Integrated assessment of in vitro regeneration, ISSR-Based genetic fidelity, and antifungal properties in Holmskioldia sanguinea

Abstract

Holmskioldia sanguinea is valued for its ornamental appeal and traditional medicinal uses for treating rheumatism, dysentery, headaches, hypertension, ulcers, and gynecological disorders, but propagation challenges limit wider cultivation and conservation efforts. To overcome the limitations associated with conventional propagation methods, an effective in vitro regeneration protocol was established using nodal explants. Following surface sterilization, explants were cultured on half strength Murashige and Skoog (MS) medium supplemented with varying concentrations of plant growth regulators for shoot bud initiation and multiplication. The best response was observed with 1.0 mg L−1 BA (Benzyladenine) particularly when combined 0.5 mg L−1 2,4-D (2,4- Dichlorophenoxyacetic acid) Shoot formation was evident within 2 to 3 wk, and rooting was successfully achieved in two wk after transferring the explant with initiated shoots on half strength MS medium supplemented with 1.0 mg L−1 IBA (Indolebutyric acid). Acclimatization involved a two-phase hardening process, beginning with a gradual reduction in external nutrients and followed by transfer to sterile peat moss + sand (2:1, v/v) potting mix, resulting in an impressive 98% survival rate of regenerated plantlets within 8 to 10 wk. The genetic fidelity of regenerants was evaluated using ISSR amplification with ten primers, out of which 6 primers yielded consistent and reproducible monomorphic profiles across wild-type and in vitro derived samples, demonstrating the absence of somaclonal variation and confirming genetic fidelity. In addition to micropropagation and ISSR-based genetic fidelity assessment, the antifungal potential of H. sanguinea was also evaluated. The methanolic leaf extract demonstrated the highest antifungal activity, inhibiting mycelial growth of tested fungi by 98%, indicating its strong potential as a natural antifungal agent. This study established a robust and scalable tissue culture regeneration protocol for H. sanguinea, supporting its conservation and offering opportunities for pharmaceutical applications as a source of natural antifungal compounds.