Studies On Genetic Transformation Of AFP Gene Into Maize

In this research, nine varieties of maize (Zea mays L.) were chosen and the immature embryos of their varieties were used to induce embryogenic callus. The factors affecting embryogenic callus regeneration, the Agrobacterium-mediated transformation and particle bombardment were studied. This study established a highly efficient regeneration system of maize and then transferred the AFP gene into maize, which mediated by Agrobacterium tumefaciens. Four positive plants were obtained. The positive results of PCR test show that the AFP gene was integrated into maize genome.The results were summarized as follows:1. The establishment of regeneration system(1)The high regeneration frequency genotypes were Qi319, 81162, Ji846, Mol7. (2)The concentration of 2,4-D varies from genotypes , the results were:81162, Ji846: 2.0 mg/L2,4-DMo 17: 3.0 mg/L 2,4-D.(3)The suitable immature embryo was 12-14 after pollination and 1.0-2.0 mm in length. (4)The low-temperature pretreatment can increase the frequency of embryogenic callus. The suitable time of low-temperature pretreatment was: 81162, Ji846 three days; Mol7 one day. (5)The differentiation mediums of different genotypes was:MB + 6-BA 1.0 mg/L + KT 0.5 mg/L. (6)The root regeneration medium of shoot was: MB + IBA 2.0 mg/L + NAA 0.5 mg/L.2. The establishment of transformation system(1)Definition of selective agent and concentrationThe selection results of Km, Neo and G418 were analysed, G418 was a more effective selective agent than Km and G418. The concentration of G418 on Qi319 was 60 mg/L in the period of callus develepment and in the callus differentiation period was 30 mg/L for Qi 319 and 81162, 10mg/Lfor Ji846 and Mol7.(2)Transformation of callus by Agrobacterium tumefaciensâ‘ Among the three genotypes, 81162, Ji846 and Mo 17, 81162 was the best target forAgrobacterium-mediated transformation.â‘¡The result of orthogonal experiment shows that the group of precultured for 7 days, infected 15min. 20g/L of Glu. , Co-cultured time 4 days was the better group. The rate of GUS-positive was 27.1% under this condition. The main factor was the time of preculture.(3)Transformation of callus by particle bombardmentOptimization of parameters for gene transformation was analyzed. The result showed that the group of 1 100psi helium pressure and 8cm of bombardment distance was the best group to transfer AFP gene into Qi319 calli. The rate of resistant callus and the rate of resistant shoots were 14.5% and 0.84% respectively.3. The PCR test of resistant plantsTen resistant plants and four positive plants were obtained, and the result of PCR test showed that the target gene was integrated into maize genome primarily.