Establishment Of Efficient Regeneration System Of Grape Jingya And Crimson Seedless

Transgene technology has been developed a kind of most important instrument of plant breeding and molecular design.However,too low regeneration frequency restricted transgene project developing of grape now.Although there were many reports in grape tissue culture,they used axillary bud as explants,these is not fit to transgene regeneration. In this study,Jingya and Crimson Seedless were used as the materials,after sterilize,subculture and multiplication,leaves of plants in vitro were used as explants,established leaf disc regeneration system,a portion prophase research for transgene was done also, and get following conclusion:1.To sterilize single bud stem with 0.1%HgCl and 2%NaClO solution,and a variety of combinations with unequal sterilization time were designed.The test shows that the 2%NaClO sterilize 20 min has the best effect for eliminating bacterium.The lowest contamination was obtained at April and May.2.The first generation culture medium:MS + 6-BA 1.0 mg/L is the most suitable for Jingya;MS + 6-BA 2.0 mg/L is the most suitable for Crimson Seedless.The subculture medium:MS + 6-BA 1.5 mg/L + IBA 0.1 mg/L is the most suitable medium for both of them,and the Multiplication times are up to 5.3.Inhibit Vitrification:Reduce the temperature and increase agar content can delay the occurrence of vitrification,but it cann't stop the occurrence of vitrification as rising of the temperature and the times of the subculture.With 0.4 g/L active carbon in subculture medium cann't get more multiplication,but it can totally stop vitrification, and can provide stabilization and strong explants for leaf disc regeneration.4.Leaf regeneration system:The leaf best hormone match of Crimson Seedless was MS + 6-BA 6.0 mg/L + IBA 0.05 mg/L regeneration rate was 11.2%;Jingya was:MS + 6-BA 3.0 mg/L + IBA0.05 mg/L,regeneration rate was 65.2%.we also found that the top 2 leaves is the best explants,Leaf abaxial to medium and partially cut in the leaf vein can improve regeneration rate too.5.Best of the radication medium of adventitious bud was 1/2MS + IBA 0.5 mg/L+ sucrose 30 g/L + agar 7 g/L.The taproot distributes radially,with strong stem and developed lateral root.It is the ideal result for rhizogenesis.6.Kanamycin was used in the rooting select but not the leaf select because of the sensitivity,and the selecting concentrate was 10 mg/L,and the concentrate of Cef was 300 mg/L which can inhibit the Agrobacterium effectively.