Studies On Introducing Bt-toxin Gene Into Rice Via Agrobacterium Tumefaciens

Two aspects of research were performed on two varieties of indica rice (Oryza sativa L.) Pei'ai64S(PA64S) and 9311 which were employed extensively as outstanding selectable parents of current super hybrid in this article. The main results were as follows:Optimization of regeneration system suitable forgenetic transformation of predominant indica riceSeveral factors affecting indica rice Pei'ai64S and 9311callus induction, subculture, bud differentiation and plant regeneration were investigated with mature embryos as explants. The results indicated that high ratio of [NO3+-N] / [NH4+-N], numerous inorganic micro-elements with larger amount of vitamineB) and inositol were the optimal medium elements. With the optimal 2,4-D concentration, supplementing the induction and subculture medium with NAA, ABA and some cytokinin cannot remarkably improve callus induction frequency. A certain concentration of these plant growth regulators may, however, make for improved and increased embryogenic callus. Application of different medium and varied concentration of hormone alternative plays an important role on the maintenance of embryogenic state of long-term cultured calli. Pre-differentiation and proper partial desiccation of calli before transferred to regeneration medium was found to apparently improve the frequency and quality of plant differentiation.With our optimized culture condition and treatment style, induction frequency of Pei'ai64S and 9311 can be reached 62.45% and 85.30% respectively and after 3 months of subculture calli can remain high-quality embryogenic state, when high-quality embryogenic calli after two times of subculture were used asacceptor, callus differentiation frequency can arrive at 85.5% and 87.7% respectively.Studies on transformation of indica rice with Bt-toxin genemediated by Agrobacterium tumefaciensPrecultured immature embryo and callus derived from young panicle, immature embryo and mature embryo were used as acceptor for genetic transformation mediated by Agrobacterium tumefaciens, the transformation rate of the above acceptor was investigated respectively. The results showed that immature embryo after precultured for 4~6d was the best.In respect to the concentration of Agrobacterium tumefaciens when calli were cotransformated in medium YEB, to Agrobacterium tumefaciens EHA 105, OD value of 0.8 was the best.Negative pressure disposition in the process of Agrobacterium tumefaciens infecting callus was found to be able to improve transformation frequency of indica tested rice apparently. With the 60ml injector, drawing distance being 6cm and drawing times being 40 can perform the greatest effect.Ca2+ and membrane surface activator being used to promote transformation may be innovation of our research. After immerged and infected in coculture medium YEB, Calli being immerged in 20mmol/L and 30mmol/LCaCl2 with additive 0.1%Tween20 can most remarkably increase transformation percentage of calli derived from Pei'ai64S and 9311 respectively. The transformation rate and regeneration rate of herbicide-resistance rice plants of 9311 variety can reach 58.33% and 33.33% respectively.PCR (polymerase chain reaction), southern blot analysis performed toward 10 transgenic rice plants revealed that foreign genes were integrated into the genome of these plants.