INTRODUCTION: Chrysanthemum naktongense is a wild species of the genus Chrysanthemum in the Asteraceae family, characterized by its remarkable capacity to rapidly form rhizomes within 10 days after cutting. Nevertheless, the absence of an effective in vitro regeneration system has impeded the advancement of genetic studies on this species. 

RATIONALE: The utilisation of leaf discs as explants constitutes a well-established and effective strategy for plant regeneration. The core of this methodology lies in optimizing the combinations and concentrations of plant growth regulators (PGRs) in the culture medium, which critically demonstrated to influence the processes of callus induction, adventitious bud differentiation, and root formation. This study was conceived with the objective of systematically determining the effects of different PGRs, with a view to establishing a robust regeneration protocol for C. naktongense. 

RESULTS: This study found that C. naktongense can form rhizomes after 10 days of cuttings and grow to 10 cm within 50 days. In this study, the results demonstrated that the optimal medium of leaf disc callus induction and adventitious bud differentiation was MS basal medium supplemented with 0.5 mg∙L–1 6-BA and 2.0 mg∙L–1 NAA. Following a 45-day treatment period, resulted in the induction of regenerated shoots that exceeded 1 cm in length, exhibiting a 100% regeneration rate. For the rooting phase, the optimal medium was identified as 1/2MS medium supplemented with 0.1 mg∙L–1 IBA, which also resulted in a rooting rate of 100%. 

CONCLUSION: This study successfully established a highly efficient and stable regeneration system for C. naktongense, encompassing the stages from callus induction to plantlet rooting. The developed system, characterized by its 100% regeneration and rooting rates, provides crucial technical support for subsequent research.