Study On The Genetic Transformation Of Bt Gene Into Maize Inbred Lines In Northeast

Maize was an important grain crops and forage crops, it was also a modem food industry and an important raw material of medical and chemical industry, so each country paied more attention to it's genetic transformation. Owing to the cofinement of the host of agrobactrium tumefliens and the difficulties of the maize inbred lines for obtaining embryogenic calli, the genetic system of maize wasn't ideal enough. In this experiment, the immature embryos were used as explant to induce calli and Bt gene was tranfered into Mo17, 81162, Ji 846 and 7922 by the Agrobactrium-mediated. We have gained the positive plants determined by PCR. Meanwhile, the major factors influencing the regeneration system and transformation system for agrobacterium-mediated method were optimized. The main results were showed as following:1.Induction of embryogenic calli was inheritable. Different maize genotypes showed different ability of emberyogenic calli establishment and plant regeneration. Mol7, Ji846, 81162 and 7922 had the most high rate of induction, Ye 107, 5416 and 444 were high, but LongKang11 and DongNong46 had little rate of it; The concentration of 2,4-D had an enfluence on the mopha, growth and differentiation of calli. On the whole, 2 mg/L 2,4-D was found to be most favorable for the induction of embryogenic callli. But the sensitivity to 2,4-D varied among genotypes, Mo 17 and 5416 need 3 mg/L 2,4-D in the course of induction.2.The resistence of transformators were selected by G418 after co-culture with agrobactrium tumeflien, which was 40 mg/L in subcultured medium and 50mg/L in differeniated medium. In the process of killing agrobactrium, Cef, Amp or Carb were also useful and the time of inhibition was long too. As the concentration of three antibiotic was 50mg/L, 300mg/L, 400mg/L respectively, we chose 50mg/L Carb as antibiotic.3.The factors influencing the transformation efficiency for agrobacterium-mediated methods were mainly studied the pre-culture, the concentration of agrobacterium, the time of infection and the effect of AS. The effect of the days of pre-culture on the rate of transformation had no notable difference; The efficiency of transformation was high when OD600 was 0.5 and the time of infection was five or ten minutes; AS made a notable impact on transfermation, especially, when AS was added into co-culture medium.4. Results of PCR and paraffin wax section of the transgenic calli confirmed that Bt gene had been transferred into maize calli. Results of PCR for the resisted shoot and transgenic plants showed that Bt gene had intergrated into maize genome. By now, transgenic plants had obtained, Southren bloting for transgenic plants was still be doing, which would pave the way for further maize agronomic improvement through genetic engineering.