农杆菌介导的菜心遗传转化体系的建立

Establishment of efficient transformation system of Brassica parachinensis mediated by Agrobacterium tumefacien

以建立的菜心高频再生体系为基础,利用GU S染色组织分析法研究了根癌农杆菌菌株、乙酰丁香酮(A S)、预培养时间、浸染时间和共培养时间等因素对菜心((B rassica camp estris L.var.p arach inesis)子叶遗传转化的影响,探讨了适宜菜心转化的卡那霉素与抑菌抗生素使用浓度。结果表明,农杆菌菌株AGL 0对菜心的浸染能力最强,添加100μm o l/L的乙酰丁香酮有利于菜心转化;子叶外植体预培养2 d后浸染(菌液浓度OD600值为0.8)15m in,共培养2 d,然后转移到含15 m g/L卡那霉素和100 m g/L T icarc illin的筛选培养基上,进行转化植株的再生,植株再生率及外植体GU S阳性率均较高,是较优的转化条件;本试验共获得8株转化植株,转化率达2.44%,经PCR分析和Sou thern杂交检测表明,GU S基因已整合进入菜心基因组。

Based on the established regeneration system,an efficient transformation and screening system of Brassica parachinensis mediated by Agrobacterium was developed. Four-day-old cotyledon served as excellent explants for transformation,and the factors affecting the transformation frequency,such as the kinds and concentrations of antibiotics to inhibit the growth of Agrobacterium, Agrobacterium strains, precuhure time of cotyledon explants,concentration of Agrobacterium ,infection time and co-infection time etc. were also discussed by GUS coloration analysis. 2 days precuhuration, 15 min Agrobacterium （OD600= 0.8） infection with 100μmol/L acetosyringone （AS）,and 2 days＇ co-infection would lead to an increase in transformation efficiency. The differential medium with kanamycin 15 mg/L and Ticarcillin 100 mg/L was found to be most optimal for transgenic plantlets selection. Plantlets resistant to kanamycin were obtained and 8 transgenetic plants were obtained. Stable integration of GUS gene in these plants were confirmed by PCR and Southern blotting analysis.