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目的:在建立灰毡毛忍冬再生体系的基础上,建立其遗传转化体系。方法:组织培养方法培养幼苗,根癌农杆菌介导方法转化外植体,gus染色和PCR检测报告基因。结果:侵染时间8 min能获得较理想的转化效果。35 mg/L的卡那霉素和600 mg/L头孢霉素能得到很好的筛选效果,起主导作用的因素是卡那霉素浓度,其不同的浓度间抗性芽诱导率达到了极显著差异。对转基因植株进行gus染色和PCR检测,结果表明gus基因已整合到灰毡毛忍冬基因组中。结论:首次建立了根癌农杆菌(Agrobacterium tumefaciens,EHA105菌株)介导的叶片为受体的遗传转化体系。
OBJECTIVE: To establish a genetic transformation system based on the establishment of regeneration system of Lonicera macranthoides. Methods: Tissue culture was used to cultivate seedlings, Agrobacterium tumefaciens-mediated transformation of explants, gus staining and PCR detection of the reporter gene. Results: Infection time 8 min can achieve better conversion effect. 35 mg / L of kanamycin and 600 mg / L of cefotaxime can get a good screening effect, the leading factor is the concentration of kanamycin, the different concentrations of resistant bud induction rate reached the pole Significant differences. The transgenic plants were gus stained and PCR, the results showed that the gus gene has been integrated into the genome of Lonicera macaulus. Conclusion: The genetic transformation system mediated by Agrobacterium tumefaciens (strain EHA105) was established for the first time.