Study On Agrobacterium-mediated Transformation Of Brassica Oleracea Var.acephala With Cold Resistance Gene

Ornamental kale(Brassica oleracea var.acephala) is an excellent landscape plant, belonging to cabbge's horticulture varieties of Cruciferae Brassiea. Low temperature is the key factor that limits the growth, colour and appreciatability in winter in the northern of China. Introducing foreign gene into kale through genetic engineering may create novel variety with cold tolerance. In this study, BADH(encoding betaine aldehyde dehydrogenase) and TCH(encoding transcription regulation factors) were used to be transformed into kale with Agrobacterium-mediated transformation. The results are as follows:1. Get 3 high efficient regeneration genotypes and optimize the regeneration system of kale cotyledonTrough the analysis of 9 pairs of genotypes, choose 3 high efficient regeneration genotypes and the highest induction rate is 100%. The best medium are MS+6-BA1.0mg/L+NAA0.1mg/L, MS+6-BA1.4mg/L, MS+6-BA1.0mg/L. Cut out for kale cotyledons when germinated 3 days, put them on the culture medium which suit in. When the buds are about 1 cm, transfer them to the culture medium MS+0.1 mg/LNAA to induce roots. After roots being 1cm, the seedling is cultivated on the sterilized soil.2. The agrobacterium transformation system has been establishedThrough the comparative test of the factors that influence conversion, the optimum transformation conditions are cutting out for transformation materials and cultivating on MS+BA 1.0mg/L+NAA 0.1mg/L for 2 days'pre-culture. Centrifugation of bacterial liquid with OD₆₀₀=0.3 which get the second activation, then adding the same volume of MS(pH=5.2)and pre-culture leaves. After 5 minutes, the leaves which remove liquid cultivate in MS+BA1.0mg/L+NAA 0.1mg/L for 2 days. Then take the leaves in MS+BA 1.0mg/L+NAA 0.1 mg/L+Carb500mg/L delay cultured for 3 days. And transfer leaves into differentiation culture which is MS+BA 1.0mg/L+NAA 0.1mg/L+Carb500mg/L+0.05%oGly. Rooting medium is MS+NAA 0.1mg/L+Carb500mg/L+0.05%。 Gly3. The identification of kale transferred with encoding transcription regulation factors geneOn the transformation systems of optimization kale's cotyledon, transferred with encoding transcription regulation factors TCF. After transferring, we got 4.8% induction rate of positive-resistant bud. We obtained 28 green buds by selection of glyphosate and 4 buds are positive by PCR.