茶树原位转化方法的建立

Establishment of an Efficient in planta Transformation Method for Camellia sinensis

解决茶树缺乏高效、稳定的遗传转化体系问题,建立了一种基于根癌农杆菌介导的茶树原位转化转基因方法,为茶树基因功能研究和种质创新奠定坚实的基础。以茶树品种‘铁观音’、‘白叶1号’和‘龙井43’的实生幼苗为受体材料,进行去顶芽和腋芽处理。利用根癌农杆菌菌液侵染实生苗伤口,通过抗性筛选获得再生芽,经分子生物学鉴定后,采用短穗扦插法获得茶树转基因植株。结果表明,再生芽中GUS(β-glucuronidase)和HYG(hygromycin)标记基因经多次PCR检测均为阳性,经测序验证PCR产物序列与标记基因序列一致。‘铁观音’、‘白叶1号’和‘龙井43’的转化率分别为8.14%、2.99%和2.53%。建立了不依赖茶树组织培养的原位转化转基因体系,具有操作简便、转化率高、成本低、试验周期短的特点。

To address the lack of an efficient and stable genetic transformation system for tea trees,an in planta transformation transgenic method based on Agrobacterium tumefaciens-mediated transformation of tea trees was established,laying a solid foundation for gene function research and germplasm innovation of tea tree.The seedlings of tea cultivar ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ were used as receptor plants,and the apical bud and axillary bud were removed.Using the A.tumefaciens broth infecting the wounds of the seedling,regenerative buds were obtained through resistance screening.After molecular biological identification,transgenic plants were obtained by short-ear cutting method.The results demonstrated that the GUS(-glucuronidase)and HYG(hyglymycin)labeled genes were tested to be positive with multiple PCR,and the PCR product sequence was consistent with the labeled gene sequence.The transformation efficiency of ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ was 8.14%,2.99%,and 2.53%,respectively.This study establishes an in planta transformation method of tea plant that does not rely on tissue culture,and the transformation protocol characterizes of simple operation,high transformation rate,low cost,and short experiment cycle.