影响农杆菌介导的大豆转化效率的因素研究

Assessment of Factors Affecting the Transformation Efficiency of Soybean Cotyledonary-node Agrobacterium-mediated Transformation System

本研究运用农杆菌介导的大豆子叶节转化的方法,探讨了包括基因型和抗生素在内的对大豆转基因效率有重要影响的多个因素。研究发现通过侵染4周后丛生芽报告基因表达的检测可以早期评估农杆菌的侵染效率,而侵染4周后丛生芽的出芽率刚能准确地反映大豆的再生能力。通过比较9个大豆品种的再生能力和对农杆菌EHA101的敏感性,得出对该法侵染效果最好的受体品种为黑农37;通过对转基因组培系统中广泛运用的4种抗生素组合的抑菌效果及对转化效率的影响的比较,得出最优的抗生素组合为头孢噻肟(cefotaxime)100mg/L加羧苄青霉素(carbenicillin)500mg/L。实验还发现,农杆菌侵染大豆子叶节的最佳侵染时间为30￣60min,共培养时间为3￣5d,组培筛选体系选用草丁膦代替潮霉素效果更好。

In this study, we assessed factors affecting the transformation efficiency of soybean cotyledonary-node A grobacterium-mediated transformation system, including genotypes, antibiotics, A grobacterium infection time, and other tissue culture conditions. Results showed that the infectionability and regenerability of transforming soybean explants could be estimated at the early stage of soybean transformation by investigating the expression of report gene expression or the regeneration rates of the inducted shoots in 4 weeks after A gobacterium infection. Heinong 37, the elite variety from Heilongjiang province, was selected as the best cultivar for the transformation system based on its regenerability and sensitivity to A grobacterium infection. In addition, we also compared 4 different antibiotics combinations for A grobacterium-overgrowth control. Results showed that the combination of 100 mg/L cefotaxime plus 500 mg/L carbenicillin had the best effects on both A grobacterium-overgrowth control and transformation effi- ciency. No significant difference was observed in the transformation efficiencies of different A grobacterium infection times between half hour to 2 hours, co-cultivation duration between 3 to 5 days. The selectable marker gene bar, conferring resistance to the herbicide glufosinate, was found to have higher selection efficiency than the hpt gene conferring hygromycin resistance in the system.