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渤丰3号杨无菌苗叶片高效再生体系的建立和优化

Establishment and Optimization of an Efficient Plant Regeneration System for leaves of in vitro P. xeuramericana 'Bofeng 3 hao' Plants

    摘要
  • 摘要:
    目的 通过5-氨基尿嘧啶（5-AU）和暗培养处理，建立渤丰3号杨（Populus x euramericana 'Bofeng 3 hao'）无菌苗叶片高效植株再生体系，为提高杨树遗传转化和基因编辑效率奠定基础。
    方法 本研究以渤丰3号杨无菌苗为材料，在叶片分化培养基中添加0.4、0.8、1.6和3.2 mmol·L−1四个浓度的5-AU，于暗培养处理6 h~6 d后，将叶片转移到不含5-AU的分化培养基中继续进行光下培养，统计叶片分化率、再生芽数和生根率，比较不同浓度5-AU和暗培养不同时长对杨树再生体系关键指标的影响，并采用隶属函数综合分析法筛选渤丰3号杨无菌苗叶片最优再生体系。
    结果 与对照相比，0.4~3.2 mmol·L−1 5-AU结合暗培养12 h~3 d，可使叶片不定芽再生提前2~3 d，4~6 d暗培养可使叶片不定芽再生提前4~5 d。双因素方差分析结果表明，5-AU浓度与暗培养时长极显著影响叶片分化率、再生芽数和不定芽生根率，5-AU浓度与暗培养交互作用极显著或显著提升再生芽数和生根率。隶属函数分析结果显示，3.2 mmol·L−1 5-AU浓度结合暗培养2 d处理下，无菌苗叶片再生效果最佳，叶片分化率达到100%，再生不定芽数达36（个/叶），生根率达96.65%。
    结论 5-AU结合暗培养处理不仅能够诱导渤丰3号杨无菌苗叶片提前分化，而且显著提高不定芽再生数及生根率。本研究通过化学药剂处理和培养条件优化，成功建立了渤丰3号杨无菌叶片高效植株再生体系，为该品种的遗传转化及基因编辑奠定基础，也为其他树种的组织培养体系的建立提供参考。

     

    Abstract:
    Objective To develop a high-efficiency regeneration system for leaves of in vitro P. x euramericana 'Bofeng 3 hao' plants using 5-aminouracil (5-AU) and dark culture treatments, providing a robust foundation for genetic transformation and gene editing in poplars.
    Methods In this study, wounded leaves of P. x euramericana 'Bofeng 3 hao' were cultured on differentiation media supplemented with different concentrations of 5-AU(0.4, 0.8, 1.6, and 3.2 mmol·L−1), dark cultured for 6 h to 6 d, and then transferred to 5-AU-free differentiation media under photoperiodic culture. To evaluate the effects of 5-AU concentration and dark culture duration on the regeneration system of in vitro leaves, key regeneration parameters-including leaf differentiation rate, the number of buds and root induction efficiency were statistically analyzed. The optimal regeneration system was determined via membership function-based comprehensive evaluation.
    Results Compared with the control treatments, the application of 0.4 to 3.2 mmol·L−1 5-AU combined with 12 h to 3 d dark incubation shortened the adventitious shoot regeneration time by 2 to 3days. Extending the dark incubation to 4–6 days further reduced the regeneration time by 4 to 5 days. Two-way ANOVA analysis revealed highly significant effects of both 5-AU concentration and dark incubation duration on leaf differentiation rate (p < 0.01), bud regeneration capacity (p < 0.01) and root induction efficiency (p < 0.01). Moreover, the interaction between 5-AU concentration and dark incubation significantly enhanced bud formation (p < 0.05) and root induction (p < 0.01). According to membership function analysis, the 3.2 mmol·L−1 5-AU with 2 d dark incubation was the best treatment for plant regeneration of P. x euramericana 'Bofeng 3 hao' in vitro leaves of plants, yielding 100% leaf differentiation rate, 36 buds per explant, and 96.65% root induction rate.
    Conclusion 5-AU supplementation and dark culture treatments synergistically promoted precocious leaf differentiation, enhanced bud proliferation, and improved root induction of in vitro leaves of P. x euramericana 'Bofeng 3 hao'. This highly efficient plant regeneration system not only provides a critical technical foundation for genetic transformation and genome editing of P. x euramericana 'Bofeng 3 hao', but also provides a valuable methodology for tissue culture and plant regeneration studies in other woody species.

     

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