Transfer Of Insecticidal Protein Gene From Bacillus Thuringiensis Into Conventional Maize Inbred-line

Extablishment of genetic transformation acceptor system has been investigated, and insecticidal protein gene from Bacillus thuringiensis was transferred into conventional maize inbred-lines through technologies such as Agrobacterium twnefaciens, Microprojectile Bombardment and Pollen-tube pathway.Extablishment of acceptor system Embryogenic callus initiation, subculture and plant regeneration from immature embryos of five conventional maize inbred-lines have been studied. An efficient embryo culture system suitable to different genotypes was established and differentiation frequency was beyond 70% which was suitable to genetic transformation. Furthermore, we brought out that callus initiation and subculture were mainly controlled by medium. The difficulty of difference among genotypes could be overcome in some grade adjustment of ingredient and concentration of media.Mediated by Agrobacterium tumefaciens. It was proved that the target Bt gene had been integrated into the genome of regenerated plants after PCR, PCR-Southern and Southern blotting analysis. Transformation frequencies(calculated from independent transgenic plants/embryos) were from 1 .63% to 4.44% The factors influencing maize transformation by A. twnefaciens have been studied. The bacterial concentration used in transformation was determined as 0.5 since the frequency of resistant calli reached the highest when OD6oo of engineering bacterial suspension is this value.. The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.8 7The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22 癈. The transgenic plants were examined for phosphinothricin(PPT) resistance. Resistant and sensitive seedlings were distinguishable 6 days after application of Basta. The positive plants were as healthy as non - treated plants while the negative plants were quickly withered and died.Via Microprojectile Bombardment. Type II embryogenic calli from 7922's immature embryos were used as acceptor via Microprojectile Bombardment. Half of the gold particles according to statard protocol were applied after pre-experiment. A large number of resistant plants have been regenerated and regeneration frequency reached to 78.40%. After PCR analysis, it was proved that the target Bt gene had been integrated into regenerated plantsand the frequency was as high as 3.09%.Through pollen-tube pathway. The plasmid DNA liquid was dropped 5-25 hours after pollination. After Basta selection and PCR analysis, among 1403 transformed plants of TO generation, there were 5 PCR positive plants which were obtained from 8,12,14,18 hours after pollination respectively. The total average transformation frequency was 0.36% and the highest frequency reached 8.3% when dropping of the plasmid DNA liquid was at 14 hours after pollination. The results confirmed that genetic transformation through pollen-tube pathway in maize was completely effective when the time of transferring was suitable.