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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 × euramericanac’v.Nanlin895 ’was used as the transgenic material, and the regenerated plants were made of shoots or axillary buds as explants. Agrobacterium-mediated transformation Bt gene and CpTI gene. The results showed that the more appropriate system for tissue regeneration and genetic transformation was: leaf differentiation medium MS + 6-BA0.5mg / L + TDZ0.002mg / L, bud elongation medium MS + 6-BA0.2mg / L + TDZ0.001mg / L + Km10mg / L + Carb500mg / L, rooting medium was1 / 2MS; pre-culture 3d, bacterial concentration OD6001.0 ~ 1.3, infection time20min, co- Up to 28.7%. 18 Kmr plants were screened by PCR and Bt genes and 1 CpTI gene-integrated transgenic plants were screened. Preliminary experiments of feeding insects on some transgenic plants showed that the feeding of transgenic poplar leaves could significantly inhibit the growth and development of A. niloticus.