Comparative Effects of Cytokinins and Spermidine on In Vitro Organogenesis of Ruscus hypoglossum L. and Rooting of Regenerated Shoots

Ruscus hypoglossum L. is an evergreen ornamental species valued as cut foliage, but its commercial production is constrained by slow vegetative propagation and limited availability of uniform planting material. This study aimed to develop an efficient in vitro propagation protocol for R. hypoglossum by evaluating the effects of different cytokinins and spermidine on shoot organogenesis and subsequent rooting. Thidiazuron (TDZ), zeatin, kinetin, and spermidine were applied at 0.5 and 1.0 mg L−1 to rhizome explants derived from in vitro-grown seedlings. Explant length, shoot and cladode number, fresh weight, shoot diameter, chlorophyll a and b, total carotenoid content, SPAD value, and rooting performance were assessed. Significant treatment effects were observed for most traits. Kinetin at 1.0 mg L−1 produced the highest shoot proliferation (4.66 shoots explant−1), cladode number (12.00 explant−1), fresh weight (0.31 g), chlorophyll a (0.72 mg g−1 FW), chlorophyll b (0.29 mg g−1 FW), and SPAD value (40.25). Spermidine at 1.0 mg L−1 resulted in the highest carotenoid content (0.59 mg g−1 FW) and promoted greater explant elongation, whereas TDZ at 1.0 mg L−1 reduced cladode formation and pigment accumulation. Multivariate analyses consistently identified kinetin as the most favorable treatment during multiplication. Shoots regenerated with 1.0 mg L−1 kinetin also showed the highest rooting response following a 10 s pulse treatment with 2.0 mg L−1 IBA and transfer to hormone-free half-strength MS medium. Overall, kinetin was the most effective treatment under the conditions tested, providing a practical basis for efficient clonal propagation and ex situ conservation of R. hypoglossum.