农杆菌介导薄皮甜瓜遗传转化体系建立

摘要

以薄皮甜瓜"M15"和"M43"子叶节为外植体,研究激素浓度、卡那霉素浓度和消毒时间等对甜瓜子叶节再生及农杆菌介导遗传转化的影响.结果表明,诱导甜瓜子叶节不定芽分化最佳激素组合为0.5 mg·L-16-BA+0.05 mg·L-1IAA,芽伸长激素为0.05 mg·L-16-BA,生根激素为0.1 mg·L-1IAA.农杆菌介导甜瓜转化最佳条件为预培养48 h,OD600=0.6农杆菌菌液侵染15 min,黑暗条件下共培养48～72 h,头孢抑菌浓度500 mg·L-1,卡那霉素浓度75 mg·L-1.PCR检测卡那霉素抗性苗,验证外源基因已整合进入甜瓜基因组,"M15"和"M43"抗性植株阳性率分别为84.2％和77.4％.%Using cotyledon nodes of muskmelon "M15" and "M43" as explants, the factors that affected melon cotyledon regeneration and the efficiency of genetic transformation were studied, such as hormone concentration, kanamycin concentration, and disinfect time. The results showed that the best hormone combination of adventitious bud differentiation in cotyledons of muskmelon was 0.5 mg· L-16-BA+0.05 mg· L-1 IAA, shoot elongation hormone was 0.05 mg· L-16-BA and rooting hormone was 0.1 mg· L-1 IAA. The best condition for Agrobacterium mediated transformation of muskmelon was pre-culture for48 h, OD600=0.6 Agrobacterium microbial infection for 15 min, co-cultivate for 48-72 h in the dark, cephalosporins bacteriostatic concentration of 500 mg· L-1, kanamycin concentration of 75 mg· L-1. It was originally proved by PCR that expression cassettes had been transferred into the genome, the positive rate of "M15" and "M43" were 84.2％ and 77.4％, respectively.