‘44号’抗虫黑杨基于根段组培再生和遗传转化体系的建立  

Establishment of Regeneration Tissue Culture and Genetic Transformation System by Roots of P.deltoides cl.‘Danhong’×P.nigra cl.‘44’

作　　者：杨松 宋学勤[2] 赵树堂[2] 胡建军[2] 卢孟柱 YANG Song;SONG Xue-qin;ZHAO Shu-tang;HU Jian-jun;LU Meng-zhu(Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China)

机构地区：[1]浙江农林大学,浙江杭州311300 [2]中国林业科学研究院林业研究所,北京100091

出　　处：《林业科学研究》2024年第5期74-84,共11页Forest Research

基　　金：国家重点研发计划“林木多性状高效聚合育种技术”(2021YFD2200205)国家级;浙江省“十四五”育种专项林木协作组课题(2021C02070-1)省市级。

摘　　要：[目的]以美洲黑杨和转BtCry1Ac欧洲黑杨的杂交子代‘44号’抗虫黑杨作为试验材料,采用无菌苗根基部为外植体,建立高效稳定的组培再生体系和黑杨遗传转化体系。[方法]以‘44号’抗虫黑杨无菌苗0.5~1.0 cm的根基部为试验材料,研究不同激素配比对根系不定芽的诱导和生根的影响,探索根系对草铵膦的临界耐受浓度,以农杆菌介导法进行遗传转化,并通过β-葡萄糖苷酸酶(GUS)基因染色实时监控和鉴定阳性植株。[结果]筛选出‘44号’抗虫黑杨根系诱导不定芽最优培养基为:WPM+20 g·L^(-1)蔗糖+7.8 g·L^(-1)琼脂+0.25 mg·L^(-1)6-BA+0.05 mg·L^(-1) NAA,分化率为86.67%;不定芽生根的最优培养基为:WPM+0.075 mg·L^(-1) NAA+7 g·L^(-1)琼脂+0.1 g·L^(-1) AC,生根率为75.00%。通过根系草铵膦抗性筛选梯度试验,确定根系对草铵膦筛选最适遗传转化筛选浓度为0.8 mg·L^(-1)。采用农杆菌介导法将外源基因转入到根中,经不定芽诱导和生根两个阶段获得再生植株苗,经特异引物进行PCR鉴定,31个外植体中阳性植株为9株,转化效率为29.0%。[结论]建立了‘44号’抗虫黑杨根系的再生和遗传转化体系,为黑杨派杨树的高效基因转化提供了途径的同时,也为以‘44号’抗虫黑杨为基础,通过转基因技术聚合更多的优良性状,提供了重要的技术支撑。[Objective]P.deltoides cl.‘Danhong’×P.nigra cl.‘44’was used to establish an efficient and stable regeneration tissue culture system and genetic transformation system for black poplars(P.deltoids×P.nigra),based on the roots as explants.[Method]The 0.5~1.0 cm root segments from clone‘44’seedlings were used to induce adventitious buds.The effects of different hormone ratios on the induction and rooting of adventitious buds were investigated and the critical screening concentration of phosphine oxalate for genetic transformation of the root segments was explored.Agrobacterium-mediated method was employed to introduce foreign genes into the clone‘44’root cells,and the transformed seedlings were identified by β-glucuronidase staining.[Result]The optimal medium for bud differentiation of clone‘44’was WPM+20 g·L^(-1) sucrose+7.8 g·L^(-1) agar+0.25 mg·L^(-1)6-BA+0.05 mg·L^(-1) NAA,and the differentiation rate was 86.67%.The optimal medium for seedling rooting was WPM+0.075 mg·L^(-1) NAA+7 g·L^(-1) agar+0.1 g·L^(-1) AC,and the rooting rate was 75.00%.The optimal genetic transformation screening concentration for glyphosate resistance in roots was determined to be 0.8 mg·L^(-1) through a gradient test.Exogenous genes were introduced into roots by Agrobacterium-mediated method,and the specific PCR primers were designed for identification of the transgenic lines.Nine transgenic plants were obtained from 31 explants,with a transformation efficiency of 29.0%.[Conclusion]The efficient regeneration and genetic transformation system for clone‘44’has been established,which provides a technical support for transformation of black poplar species and adding excellent traits through genetic transformation to clone‘44’in particular.

关 键 词：‘44号’抗虫黑杨(P.deltoides cl.‘Danhong’×P.nigra) 美洲黑杨 欧洲黑杨 根系侵染 遗传转化 组培再生 GUS染色 

分 类 号：S722[农业科学—林木遗传育种] S792.11[农业科学—林学]