发根农杆菌介导的虎杖转基因体系的优化

为建立虎杖高效遗传转化体系，用携带有植物表达载体pCAMBIA1301的发根农杆菌ATCC15834侵染虎杖无菌苗获得毛状根。对影响虎杖毛状根诱导的因素如外植体种类、农杆菌活力、培养基种类、超声波辅助时间进行优化以获得最佳培养条件。通过潮霉素筛选阳性毛状根并确定最佳潮霉素浓度。结果表明以叶片为外植体，1/2MS为培养基，菌液OD600=0.8，超声波辅助5s时，虎杖遗传转化毛状根效果最佳。筛选出的毛状根经GUS染色和PCR分子鉴定表明，该方法的阳性转化率为92%。该研究优化了虎杖毛状根的培养条件，为后期实验探究建立了更加高效的虎杖转基因体系。

Optimization of Agrobacterium rhizogenes-mediated Polygonum cuspidatum transgenic system

In order to establish a highly efficient genetic transformation system of Polygonum cuspidatum, Agrobacterium rhizogenes ATCC15834 carrying gene expression vector pCAMBIA1301 was used to infect the aseptic seedling of Polygonum cuspidatum to obtain hairy roots. The factors affecting the induction of hairy roots were optimized, such as explants types, agrobacterium activity, culture medium and ultrasonic assisted time. The positive hairy roots were screened by hygromycin and the optimal hygromycin concentration was determined. When leaves were used as explants, 1/2MS as culture medium, OD600 is 0.8, ultrasonic-assisted for 5s, Polygonum cuspidatum genetic transformation effect was the best. The hairy roots were identified by GUS staining and PCR. The positive transformation rate was 92%. This study optimized the culture conditions of Polygonum cuspidatum hairy roots, and established a more efficient transgenic system for later experimental exploration.