农杆菌介导小黑杨花粉植株转化体系的建立

Establishment of agrobacterium-mediated transformation System for Populus simonii×P.nigra plants

通过对影响根癌农杆菌介导小黑杨花粉植株转化效果各种因素的研究,建立了小黑杨花粉植株高效稳定的转化体系。研究结果表明:小黑杨花粉植株叶片外植体在分化培养基中预培养2 d,用OD600值为0.1的工程菌液侵染3～5min,黑暗条件下共培养2～3 d,采用适宜的脱菌方法有利于提高遗传转化率,而用在培养基中加入CaCl2则不利于遗传转化;叶片外植体与农杆菌共培养后,经过抗性芽的诱导、叶片的再分化和根的再生,3个月后即可获得抗性苗;抗性植株经PCR和Southern斑点杂交检测,表明外源基因已转入小黑杨花粉植株的基因组中。

An efficient and stable Agrobacterium-mediated genetic transformation system for Populus simoniixP.nigra plants was established through investigating the factors influencing the transformation efficiency.The results showed that the leaves were pre-cuhured in differentiation medium for 2 days,and dipped the leaves in the Agrobacterium （OD600=0.1） for 3-5 minutes,and co-cultured in darkness for 2-3 days,then it could get the highest transformation frequency;CaCl2 had an adverse effect on transformation efficiency.In addition,the optimum method of disinfection could improve transformation efficiency.By using the optimized conditions,hygromycin resistant plants could be obtained within 3 months from the infection of the leaf explants with Agrobacterium. PCR and Southern-blot analysis confirmed that transgene were integrated into the genome of Populus simoniixP. nigra plants.