Agrobacterium-mediated Genetic Transformation Of Maize Immature Embryos Of 2mG₂-epsps

China ranks second in the world corn production. It’s corn acreage and production continues to increase, currently has more than rice become China’s first major food crops. China’s corn production in the long-term self-sufficiency of equilibrium, in 2010, in 2012 China has become a net importer of corn.China’s shift on the relationship between import and export of corn, increase domestic corn production to make the task even more urgent. Maize growth at high temperature, high humidity season, weeds harm is serious, chemical weed control is an important way of weeding to reduce weeds and ensure increase production of corn. As the destructive herbicides, glyphosate is currently the most widely use scope of herbicides, killing crops at the same time of killing weeds. Genetic engineering breeding of maize get glyphosate resistance, is an effective way to solve this contradiction.This study through enzyme digestion and ligation modified plant expression vector pCAMBIA3301-epsps, made the glyphosate gene 2mG2-epsps behind corn ubiquitin promoter.Agrobacterium-mediated transformation of maize inbred 18-599R and Hi-II immature embryos, and the embryo transformation system has been optimized, obtained the TO generation of transgenic plants.Detected by PCR and sequencing of transgenic plants indicated that the gene 2mG2-epsps been integrated into the maize genome.The main results are as follows:1. The mediated of plant expression vector pCAMBIA3301-epsps.Changed CaMV35S promoter before gene 2mG2-epsps to maize ubiquitin promoter. As a promoter of maize gene itself, when it drives exogenous gene expression in maize can reduce exogenous gene copy number in transgenic individuals and avoid the occurrence of gene silencing.2. Maize immature embryos as explants for genetic transformation, by affecting the rate of callus induction affect Agrobacterium transformation efficiency of maize. In this study, the size 0.8-2.0 mm immature embryos in bacterial concentration of OD600=0.4-0.6, infection time of lOmin, statistics callus formation rate after recovery training. As the results, the rate of callus formation of 0.8-1.2 mm,1.2-1.5 mm,5-2.0 mm immature embryos was 93%,74% and 38%, prove 0.8-1.5 mm immature embryos could be the receptors of Agrobacterium-mediated genetic transformation of maize.3. After infection, using 400 mg/L of cephalosporins to inhibit the growth of Agrobacterium. In order to ensure the formation of resistant callus, screening media also need to add cephalosporins. Experiments were treated with 200 mg/L,400 mg/L, and 600 mg/L of cephalosporins, screening statistics resistant callus rate after 15 days in 1.2 mmol/L glyphosate screening medium. The results indicate that the use of cephalosporins is still 400 mg/L in the screening medium to ensure a high rate of resistant callus.4. Bacterial concentration, infection time and their interaction were significant impact on the efficiency of infection. According to two factors and three levels designed nine handle, repeated three times. Experimental results show that bacterial concentration OD600= 0.6, infection time was 10 min,15days later in 1.5 mmol/L glyphosate screening medium, the average resistant callus rate up to 37.33%, was the highest. Bacterial concentration of OD600= 0.4, may be appropriate to extend the time to improve the infection efficiency.5. For 18-599R and Hi-II immature embryos of Agrobacterium-mediated method were compared, found that Hi-II earlier than 18-599R to form callus, and callus growth faster, and screening for glufosinate faster response. Experiment to get 7818-599R regenerated plants, nine PCR test was positive, the positive rate was 11.5%.And get 18 Hi-II plant regeneration, five PCR test was positive, the positive rate was 27.8%. So, Hi-II is more suitable than 18-599R to be the acceptor material of genetic transformation of maize, but pollination difficult. Select the appropriate planting conditions to ensure a stable supply of Hi-II immature embryos is its prerequisite to be the receptor for genetic transformation off maize.6. Transgenic regenerated plants were serious deformities, growth weak because of conditions changed from laboratory to field, and uncoordinated silking flowering, pollination caused by difficulties in obtaining fertile offspring. In practice, through sister hybridization, or hybridized with the parental to get the T1 generation seeds.