技术方法

发根农杆菌介导的芙蓉菊高效遗传转化体系的建立

  • 郝梦冉 ,
  • 柳志勇 ,
  • 孙艳艳 ,
  • 孙明秀 ,
  • 张静 ,
  • 宋爱萍 ,
  • 王海滨 ,
  • 陈发棣 ,
  • 王振兴
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  • 1南京农业大学园艺学院, 南京 211800; 2国家南方草本花卉种质资源圃(南京), 南京 211800

收稿日期: 2025-09-10

  修回日期: 2025-11-20

  网络出版日期: 2025-12-08

基金资助

国家重点研发计划项目(No.2024YFD1200503)、江苏省农业科技自主创新项目(No.CX(23)3002)和中央高校基本业务费(No.YDEX2025038)

Establishment of an Agrobacterium rhizogenes-mediated Genetic Transformation System in Crossostephium chinense

  • HAO Meng-Ran ,
  • LIU Zhi-Yong ,
  • XUN Yan-Yan ,
  • XUN Meng-Xiu ,
  • ZHANG Jing ,
  • SONG Ai-Ping ,
  • YU Hai-Bin ,
  • CHEN Fa-Ti ,
  • YU Zhen-Xin
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  • 1College of Horticulture,Nanjing Agricultural University, Nanjing 211800, China; 2National Herbaceous Flower Germplasm Repository (Nanjing), Nanjing 211800, China

Received date: 2025-09-10

  Revised date: 2025-11-20

  Online published: 2025-12-08

摘要

芙蓉菊(Crossostephium chinense)为菊科芙蓉菊属多年生银叶植物, 作为典型的药赏兼用植物, 其表现出极强的环境适应性, 如耐盐、耐旱、抗虫, 是研究植物抗性机制和观赏性状遗传改良的重要材料。但芙蓉菊遗传转化体系尚不完善, 严重制约了其种质创新和功能基因组学研究的深入开展, 以及优异性状功能基因的挖掘与利用。该研究以芙蓉菊为实验材料, 通过组织培养和非组织培养的方法建立了发根农杆菌(Agrobacterium rhizogenes) K599介导的高效遗传转化体系。结果表明, 以茎段为外植体, 组织培养与非组织培养条件下的毛状根诱导率均可达18%; 而以无根苗为外植体, 在Wus2-IPT辅助下, 无需植物生长调节剂即可直接获得再生植株, 且毛状根诱导率提高至38.95%, 显著提升了转化效率与操作便捷性。该系统整合了Wus2-IPT发育调控模块和可视化报告基因RUBY, 实现了转基因毛状根的高效诱导与阳性材料的快速筛选, 突破了传统方法依赖植物生长调节剂和基因型限制的局限, 为芙蓉菊基因功能解析和种质创新提供了重要技术支撑, 并具有推广至其它菊科植物遗传改良的应用潜力。

本文引用格式

郝梦冉 , 柳志勇 , 孙艳艳 , 孙明秀 , 张静 , 宋爱萍 , 王海滨 , 陈发棣 , 王振兴 . 发根农杆菌介导的芙蓉菊高效遗传转化体系的建立[J]. 植物学报, 2026 , 61(6) : 1 -0 . DOI: 10.11983/CBB25165

Abstract

INTRODUCTION: Plant genetic transformation systems serve as fundamental technical platforms for functional genomics research and molecular breeding. Crossostephium chinense, a valuable medicinal-ornamental species within the Asteraceae family, is characterized by its distinctive silvery-white foliage and exhibits multiple stress-resistant traits, including salt tolerance, drought resistance, and insect resistance. However, the absence of an efficient genetic transformation system has significantly impeded progress in functional gene studies and molecular breeding of this species. This study reports the first establishment of an Agrobacterium rhizogenes-mediated genetic transformation system for C. chinense, aiming to overcome this technical barrier. 

RATIONALE: Agrobacterium rhizogenes-mediated transformation has been widely adopted in diverse plant species due to its operational simplicity and short experimental cycle. Nevertheless, its application in Asteraceae plants, particularly in C. chinense, remains largely unexplored. In this study, we innovatively integrated the visual reporter system RUBY with developmental regulators Wus2 and IPT. A systematic comparison was conducted between tissue culture and non-tissue culture approaches, and the transformation efficiencies of different explant types—including rootless seedlings with attached cotyledons and stem segments—were evaluated. This work provides a comprehensive technical framework for establishing an efficient and stable genetic transformation system in C. chinense. 

RESULTS: Under tissue culture conditions, rootless seedlings with cotyledons attached exhibited superior transformation efficiency as explants, achieving a positive hairy root induction rate of 38.95% and successfully yielding transgenic regenerated plants. This efficiency was significantly higher than that of stem segment explants (17.45%). In non-tissue culture settings, the cut-dip-budding (CDB) method resulted in a (18.8±5.46)% hairy root induction rate using stem segments. Notably, the application of Wus2-IPT developmental regulators effectively promoted direct shoot organogenesis independent of plant growth regulators, thereby streamlining the regeneration process. 

CONCLUSION: This study successfully establishes the first efficient Agrobacterium rhizogenes-mediated genetic transformation system for C. chinense, identifying tissue-cultured rootless seedlings with attached cotyledons as the optimal explant type. The developed system not only provides a critical tool for gene functional analysis and molecular breeding in C. chinense but also offers a valuable reference for genetic transformation studies in other Asteraceae species. This breakthrough is expected to significantly accelerate the exploitation of elite gene resources and enhance germplasm innovation in C. chinense.

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