农杆菌介导玉米幼胚遗传转化体系的建立

摘要

In order to establish genetic transformation system of maize immature embryos mediated by Agrobacterium tumefaciens, factors including size of explants, concentration of Agrobacterium tumefaciens, temperature of thermal pretreatment, infection time, cultivation and recovery time, antibiotics, screening agent affecting genetic transformation system were optimized using immature embryos of HiII as explants and GUS as reporter gene. The results showed that the size of the embryos was 1.0 to 1.5 mm in length. The OD600nm of Agrobacterium was adjusted to 0.5 before inoculation. Pretreatment was 3 min at 40℃. Infection time was 8 min. It was co-cultivated 3 days and rested 4 days. 100 mg/L carbenicillin was used to eliminate Agrobacterium and bialaphos was used as selective agent. Transgenic plants were obtained by the optimized system, which was a highly and reliable system.%以玉米(Zea mays L.)HiII的幼胚为外植体，β-葡萄糖苷酸酶基因(GUS)为报告基因，通过农杆菌介导转化法对影响遗传转化体系的外植体大小、农杆菌浓度、热预处理温度、侵染时间、共培养及恢复培养时间、抗生素、筛选剂等因素进行优化，以建立农杆菌介导玉米幼胚遗传转化体系。结果表明，幼胚大小为1.0~1.5 mm，农杆菌菌液的OD600 nm为0.5，40℃热处理3 min，侵染8 min，共培养3 d和恢复培养4 d，100 mg/L羧苄青霉素作为抑菌剂，双丙氨膦作为筛选剂时为最佳遗传转化条件。通过该优化体系已获得多种转基因玉米材料，表明该体系具有较高的可重复性和可靠性。