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<p><?tk 2?><i>Hemsleya zhejiangensis</i> C. Z. Zheng, a medicinal plant in the Cucurbitaceae family and a plant species with extremely small populations in China, faces significant propagation challenges. Seed-based propagation presents challenges of genetic instability and reproductive barriers, while vegetative propagation is limited by its low multiplication efficiency. Previous tissue culture attempts produced plantlets lacked sex-specific protocols. In this study, we established an efficient, sex-specific in vitro micropropagation system using nodal segments as explants. A 60% survival rate was achieved with a 7-min surface sterilization using 0.1% HgCl₂. Axillary bud induction rate of 66.7 ± 4.2% was achieved on Murashige and Skoog (MS) medium with 2.0&#xa0;mg/L 6-benzylaminopurine (BA). Male and female explants exhibited distinct optimal media for shoot multiplication and root induction. For shoot multiplication, male explants performed best (4.96 ± 0.29) on half strength MS (1/2MS) containing 0.5&#xa0;mg/L Zeatin (ZT), whereas female explants exhibited maximum proliferation (4.85 ± 0.29) on MS with 0.3&#xa0;mg/L BA. For root induction, male shoots achieved the highest rooting rate (86.50 ± 9.50%) on MS with 2.0&#xa0;mg/L indole-3-butyric acid (IBA), whereas female shoots demonstrated 100% root induction on MS medium with 0.2&#xa0;mg/L 1-Naphthaleneacetic acid (NAA). All rooted plantlets were successfully acclimatized in the greenhouse with complete survival. Genetic fidelity of the regenerants was evaluated using Simple Sequence Repeat (SSR) markers in capillary electrophoresis, revealing no polymorphism between the regenerants and the mother plant, indicating high genetic stability. This protocol minimizes physiological disorders and somaclonal variation, enabling efficient production of healthy, genetically uniform plantlets of both sexes. It offers a reliable system for mass propagation, germplasm conservation, and reintroduction of <i>H. zhejiangensis</i>. The sex-specific optimization is a critical advancement for breeding and restoration programs requiring balanced sex ratios, supporting both <i>ex situ</i> conservation and sustainable use of this endangered medicinal species.</p>

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Efficient in vitro protocol for female and male nodal segments multiplication and genetic fidelity assessment of micropropagated plants in Hemsleya zhejiangensis C. Z. Zheng

  • 期刊论文

  • 发表日期：

    2025年11月18日

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  • Xixi Wang,
  • Jian Yao,
  • Xi Liu,
  • Zupei Lei,
  • Fangdong Zheng,
  • Sheng Teng,
  • Weimei Jiang

摘要

Hemsleya zhejiangensis C. Z. Zheng, a medicinal plant in the Cucurbitaceae family and a plant species with extremely small populations in China, faces significant propagation challenges. Seed-based propagation presents challenges of genetic instability and reproductive barriers, while vegetative propagation is limited by its low multiplication efficiency. Previous tissue culture attempts produced plantlets lacked sex-specific protocols. In this study, we established an efficient, sex-specific in vitro micropropagation system using nodal segments as explants. A 60% survival rate was achieved with a 7-min surface sterilization using 0.1% HgCl₂. Axillary bud induction rate of 66.7 ± 4.2% was achieved on Murashige and Skoog (MS) medium with 2.0 mg/L 6-benzylaminopurine (BA). Male and female explants exhibited distinct optimal media for shoot multiplication and root induction. For shoot multiplication, male explants performed best (4.96 ± 0.29) on half strength MS (1/2MS) containing 0.5 mg/L Zeatin (ZT), whereas female explants exhibited maximum proliferation (4.85 ± 0.29) on MS with 0.3 mg/L BA. For root induction, male shoots achieved the highest rooting rate (86.50 ± 9.50%) on MS with 2.0 mg/L indole-3-butyric acid (IBA), whereas female shoots demonstrated 100% root induction on MS medium with 0.2 mg/L 1-Naphthaleneacetic acid (NAA). All rooted plantlets were successfully acclimatized in the greenhouse with complete survival. Genetic fidelity of the regenerants was evaluated using Simple Sequence Repeat (SSR) markers in capillary electrophoresis, revealing no polymorphism between the regenerants and the mother plant, indicating high genetic stability. This protocol minimizes physiological disorders and somaclonal variation, enabling efficient production of healthy, genetically uniform plantlets of both sexes. It offers a reliable system for mass propagation, germplasm conservation, and reintroduction of H. zhejiangensis. The sex-specific optimization is a critical advancement for breeding and restoration programs requiring balanced sex ratios, supporting both ex situ conservation and sustainable use of this endangered medicinal species.