白城小黑杨遗传转化体系建立及其应用

Establishment and Application of Genetic Transformation System for Populus simonii×P.nigra‘Baicheng’

以白城小黑杨(Populus simonii×P.nigra‘Baicheng’)组培苗茎段为外植体,MS为基本培养基,通过调节不同植物生长激素6-BA、NAA、TDZ和IBA浓度诱导其离体再生。基于组织培养系统,经过卡那霉素浓度筛选、侵染时间确定,最终建立适用于白城小黑杨的农杆菌(Agrobacterium tumefaciens)介导的遗传转化体系。利用该系统成功创制了杨树应拉木形成关键调控基因LBD39(Lateral Organ Boundaries Domain)的过量表达转基因植株。结果表明:白城小黑杨组织培养体系包括不定芽分化诱导、抽茎诱导、生根诱导3个阶段,不定芽分化诱导最适培养基为MS+0.5 mg·L^(-1)6-BA+0.1 mg·L^(-1)NAA+0.001 mg·L^(-1)TDZ,分化率92.6%;抽茎诱导最适培养基为MS+0.2 mg·L^(-1)6-BA+0.05 mg·L^(-1)NAA,抽茎增值系数6.5;生根诱导最适培养基为1/2 MS+0.4 mg·L^(-1)IBA,生根率100%。遗传转化最适卡那霉素质量浓度为30 mg·L^(-1)、最佳侵染时间为20 min,经30 d不定芽诱导、15~30 d抽茎诱导、25 d生根诱导即可成功获得转基因植株,GUS基因的转化效率为2%。应用此遗传转化体系,对杨树应拉木关键调控基因LBD39进行了过表达植株创制,最终获得5株过表达植株,转化效率为3.3%。

Stems of Populus simonii×P.nigra‘Baicheng’in vitro plants were selected as explants and MS medium was used to establish tissue culture system by adjusting the hormone concentrations of 6-BA,NAA,TDZ and IBA.Based on the tissue culture system,the optimal concentration of kanamycin and infection time were confirmed to establish Agrobacterium tumefaciens-mediated genetic transformation system for P.simonii×P.nigra‘Baicheng’.By using this system,the transgenic plants overexpressed a key tension wood formation regulator LBD39(Lateral Organ Boundaries Domain)were created successfully.The results showed that the tissue culture system consisted of three stages,including adventitious bud differentiation induction(MS+0.5 mg·L^(-1)6-BA+0.1 mg·L^(-1)NAA+0.001 mg·L^(-1)TDZ,shoot differentiation rate=92.6%),stem induction(MS+0.2 mg·L^(-1)6-BA+0.05 mg·L^(-1)NAA,multiplication coefficient=6.5)and rooting induction(1/2 MS+0.4 mg·L^(-1)IBA,rooting percentage=100%).The optimal kanamycin concentration for genetic transformation was 30 mg·L^(-1)and the optimal infection time was 20 min,and the transgenic plants were obtained successfully after 30 d of adventitious bud differentiation induction,15-30 d of stem induction and 25 d of rooting induction with 2%transformation efficiency respectively.By using this system,five overexpressed plants of LBD39 were obtained,and the transformation efficiency was 3.3%.