Liquid overlaying with calcium supplementation triggers antioxidants and secondary metabolites accumulation during somatic embryogenesis in Catharanthus roseus (L.) G. Don

Catharanthus roseus is a valuable medicinal plant and produce more than 200 alkaloids, including the cancer-fighting drugs vincristine (VCR) and vinblastine (VLB). These alkaloids are rare with low concentration in the plant. Therefore, efforts are made to increase their production through biotechnological approaches. The present study elucidates the effect of CaCl₂ supplementation (5–100 mM) on C. roseus embryoids cultured in vitro using a liquid overlay system. The responses were evaluated in terms of growth characteristics, biochemical changes, and alkaloid accumulation. CaCl₂ treatment significantly enhanced callus growth and embryoid formation, with the most effective responses observed at 15–25 mM. Biochemical analysis showed increased accumulation of sugars, proline, and proteins in CaCl₂-treated cultures. In addition, the activities of antioxidant enzymes, including glutathione reductase (GR), ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), were elevated at higher CaCl₂ concentrations during both induction and proliferation phases. It suggests their putative involvement in the modulation of primary metabolism and cellular redox status and may lead to enhanced indole alkaloid biosynthesis. High-performance thin-layer chromatography (HPTLC) analysis further confirmed that CaCl₂ supplementation increased VCR and VLB contents by up to 14% and 17%, respectively. Overall, the findings indicate that CaCl₂ supplementation can improve the production of valuable anticancer alkaloids and may provide a useful basis for future scale-up studies. Further investigations are required to elucidate the influence of CaCl₂ on production pathways and to determine the optimal concentration for maximizing yield. • Optimal CaCl₂ concentrations (15–25 mM) improve callus growth and embryo development. • CaCl₂ supplementation/elicitation increases accumulation of sugars, proteins, and proline in tissues. • Antioxidant enzyme activity (SOD, CAT, APX, GR) rises with CaCl₂ concentration. • Vinblastine and vincristine production increases by up to 17% and 14%, respectively. • CaCl₂ supplementation/elicitation offers a promising approach to boost somatic embryogenesis and alkaloid yield in C. roseus cultures.