In vitro Propagation and Endophyte Exploration of Berberis aristata DC: Standardization of Sterilization and Culture Protocols

Abstract

Berberis aristata DC (Berberidaceae), an endangered Himalayan shrub of significant ethnopharmacological importance, is a rich source of bioactive alkaloids principally berberine with documented antimicrobial, antidiabetic, anti-inflammatory, and anticancer properties. Escalating pharmaceutical demand has driven severe overharvesting of wild populations, necessitating the development of sustainable propagation and bioprospecting strategies. The present study aimed to standardize surface sterilization and culture protocols for in vitro micropropagation of B. aristata, and to establish protocols for the isolation of endophytic microorganisms as potential biofactories for plant-analogous bioactive metabolites. Three explant types leaf sections, nodal segments, and seeds were evaluated on Murashige and Skoog (MS) basal medium supplemented with varying concentrations of 6-Benzylaminopurine (BAP) and Indole-3-acetic acid (IAA). Surface sterilization was optimized using sequential treatments of Bavistin® (carbendazim) and mercuric chloride (HgCl₂). Endophytic fungi and bacteria were isolated from surface-sterilized stem bark, leaves, and roots on Potato Dextrose Agar and Nutrient Agar, respectively. The combination of 3.0 mg/L BAP and 2.0 mg/L IAA yielded optimal results, achieving 85% shoot initiation, 95% shoot proliferation, and excellent rooting within a 37-day culture period, consistent with the Skoog and Miller cytokinin-to-auxin morphogenesis hypothesis. Among sterilization regimens, Bavistin® at 1.0% for 20 minutes (pre-treatment) followed by 0.05% HgCl₂ for 4 minutes produced the most favorable outcome, reducing contamination to 15% while maintaining 85% explant survival. Higher concentrations of both sterilants caused measurable phytotoxicity. Progressive plantlet development was confirmed across a 60-day culture period, culminating in well-rooted, multi-leaved plantlets suitable for acclimatization. Endophytic isolation protocols were successfully standardized using antibiotic-supplemented selective media. These findings establish a reproducible and optimized framework for the micropropagation and endophyte exploration of B. aristata, contributing to conservation efforts for this endangered species while providing a foundation for microbial bioprospecting of novel plant-analogous bioactive compounds.