农杆菌介导的eGFP基因转化瓜叶菊技术体系的建立

Establishment of Agrobacterium Tumefaciens-mediated Genetic Transformation System of eGFP Gene in Pericallis hybrida

为了建立稳定、高效的瓜叶菊遗传转化体系,本试验以瓜叶菊无菌苗茎段作为外植体,采用农杆菌EHA105介导法转化eGFP基因。探讨了不同菌液浓度,共培养时间,硫酸卡那霉素(Kan)和特美汀(Tim)浓度,乙酰丁香酮(AS)和表面活性剂Silwet L-77浓度,以及浸泡时间、超声波和真空渗入处理对外植体生长与转化率的影响。结果表明:在瓜叶菊叶片中成功检测到eGFP基因的瞬时表达,农杆菌菌液OD600为1.0,共培养3 d时最有利于农杆菌与外植体的融合生长;40 mg/L Kan和200 mg/L Tim可以分别作为有效的外植体筛选压和抑菌浓度;100μmol/L AS和0.03%Silwet L-77能有效地诱导愈伤组织和提高转化率;浸泡30 min、超声波60 s和真空渗入30 s都能促进愈伤组织的形成及提高转化率。

In order to establish a stable and efficient genetic transformation system of Pericallis hybrida B.Nord.,this experiment used the stem segment of sterilized cineraria as an explant,and transformed the eGFP gene by Agrobacterium EHA105-mediated method.The effects of different concentration of bacterium solution,co-culture time,concentration of Kanamycin sulfate(Kan)and Timentin(Tim),concentration of Acetosyringone(AS)and surfactant Silwet l-77,soaking time,ultrasonic wave and vacuum infiltration treatment on the growth and conversion of external grafts were discussed.The results showed that the transient expression of eGFP gene was detected successfully in Pericallis hybrida B.Nord.leaves.The Agrobacterium solution OD600 was 1.0,co-culture time of 3 d is most beneficial to the fusion growth of Agrobacterium tumefaciens and explants.40 mg/L Kan and 200 mg/L Tim can respectively as explants effective to screen pressure and bacteriostatic concentration.100μmol/L AS and0.03%Silwet l-77 can effectively induce callus and improve the conversion rate.Soaking for 30 min,ultrasonic wave for 60 s and vacuum infiltration for 30 s can promote callus formation and increase conversion rate.