Standardizing in vitro callus induction and indirect organogenesis of Gloriosa superba L. leaf explants using exogenous phytohormones

Abstract

Gloriosa superba L. is classified as an endangered species owing to slow natural propagation and widespread exploitation in the wild. Therefore, we aim to develop an efficient protocol for the in vitro regeneration of G. superba L. using leaf explants. Optimal callus induction was achieved using a combination of 1-naphthalene acetic acid (NAA) and kinetin (KN) [1.5 mg L-1 NAA + 0.5 mg L-1 KN was supplemented with 10 mg L-1 casein hydrolysate (CH)]. This formulation resulted in the swiftest initiation of callus formation (within 12 d) and yielded the highest callus induction rate (71.88%). Furthermore, addition of 5 mg L-1 CH and 20% (v/v) coconut water to Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 6-benzyl-aminopurine and 0.5 mg L-1 NAA facilitated the formation of shoot primordia within 14 d, achieving the highest average number of shoots per callus (5.25). For root development, use of half-strength MS medium supplemented with 1.0 mg L-1 indole-3-butyric acid 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 had a 100% survival rate within three weeks of placement in culture rooms and shade net enclosures. After transplantation into a substrate comprising garden soil, sand, and vermiculite and exposure to direct sunlight, the plantlets achieved a 76% survival rate by the fifth week, thereby maintaining their typical growth characteristics. Our protocol enables large-scale production of genetically uniform G. superba L. plants. This demonstrates the potential of tissue culture techniques in plant propagation and biotechnological applications, thereby contributing to current understanding and paving the way for future research.