'44号'抗虫黑杨基于根段组培再生和遗传转化体系的建立

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中文摘要：[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.

外文标题：Establishment of Regeneration Tissue Culture and Genetic Transformation System by Roots of P.deltoides cl.'Danhong'×P.nigra cl.'44'

外文摘要：[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'seed-lings 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 oxal-ate 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 differenti-ation 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 con-centration for glyphosate resistance in roots was determined to be 0.8 mg·L-1 through a gradient test.Exo-genous 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 trans-formation of black poplar species and adding excellent traits through genetic transformation to clone'44'in particular.

外文关键词：

insect-resistant P.deltoides cl.'Danhong'×P.nigra cl.'44'P.deltoidesP.nigraroot infectiongenetic transformationregeneration tissue cultureGUS staining

作者：

杨松、宋学勤、赵树堂、胡建军、卢孟柱

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作者单位：

浙江农林大学,浙江杭州 311300

中国林业科学研究院林业研究所,北京 100091

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

基金：

国家重点研发计划浙江省"十四五"育种专项林木协作组课题

项目编号：

2021YFD22002052021C02070-1

出版年：

2024

DOI：

10.12403/j.1001-1498.20230502

林业科学研究

中国林业科学研究院

林业科学研究

CSTPCD北大核心

影响因子：0.996

ISSN：1001-1498

年,卷(期)：2024.37(5)

参考文献量21