潮霉素浓度和农杆菌浸泡时间对黄瓜外植体再生的影响

摘要

Based on the regeneration system established,the Cucumis sativus cv.inbred line M8 which has strong capability of regeneration was used as experiment material to transform invertase inhibitor INH gene into cucumber by using Agrobacterium tumefaciens-mediated gene transfer.Cotyledon explants were immersed in an Agrobacterium suspension and inoculated in the select medium that was on the concentration gradient of 1 mg·L-1 to 10 mg·L-1 to study the resistance and sensitivity of the cotyledon explants to hygromycin.The effect of immersing time on the conversion efficiency for 5 min,10 min,15 min,20 min with Agrobacterium suspension was studid.The results showed that the highest regeneration frequency was 18.9％ when the choice of the HygB pressure levels was 6 mg·L-1 and the infection time was 15 min.The PCR analysis showed that the conversion of INT gene had been integrated into the cucumber genome with the conversion rate of 2.0％.%选用再生能力较强的黄瓜自交系M8为试验材料,在再生体系建立的基础之上,采用根癌农杆菌介导法将转化酶抑制子基因转入黄瓜中.将农杆菌菌液浸泡过的子叶节分别接种在浓度梯度为1 mg· L-1到10 mg·L-1的选择培养基上,研究子叶节对潮霉素的耐受性和敏感度.使用农杆菌菌液浸泡5,10,15,20min,分别接种到筛选培养基上,研究浸泡时间对转化效率的影响.结果表明:HygB的选择压浓度为6mg· L-1,侵染时间为15min时,再生频率最高,为18.9％.经PCR检测表明转化酶抑制子基因已经整合到黄瓜基因组中,转化率为2.0％.