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<p>This study aimed to optimise an Agrobacterium-mediated transient transformation system for <i>Taxus × media</i> callus tissue, and, for the first time, establish an efficient transient transformation system for its leaves. This seeks to provide reliable technical support for gene functional studies in this species. <i>Agrobacterium</i> GV3101 carrying the pBI121-<i>GUS</i> reporter gene was used to infect callus tissue and young leaves of <i>Taxus</i> × <i>media</i>. For callus tissue, an L₉(3³) orthogonal experimental design was employed to optimise three factors: <i>Agrobacterium</i> suspension concentration (OD<sub>600</sub>), infection time, and co-cultivation time. For leaf tissue, an L₉(3⁴) orthogonal design was employed, incorporating an additional factor: acetylcinnamaldehyde (AS) concentration. Transduction efficiency was comprehensively evaluated by quantifying relative <i>GUS</i> gene expression via qPCR and measuring GUS enzyme activity through an enzyme assay. The optimal conditions for transient transformation of callus tissue were determined to be: bacterial suspension concentration OD<sub>600</sub> = 0.8, infection duration 30&#xa0;min, and co-cultivation time 72&#xa0;h. Under these conditions, <i>GUS</i> gene expression reached its highest level, being five times that of the control group, while GUS enzyme activity peaked at 89.084 U/mg. The optimal conditions for leaf transient transformation were: bacterial suspension concentration OD<sub>600</sub>= 1.0, infection duration 16&#xa0;min, AS addition concentration 200 µmol/L, and co-cultivation for 48&#xa0;h. Under these conditions, <i>GUS</i> gene expression was highest, 5.65 times that of the control group, with GUS enzyme activity reaching a maximum of 58.06 U/mg. This study successfully optimised the transient transformation system for <i>Taxus × media</i> callus tissue and, for the first time, established an efficient transient transformation system for its leaves. This lays a solid foundation for utilising transient transformation technology to investigate gene function and metabolic regulation in <i>Taxus × media</i>.</p>

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Establishment and optimisation of a transient transformation system for Taxus × media

  • 期刊论文

  • 发表日期：

    2026年4月2日

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  • Yang Menglu,
  • Du Yuqing,
  • Xu Fan,
  • Wu Chenwei,
  • Xia Siyu,
  • Fan wei,
  • Liu Shuai,
  • Qiu Deyou,
  • Yang Yanfang

摘要

This study aimed to optimise an Agrobacterium-mediated transient transformation system for Taxus × media callus tissue, and, for the first time, establish an efficient transient transformation system for its leaves. This seeks to provide reliable technical support for gene functional studies in this species. Agrobacterium GV3101 carrying the pBI121-GUS reporter gene was used to infect callus tissue and young leaves of Taxus × media. For callus tissue, an L₉(3³) orthogonal experimental design was employed to optimise three factors: Agrobacterium suspension concentration (OD600), infection time, and co-cultivation time. For leaf tissue, an L₉(3⁴) orthogonal design was employed, incorporating an additional factor: acetylcinnamaldehyde (AS) concentration. Transduction efficiency was comprehensively evaluated by quantifying relative GUS gene expression via qPCR and measuring GUS enzyme activity through an enzyme assay. The optimal conditions for transient transformation of callus tissue were determined to be: bacterial suspension concentration OD600 = 0.8, infection duration 30 min, and co-cultivation time 72 h. Under these conditions, GUS gene expression reached its highest level, being five times that of the control group, while GUS enzyme activity peaked at 89.084 U/mg. The optimal conditions for leaf transient transformation were: bacterial suspension concentration OD600= 1.0, infection duration 16 min, AS addition concentration 200 µmol/L, and co-cultivation for 48 h. Under these conditions, GUS gene expression was highest, 5.65 times that of the control group, with GUS enzyme activity reaching a maximum of 58.06 U/mg. This study successfully optimised the transient transformation system for Taxus × media callus tissue and, for the first time, established an efficient transient transformation system for its leaves. This lays a solid foundation for utilising transient transformation technology to investigate gene function and metabolic regulation in Taxus × media.