harmone . (after this spray, it shows complete male sterility). Attractive alternative to 3- line CMS or barnase - barstar system , as male sterile lines are self maintainable and any line would act as fertility restorer.

29. Pollen Self-Destructive Engineered Male Sterility Theoretically, it is feasible to genetically engineer plants having altered endogenous auxins indole acetic acid (IAA) levels with pollen exhibiting self-destructive mechanisms. To achieve this, Mc Cormick et al. (1989) and Wood (1990) transformed plants with a chimeric gene consisting of pollen-specific promoter (LAT59) and a gene(fins2) that converts indole acetamide (IAM) into IAA. Although the detailed characterization of such a transgenic was not reported, it was argued that, if this system works, plants carrying the LAT59-fms2 gene when sprayed with IAM will selectively convert IAM into IAA at very high concentrations to kill the pollen and render the plants male sterile.

30. Another possibility of inducing such male sterility is the transformation of plants with chimeric gene involving TA-29 promoter and coding region of β- glucuronidase (GUS). Such transformants , when sprayed with protoxins like sulfonyl urea or maleic hydrazide , cause malesterility through their breakdown in the tapetum by the β- glucuronidase enzyme.

31. The transgenic plants not sprayed with protoxins remain fertile, so a fertility restoration system like TA29-barstar is not required. Shihshieh et al (2003) used tissue-specific promoters expressing CKX1 and gai  , genes involved in oxidative cytokinin degradation and gibberellins (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development.

32. Male Sterility through Hormone Engineering . Drastic changes in endogenous levels of auxins have been demonstrated to cause male sterility in tomato ( Sawhney 1997) and several other crops. Induction of male sterility by manipulating endogenous hormone levels was reported in transformed tobacco plants having the “ rol C” gene of Agrobacterium rhizogenes under the control of 35 S CaMV promoter and flanked with a marker gene ( Spena et al. 1987).

33. Such male sterile can be maintained by linking the gene for herbicide resistance to the male sterilizing “ rol c”. Male fertile segregants can be chemically rouged out by selective elimination in maintainer population.

34. In another experiment, Spena et al. (1992) used the tissue specific promoter “tap 1” and found that the inserted gene “ rol b” from A. rhizogenes greatly impaired the flower development of transgenic tobacco, due to an increase in the levels of indole acetic acid and decreased levels of gibberillin in anthers.

35. Male Sterility Using Pathogenesis-Related Protein Genes Pathogenesis-related (PR) protein β-1,3 glucanase ( callase ) is known to dissolve specific cell wall made of callase , a β-1,3-linked glucan between cellulose cell wall and plasma membrane and tetrads synthesized by microsporocyte. The β-1, 3 glucanase ( callase )secreted by the tapetum helps to release free microspores into locular space by breaking down the callase wall. The genetic alteration of this mechanism in plants caused male sterility.

36. Callase appearance and distribution was normal in male sterile transgenic plants up to prophasI , whereupon callase was prematurely degraded. Electron microscopy revealed a thick callase wall surrounding each microspore of the tetrad in fertile anthers, whereas it was clearly absent in sterile microspores.

37. The premature dissolution of callase indicated that the modified glucanase is secreted from the tapetum and is active within the anther locule . Transgenic tobacco plants expressing the β-1, 3 glucanase under the control of CaMV35S promoter showed normal fertility despite the expression of modified glucanase . This was attributed to either lower expression of the modified gene in the tapetum or inappropriate timing of the modified callase synthesis during the process of microsporogenesis , stronger (Rf-WA-1) being located on chromosome 10.

38. It has also been possible to identify 6 RAPD markers linked to another gene for fertility restoration (Rf-WA-3), three of these mapped on chromosome 1. These marker-aided selections are for fertility-restoring ability.

39. Male Sterility through Modification of Biochemical Pathways 1 . Flavonoids Among the three major flower pigments, ( flavonoids , carotenoids , and betalains ), the flavonoids are the most common and most important. Apart from their role in color development, flavonoids are important in plant reproduction and defence -related mechanisms. Vander Meer et al . (1992) induced male sterility by antisense inhibition of flavonoid biosynthesis.

40. A large number of genes involved in the biochemical pathway of flavonoids synthesis have been cloned and characterized ( Forkmann 1991). However, it was not concluded whether the anther box on its own directs the expression in the tapetum cells. It was argued that modules that confer organ specificity to the CaMV35S promoter may act in concern with anther box.

41. About 14% of transgenic plants showed a clear reduction of anther and pollen pigmentation, which confirmed the involvement of anther box in tapetum -specific expression. In Maize and Petunia the pollen lacking flavonoids are unable to produce functional pollen tubes

42. Flavonoids are essential for normal pollen development and function and flavonoids deficiency prevents pollen maturation Chalcone synthase is a key enzyme of Flavonoid biosynthesis In petunia, antisense construct of the gene encoding CHS has been transferred and transgenic plants carrying this construct have been regenerated These plants shows negligible chalcone synthase activity white flowers and non-functional pollen

43. However this pollengrains become functional when certain flavonoids are either applied to the stigmas or mixed with the pollen Antisense approach has also been used to restore fertility in the case of MS induced by the role of C gene of A. rhizogenes

44. 2. Jasmonic acid Jasmonic acid (JA), synthesized from linolenic acid (LA) through octadecanoid pathway plays an important role in anther dehiscence and pollen maturation . The triple fad (fad3,fad7,fad8) mutants lacking LA had indehiscent anthers and hence showed functional male sterility. Ishiguru et al. (2001) linked impaired dehiscence in Defective Anther Dehiscence I (DAD I) mutant (Sanders et al. 2000;Stintzi and Browse 1996) to mutation in DAD I gene that encoded a chloroplastic phospholipase AI to supply free LA at the initial step in JA biosynthesis, they further suggested that JA also regulated flower opening, anther dehiscence as well as pollen maturation. Exogenous application of JA reversed the impaired dehiscence

45. The male sterile phenotype could be reversed by application of JA (0.5 μm ) as well as LA. Genetic male sterility based on DAD I gene has many advantages; most important being the ability to induce normal pollen production and dehiscence. This is critical for developing lines homozygous for the male sterilizing allele. Moreover, this system involves suppression of a single gene and floral morphology is not adversely affected. The stability of expression over a range of environmental conditions, however, remains to be investigated.

46. 3. Carbohydrates Carbohydrates play a critical role in the anther and pollen development by sustaining growth as well as signal pathways. Their transportation from photo synthetically active source tissues to developing sinks is regulated by extra cellular invertase . This class of  invertase is encoded by small gene familiar with differential regulation and expression patterns. The extra cellular invertase Nin 88 of tobacco shows specific temporal and spatial expression patterns in developing anthers.

47. At early stages of flower development, the invertase is present exclusively in the tapetum cell layers of the anthers followed by a distinct expression pattern during pollen development. The tissue specific antisense repression of Nin endothecium , and vacuolation decreased the inner space of the locule , affected pollen grain, and resulted in the irregular shape or collapsed phenotype.

48. Reversibility of the male sterile phenotype was observed under continuous illumination, resulting in viable Pollen and a copious amount of seeds. This study offers a new tool for transgene containment for both nuclear and organelle genomes and provides an expedient mechanism for F1 hybrid seed production.

49. Why would mitochondrial dis -unction specifically affect pollen development Mitochondrial gene functions are essential to all cells-electron transport, ATP formation, and translocation of mitochondrial Mzna Interuption of any of these functions would be expected to be lethal Confers both male sterility and disease resistance

50. Christine D. Chase plant mitochondrial genomes are not currently amenable to genetic transformation, but genetic manipulation of the plastid genome allows engineering of maternally inherited traits in some species. A recent study has shown that the  Acinetobacter  β- ketothiolase gene, expressed in the   Nicotiana tabacum  plastid, conditions maternally inherited male sterility, laying the groundwork for new approaches to control pollen fertility in crop plants.

51. We constructed two chimaeric ribonuclease genes that are specifically expressed in anthers of tobacco and rapeseed plants. The expression of these genes affects the production of functional and viable pollen yielding plants that are male sterile. This dominant gene for nuclear male sterility should facilitate the production of hybrid seed in various crops.

52. Induction of male sterility in plants by a chimaeric ribonuclease gene Nature 347 , 737-741 (25 October 1990) Chimaeric ribonuclease genes that are expressed in the anthers of transformed tobacco and oilseed rape plants were constructed. Chimaeric ribonuclease gene expression within the anther selectively destroys the tapetal cell layer that surrounds the pollen sac, prevents pollen formation, and leads to male sterility. These nuclear male sterility genes should facilitate the production of hybrid seed in various crop plants. Isoltaion of a tapetum-specific gene, TA29, facilitated tapetum specific expression of RNase gene Tetrad stage: maturation of microspores depends on tapetal cells SMC= spore mother cells V= vascular bundle Fi= filament w= anther inner wall t= tapetum

53. Mechanism for CMS Pollens of untransformed plant Pollens of transgenic plant Microspores and surrounding tapetal cells are particularly active in lipid metabolism which is especially needed for the formation of the exine pollen wall from sporopollenin . High demand for fatty acid in tapetal cells cannot be satisfied because of the depletion of acetyl-coA.

54. Reversibility of Male Fertility Acetoacetyl-CoA Acetyl- CoA Malonyl-CoA Fatty acid Acetyl-CoA carboxylase Illumination for 8 ~ 10 days Male fertility b -ketothiolase

55. Advantages of CMS Engineering Male sterile parent can be propagated without segregation. Transgene is contained via maternal inheritance. Pleiotropic effects can be avoided due to subcellular compartmentalization of transgene products. Non-transgenic line can be used as maintainer.

56. Prospects for CMS Engineering In present, chloroplast transformation is not efficient for most of the crops except for tobacco. Although mitochondrial transformation has been reported for single-celled Chlamydomonas and yeast, there is no routine method to transform the higher-plant mitochondrial genome. If the routine methods to transform organellar DNA of crops are prepared, various systems for the CMS engineering may be attempted.

57. CASE STUDY

58. In case of RAPESEED

59. Characterization of transgenic male sterility in alfalfa Alfalfa plants obtained by genetic transformation with a construct containing the barnase gene under control of a tobacco anther tapetum specific promoter were studied. Vacuolization and degeneration of the tapetal cell cytoplasm at a premeiotic stage of development were observed in all five transfomed plants

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