Axenic leaf-derived callus induction of Gynura pseudochina (L.) DC. using combined 2,4-dichlorophenoxyacetic acid and benzylaminopurine

B ackground: Gynura pseudochina is a promising herbal plant belonging to the Asteraceae family. It contains many secondary metabolite compounds, including saponins, flavonoids, essential oils, and alkaloids. In vitro culture offers an efficient method for mass and rapid propagation of G. pseudochina. Callus induction is alternatively used to generate in vitro shoot proliferation and production of bioactive compounds. Previous study has reported that combined 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzyl Amino Purine (BAP) can effectively induce callus. However, the effect of this combination on G. pseudochina in vitro culture is still limited. Therefore, this study investigates the impact of auxin and cytokinin combination (2,4-D and BAP) on G. pseudochina callus formation in vitro. Methods: Axenic nodal segments from G. pseudochina were used as explants and placed in Murashige and Skoog (MS) medium enriched with different 2,4-D and BAP combination for 60 days of culture. Subsequently, callogenic responses, callus emergence time, callus fresh weight and morphology were observed. Results: We observed that 2,4-D was vital to induce callus formation. All axenic leaves were able to undergo callogenesis in all concentrations of 2,4-D. The combination of 1.5 mg/L 2,4-D and 1 mg/L BAP (A3B2) was the most effective treatment, producing 1.12 g fresh weight callus and showing rapid induction with callus emergence 12 days after culture. Meanwhile, calli obtained in this study were compact and friable with green, greenish-yellow and brown colors. Conclusion: Our research demonstrates the effectiveness of combining 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) in inducing callus formation in Gynura pseudochina through in vitro culture. This study underscores the critical role of 2,4-D in promoting callogenesis, with the optimal callus formation achieved using a combination of 1.5 mg/L 2,4-D and 1 mg/L BAP.