根癌农杆菌介导的马铃薯DM茎段遗传转化条件的优化

Optimization on the Factors Affecting Agrobacterium Tumefacien Mediated Transformation in Stem Segments of DM Potato

本研究利用以双单倍体马铃薯DM的茎段为试验材料,对影响马铃薯遗传转化的条件进行了优化,主要从对通过抗生素浓度、预培养时间、侵染时间、农杆菌浓度和共培养时间等几个因素进行了的分析,对影响马铃薯遗传转化的条件进行了优化。研究表明:头孢霉素(cef)的浓度在600 mg/L时既不影响愈伤的正常生长还能有效的抑制农杆菌的生长。潮霉素(hyg)浓度在8 mg/L时对愈伤达到半致死状态,是因为较低的潮霉素浓度无法起到对抗性愈伤的筛选作用,所以合适的浓度应为愈伤达半致死状态浓度。得到更多抗性愈伤的最佳条件为:外植体预培养时间13天,侵染时间为8 min,农杆菌浓度以OD600=0.4左右,且共培养时间3天。上述的条件的优化大大的提高了马铃薯DM的遗传转化效率。

In this research, we optimized the conditions which affected the genetic transformation of potato through analysis on the factors of the concentration of antibiotics, pre-culture time, infection time, concentration of agrobacterium tumefaciens and co-culture time etc by using stem segments of the double haploid potato DM as experimental materials. The results showed that when the concentration of cef was 600 mg/L, it could not affect the normal growth of the callus, while inhibited the growth of Agrobacterium instead. When the concentration of hyg was 8 mg/L, the callus was half lethal, because the low concentration of the hyg could not play the role in screening of antagonistic callus, then the suitable condition should be the half lethal state concentration. The best conditions for getting more resistant callus were 13 days of explant pre-culture time, 8 minutes of infection time,Agrobacterium concentration about OD_(600)=0.4 and the co-culture time 3 days. The optimization of the above conditions greatly improved the efficiency of genetic transformation of potato DM.