Genetic Transformation Of BT-CpTI Gene Into Maize

In this research, seven varieties of maize (Zea mays L.) were chosen and the immature embryos and embryo leaves were used to induce embryogenic callus. The factors affecting embryogenic callus regeneration, Agrobacterium-mediated transformation and electroporation were studied. This study established a highly efficient regeneration system of maize and then transferred the Bt-CpTI gene into maize, which mediated by Agrobacterium tumefaciens.One PCR-positive plant was obtained. The positive results of PCR test showed that the Bt-CpTI gene was integrated into maize genome. The results were summarized as follows: 1. The establishment of regeneration system (1) The ability of callus induced maize by embryo leaf was obviously different from positions and genotype . (2) The concentration of 2,4-D varies from genotypes . (3) The differentiation medium was: MB + 6-BA 1.0mg/L + KT 0.5mg/L. (4) The root regeneration medium was:MB + IBA 2.0mg/L + NAA 0.5mg/L. 2. The establishment of transformation system ⑴Definition of concentration of selective agent G418 The selection results of G418 were analysed. The concentration of G418 on DH11 was 60mg/L in the period of callus develepment and in the callus differentiation period was 40mg/L. ⑵Transformation of calli mediated by Agrobacterium tumefaciens ①Among DH11, DH9 and Ji846, DH11 was the best target for Agrobacterium-mediated transformation. ②The results of orthogonal experiment showed that the group of precultured time of 6 days, infected time of 10min, 10g/L Glu. and Co-cultured time of 3 days was the better group. The main factor was co-cultured time. ⑶Transformation of calli by electroporation Optimization of parameters for gene transformation was analyzed. The result showed that the group of capacitance of 960μF,intensity of 375v/cm and electroporation time of 61ms was the best group to transfer Bt-CpTI gene into calli. 3. PCR test of resistant plants Seven resistant plants and one PCR-positive plant were obtained, and the results of PCR test showed that the target gene was integrated into maize genome primarily.