Optimization of auxin and cytokinin concentrations for callus formation, total volatile extractables, total phenolic, and flavonoid contents in Orthosiphon aristatus leaves using response surface methodology

Abstract

Orthosiphon aristatus (Lamiaceae) is a medicinal plant widely used for its antioxidant, antimicrobial, and anti-inflammatory properties, yet efficient in vitro systems for biomass and metabolite production remain underexplored. This study aimed to optimize auxin and cytokinin concentrations for callus induction and to assess associated changes in total volatile extractables, phenolic, and flavonoid contents. Leaf explants from ex situ nursery were cultured on media containing either auxins (2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA)) or cytokinin (6-benzylaminopurine (BAP), or kinetin). Among auxins, 2,4-D was the most effective, achieving 100% induction and the highest biomass accumulation by week 4, significantly outperforming IAA and NAA. Among cytokinin, BAP was superior to kinetin in promoting callogenesis and growth, although cytokinin effects were generally weaker than those of auxins. Response surface methodology identified culture duration and 2,4-D concentration as the dominant factors influencing induction and biomass yield, with BAP exerting only minor effects. Validation experiments under optimized conditions (2,4-D 4.5 mg/L, BAP 1.5 mg/L, 4 weeks) confirmed model accuracy, as observed values closely matched predictions. Biochemical analyses revealed that optimized callus cultures produced significantly higher total volatile extractables, total phenolics, and flavonoids than leaves. These findings establish a reliable and scalable strategy for enhancing biomass and metabolite production in O. aristatus, supporting its application in medicinal plant biotechnology.