棉花农杆菌介导转化体系中影响抗性愈伤组织诱导率的因素

Influencing Factors of Induction Ratio of Resistant Callus by Agrobacterium-mediated Genetic Transformation in Cotton

以冀无2031为材料,采用正交设计方法,研究了农杆菌介导的棉花下胚轴遗传转化过程中菌液浓度、侵染时间、共培养时间和共培养温度对抗性愈伤转化效率的影响。结果表明,4个因素均对抗性愈伤的诱导率有极显著影响,其诱导效应依次是共培养温度>菌液OD值>侵染时间>共培养时间。按照正交设计的原则,最终得到农杆菌介导转化棉花下胚轴的最佳条件是:菌液OD值为0.3,侵染15 min,24℃共培养24 h,下胚轴抗性愈伤的转化效率最高。用珂312、中521和农大94-7对所选出的最佳组合进行了进一步验证,结果3个品种在优选组合下的抗性愈伤组织诱导率都很高,分别为93.85%、86.72%和85.83%。

The influences of Agrobacteriurn concentration,infection time, the time and the temperature of co-cultute on the induction ratios of kanamyein-resistant calli were studied by Agrobacterium-mediated transformation using the hypocotyls of Jiwu 2031 as explants. The factors were optimized using orthogonal design. The results showed that the transformation ratio was the highest under the regime of OD=0.3 of bacteria concentration,15 rain of infection time,24 ℃ of co-culture temperature and 24 h of co-culture time. High frequency of kanamycin-resistant calli formation was observed when infecting hypocotyls of Coker 312 ,Zhong 521 and Nongda 94-7 using the optimized combination of factors, and the induction ratios reached 93.85% ,86. 72%and 85.83% ,respectively.