A Method Based on the Induction of Stem-tipped Protobulbs to Obtain the Virus-free Plantlets of Dioscorea opposita ‘Tiegun’

Introduction: Gentiana dahurica is a valuable medicinal plant whose roots are used in traditional Chinese medicine for their anti-inflammatory and hepatoprotective properties. However, over-harvesting of wild populations, coupled with low natural regeneration rates due to seed dormancy and habitat degradation, has led to resource scarcity. Conventional propagation methods are inefficient, underscoring the urgent need for an efficient in vitro regeneration system to enable germplasm conservation and large-scale cultivation. Rationale: This study aimed to establish a complete and efficient in vitro regeneration protocol for G. dahurica. The work focused on optimizing key steps including seed germination, callus induction, adventitious bud differentiation and proliferation, root induction, and plantlet acclimatization. The effects of different basal media, plant growth regulator types and concentrations, and transplanting substrates were systematically investigated. Results: Seed germination was significantly enhanced by treatment with 50 mg∙L–1 GA₃. The optimal basal medium for overall plantlet growth was identified as 3/4-strength MS medium. The root segment is the optimal explant. The most effective callus induction (100%) was achieved from root explants cultured on 3/4MS medium supplemented with 2.0 mg∙L–1 2,4-D and 1.0 mg∙L–1 6-BA. For adventitious bud differentiation and proliferation, a medium comprising 3/4MS with 1.5 mg∙L–1 6-BA and 1.0 mg∙L–1 KT proved superior, yielding a 100% differentiation rate and an average of 44 buds per callus clump. Optimal rooting was obtained on 3/4MS medium containing 0.5 mg∙L–1 IBA. Finally, the highest survival rate (92.5%) of acclimatized plantlets was achieved using a substrate mixture of vermiculite:perlite:peat (1:1:3, v/v/v) following a 7-day gradual acclimatization period. Conclusion: A complete and efficient in vitro regeneration system for G. dahurica has been successfully established for the first time. This protocol provides a robust foundation for the germplasm conservation, rapid propagation, and potential future genetic improvement of this important medicinal species.