根癌农杆菌介导万寿菊遗传转化体系的建立

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold(Tagetes erecta)

以万寿菊(Tagetes erecta)里程碑·黄色的小叶为外植体,采用农杆菌介导法探究抗生素浓度、菌株类型、菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度和抗褐化剂种类对万寿菊遗传转化效率的影响。结果表明,头孢霉素(Cef)和硫酸卡那霉素(Kan)的适宜浓度分别为100 mg·L^(-1)和10 mg·L^(-1);EHA105菌株的稳定转化效率最高;菌液浓度OD600=0.1、侵染5分钟及共培养1天为最佳侵染条件。此外,在侵染过程中添加100μmol·L^(-1)乙酰丁香酮(AS)和筛选培养基中添加0.2 g·L^(-1)聚乙烯吡咯烷酮(PVP)均能提高出芽率。经GUS染色、PCR检测以及Southern blot检测证明转化成功,转化效率达到4%左右。研究结果为万寿菊基因功能研究和转基因育种奠定了基础。

In this study,we used the leaflets of marigold(Tagetes erecta)Milestone Yellow as explants to investigate the major factors impacting the transformation efficiency of Agrobacterium-mediated method.The factors included antibiotic concentration,strain type,bacterial concentration,infection time,co-culture time,acetosyringone concentration and anti-browning agent type.We found that the suitable concentrations of cefotaxim sodium salt and kanamycin sulfate were 100 mg·L^(-1) and 10 mg·L^(-1),respectively.We also found that the strain EHA105 had the highest transformation efficiency up to 4%.The best infection conditions for EHA105 were at bacterial concentration of 0.1 for OD600,infected for 5 minutes,and co-cultured for 1 day.In addition,the bud germination rate could be improved by both applying 100μmol·L^(-1) acetosyringone during the infection process and adding 0.2 g·L^(-1) polyvinyl pyrrolidone in the screening medium.This study laid a foundation for marigold gene function research and transgenic breeding.