In vitro Micropropagation of Asparagus racemosus Willd. Using Nodal Segment Explants Optimization of Surface Sterilization, Shoot Root Induction and Acclimatization Protocol

Abstract

Asparagus racemosus Willd. (family Asparagaceae), popularly known as Shatavari and revered as the ‘Queen of Herbs’ in Ayurveda, is a high-value medicinal plant of critical pharmacological importance, now listed as an endangered species due to over-exploitation and habitat loss. The present study was undertaken to develop an optimized in vitro micropropagation protocol using nodal segment explants (5th–6th node from shoot apex) on Murashige and Skoog (MS) basal medium. Two critical variables were systematically evaluated: (i) surface sterilization efficacy, and (ii) plant growth regulator (PGR)-mediated shoot organogenesis. For surface sterilization, Bavistin (Carbendazim, 1%) applied for 8 minutes (B5) yielded the maximum contamination-free rate of 78% without phytotoxicity; subsequent treatment with 0.01% HgCl₂ for 0.1 minutes (H7) achieved 90% contamination-free explants with fully preserved tissue viability. Among eight NAA × BAP combinations evaluated over three consecutive weeks, Treatment T4 (NAA 3.0 mg/L + BAP 4.0 mg/L) produced the maximum shoot height (6–8 cm) and shoot number (6 shoots/explant) by Week 3, while Treatment T6 (NAA 3.0 mg/L + BAP 2.0 mg/L) offered the most stable and contamination-free multiplication (4 shoots/explant, 5–7 cm). Both treatments confirmed that elevated cytokinin-to-auxin ratios drive vigorous axillary bud activation in this species. Two successive hardening trials, however, failed entirely due to the complete absence of in vitro root formation in the transferred plantlets, identifying root induction as the single most critical unresolved step in the protocol. Future work should prioritize a dedicated in vitro rooting phase using 0.5–1.0 mg/L IBA on half-strength MS medium, followed by evaluation of acclimatization substrates, to complete a commercially scalable and reproducible micropropagation protocol for this medicinally essential species.