美洲黑杨杂种优良无性系转抗虫基因(Bt和CpTI)的研究

Transformation of Populus×euramericana cv. ‘Nanlin895' Using Bt and CpTⅠ Insect-resistant Genes

以美洲黑杨杂种优良无性系南林895杨(Populus×euramericanac‘v.Nanlin895’)为转基因受体材料,以嫩芽或腋芽为外植体材料组织培养再生植株,利用农杆菌介导法转化Bt基因和CpTI基因。结果显示较合适的组培再生与遗传转化系统为:叶分化培养基为MS+6-BA0.5mg/L+TDZ0.002mg/L,芽伸长培养基为MS+6-BA0.2mg/L+TDZ0.001mg/L+Km10mg/L+Carb500mg/L,生根培养基为1/2MS;预培养3d,菌液浓度OD6001.0～1.3左右,侵染时间20min,共培养4d,叶盘转化频率可达28.7%。对Kmr植株经PCR分析,筛选获得了18株整合有Bt基因和1株整合有CpTI基因的转基因植株。部分转基因植株的初步饲虫实验表明,饲喂转基因杨树叶片可明显抑制杨小舟蛾的生长发育。

Populus×euramericana cv.‘Nanlin895 ＇, an elite clone of Populus deltoides hybrid was choose as a receipt for gene transformation. Popar buds were used as explants for tisse culture. Bt and CpTI gene were transferred into Nanlin895 by Agrobacterium mediated transformation method. The regeneration system was MS＋6-BA 0.5mg/L ＋TDZ 0.002mg/L for bud differentiation, MS＋6-BA 0.2mg/L＋TDZ 0.001mg/L＋Km 10mg/L＋Carb 500mg/L for shoot growth and 1/2MS for rooting. An effective protocol in optimized conditions was developed which were 3 days for pre-culture time, OD600 1.0-1.3 for bacterium liquid concentration, 20 minutes for infection time and 4 days for co-culture time and the efficiency of transformation reached to 28.7%. 18 transgenic plants with presence of Bt gene and 1 transgenic plants with presence of Cp TI gene were obtained from Kanamycin resistant （Km^r） plantlets by PCR analysis. The result of insect bioassay showed that some transgenic plants were extremely resistant to development of the larvae of Micrornelalopha troglodyte.