利用农杆菌菌株EHAl05,对17个栽培大豆品种的大豆子叶节进行了侵染,从大豆基因型、氯气灭菌时间、外植体状态、菌株活力、侵染浓度、侵染时间、共培养时间等方面进行了优化。结果表明:氯气灭菌14~18h,可在不影响大豆种子活力的同时最大限度的减少污染;暗处理1d的外植体活力高于光照处理5~7d的外植体;菌液浓度(OD600nm)在0.8~1.0且侵染浓度OD600nm)为0.6~0.8时GUS瞬时表达率最高;适宜的侵染时间和共培养天数分圳为30min和4d。在上述优化研究基础上,形成一套综合的转基因体系,该体系的最高转化率可达3.33%。不同处理下的GUS瞬时表达率以及17个大豆品种的丛生芽诱导率综合评价显示,适宜转化的基因型为TL-1、HC-3、HC-6、Wil-liams82。
In the present study, cotyledon nodes obtained from 17 soybean cuhivars were infected with Agrobacterium tume- facines EHA105. Subsequently, we optimized the soybean genotype, chlorine slerilization lime, explant state, vitality of bac- terial strain, concentration of infection liquid, infection time and co-cultivation time. As a result, when the chlorine steriliza- tion time was 14-18 h, the minimal pollution and the highest seed vigor was observed. The vitalily of overnight-treated explants in the dark were better than those germinated in the light for 5-7 d. The highest GUS transient rate was achieved when the bac- terial concentration at OD600nm was 0. 8-1.0 and the infection liquid at OD600nm was 0. 6-0.8. We also found that the best infec- tion and co-cultivation time were 30 rain and 4 d, respectively. Based on the above optimized research, we established a com- prehensive transgenic system which resulted in the maximum transformation efficiency of 3.33%. For the comprehensive eval- uation about the transient GUS expression under different treatments and the shoot induction rate of 17 soybean varieties, TL- 1, HC-3, HC-6 and Williams 82 were the ideal genotypes for transformation.