Study On Agrobacterium-mediated Genetic Transformation Of Maize (Zea Mays L.) Immature Embryos

Maize (Zea mays,L.) is a widely grown cereal crop and the most important fodder crop in the world today. It is for engineered breeding for maize to establish an Agrobacterium-mediated transformation system for maize. In this dissertation, a system of high frequency regeneration and Agrobacterium-mediated transformation of maize inbred lines is developed with the immature embryos of maize inbred lines(Zheng58,Zong3,87-1,137,K12 and Chang7-2). It provides some important theoretical basis for the further study maize transgenic technology; the main results are as following1. It is believed that the immature embryos is the best source as the explants in tissue culture and genetic transformation research in maize. In this study, immature embryos of six maize inbred lines(Zheng58,Zong3,87-1,137,K12 and Chang7-2) are used as explants in inducing the callus and filtering the best subculture medium. The results showed that the D-medium and N6B5 basic medium added with different ingredients turned to be different both in inducing and sub-culturing the callus. Meanwhile, the D- medium can effectively induce the primary callus for Zheng58 and Zong3, and the N6B5 basic medium was more suitable for the primary callus induction of 87-1 and 137.2. In this dissertation, maize inbred Zong3 is used to analyze the effects of different factors and optimize the experiment system in the induction of callus with the method of orthogonal. The results showed that in the course of the primary callus induction, proline (L-pro) was the most significant factor, while 2,4 - dichlorophenoxyacetic acid (2,4-D) ,silver nitrate (AgNO₃) and asparagine ( L-Asn ) played a significant role, but the effects of hydrolyzed casein (CH) was not so important, and the optimized system was L-pro 500mg/L,L-Asn 800mg/L,CH 1000mg/L,2,4-D 3.0mg/L,AgNO₃ 12mg/L; In the course of embryonic callus induction, the best match for the various factors is L-pro 500mg/L,L-Asn 500mg/L,CH 2000mg/L,2,4-D 2.5mg/L,AgNO₃ 8mg/L;In the course of subculture,by adjusting the pH value, changing the concentration of silver nitrate and hydrolyzed casein,some problems such as callus water-soaking, browning can be prevented and cell osmotic pressure can be regulated, so the compact embryogenic callus can be stably obtained. 3. Maize inbred lines Zheng58,137 and 87-1 were used in the dissertation to develop a excellent medium system which the embryogenic callus and makes it possible in the long-term subculture. With the combination of 500mg/L×800mg/L×/500mg/L×1.5mg/L×8mg/L×0.3mg/L for the concentration of praline, asparagine, hydrolyzed casein, 2,4-D,AgNO₃ and 6-BA,and 16℃×30d on the subculture temperature and period, the embryogenic callus can be stably maintained and effectively subcultured for a long term.4.The immature embryo of excellent maize inbred zheng-58 is used in the experiment, taking the embryogenic callus as the receptor for the transformation of Trivalent plant expression vector which is mediated by Agrobacterium tumefaciens. Important factors which influence efficiency of the transformation is identified and an efficient transformation system is developed for maize immature embryo calli. The result shows that the resistant calli rate is improved remarkably (17.39% higher than other concentration) when the concentration of acetosyringone (AS) is 150 mg/L both in the infection solution and co-culture medium. The high efficiency system for transformation was established with the combination of 0.3 OD×20 minutes for Agrobacterium tumefaciens concentration and incubation-time,20℃×60h for the co-culture temperature and time, 400 mg/L for the cefotaxime (Cef) concentration , and the pH was 5.4. By this optimized system, the rate of the resistant calli can be highly increased to 17.26%.