This study aims to develop an efficient protocol for the in vitro regeneration of Gloriosa superba L. using leaf explants, addressing the species' endangered status arising from slow natural propagation and widespread exploitation in the wild. Optimal callus induction was achieved with a combination of 1-naphthalene acetic acid (NAA) and kinetin (KN) [1.5 mg L-1 NAA + 0.5 mg L-1 KN, supplemented with 10 mg L-1 casein hydrolysate (CH)]. This formulation demonstrated the swiftest initiation of callus formation (within 12 days) and yielded the highest callus induction rate (71.88%). Furthermore, the addition of 5 mg L-1 CH and 20% (v/v) of coconut water (CW) to Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 6-benzylaminopurine (BAP) and 0.5 mg L-1 NAA, facilitated the formation of shoot primordia within 14 days, achieving the highest average number of shoots per callus (5.25). For root development, half-strength MS media with 1.0 mg L-1 indole-3-butyric acid (IBA) resulted in the highest root-to-shoot ratio (5.75), root fresh weight (143 mg), and root dry weight (22.2 mg). The in vitro-cultivated plantlets showed a 100% survival rate within three weeks of placement in culture rooms and shade net enclosures. After being transplanted into a substrate of garden soil, sand, and vermiculite and exposed to direct sunlight, they achieved a 76% survival rate by the fifth week, maintaining their typical growth characteristics. The protocol enables large-scale production of genetically uniform Gloriosa superba L. plants, demonstrating the potential of tissue culture techniques in plant propagation and biotechnological applications, contributing to current understanding and paving the way for future research.

Keywords: Acclimatization, Callogenesis, Leaf explant(s), Micropropagation, Ornamental plant