Oldenlandia umbellata L. is widely used for its medicinal properties, particularly in pharmaceutical industries and conservation endeavors. Our study aimed to enhance in vitro plant regeneration techniques by supplementing thidiazuron (TDZ) and cytokinins (CKs) (KI; kinetin, BAP; 6-benzylaminopurine), bolstering conservation efforts and therapeutic applications. The results revealed concentration-dependent responses, with KI (6.96 µM) and BAP (4.44 µM) significantly promoting callus formation, while TDZ (12-18 days) notably accelerated shoot induction. Root induction demonstrated variability based on indole-3-acetic acid (IAA) concentrations, whereas 1-naphthaleneacetic acid (NAA) influenced root formation. Particularly noteworthy were the substantial shifts in growth parameters induced by CK supplementations, including enhanced shoot length, biomass, and modifications in leaf-root ratios. BAP (6.66 µM) notably augmented leaf growth, whereas TDZ (4.54 µM) facilitated root elongation. Additionally, CK supplementation exhibited a stimulating effect on secondary metabolites, thereby enhancing shoot biochemistry. These results shed light on the intricate regulatory mechanisms underlying O. umbellata tissue culture, providing valuable insights for tailored conservation strategies and pharmaceutical innovations. Notably, identifying optimal TDZ concentrations underscores its potential for stress-free growth promotion. These findings propel the refinement of tissue culture methodologies, unlocking the therapeutic potential of O. umbellata while safeguarding its genetic diversity. Furthermore, the elucidation of such mechanisms serves as a cornerstone for future research endeavors aimed at harnessing the full medicinal potential of this invaluable plant species.
