Study On Factors Effectting Transformation Of Capsicum In T-DNA Insertional Mutagenesis Library Construction

Using T-DNA tagging approach for transforming plant is a powerful tool for the study of plant functional genome. Capsicum is the crucial vegetable crop in our province, for researching its functional genome, we developed T-DNA tagging transformation used capsicum inbred line H8214 as material and pUC18,pWMl0l as original plasmid to construct T-DNA tagging plasmid pWM101+Amp^r for plasmid rescue, selected universal activated expressed tags plasmid pSKI015 along with two kinds of Agrobacterium Tumefactions strain: LBA440,GV3101 to forming four kinds of Engineering Agrobacterium Tumefaciens:LBA4404 (pWM101+Amp^r ),GV3101 (pWM101+Amp^r ),LBA4404 (pSKI0l5) andGV3101 (pSKI015) to seek for suitable conditions of genetic transformation and the construction of mutant library about H8124 T-DNA.Using MS basic medium to induce differentiation of capsicum bud and studying the effect to differentiation of bud by factors like explant type, ratio of 6-BA/NAA and DJ,AgNO3,seedling age, dark treatment, the PH of medium, sucrose concentration, explant inoculum density atc. The results showed that using cotyledon as explant is better than hypocotyls, and medium added with NAA as well as dark treatment go against the process, also relatively easy to root. The differentiation rate as high as 52% when the medium is MS+6-BA(5.0mg/L)+NAA(0mg/L) and the ratio increase to 62% by adding DJ,AgNO₃. We finally find the optimum culture medium of bud elongation is 6-BA (3 .0 mg/L)+ GA₃(4mg/L). Bud elongation was strongly improved by the combination of DJ(4mg/L)+AgNO₃(10mg/L). The condition will be better if adding agarose to 4.5 g/L and keep erlenmeyer flask sealing by plastic film. NAA(0.5 mg/L) is good for radication.It is found by study that cephalothin(400 mg/L) can restrain Agrobacterium Tumefaciens growth effectively and do not affect differentiation of cotyledon, the bud differentiation can totally be restrained when concentration of PPT is 0.5 mg/L. Explant pre-cultured for 2 d, Tumefaciens in logarithmic growth phase was diluted to half as original concentration plus cotyledon infection in 5 min , then co-culture 1d in solid medium in four days delayed selection, resistant buds were obtained with the topregeneration frequency of 20%.