Male Sterility

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Slide 1: 

AK Chhabra Male Sterility & its utilization in Plant Breeding DISCLAIMER: Copyright of some of the figures used from internet and different web sites is duly acknowledged. The copyright stands with its original developer. The information has been gathered here for educational purpose and not for any kind of commercial purpose.

Introduction : 

Introduction Reported by Koelreuter in 1763

Introduction : 

Introduction Production of non-functional pollen grains while normal female gamete function nature sporadically due to mutation & environmental factors a, In chloroplasts, acetyl-CoA, the substrate for the synthesis of fatty acids, is normally converted by acetyl-CoA carboxylase to yield malonyl-CoA. This pathway results in the correct development of anthers, pollen grains and seeds. b, In the transgenic chloroplasts, -ketothiolase out-competes acetyl-CoA carboxylase for acetyl-CoA, with acetoacetyl-CoA being produced instead. The upshot is distorted anthers and failure of pollen development. Fertility in plants grown from the resulting hybrid seeds is restored under continuous illumination, with reversion to the normal pathway. FIGURE 1. Engineering cytoplasmic male sterility with -ketothiolase1. From the following article: Plant biology:  Engineered male sterility Muhammad Sarwar Khan Nature 436, 783-785 (11 August 2005) doi: 10.1038/436783a

Slide 4: 

Male Sterile Male Fertile

Slide 5: 

Potential strategies for restricting gene flow.(A) By restricting flower opening and floral development (cleistogamy) in a GM crop, pollen dispersal and outcrossing could be prevented, thereby mitigating gene flow. (B) Gene flow through pollen could be restricted via chloroplast engineering, male-sterility approaches (including tapetum-specific excision of transgenes), and genome incompatibility. (C) Gene flow through seeds could be restricted by seed-sterility approaches (including seed-specific excision of transgenes) and apomixis.

Phenotypic expressions of MS : 

Phenotypic expressions of MS Absence , atrophy or malformation of androecium Lack of normal anther sac or anther tissues Inability of the pollen to mature or to be released from anther sac Inability to develop normal microspores or pollen PB 406A3 PB 406A3 NORMAL FERTILE

STERILE AND FERTILE POLLEN-INTACT ANTHERS : 

STERILE AND FERTILE POLLEN-INTACT ANTHERS

Slide 8: 

STERILE FERTILE

Slide 9: 

ANTHER LOBES-NORMAL AND ABNORMAL

Slide 10: 

ANTHER ABNORMAL TAPETUM

Slide 11: 

ANTHER LOBES-NORMAL AND ABNORMAL

Classification of male sterility : 

Classification of male sterility Genetic Male Sterility Temperature-sensitive Genetic Male Sterility Photoperiod-sensitive Genetic Male Sterility Transgenic Genetic Male Sterility Cytoplasmic Male Sterility Cytoplasmic-Genetic Male Sterility Chemically induced Male Sterility

Genetic Male Sterility : 

Genetic Male Sterility Ordinarily governed by single recessive gene but sometimes by dominant genes e.g. Safflower Alleles arise spontaneously by mutation or may be artificially induced by use of mutagens MS x MF = MF in F1 while a ratio of 3(MF):1(MS) in F2

Molecular mechanism of ms Action : 

Molecular mechanism of ms Action Many possible causes viz. Proline deficiency Delayed tapetum degeneration Disturbed balance of endogenous growth regulators e.g. gibberellins, cytokinins, auxins, ABA, ethylene etc. But no single reason clearly explains the molecular mechanism of ms Action, So ms genes are expected to act in more than one biochemical pathways.

Types of GMS : 

Types of GMS Environment insensitive GMS : ms Gene expression is much less affected by the environment Environment sensitive GMS : ms Gene expression occurs within a specified range of temperature and/or photoperiod regimes i.e. TGMS : sterility is at a particular temperature e.g. in Rice TGMS line Pei-Ai645, at 23.3*C PGMS : Sterility is obtained in long day conditions while in short day, normal fertile plant. Transgenic male sterility

Transgenic Genetic Male Sterility : 

Transgenic Genetic 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.

TRANSGENIC-GENETIC MALE STERILITY : 

TRANSGENIC-GENETIC MALE STERILITY

Slide 21: 

Barnase gene…produces RNAse Barnase gene…produces RNAse TA29 Barnase gene Tissue specific (tapetum layer) promoter Herbicide Resistance (Phosphinothricin) (Gene construct) Endothecium Middle layer Tapetum Bacillus amyloliquefaciens Tapetum degenerates PMCs/pollen become sterile BARNASE-BARSTAR SYSTEM TRANSGENIC GENETIC MALE STERILITY PRINCIPLE © A.K. Chhabra 2007 PMCs

Slide 22: 

Tapetum persistant PMCs/pollen are fertile Barnase gene…produces RNAse Barstar gene TA29 Barnase gene Tissue specific (tapetum layer) promoter Herbicide Resistance (Phosphinothricin) (Gene construct) Inhibits RNAse Endothecium Middle layer Tapetum Bacillus amyloliquefaciens BARNASE-BARSTAR SYSTEM TRANSGENIC GENETIC MALE STERILITY PRINCIPLE © A.K. Chhabra 2007 PMCs

Slide 23: 

Barnase gene…produces RNAse Barstar gene (fertility restorer) TA29 Barnase gene Tissue specific (tapetum layer) promoter Herbicide Resistance (Phosphinothricin) (Gene construct) Inhibits RNAse Endothecium Middle layer Tapetum Bacillus amyloliquefaciens Tapetum degenerates PMCs/pollen become sterile Tapetum persistant PMCs/pollen are fertile BARNASE-BARSTAR SYSTEM TRANSGENIC GENETIC MALE STERILITY PRINCIPLE Dominant male sterility Barstar-Barnase interaction (binding) © A.K. Chhabra 2007 PMCs

Utilization of GMS in Plant Breeding : 

Utilization of GMS in Plant Breeding Hybrid seed production Two isogenic lines ms ms (MS) & Ms ms (MF) are used, MF are to be identified ( Closely related genes having phenotypic expression) & removed before pollen shedding (Rouging). Generally, pollen dispersal is poor and Identification is tough

Cytoplasmic Male Sterility : 

Cytoplasmic Male Sterility Determined by Cytoplasm Result of mutation in mitochondrial genome (mtDNA) Includes ‘A’ (MS) and ‘B’ (Maintainer) line system CMS would be included in CGMS system when restorer genes are discovered.

Slide 26: 

CYTOPLASMIC MALE STERILITY Cytoplasmic sterile nuclear gene non restorer Cytoplasmic fertile nuclear gene non restorer x Male sterile Male fertile Male sterile

Slide 27: 

A system for the inducible inactivation of plant tissues based on a deacetylation of the non toxic phosphinothricin derivative N-acetyl-Phosphinothricin (N-ac-Pt) has been used to induce male sterility in transgenic plants. The induced cell death was achieved by the tapetum specific expression of the argE gene product (E.coli) which was identified to remove the acetyl group from the substrate resulting in the release of the herbicide phosphinothricin (glufosinate). Deacetylation was demonstrated in vivo by the incubation of transgenic plants with radioactively labeled N-ac-Pt prior to TLC of the crude protein extracts. Transgenic plants constitutively expressing the argE gene were constructed. No effect of the enzyme on plant growth and reproductivity could be traced. The tissue specific expression of the chimaeric gene leads to a destruction of the tapetum cells only in the presence of N-ac-Pt, the developing anthers are empty thus creating male sterile plants. The untreated plants are completely fertile. The fact, that the male sterility is inducible by the application of N-ac-Pt makes the presence of a second gene to restore fertility in the F1 generation superfluous. The system presented here is therefore easier to handle and less time consuming then those formerly described since only the female partner of the crossing has to be transgenic. It was primarily developed in Nicotiana tabacum but proved to be also functional in Brassica napus. Induced male sterility in transgenic rape seed Using the deacetylase system to induce male sterility in transgenic rape seed, the breeder is free to use any pollen donor for the crossings.

Utilization in Plant Breeding : 

Utilization in Plant Breeding Utilized in hybrid seed production in certain ornamental species, or in the species where a vegetative part is pf economic value Not of use where seed is value part as hybrid progeny would be sterile.

Cytoplasmic Genetic Male Sterility : 

Cytoplasmic Genetic Male Sterility Case of CMS where a nuclear gene for restoring fertility is known Restorer is dominant Also known as Nucleoplasmatic Male Sterility

Slide 32: 

CYTOPLASMIC - GENETIC MALE STERLITY Male fertile Male fertile Male fertile

Utilization in Plant Breeding : 

Utilization in Plant Breeding Hybrid seed production in maize, bajra, cotton, Rice, sunflower, jowar etc. ABR line system is being used for hybrid seed production

Hybrid seed production in canola : 

Hybrid seed production in canola AVOIDED

Slide 36: 

A-line showing no pollen production

Slide 37: 

B-line

Slide 38: 

Collection of pollen from B-line

Slide 39: 

Dusting B-pollen on A-line head Maintenance of A-line

Slide 40: 

Head of R-line showing abundance of pollen grains

Slide 41: 

Multi shoot Plant of Restorer line in Sunflower

Slide 42: 

Bagging (Selfing or sib mating) in Sunflower

Origin of male sterile cytoplasm : 

Origin of male sterile cytoplasm Spontaneous mutation Interspecific hybridization Mutation indution by ethidium bromide

Limitations of CGMS system : 

Limitations of CGMS system Undesirable effects of the cytoplasm Unsatisfactory fertility restoration Unsatisfactory pollination Spontaneous reversion Modifying genes Contribution of cytoplasm by sperm Environmental effects Non-availability of suitable restorer line

Chemically induced male sterility : 

Chemically induced male sterility Chemical Hybridizing Agents are commonly used Several CHA’s are being used viz. maleic hydrazide, NAA, IAA, FW450, Ethrel, RH531, MSMA, ZMA etc. CHAs

An Ideal CHA : 

An Ideal CHA Must be highly selective Should be easily applied & economical in use Time of application must be flexible Must not be mutagenic Must not be carried over in F1 seed Must constantly produce complete (95% or more) male sterility Should not be hazardous to the environment

Hybrid seed production using CHA’s : 

Hybrid seed production using CHA’s Proper environmental conditions Synchronization of flowering of male & female parents Effective chemical emasculation and cross pollination

Advantages of CHA’s : 

Advantages of CHA’s Any line can be used as female parent No need to produce cumbersome CMS or GMS lines Restorers are not required as any line can be used as male parent Hybrid seed production program is based on two lines only Maintenance is readily achieved by selfing of parents CHA based hybrids are fully fertile, so F2 of such hybrids showing high vigour can be used to reduce the cost of hybrid seed production

Limitation of CHA’s : 

Limitation of CHA’s Expression of MS is stage specific MS is vulnerable to prevailing environmental conditions Incomplete MS can lead to selfing on female parent Many CHAs are toxic to animals & plants Some CHAs e.g. Arsenicals can have carry-over effect for next generations Certain CHAs e.g. RH531 interfere with cell division CHAs are generally genotype, dose & stage specific

Slide 50: 

THANKS

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