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<p><i>Blumea balsamifera</i> (L.) DC. is a high-value aromatic medicinal plant, known for its content of L-borneol and blumeatin, compounds that demonstrate substantial therapeutic potential for conditions such as stroke. However, the absence of an efficient and robust genetic transformation protocol has significantly impeded advances in functional genomics and metabolic engineering research in this species. Herein, we report the establishment of an efficient, single-step, non-aseptic hairy root transformation protocol mediated by <i>Rhizobium rhizogenes</i> strain K599, achieving PCR-confirmed transgenic hairy roots within 40 days post-inoculation. Two distinct inoculation methodologies – surface application onto apical bud incisions (smearing method) and injection into axillary buds (injection method) – were systematically evaluated at three bacterial optical densities (OD<sub>600</sub> = 0.4, 0.6, 0.8). The enhanced yellow fluorescent protein (<i>eYGFPuv</i>) reporter gene facilitated single-step visual screening under ultraviolet illumination. Results demonstrated that injection at an OD<sub>600</sub> of 0.8 yielded optimal transformation efficiency. Plant survival rate, root induction rate, and transformation efficiency all surpassed 95%. Approximately 34% of the induced hairy roots exhibited reporter gene expression, with a mean of 17 roots per transformed plant. In comparison with the surface application method, injection enhanced the proportion of transformed plants and the number of transgenic roots by approximately 1.6-fold and 1.3-fold, respectively, and eliminated the need for sterile tissue culture conditions. This transformation system exhibits compatibility with CRISPR/Cas-based gene-editing vectors and multi-gene synthetic expression cassettes, making it suitable for high-throughput functional gene characterization and the targeted metabolic engineering of terpenoid and flavonoid biosynthetic pathways in <i>B. balsamifera.</i> Furthermore, this optimized workflow establishes a transferable paradigm for functional genomics investigations and the metabolic studies of bioactive compounds in other recalcitrant medicinal plant species.</p>

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Rhizobium rhizogenes-mediated genetic transformation for hairy root induction of Blumea balsamifera

  • 期刊论文

  • 发表日期：

    2025年10月30日

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  • Qiumei Luo,
  • Sihong Sang,
  • Minghui Huang,
  • Kailang Mu,
  • Fei Ran,
  • Shan Sha,
  • Changmao Guo,
  • Jie Zou,
  • Yuchen Liu,
  • Yuxin Pang

摘要

Blumea balsamifera (L.) DC. is a high-value aromatic medicinal plant, known for its content of L-borneol and blumeatin, compounds that demonstrate substantial therapeutic potential for conditions such as stroke. However, the absence of an efficient and robust genetic transformation protocol has significantly impeded advances in functional genomics and metabolic engineering research in this species. Herein, we report the establishment of an efficient, single-step, non-aseptic hairy root transformation protocol mediated by Rhizobium rhizogenes strain K599, achieving PCR-confirmed transgenic hairy roots within 40 days post-inoculation. Two distinct inoculation methodologies – surface application onto apical bud incisions (smearing method) and injection into axillary buds (injection method) – were systematically evaluated at three bacterial optical densities (OD600 = 0.4, 0.6, 0.8). The enhanced yellow fluorescent protein (eYGFPuv) reporter gene facilitated single-step visual screening under ultraviolet illumination. Results demonstrated that injection at an OD600 of 0.8 yielded optimal transformation efficiency. Plant survival rate, root induction rate, and transformation efficiency all surpassed 95%. Approximately 34% of the induced hairy roots exhibited reporter gene expression, with a mean of 17 roots per transformed plant. In comparison with the surface application method, injection enhanced the proportion of transformed plants and the number of transgenic roots by approximately 1.6-fold and 1.3-fold, respectively, and eliminated the need for sterile tissue culture conditions. This transformation system exhibits compatibility with CRISPR/Cas-based gene-editing vectors and multi-gene synthetic expression cassettes, making it suitable for high-throughput functional gene characterization and the targeted metabolic engineering of terpenoid and flavonoid biosynthetic pathways in B. balsamifera. Furthermore, this optimized workflow establishes a transferable paradigm for functional genomics investigations and the metabolic studies of bioactive compounds in other recalcitrant medicinal plant species.