Nanoparticle-induced modulation of indole alkaloid biosynthesis and gene expression in Catharanthus roseus in vitro cultures

Catharanthus roseus (L.) G. Don is a key medicinal plant producing pharmacologically important terpenoid indole alkaloids (TIAs). This study aimed to enhance the biosynthesis of three major TIAs; catharanthine (CAT), vincristine (VCR), and vinblastine (VBL) through a stepwise in vitro approach involving callus culture, hairy roots (HRs) induction via Agrobacterium rhizogenes, and elicitation with zinc oxide (ZnO) and chitosan (CH) nanoparticles (NPs) in solid and liquid media. Two HR lines (L1, derived from K599/p35SGFPGUS⁺, and L2, from disarmed K599) and stem-derived calli were treated with 50 and 100 mg/L of ZnO- and CH-NPs. Nanoparticle treatments had limited effects on callus growth, except 100 mg/L CH-NPs, which reduced biomass in solid medium. In suspension culture, 50 mg/L ZnO-NPs enhanced fresh weight, whereas CH-NPs reduced it. Among HRs, the highest biomass occurred with 100 mg/L ZnO-NPs. HPLC analysis revealed higher VCR levels in cultured tissues than in field-grown leaves. In solid-grown HRs, CAT increased with 100 mg/L CH-NPs, and VCR rose significantly with 100 mg/L of either NP type. In liquid-grown HRs, CAT increased with 100 mg/L CH-NPs, VCR with 100 mg/L ZnO-NPs, and VBL with 50 mg/L CH-NPs. In callus cultures, ZnO-NPs promoted CAT and VCR accumulation in solid media, while VCR increased in suspension cultures treated with 100 mg/L ZnO-NPs or 50 mg/L CH-NPs. Gene expression analysis confirmed NP-induced modulation of STR, DAT, PRX1, and SLS, correlating with alkaloid accumulation. Overall, ZnO and CH nanoparticles effectively elicited TIA biosynthesis in C. roseus cultures, highlighting their potential for enhanced alkaloid production. Zinc oxide and chitosan nanoparticles differentially modulate terpenoid indole alkaloid production in Catharanthus roseus callus and hairy roots, depending on nanoparticle type, concentration, culture system, and target alkaloid.