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Influence of plant growth regulators on indirect regeneration and secondary metabolite production in Aconitum violaceum Jacq. was evaluated. Among the different plant growth regulators studied, 2.5 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.25 μM kinetin (Kn) promoted the highest frequency of callus production for indirect regeneration. 6-Benzyl aninopurine (BAP) was more effective in improving shoot regeneration and secondary metabolite production compared to thidiazuron (TDZ). The highest frequency of regeneration (61.8%) was obtained when calli were transferred to Murashige and Skoog medium supplemented with 1 μM BAP and 0.5 μM α-naphthalene acetic acid (NAA) and was more than two-times higher when compared to the treatments with cytokinin only. Supplementation with low NAA concentrations resulted reduction in in vitro secondary metabolite production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on indirect shoot regeneration and the production of secondary metabolites in A. violaceum.