COMPREHENSIVE EVALUATION OF IN VITRO ORGANOGENESIS AND HPTLC BIO-ANALYTICAL VALIDATION OF STIGMASTEROL AND PHYTOCHEMICAL FINGERPRINTING IN BIXA ORELLANA L

Abstract

This study developed a reliable micropropagation and phytochemical analysis system for Bixa orellana L. (annatto), an economically important tropical shrub valued for its natural pigments, bixin and norbixin, and medicinal properties. Conventional propagation is limited by poor seed viability and high genetic variation; therefore, an efficient in vitro propagation method is valuable for conservation and metabolite production. Shoot apex and leaf explants were cultured on Murashige and Skoog (MS) medium with plant-growth regulators. Leaf explants showed 100% callus induction on MS medium containing 1.0 mg/L BAP. The same BAP concentration gave the best direct shoot regeneration, producing an average of 23.2 ± 0.20 shoots per explant, with shoots reaching 9.10 ± 0.35 cm long. Rooting was most successful on MS medium supplemented with 2.0 mg/L NAA, achieving an 84.00 ± 2.44% rooting rate. Following acclimatization under net-house conditions, 62% of plantlets survived. The researchers also established and validated an HPTLC method to identify and quantify stigmasterol in wild and micropropagated tissues. Separation was performed on silica gel 60 F254 plates using a toluene:ethyl acetate:methanol (17:2:1, v/v/v) mobile phase, followed by anisaldehyde–sulfuric acid derivatization and densitometric analysis at 540 nm. The method met ICH validation requirements, demonstrating strong linearity (r² = 0.99551), acceptable accuracy (90.0–94.8% recovery), and good precision with relative standard deviation below 2.21%. Stigmasterol levels were substantially higher in in vitro tissues than in wild leaves. In vitro leaves contained 18.73 µg/g, followed by shoots at 17.26 µg/g and roots at 16.60 µg/g, compared with 0.994 µg/g in wild leaves. Overall, micropropagation offers a practical platform for germplasm conservation and enhanced stigmasterol production.