Studies On The Regeneration System From Leaves Of Grape In Vitro

It is hard to improve grape by traditional breeding technic as it has a complex descendbackground and long breeding term. As the appearance of transgenic technic and it'simprovement, transiting the aimgene into the plant can improve specific character andbreeding efficiency in the base of keeping some choiceuess charactor, it proves a new wayfor breeding grape. The key difficulty for grape's decend and transform is it's lowregeneration rate, so the mainly problem of grape biotechnology breeding is how to makeit regenerate effectively.This article was mainly involved in the breeding of grape named "Mellisa" and"Summer Royal". Regeneration system was studied using the in vitro leaves. Theadventitious bud regeneration system had been modified by studying the effect factors onregeneration such as the basic medium componet, the types and concentration of plantgrowth regulators, cultivate condition etc.The main results were as followed:1. Genotype plays an important role in the efficiency of regeneration. The results showedthat the regeneration rate and the adaptability to cultivate condition, plant growthregulators and antibiotics of Mellisa is abviously higher than Summer Royal.2. As to sieving the different mediums type, B5 was confirmed as the best suitable medium.The applying of 10～20g/L Glucose or commercial granulated sugar as the carbon sourcewas adopted.3. The different plant growth regulators had significant effects on leaf regeneration. Itshowed that both of cytokinin BA and TDZ could induce regeneration of in vitro leaves ofMellisa and Summer Royal. Although the regeneration rate of TDZ was higher than that ofBA, But the adventitious buds was usually abnormal, so the optimum cytokinin for Mellisaand Summer Royal was BA, the optimum concentrations was 4.0mg/L and BA 3.0 mg/L.Lower concentration of auxins could induce Mellisa and Summer Royal regenerateadventitious buds, IBA is hard to induce regeneration be used with TDZ, and theregeneration rate is lower. So the optimum auxins for Mellisa was IAA 0.10mg/L, and forSummer Royal was 0.05mg/L.4. The top three young leaves were the best sutiable explant, different parts of leaves had not obvious difference.; and different inoculated fashion had not obvious difference too.5. Dark incubation was necessary for leaf regeneration. The regeneration rate roseobviously aider incubation in the dark. The best dark time is about 21 days.6. It has shown that grape subculture plantlets was more sensitive to hygromycin thankanamycin. For Mellisa, the supplying of hygromycin0.8mg/L in the media could restrictthe adventitious bud regeneration in a greatest extent, and it could be used as a selectingpressure for the regeneration of adventitious buds with antibiotic-resistant. The supplyingof optimal concentration of hygromycin3.0mg/L in the media could led the subcultureplantlets to death, and it could be used for the selection of transgenic plantlets withantibiotic-resistant. For Summer Royal, was hygromycin0.8mg/L and hygromycinl.0mg/L.The hygromycin is considered to be more useful than kanamycin as a selecting markerduring gene transformation in grap, since the difference of the concentrations ofHygromycin used for adventitious bud regeneration and the plantlets selection is less thanthat of Kanamycin. The optimum concentration of Carbenicillin was 400mg/L, andCefotaxime was 300mg/L.