Micropropagation and ISSR-based genetic fidelity assessment of the medicinal legume Butea monosperma (Lam.) Taub

Butea monosperma (Lam.) Taub., a leguminous tree of high ecological, medicinal and socio-economic importance, suffers from poor seed viability, dormancy and unreliable vegetative propagation, which restricts its large-scale cultivation and conservation. In this study, an efficient, reproducible indirect organogenesis protocol, combined with genetic fidelity analysis based on ISSR markers, was established for mature leaf and internodal explants of B. monosperma, providing an integrated system for clonal multiplication and quality control. Explants cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D exhibited a clear dose-dependent response, with 5.0 µM 2,4-D inducing maximum callus formation (88.89% in leaf and 77.78% in internodes) and producing profuse, compact, organogenic callus within 4 weeks, whereas hormone-free controls remained non-responsive. Subsequent transfer of this callus to MS media containing individual cytokinins identified 2.5 µM 6-benzyladenine (BA) and 2.5 µM meta-topolin (mT) as optimal, yielding 13.56 ± 0.10 and 13.30 ± 0.36 shoots per callus clump with mean shoot lengths of 4.45 ± 0.15 and 4.08 ± 0.02 cm, respectively, while higher cytokinin levels reduced shoot number and elongation. Combining 2.5 µM BA or mT with low auxin concentrations further enhanced shoot multiplication; BA + 0.5 µM NAA produced 18.42 ± 0.05 shoots (5.34 ± 0.02 cm), and mT + 0.5 µM NAA yielded 18.64 ± 0.07 shoots (5.38 ± 0.03 cm), highlighting a narrow optimum for cytokinin-auxin balance. For rooting, half-strength MS containing 1.0 µM IBA produced the most vigorous root systems (94.44% response, 12.56 ± 0.07 roots, 5.26 ± 0.08 cm). Acclimatized plantlets transferred to a sterilized garden soil: organic manure (3:1) mixture exhibited normal morphology, rapid growth and about 80% survival under greenhouse and field conditions, comparable to earlier reports in B. monosperma var. lutea. Using 20 primers, ISSR profiling of the donor and nine regenerants yielded 71 scorable bands, 69 of which were monomorphic (97.18%) and only 2 polymorphic, indicating very high clonal uniformity despite the callus phase. The optimized micropropagation protocol and ISSR-based fidelity assessment together provide a promising system for large-scale propagation. However, further field validation is required, as well as for germplasm preservation and future genetic improvement and biotechnological applications in B. monosperma.”