In Vitro Regeneration and bioactivity evaluation of Plumbago indica for enhanced propagation and plumbagin production

Plumbago indica, a medicinal plant, is valued for its therapeutic potential due to secondary metabolites, particularly plumbagin. Conventional propagation methods are limited by slow growth and environmental constraints. This study aimed to optimize in vitro regeneration protocols for P. indica to enhance propagation efficiency, conservation, and secondary metabolite production. An indirect organogenesis protocol was developed, encompassing surface sterilization, callus induction, shoot and root development, acclimatization, and bioactivity assessment. The highest survival rate (83.34%) with minimal contamination was achieved using 0.3% Carbendazim® and 10% Clorox. Optimal callus induction occurred on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L NAA and 2.5 mg/L BAP, yielding the highest callus weight (0.60 ± 0.18 g). Shoot induction was most effective with 4.0 mg/L BAP and 1.5 mg/L NAA, while the highest rooting was achieved in half-strength MS medium with 0.4 mg/L IBA, producing the longest roots (2.49 ± 0.31 cm) and the highest number (26.50 ± 3.54). Plants from Sabaragamuwa province exhibited the greatest height (104.60 ± 8.35 cm) and plumbagin content (42.90 ± 3.25 µg/mL), indicating environmental influence on growth and metabolite accumulation. Antioxidant activity (DPPH assay) showed IC50 values of 30.88 ± 1.68 μg/mL for tissue-cultured plants versus 31.73 ± 1.53 μg/mL for conventionally propagated plants. Brine shrimp lethality assay indicated LC50 values of 37.46 ± 2.90 μg/mL (tissue-cultured) and 44.53 ± 0.69 μg/mL (conventional). Anticancer activity (MTT assay) showed IC50 values of 117.18 ± 2.93 μg/mL (RD cells) and 313.94 ± 29.84 μg/mL (Vero cells) for tissue-cultured plants, compared to 103.07 ± 3.35 μg/mL (RD) and 200.63 ± 2.90 μg/mL (Vero) for conventional plants. These findings highlight the potential of in vitro propagation for sustainable plant production and enhanced bioactive compound yield, supporting eco-friendly and economically beneficial strategies.