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  1. MALE-STERILE Taryono Faculty of Agriculture Gadjah Mada University

  2. 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

  3. Plant that do not produce viable, functional pollen grains • An inability to produce or to release functional pollen as a result of failure of formation or development of functional stamens, microspores or gametes Male-sterile Three types of sterility: 1. “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) 2. “Structural or staminal male sterility” in which male flowers or stamen are malformed and non functional or completely absent 3. “Functional male sterility” in which perfectly good and viable pollen is trapped in indehiscent anther and thus prevented from functioning

  4. Type of Male-sterile • Based on its inheritance or origin • Cytoplasmic male sterility (CMS) = sterile cytoplasm (S) • Male steril comes about as a result of the combined action of nuclear genes and genic or structural changes in the cytoplasmic organellar genome • maternally inherited • Nuclear male sterility (NMS) = Genic, genetic, mendelian Male sterility is governed solely by one or more nuclear genes Nuclear inherited • Non genetic, chemically induced male sterility Application of specific chemical (gametocides or chemical hybridizing agents)

  5. Flower phenotypes in carrot a) Normal (N-cytoplasm, restored CMS plants) b) Brown anther CMS (Sa) c) Petaloid CMS (Sp)

  6. Stamen (anther and filament) and pollen grains are affected • It is divided into: 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, 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 Cytoplasmic male-sterile

  7. The nuclear genetic control of CMS is predominantly governed by one or more recessive genes, but can be also dominant genes as well as polygenes • The different mtDNA restriction endonuclease digestion patterns are reflections of aberrant intra- or inter molecular DNA recombination events in the mitochondrial genome which have either modified existing genes or related new genes some of which are more or less related to the male sterile phenotypes Cytoplasmic male-sterile • Some drawback: • 1. insufficient or unstable male sterile • 2. Difficulties in restoration system • 3. Difficulties with seed production • 4. Undesirable pleitropic effect

  8. Origins: 1. Intergeneric crosses 2. Interspecific crosses 3. Intraspecific crosses 4. Mutagens (EMS, EtBr) 5. antibiotic (streptomycin and Mitomycin) 6. Spontaneus Cytoplasmic male-sterile CMS Characterization • It has been traditionally characterized by the restore genes required to overcome the CMS and to provide male sterile progeny in the male sterile system • CMS restoration is by nuclear genes, frequently dominant in action, in many cases, few in number • The CMS restore genes temporarily suppress the expression of the CMS permitting normal or near-normal pollen production

  9. CMS mechanism of action • Abnormal behavior of the tapetum in the anther • Genetic determinant of CMS reside in mitochondria • Nuclear gene control the expression of CMS CMS Limitation • Pleiotropic negative effect of the CMS on agronomic quality performance of plants in the CMS cytoplasm • Enhanced disease susceptibility • Complex and environmentally unstable maintenance of male sterility and/or male fertility restoration • Inability to produce commercial quantities of hybrid seed economically because of poor floral characteristic of cross pollination

  10. It provides a possible mechanism of pollination control in plants to permit the easy production of commercial quantities of hybrid seeds • It consists of a male sterile line (the A-line), an isogenic maintainer line (The B line), and if necessary also restore line (the R-line) • A lines are developed by back-crossing selected B-lines to a CMS A-line for 4 – 6 times to generate a new A-line • B and R-lines are developed by similar back cross procedures using a CMS R-line as female in the original cross and a new line as the recurrent parent in 4 – 6 backcrosses CMS Utilization

  11. Fertility restoration in maize

  12. x N2 N1 N1 N1 C1 C2 C1 C1 C1 Simple hybrid with cms and restoration CMS line (A-line) CMS, rfrf Maintainer line (B-line) N, rfrf x Large amounts of CMS line Male line (C-line) N and RfRf Fertile F1 hybrid CMS, Rfrf

  13. Breeding hybrid carrots

  14. Selfing the last backcross generation two successive times and selection of pure breeding male fertility restore line is required to complete the development of the new R-lines developed in the CMS • Current commercial hybrid seed production relies entirely on the block method (alternating strips of female and male genotypes CMS Utilization

  15. Nuclear male sterility • Originated through spontaneous mutation or mutation by ionizing radiation and chemical mutagens such as ethyl methane sulphonate (EMS) and ethyl imine (EI) or by genetic engineering, protoplast fusion, T-DNA transposon tagging and affecting the synthesis of flavonoids • can probably be found in all diploid species • Usually controlled by mutations in genes in the single recessive genes affect stamen and pollen development, but it can be regulated also by dominant genes

  16. Morphology • Variable (complete absence of male reproductive organs to the formation of normal stamen with viable pollen that fail to dehisce) • It is not distinguishable from parent fertile plants with the exception of flower structure • Male sterile flowers are commonly smaller in size in comparison to the fertile • The size of stamens is generally reduced

  17. Determining factor • Temperature Changing the optimal temperature can induce sterility • Photoperiod It has a strong influence (Photoperiod sensitive) Changing the growth habit can stimulate the sterility

  18. Cytological Changes • Breakdown in microsporogenesis can occur at a number of pre-or postmeiotic stages • The abnormalities can involve aberration during the process of meiosis, in the formation of tetrads, during the release of tetrad (the dissolution of callose), at the vacuolate microspore stage or at mature or near-mature pollen stage

  19. Biochemical Changes • Male sterility has been shown to be accompanied by qualitative and quantitative changes in amino acids, protein, and enzymes in developing anther • Amino acids The level of proline, leucine, isoleucine, phenylalanine and valine is reduced, but asparagine, glycine, arginine, aspartic acids is increased • Soluble proteins Male sterile anthers contain lower protein content and fewer polypeptide bands Some polypeptides synthesized in normal stamens were absent in mutant stamens

  20. Biochemical Changes • Enzymes Callase is required for the breakdown of callose that surrounds PMCs and the tetrad. Mistiming of callase activity results in premature or delayed release of meiocytes and microspore Esterases have also been related to pollen development. The activity of esterase is decreased The activity of amylases is decreased and it corresponds with high starch content and reduced levels of soluble sugars Accumulation of adenine due to the decrease of adenine phosphoribosyltransferase (APRT) activity may be toxic to the development of microspores

  21. Hormones and male Sterility • Plant growth substances play an important role in stamen and pollen development. Aberrant stamen and pollen development is known to be accompanied by changes in endogenous PGS • GMS line was related to a change in the concentration of gibberellins (rice), IAA (Mercurialis annua), ABA (soybean), and cytokinin (Mercurialis annua) • Male serility is associated with changes in not one PGS but several PGS

  22. Use of genic male sterility in hybrid programs • Male sterile plants of monoecious or hermaprodite crops are potentially useful in hybrid program because they eliminate the labor intensive process of flower emasculation

  23. Constraint of the use of genic male sterility • The maintenance of the male sterile line. Normally, a GMS line (A-line) is maintained by backcrossing with the heterozygote B-lines (Maintainer lines), but the progeny produced are 50% fertile and 50% male sterile • Solution: • Identify marker genes that are closely linked to ms genes and affect some vegetative characters • Use of environmental and chemical methods that can lead to production of 100% male-sterile seed

  24. CHEMICAL INDUCED MALE-STERILE Taryono Faculty of Agriculture Gadjah Mada University

  25. Biochemical means of producing male sterile plants • Feminizing hormones • Inhibitors of anther or pollen development a. acting on sporophytic tissue b. acting on gametophytic tissue (gametocides) • Inhibitors of pollen fertility

  26. Chemical hybridizing agent (CHA) • Could be used in the large scale commercial production of hybrid seed • Are applied to plant only at certain critical stage of male gametophyte development • Their action could result from a range of mechanism: • Inherently selective action as male gametocides or inhibitors of anther development • Selective transport of generally toxic or growth-inhibitory substances to the anthers during these periods • Metabolic detoxification of generally toxic or growth-inhibitory substances after they have suppressed male fertility

  27. The logic of chemical hybridization • High degree of efficacy and developmental selectivity • Persistence during the development of flower or spikes • Low cost • Acceptable levels of toxicity to people and the environment • Low general phytotoxicity • Agronomic performance of hybrid seed produced is not inferior to equivalent crosses produced by genetic methods

  28. CHAs and pollen development • Chemical inhibitors of pollen development are not familiar topic to the majority of academic scientists. The most likely explanation for the unfamiliarity is that these substances have been identified and developed almost entirely within the industry • Pollen comes into being through a sequential and determinate program within the central cavity or locule of anther. These programmes are biochemically controlled and may be affected by one or more chemical agents • There are at least 4 classes of chemical agents: a. Plant growth regulators and substances that disrupt floral development b. Metabolic inhibitors c. inhibitors of microspore development d. inhibitors of pollen fertility These categories have considerable conceptual overlap and do not address the molecular action of the chemical male sterilants

  29. Plant growth regulators and substances that disrupt floral development • Plant hormones/hormones antagonists a. auxins and auxin antagonists (NAA, IBA, 2,4-D, TIBA, MH) b. Gibberellins and antagonist (GA3, GA4+7, CCC: 2-chloroethyl-trimethyl ammonium chloride) c. Abscisic acid • Other substances a. LY195259 b. TD1123

  30. Auxins and antagonists • It may differently affect some far-reaching process, such as blockade of nutrient transport to the development anthers • Male sterility induced was expressed in several ways in situ pollen germination, in situ exudation of pollen cytoplasm, modification of certain stamens into staminodes Tapetum fails to enlarge (MH and IBA) or tapetal cells enlarges atypically and was persistent

  31. Gibberellins and antagonists • GA affects on sexual determination and floral development • The response varies by species • GA interferes with the development of male floral organs or promotes feminization • Gibberellin-synthesis inhibitors (CCC) at certain concentration, selectively inhibits the development of stamen or otherwise suppresses pollen development . These effects are not sufficiently selective

  32. Abscisic acid • ABA caused effect on developing floral buds similar to CCC • ABA caused male sterility if applied to plant just prior to or during meiosis of pollen mother cells (wheat). ABA may cause male sterility through more than one mechanism

  33. LY195259 • It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-pyrazole-4-carboxylic-acid • It is an effective chemical hybridizing agent • It is applied when the flower was quite short with high application rates, whereas lower dosages resulted in progressively reduced inhibition • Sterility at lower dosages was associated with smaller, abnormally twisted and intensively pigmented locules • The hybrid seed appeared normal, and no other phytotoxic effects were visually evident from rates • Uptake from soil was particularly effective

  34. TD1123 • It is potassium 3,4-dichloro-5-isothiocarboxylate • When applied underdeveloped anthers, they will fail to dehisce • A variety of morphological effects were observed at higher treatment levels

  35. Metabolic Inhibitors • There are halogenated aliphatic acids (alpha, beta-dichloroisobutyrate and 2,2-dichloropropionate salts) and arsenicals (methanearsonate salts) • They affect mitochondrial protein by reducing the efficiency of normal metabolic processes

  36. Inhibitors of microspore development • Copper chelators Copper deficiency causes the irregular or absent of pollen development Copper deficiency exerts the effects by inhibiting copper-requiring oxidases that function in auxin metabolism • Ethylene It is a natural regulator of the development and maturation of several floral organs. Filament and corolla growth (unfolding and senescence) are inhibited by ethelene production • Fenridazon It is 1-(-4chlorophenyl-1,4-dihydro-6-methyl-4-oxopyridazine-3-carboxylic-acid. The treated microspores had wavy surfaces and progress to plasmolysis and abortion with the onset of the microspore vacuolation stage Pollen wall was 80% thinner in treated plants

  37. Inhibitors of microspore development • Phenylcinnoline carboxylates (SC-1058, SC-1271 and SC-2053) All capable of producing complete male sterility with minimal phytotoxicity and loss of seed yield when applied just prior to meiosis They cause a general retardation of anther development Pollen development was generally arrested in the late prevacuolate or early vacuolate microspore stage The microspore often becomes wavy or wrinkled and the cytoplasm degenerates and the cells become collapsed. SC-1058: 1-(4’-trifluoromethylphenyl)-4-oxo-5-fluorocinnoline-3-carboxylic acid SC-1271: 1-(4’-chlorophenyl)-4-oxo-5-propoxycinnoline-3-carboxylic acid SC-2053: 1-(4’-chlorophenyl)-4-oxo-5(methoxyethoxy) cinnoline-3-carboxylic acid

  38. Inhibitors of microspore development • Genesis ® (MON 21200) It provides good CHA activity over a very diverse range of genotypes, geographic regions and growing condition Seed production has provided a high and reliable level of outcrossing Hybrids produced with the aid of genesis are equivalent to conventional hybrids based on CMS technology

  39. Inhibitors of pollen fertility • Azetidine-3-carboxylate (A3C, CHA™) It effectively induces male sterility in small grains, particularly wheat The major effect of mature pollen is a structural alteration of cell wall precursor vesicles Only 10% of the pollen grains showed normal pollen tube growth in the first hour after pollination and none penetrated the secondary stigmatic branch

  40. MALE-STERILITY THROUGH RECOMBINANT DNA TECHNOLOGY Taryono Faculty of Agriculture Gadjah Mada University

  41. 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 • Used antisense or cosuppression of endogenous gene that are essential for pollen formation or function • Reproducing a specific phenotype-premature callose wall dissolution around the microsporogenous cells • Reproducing mitocondrial dysfunction, a general phenotype observed in many CMS

  42. 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 • Served to protect the bacterium from its own RNAse activity by forming a diffusion-dependent, extreemely one to one complex which is devoid of residual RNase activity 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

  43. 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)

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

  45. How to induce sterility? How to restore fertility? How to propagate male-sterile plants? Induced GMS System X Sterlie (Ss, rfrf) Fertile (ss, RfRf) (50%) F1 (Ss, Rfrf) fertile (50%) F1 (ss, Rfrf) fertile Sterile (Ss, rfrf) X Fertile (ss, rfrf) (50%) Sterile (Ss, rfrf) (50%) Fertile (Ss, rfrf)

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

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

  48. Selection by Herbicide Application X A (SH/-) 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/-)

  49. Inducible Sterility • Male sterility is induced only when inducible chemical is applied. NH4+ accumulation in tapetal cell Male sterility Glutamate Glutamine N-acetyl- L-phosphinothricin (non-toxic) Glufosinate (toxic) N-acetyl-L-ornithine deacetylase (coded by argE) • 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.

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