Abstract
Artemisia vulgaris L. has attracted considerable interest for its pharmaceutical, agricultural, and cosmetic applications due to its rich bioactive metabolites. However, low metabolite yields and challenges in large-scale cultivation constrain its commercial potential. Therefore, this study aimed to optimize a callus regeneration system, develop a comprehensive metabolite profile of the callus culture, and assess the effects of elicitor treatments on metabolite biosynthesis pathways to identify optimal concentrations for enhanced metabolite production. In vitro callus cultures were initiated on Murashige and Skoog (MS) medium with varying levels of benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) phytohormones. Callus cultures were evaluated for colour, texture, moisture content, and biomass, and metabolite profiling was conducted using nuclear magnetic resonance (NMR) platform. Elicitor treatments of the callus cultures with different concentrations of methyl jasmonate (MJ) and salicylic acid (SA) were analysed via NMR-based metabolomics. MS medium containing 2.0 mg L⁻ 1 BAP and 0.2 mg L⁻ 1 NAA was optimal for callus culture establishment and maintenance. A total of eighteen compounds were identified, including dihydroartemisinic acid, caffeic acid, and gallic acid. MJ and SA functioned as stress elicitors, modulating the tricarboxylic acid cycle, phenylpropanoid, and artemisinin biosynthesis pathways, although they had limited direct effects on artemisinin concentrations. Overall, this study optimized callus culture production, generated the first detailed metabolite profile, and highlighted the potential of elicitation strategies to enhance bioactive metabolite yields, providing a foundation for future metabolic engineering and industrial applications.