Studies On Plant Regeneration Of Muskmelon "Huang Hemi" And Methods Of CMV CP Gene Transformation Mediated By Agrobacterium Tumefaciens

In this studies, the optimum regeneration system of Muskmelon "Huang Hemi " was established. On the base of regeneration system, the effects of various factors on transformation efficiency were discussed and the transformation system was established. It was the first time that CMV CP gene was introducted into Muskmelon "Huang Hemi "mediated by Agrobacterium tumefaeiens.The calli were obtained from cotyledon and hypocotyls, but the regeneration potention of cotyledon was better than that of hypocotyls. Using cotyledon as explant, in the process of calli induction, MS medium supplemented with 6-BA0.4mg/L, NAA 0.1mg/L and sucrose 30g/L was the most effective medium, and the calli induction rate was as high as 87.2%. MS medium added to 6-BA0.8ng/L, IAA0.2mg/L and sucrose 30g/L was the most suitable medium for differentiation of buds, the average number of buds differentiated from calli was up to 10.8. The buds cultivated 2 days under darkness was easy for rooting, and 1/2MS supplemented with IAA 0.2mg/L, sucrose 20g/L was very efficient in the rooting process, rooting frequency was higher than 73.2%.Using hypocotyls as explant, MS medium added to 6-BA 0.4mg/L, NAA 1 .Omg/L and sucrose 30g/L was the most suitable medium for calli induction, and the calli induction rate was 73.8%. In the process of differentiation buds, MS medium supplemented with 6-BA2.0mg/L, NAA 0.1mg/L and sucrose 30g/ L was the effective medium, and the average number of buds differentiation from calli was 9.4. The treatments and condition of rooting was as same as cotyledon, rooting rate was 67.2%.The dismard Agrobacterium tumefaeiens Ti plasmid pBin438 integrated CMV CP gene was used in the present studies to transfer Ti-T-DNA into cotyledon and hypocotyls explants of Muskmelon "Huang Hemi". In the process of transformation, the selecting pressure of Kan concentration was 100mg/L; while Carb 500mg/L inhibited effectively bacterial growth. Through testing factors of pre-cultivation, vigor of bacterium and concentration of AS and NAA, the results showed as follows: The cotyledon pre-cultivated 2 days had better transformation rate. Transformation rate with AS 40umol/L added to pH5.6 YEP solution was 2.1 higher than that of control. The transformation rate by infection of bacteria cultivated in pH5.6 YEP solution for 6 hours for activation was higherthan that of the bacteria in pH6.5YEP treatments. Different concentration of NAA had different effect on transformation rate of explants,'the concentrations of NAA for cotyledon and hypocotyls suitable for transformation were 0.1mg/L, 1.0mg/L respectively. The highest transformation rate was 12.8% in all of these treatments. The transformated calli were obtained through these treatments, and transgenic plants were obtained from the transformated calli cultivated on the medium of differentiation and rooting. The PCR-Southern assay showed that CMV CP gene had integrated into chromosome of Muskmelon "Huang Hemi" and transgenic plants ware obtained.