海滨雀稗以hpt与bar基因为筛选标记的转化体系比较研究

海滨雀稗作为耐盐性较强的暖季型草坪草，具有极大的应用潜力。为建立高效稳定的海滨雀稗遗传转化体系，本研究以海滨雀稗成熟种子为外植体，确定了筛选剂潮霉素和草丁膦在海滨雀稗的愈伤组织继代培养和再生阶段的最佳筛选压。在进一步优化遗传转化条件的基础上，比较了以hpt与bar基因作为转基因筛选标记对海滨雀稗农杆菌介导的遗传转化效率的影响。结果表明：愈伤组织的再生率在继代36周后显著下降，继代48周的最高再生率为67.9%。潮霉素的最佳筛选压在继代培养阶段为120 mg·L^-1，再生阶段为80 mg·L^-1。草丁膦的最佳筛选压均为1.2 mg·L^-1。选取继代24周的愈伤组织用于农杆菌介导的遗传转化，最佳的转化条件为菌液浓度OD600=0.6，添加100μmol·L^-1乙酰丁香酮和0.01%的Silwet L-77，并辅以超声波处理20 min或真空处理10 min，浸染30min后共培养2 d。通过多次抗性愈伤组织和抗性再生苗的筛选，2种载体均获得了转基因再生植株。通过PCR检测证明hpt和bar基因分别成功在...

Comparative study on transformation systems of seashore paspalum using hpt and bar genes as selection markers

Seashore paspalum is a warm-season turfgrass with strong salt tolerance， and it has a wide range of potential applications. To establish an efficient and stable genetic transformation system for this plant， we used its mature seeds as explants， and determined the optimal selection pressures of hygromycin and glufosinate during callus subculture and the regeneration stage. After optimizing the genetic transformation conditions， we compared the transformation efficiency of hpt and bar genes as selection markers in Agrobacterium-mediated transformation of seashore paspalum. The results showed that the regeneration rate of callus decreased significantly after subculture for 36 weeks， and the highest regeneration rate was 67.9% after subculture for 48 weeks. The optimal concentration of hygromycin for screening was 120 mg·L^-1 at the subculture stage and 80 mg·L^-1 at the regeneration stage. The optimal concentration of glufosinate for screening was 1.2 mg·L^-1. Calli subcultured for 24 weeks were selected for Agrobacterium-mediated genetic transformation. The optimal transformation conditions were as follows： ultrasonic treatment of the explant for 20 min with bacterial solution （concentration OD₆₀₀=0.6） containing 100 μmol·L^-1 acetosyringone and 0.01% Silwet L-77， followed by vacuum treatment for 10 mins， then soaking for 30 mins and co-culturing for 2 days. Through screening multiple resistant calli and resistant regenerated seedlings， we obtained regenerated transgenic plants expressing each vector. Analyses by PCR confirmed that hpt and bar genes were successfully expressed in the respective transformed seashore paspalum materials. The average efficiency of GUS stable expression and PCR detection with hygromycin as the screening agent was 18.9%， and the highest transformation efficiency was 20.3%， which were higher than those obtained using glufosinate as the screening agent. Our results indicate that the hpt gene is more suitable as a selectable marker gene for the genetic transformation of seashore paspalum， as it achieves a higher transformation efficiency.