Explant origin and growth regulators as determinants of callus performance and terpenoid indole alkaloid accumulation in Catharanthus roseus.

Catharanthus roseus (L.) G. Don (Apocynaceae) is a highly important medicinal plant and a valuable source of diverse pharmacologically active compounds particularly anticancer terpenoid indole alkaloids (TIAs), such as catharanthine (CAT), vinblastine (VBL) and vincristine (VCR). This study investigated the impact of various plant growth regulator (PGR) combinations on callus induction and production of CAT, VBL and VCR. Leaf and stem explants were cultured on media containing seven combinations of auxins and cytokinins, and evaluated for callus induction frequency, biomass accumulation, and alkaloid content after eight weeks. Stem explants exhibited superior responsiveness, achieving 100% callus induction under most PGR treatments, whereas the combination of GA₃ and BAP failed to induce callogenesis in both explant types. HPLC analysis revealed that the combination of 1.5 mg/L 2,4-D and 1.5 mg/L kinetin (Kin) in leaf-derived callus yielded the highest CAT and VBL concentrations (5.01 µg/gFW and 1.96 µg/gFW; respectively). However, VCR notable enhanced in the stem- derived callus grown on a medium containing a combination of 1.0 mg/L each of BAP and NAA (4.71 µg/gFW) and leaf-derived callus grown in a medium containing 1.5 mg/L each of BAP and 2,4-D (2.79 µg/gFW) as compared with the controls (leaves and stems from in vivo grown plants, 1.51 µg/gFW and 0.38 µg/gFW; respectively). Gene expression analysis revealed that the combined application of 1.0 mg/L BAP and 1.0 mg/L NAA enhanced the transcript levels of peroxidase 1 (PRX1), secologanin synthase (SLS), and strictosidine synthase (STR) in stem-derived callus. The upregulation of these genes was positively correlated with increased VCR accumulation. In contrast, the expression levels of octadecanoid-derivative Responsive Catharanthus AP2-domain protein 3 (ORCA3) and desacetoxyvindoline 4-hydroxylase (D4H) were reduced. These findings highlight the importance of tailored hormonal treatments for optimizing in vitro biomass production and secondary metabolite synthesis in C. roseus, offering insights into sustainable alkaloid production for pharmaceutical applications. This study addresses the link between tailored plant growth regulator regimes and the coordinated activation of late-pathway genes, resulting in the selective enhancement of individual TIAs in C. roseus callus cultures, thereby providing a novel molecular–biotechnological strategy for targeted alkaloid production.