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<p>This study aimed to establish an efficient in vitro regeneration system using cotyledons of <i>Juglans sigillata</i> to overcome the problem of difficult regeneration of this species. Our experiments demonstrated that cotyledons harvested 12 weeks after female flowers full bloom were the optimal developmental stage for adventitious shoot regeneration, and the cotyledon region in contact with the embryo exhibited stronger regeneration capacity. Ten days of dark culture following inoculation of cotyledons significantly increased the callus induction percentage, with a maximum of 94.44%. The optimal medium for adventitious shoot induction was Driver and Kuniyuki Walnut Medium (DKW) supplemented with 0.2&#xa0;mg/L indole-3-butyric acid (IBA) and 6&#xa0;mg/L thidiazuron (TDZ), achieving a maximum adventitious shoot induction percentage of 71.11% and an average of 6.88 shoots per explant. After single adventitious shoots were cultured for strengthening, they were transferred to the optimal double-layer rooting medium (the upper layer was DKW + 0.1&#xa0;mg/L IBA + 1&#xa0;mg/L 6-benzylaminopurine (6-BA), and the lower layer was DKW + 3&#xa0;mg/L IBA + 3&#xa0;g/L activated carbon) and cultured in the dark for 7 days to induce rooting, reaching a maximum rooting percentage of 64.44%. Subsequently, healthy rooted seedlings were successfully acclimatized and transplanted. Flow cytometry analysis of ploidy stability showed there were no ploidy differences between regenerated plants from cotyledons and their parental plants. This complete regeneration system can rapidly generate multiple seedlings within approximately 4 months, which provides a technical foundation for the rapid propagation of <i>J. sigillata</i> in agricultural production.</p>

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Establishment of an efficient cotyledon-based regeneration system in Juglans sigillata dode

  • 期刊论文

  • 发表日期：

    2026年2月18日

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  • Xiaojin Liu,
  • Chunxiang Li,
  • Wenlong Zhang,
  • Jian Peng,
  • Xuejun Pan,
  • Wen’e Zhang

摘要

This study aimed to establish an efficient in vitro regeneration system using cotyledons of Juglans sigillata to overcome the problem of difficult regeneration of this species. Our experiments demonstrated that cotyledons harvested 12 weeks after female flowers full bloom were the optimal developmental stage for adventitious shoot regeneration, and the cotyledon region in contact with the embryo exhibited stronger regeneration capacity. Ten days of dark culture following inoculation of cotyledons significantly increased the callus induction percentage, with a maximum of 94.44%. The optimal medium for adventitious shoot induction was Driver and Kuniyuki Walnut Medium (DKW) supplemented with 0.2 mg/L indole-3-butyric acid (IBA) and 6 mg/L thidiazuron (TDZ), achieving a maximum adventitious shoot induction percentage of 71.11% and an average of 6.88 shoots per explant. After single adventitious shoots were cultured for strengthening, they were transferred to the optimal double-layer rooting medium (the upper layer was DKW + 0.1 mg/L IBA + 1 mg/L 6-benzylaminopurine (6-BA), and the lower layer was DKW + 3 mg/L IBA + 3 g/L activated carbon) and cultured in the dark for 7 days to induce rooting, reaching a maximum rooting percentage of 64.44%. Subsequently, healthy rooted seedlings were successfully acclimatized and transplanted. Flow cytometry analysis of ploidy stability showed there were no ploidy differences between regenerated plants from cotyledons and their parental plants. This complete regeneration system can rapidly generate multiple seedlings within approximately 4 months, which provides a technical foundation for the rapid propagation of J. sigillata in agricultural production.