In vitro Propagation of Santalum album Linn. (Indian Sandalwood): Optimization of Plant Growth Regulators and Surface Sterilization Protocols for Micropropagation

Abstract

Santalum album Linn. (Indian Sandalwood; Family: Santalaceae) is a slow-growing, hemiparasitic tree species of exceptional economic, cultural, and pharmaceutical significance, classified as Vulnerable by the IUCN due to decades of over-exploitation. Its aromatic heartwood and essential oil rich in α-santalol and β-santalol accumulate only after 15–20 years of growth, making conventional seed-based and vegetative propagation methods inadequate for large-scale commercial and conservation demands. The present study was designed with two primary objectives: (i) to determine the optimal concentration and combination of Plant Growth Regulators (PGRs) for in vitro shoot proliferation, and (ii) to evaluate effective surface sterilization treatments to minimise microbial contamination during in vitro culture establishment of S. album nodal segment explants. Nodal segment explants were collected from healthy, field-grown S. album trees at the State Forest Research Institute (SFRI), Jabalpur, Madhya Pradesh, India (January–March 2026). Explants were surface-sterilized using sequential treatments of 1% Bavistin (Carbendazim; 0–10 min) and Mercuric Chloride (HgCl₂; 0.0–0.5%; 1–5 min). Six combinations of 6-Benzylaminopurine (BAP: 0.0–4.0 mg/L) with Naphthalene Acetic Acid (NAA: 0.0–0.5 mg/L) were evaluated on Murashige and Skoog (MS, 1962) basal medium. Growth responses — initiation, proliferation, elongation, and final shoot status were recorded at 7, 14, 21, and 28 days each of culture. Hardened plantlets were acclimatized in a 1:1:1 (soil:vermicompost:sand) substrate under greenhouse conditions. Among all PGR treatments, the combination of 3.0 mg/L BAP with 0.5 mg/L NAA on MS basal medium yielded the maximum morphogenic response, recording 90–95% shoot initiation, 95–100% proliferation, and 90–95% elongation, with the production of healthy, vigorous plantlets. Supraoptimal BAP concentrations (4.0 mg/L) suppressed organised shoot development and induced undesirable callogenesis. For surface sterilization, 1% Bavistin applied for 10 minutes reduced fungal contamination to 10% (from 100% in the untreated control), while 0.4% HgCl₂ for 2 minutes achieved the minimum contamination of 5–10% while maintaining full explant viability. Higher HgCl₂ concentrations (0.5%) caused phytotoxic tissue injury, paradoxically increasing contamination. The study establishes a reliable, reproducible micropropagation protocol for S. album using MS medium supplemented with 3.0 mg/L BAP + 0.5 mg/L NAA, with a sequential sterilization regimen of 1% Bavistin (10 min) followed by 0.4% HgCl₂ (2 min). This protocol offers a scalable foundation for clonal forestry, germplasm conservation, and commercial-scale production of Indian Sandalwood.