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目的:建立高效的半夏遗传转化体系。方法:以半夏试管苗的叶柄为外植体,采用根癌农杆菌介导法,探索了酚类物质、农杆菌菌液浓度、侵染时间、预培养时间及共培养时间等对半夏遗传转化效率的影响。结果与结论:不经过预培养阶段,用含AS 40 mg.L-1的根癌农杆菌菌液侵染15 min后共培养3 d时可有效提高遗传转化效率。将转化后的叶柄接到含有100 mg.L-1Kan和350 mg.L-1Carb的分化培养基上进行筛选培养,30 d左右在叶柄的两端分化出抗性的试管小块茎。对抗性转化植株进行Gus染色和PCR检测,结果表明外源基因sHSP已经整合到半夏基因组中。
Objective: To establish an efficient transformation system of Pinellia ternata. Methods: The petiole of Pinellia ternata tube was used as explant. Agrobacterium tumefaciens mediated method was used to explore the effects of phenanthrene, Agrobacterium tumefaciens concentration, infection time, pre-culture time and co- Effect of genetic transformation efficiency. RESULTS AND CONCLUSION: The results showed that the efficiency of genetic transformation could be effectively improved by co-cultivation for 3 days after inoculation with Agrobacterium tumefaciens containing AS 40 mg.L-1 for 15 min without pre-incubation. Transformed petioles were plated on differentiation media containing 100 mg.L-1 Kan and 350 mg.L-1Carb, and resistant tubers were differentiated around the petiole about 30 days later. The transgenic plants were tested by Gus staining and PCR. The results showed that the foreign gene sHSP had been integrated into the Pinellia ternata genome.