Optimization Of Regeneration Transformation Of Brassica Oleracea In Tissue Culture And Screening System

Brassica oleracea were used in the expriment. The study for the first time established a high efficient regeneration system by using cotyledons and hypolotyl for the explant and discussed various factors,which had effects on cotyledons and hypolotyl differentiation and growth. The optimal culture medium and conditions for high frequency regeneration had been selected under the variant hormones combinations and culture conditions. The results are as follows:MS + NAA 0.2 mg/L + 2,4-D 0.5 mg/L gave the best result for the hypolotyl callus propagation and differentiation. The cotyledons explants were transferred to MS culture medium with 0.1mg/L NAA and 0.5 mg/L 2,4-D for the differentiation. Supplements of 6-BA 2 mg/L ,NAA 0.1 mg/L and AgNO3 20 μm attached to MS medium were suitable for bud induction.and then rooted on 1/2MS medium cotentaining 0.1mg/L NAA. In the study,Bcka gene was transformed into Brassica oleracea by Agrobacterium tumefaciens. And further more,some factors and conditions which affect the transformation efficiency,were also investigated. Including kanamycin screening,different Agrobacterium tumefaciens concentration , antibiotic concentration of selection ,browning ,co-culture time and so on .The results showed as follows: AgNO3 was helpful for against browning and induction of regeneration .The optimal selected concentration of Km and Cab is 25 mg/L and 500 mg/L,Transgenic plants have resistance against Cab , during the experient ,antibiotic concentration should be grandual decreased.