Studyonoptimizationofagrobacterium-mediated Transformation System And Aphid-resistant Gene Transformation In Maize

As the important cereal, fodder crop and industrial raw material, Maize has played an important role in the development of national economy. In recent years, the pest harm of aphid partellus was becoming the major crisis which leads to a great lose of maize yield. Based on maize immature embryo regeneration systems, the Agrobacterium-Mediated transformation method was used to analyze the aphid-resistant genetic transformation. The objective was to transfer aphid-resistant gene to maize through optimal transformation systems, thus gain new maize germplasm with high aphid-resistance and build a foundation for maize insect-resistant breeding by transgenic operation.This study illustrated the technological operations in Agrobacterium-mediated system in maize transformation concerning embryonic callus induction, regeneration, antibiotics concentration and transformation technology. The main results were as follows:The genotype was one of important factors that affected the regeneration efficiency in the maize transformation via Agrobacterium tumefactions. The significant differences in transformation efficiency had been found among the different genotype. Most genotypes were difficult to inducing the high quality callus and hard to get the subsequent regenerate plantlets, especially for maize inbred lines. The characters of immature embryo were analyzed using 4 maize inbred lines and 8 hybrids. The result showed that the immature embryos derived from 87-1 inbred and 7 hybrids were the optimal genotypes for maize transformation and inducing the high quality callus, among them the hybrid Chang7-2×HiⅡA was the best genotype. Different concentration of 2,4-D was added in the media to evaluate its affection before callus initiation. The results demonstrated that low concentration of 2,4-D treatments had the effects on increasing the regeneration frequency and speeding up regeneration course.The optimal antibiotic is the important factor to screening transformation. The study analyzed the effect of different antibiotic including cephalosporins, G418, Kanamycin and PPT on maize embryogenic callus induction and plant regeneration. The result showed that the optimal concentration of cephalosporins, Kanamycin, G418 and PPT was 100mg·L-1, 6-10mg·L-1,15-25mg·L-1 and 9mg·L-1, respectively.We analyzed the effect factors on transformation efficiency such as infection time, acetosyringone(AS) and optimal desiccation using GUS expression. The results suggested that the GUS positive ratio was increased when the medium supplemented with 100μM AS. The negative effect of Agrobacterium to callus was least when the infection time set to 10 minutes and desiccation 4 hours. The GUS expression ratio was 37.6% and 28.1% from Zheng 22×87-1 and Chang7-2×HiⅡA using plantlet chemical staining detection method.The plant expression vector pBJ40-EβF was constructed and transformed into Agrobacterium LBA4404 and subsequently induced the maize callus. The vector pNCX-EβF with self- excised system was set as a control. The PCR screening from the positive callus indicated that the resistant gene has been integrated into maize genome.