| Tomato (Lycopersicon esculentum Mill) belonged to Solanaceae Lycopersicon,widely cultivated throughout the world. However,progress in breeding new varieties only by conventional breeding had been slowed. Production of haploids in tomato anther or microspore culture would allow tomato breeder to release new lines more quickly and screen for particular traits more efficiently. At the same time, tomato was self-pollination crop, its heterosis was very clearly. The study on male sterility of tomato was very important. In this paper, anther culture and isolated microspore culture in tomato were studied. And regenerated plants were obtained from 'Liger87-5', 'Sf9' and 'Hp' through anther. Lots of Globular embryoids,Heart-shaped embryoids were produced from 'Liger87-5','Jg', 'Zs' and 'Hp' through isolated microspore for the first time. At the same time, Agrobacterium-mediated genetic transformation system was adoptted for tobacco and tomato.We obtained 25 trangenic tobacco and 20 transgenic tomato. Specific results were as follows:1. We established a stable and high efficient embryogenic callus induction and plants regeneration system in tomato. Six tomato varieties were used in the experiment.The results showed that the callus was related to the genotypes;The callus inducement frequency with addition of potato milk and active carbon decreased 35.14%,95.48% in the six different materials respectively. The optimized hormone combination of callus inducement was 1.Omg/L 2,4-D and 0.5mg/L KT.30g/L sucrose could enhance the callus inducement about 20.24% on 'Liger87-5'. 10μmol/L silver nitrate could reduce browning of the tissues about 70.59%. When the concentration of silver nitrate exceeded 50μmol/L, the browning of the tissues would increased and the callus inducement would decreased imperceptibility. Medium of MS+ZT1.5mg/L+ IAA0.2mg/L could make callus dedifferentiate into adventitious bud, and the rate of callus inducement was 40%, this hormone for tomato anther callus differentiated into adventitious buds again optimum formula. Callus induction in tomato shoot the medium with 2mg/L triacontanol, increased the rate of bud differentiation, but also could improve the quality of adventitious buds, adventitious buds significantly increased the percentage of effective bud. The adventitious buds were accessed to 1/2MS+2.0 mg/L IBA for rooting when they grew to 3-4cm size.We obtained plants through anther culture.2. Tomato microspore culture of producing of embryoid. This study used two different methods of microspore isolation and culture system, WM technology and SMC technology, the results showed that WM technology microspore isolation and culture system was the optimistic system. Comparing to WM technology,'Liger 87-5'and'Jg'of the embryo yield decreased by 53.56%,49.86%. Embryogenesis ability of different genotypes varied widely, and the embryo was closely related to the rate. The three tomato varieties in the same culture conditions, microspore embryo production from high to low were:'Liger 87-5','Jg','Zs','Hp','002'.Among them,'Liger 87-5','Jg' and 'Zs' were easier to form embryoid bodies, and in the same culture conditions, '002' of embryo yield was only 0.13, not easy to produce embryoids. Free microspore culture in the process of tomato, adding tomatoes immature ovary, five varieties of microspore embryo yield was higher than the control.3.Tobacco and tomato plants transfected male sterility Barnase gene. pBITBAS-BAR was transferred into Agrobacterium tumefaciens LB4404 through freeze-thaw method, tobacco and tomato plants transferred Barnase gene were obtained.It preliminary indicated the Barnase gene had been integrated into the tobacco and tomato genome and could be normally transcripted and translated in the transgenic tobacco and tomato plants by kanamycin selection, PCR, RT-PCR detection. We observed that filament of transgenic tobacco reduced significantly and the anther flatted through observing flower morphology.The anther had not normal pollen. Transgenic tobacco did not fruit. |
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