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<p><i>Hosta plantaginea</i> is a perennial shade-tolerant herb of the Liliaceae family, with high ornamental and urban greening value. <i>Hosta ‘Light Star’</i> is a newly developed ornamental cultivar with yellow-margined leaves and lilac flowers, but no efficient in vitro regeneration or genetic transformation system has been established for this cultivar to date. In this study, we established a highly efficient in vitro regeneration system for <i>Hosta ‘Light Star,’</i> and developed an Agrobacterium-mediated genetic transformation protocol using the <i>RUBY</i> visual reporter gene for non-invasive screening of positive transformants. The optimal callus induction medium was MS + 2&#xa0;mg/L 6-BA + 0.3&#xa0;mg/L NAA + 0.05&#xa0;mg/L 2, 4-D, with a callus induction rate of 53.33% for leaf explants (the optimal explant for sterile seedlings). The optimal adventitious bud proliferation medium was MS + 2&#xa0;mg/L 6-BA + 0.1&#xa0;mg/L NAA, with a proliferation coefficient of 5.87. The optimal rooting medium was 1/2 MS + 0.5&#xa0;mg/L NAA + 0.5&#xa0;mg/L IBA, with a 100% rooting rate. The optimal transplant substrate was perlite:vermiculite = 2:1, with a 100% transplant survival rate after acclimatization. For Agrobacterium-mediated transformation, the optimal infection parameters were as follows: Agrobacterium suspension OD<sub>600</sub> = 0.6, infection time of 10&#xa0;min, and 200&#xa0;μM acetosyringone; the optimal selection conditions were 300&#xa0;mg/L cefotaxime for bacteriostasis and 30&#xa0;mg/L hygromycin for transformant screening. The final stable transformation efficiency was 2.50% (95% CI 1.23–3.77%), with an escape rate of 16.13%. Transgenic plants showed distinct purplish-red coloration in roots, stems, and leaves, with significantly higher betacyanin accumulation than wild-type plants (<i>p</i> &lt; 0.05). Stable integration and expression of the <i>RUBY</i> gene were confirmed by PCR, RT-PCR, and RT-qPCR. This study establishes the first efficient regeneration and Agrobacterium-mediated transformation system for <i>Hosta ‘Light Star,’</i> and validates the feasibility of the <i>RUBY</i> reporter gene as a visual marker for <i>Hosta</i> transformation. This system provides a solid technical platform for functional genomic studies, CRISPR/Cas9-mediated gene editing, and molecular breeding of ornamental traits in <i>Hosta</i>.</p>

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Development of an Efficient Regeneration and Agrobacterium-Mediated Transformation Protocol for Hosta ‘Light Star’ Using the RUBY Reporter Gene

  • 期刊论文

  • 发表日期：

    2026年4月20日

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  • Hangkuan Zhou,
  • Yidi Qin,
  • Shuang Liu,
  • Xinyue Zhang,
  • Chen Chen,
  • Jinzhu Zhang,
  • Tao Yang,
  • Wuhua Zhang,
  • Jinping Fan

摘要

Hosta plantaginea is a perennial shade-tolerant herb of the Liliaceae family, with high ornamental and urban greening value. Hosta ‘Light Star’ is a newly developed ornamental cultivar with yellow-margined leaves and lilac flowers, but no efficient in vitro regeneration or genetic transformation system has been established for this cultivar to date. In this study, we established a highly efficient in vitro regeneration system for Hosta ‘Light Star,’ and developed an Agrobacterium-mediated genetic transformation protocol using the RUBY visual reporter gene for non-invasive screening of positive transformants. The optimal callus induction medium was MS + 2 mg/L 6-BA + 0.3 mg/L NAA + 0.05 mg/L 2, 4-D, with a callus induction rate of 53.33% for leaf explants (the optimal explant for sterile seedlings). The optimal adventitious bud proliferation medium was MS + 2 mg/L 6-BA + 0.1 mg/L NAA, with a proliferation coefficient of 5.87. The optimal rooting medium was 1/2 MS + 0.5 mg/L NAA + 0.5 mg/L IBA, with a 100% rooting rate. The optimal transplant substrate was perlite:vermiculite = 2:1, with a 100% transplant survival rate after acclimatization. For Agrobacterium-mediated transformation, the optimal infection parameters were as follows: Agrobacterium suspension OD600 = 0.6, infection time of 10 min, and 200 μM acetosyringone; the optimal selection conditions were 300 mg/L cefotaxime for bacteriostasis and 30 mg/L hygromycin for transformant screening. The final stable transformation efficiency was 2.50% (95% CI 1.23–3.77%), with an escape rate of 16.13%. Transgenic plants showed distinct purplish-red coloration in roots, stems, and leaves, with significantly higher betacyanin accumulation than wild-type plants (p < 0.05). Stable integration and expression of the RUBY gene were confirmed by PCR, RT-PCR, and RT-qPCR. This study establishes the first efficient regeneration and Agrobacterium-mediated transformation system for Hosta ‘Light Star,’ and validates the feasibility of the RUBY reporter gene as a visual marker for Hosta transformation. This system provides a solid technical platform for functional genomic studies, CRISPR/Cas9-mediated gene editing, and molecular breeding of ornamental traits in Hosta.