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1. TRANSGENIC MALE-STERILITY:- ALTERANATIVE TOOL FOR HYBRID SEED PRODUCTION IN PLANTS Bharat taindujain 2013A58M 25/10/2014 http://biology.mcgill.ca/faculty/brown/male_sterility

2. Male sterility the failure of plants to produce functional anthers, pollen, or male gametes( Rick 1944) • 1763--Kölreuter observed anther abortion in some plant species and some inter-specific hybrids (Darwin, 1876) • the first male sterility system (CGMS) was developed in onion in 1943 (Jones, 1943) • The CMS in carrot by Welch and Grimball (1947) • Induced CMS in pearl millet by Ethidium bromide (Burton and hanna, 1976) • More prevalent than female sterility: • 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) Mariani et al., 1990

3. CAUSES OF MALE STERILITY • Absence or malformation of male organs (stamens) in bisexual plants or no male flowers in dioeciousplants • Failure to develop normal anther • Abnormal pollen maturation; inability to germinate on compatible stigma • Non dehiscent anthers but viable pollen • Barriers other than incompatibility preventing pollen from reaching ovule (Kaul ,1988)

4. 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 (Kaul , 1988)

5. Constraints of genetic and cytoplasmic male sterility • Absence of marker genes in GMS does not permit the sorting of male sterile orfertile plants in the progeny. • Undesirable effect of the cytoplasm • Unsatisfactory fertility restoration • CGMS only available to limited crop • Unsatisfactory pollination • Male sterility brake down due to environmental effect • No availability of suitable restorer line • (Bangaand Banga,1998)

6. Transgenic genetic male sterilty • Transgene – a gene introduced into genome of an organism by recombinant DNA or genetic engineering. • The organism that develop after transformation are known as transgenic • First transgenic plants (1983)– Tobacco line resistance to kanamycin • In 2013 , global area of GM crop 175.2 million hectare and in india is 11 million hactare (www.isaaa.org)

7. Transgenic genic male sterility • First transgenic genetic male sterile devised Mariani et al(1992) by barnase/barstarsysyem (Ananthi et al ., 2013) • The SeedLinkInvigorhas successfully introduced a GE canola hybrid, using the barnase/barstar gene system in 1996(Department of Agronomy, Iowa State University) • Canola was originally naturally bred from rapeseed in Canada by Keith and Baldur in the early 1970s • The term ‘canola’ refers to those varieties of B. napus that meet specific standards on the levels of erucic acid (below 2%)and glucosinolates( 30 µmoles/g(CODEX 1999), and are often referred to as “double low” varieties. • Indian canola varieties :- Low erucic (< 2%)and low glucosinolate B.napus (gobhisarson) (00):- Hyola-401 (Hybrid) , GSC-5 B. juncea(0) :- PusaKarishma , Pusa Mustard -22 )

8. Area of transgenic canola mustard is about 8.16 million hactare in 2013 mainly in North America and Australia (www.isaaa.org)

9. Approach for development of transgenic male sterility • Barnase/Barstar system for engineered male sterility (Mariani et al .,1992) 2. RNA interfernece for male sterility ( Tehseen et al.,2010) 3. Maize 32138 SPT maintainer used in the PIONEER seed production technology (SPT) process (reference) 4. Male sterility through modification of biochemical pathways (Marc et al.,2000 ) 5. Transgenic Reversible male sterility in plants by expression of cytokininoxidase( Huang et al.,2011)

10. 1.Targeting the tapetum • A specialized anther tissue the tapetum , play an important part in pollen develpoment • The tapetum surrounds the pollen sac early in anther development, degenerates during the latter stages of development • Tobacco TA29 gene expression in anther tapetum and that related gene exist in many other plant such as tomato , oilseed , rape, lettuce and alfaalfa (Seurinck et al.,1990 ) • TA29 tobacco tapetum specific gene can activate the ribonuclease genes Rnase T1 from Aspergillusoryzae(Mariani et al.,1990) and Barnase from Bacillus amyloliquefacienswithin tapetal cell of tobacco and Brassica napus. • Expression of chimaricRnase genes slectively destroy the tapetum during anther delopment

11. Mariani et al.,1990

12. BARNASE/BARSTAR SYSTEM FOR ENGINEERED MALE STERILITY • Barnase is extracellular RNase; barstar is inhibitor of barnase (both from bacterium Bacillus amyloliquefacienswhich uses barnase for protection from microbial predators and 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. • Both gene link to bar gene (Murakami et al., 1986 ) which confers resistance to the herbicide PPT. • Brassica napuscv. Darkar containing the TA29–barnase construct are male sterile; those with TA29–barstar are not affected by the transgene ( Male fertile ) • Cross male sterile (barnase) with male fertile (barstar) to get hybrid seed, which now has both barnase and barstar expressed in tapetum and, hence, is fully fertile. barstar is dominant over barnase (Mariani et al., 1992)

13. Martin et al.,1993

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