Introduction: Catharanthus roseus (Madagascar periwinkle) is a vital medicinal plant renowned for producing anticancer terpenoid indole alkaloids (TIAs), including vincristine and vinblastine. However, the inherently low yield of these high‑value metabolites (typically <0.001% dry weight) necessitates effective elicitation strategies. Methyl jasmonate (MJ) and salicylic acid (SA) are well‑known elicitors that activate plant defense responses and secondary metabolism through distinct but interconnected signaling pathways (COI1‑dependent jasmonate signaling and NPR1‑mediated SA pathway). While individual effects of MJ and SA have been reported, their synergistic potential on a comprehensive set of physiological, biochemical, and molecular parameters in C. roseus has not been systematically investigated.

Aims: This study aimed to (1) evaluate the individual and combined effects of MJ and SA on biomass, membrane stability index (MSI), anthocyanin, chlorophyll (SPAD), protein content, antioxidant enzyme activities (SOD, CAT, GPX), essential oil (EO) yield, and the expression of four key TIA biosynthetic genes (TDC, STR, SGD, and DAT); and (2) identify the optimal concentration combination for maximizing alkaloid production.

Material and Methods: A factorial experiment based on a completely randomized design (CRD) with three replications was conducted. Four‑month‑old C. roseus 'Pink Rose' plants were foliar‑sprayed with MJ (0, 0.5, 1, 2 mM) and SA (0, 1, 2, 3 mM), individually or in combination, at 15‑day intervals. The treatments were applied three times to ensure sustained elicitation, and all plants were maintained under controlled greenhouse conditions (16 h light/8 h dark, 25±2°C day/20±2°C night, 60±5% relative humidity). Sampling was performed 24 h after the final treatment. Physiological traits (fresh/dry weight, MSI, anthocyanin, SPAD, total protein) and antioxidant enzyme activities were measured using standard spectrophotometric methods. Total protein content was determined by the Bradford assay, and antioxidant enzyme activities (SOD, CAT, and GPX) were assayed following established protocols. Essential oil was extracted by hydrodistillation from dried leaf powder and quantified gravimetrically. Gene expression was analyzed by qRT‑PCR using 18S rRNA as the internal reference. Data were subjected to two‑way ANOVA and Duncan's multiple range test (p ≤ 0.05) using SAS 9.2.

Results: The interaction between MJ and SA was significant (P ≤ 0.01) for all measured traits. Two combined treatments (1 mM MJ + 3 mM SA and 2 mM MJ + 2 mM SA) were identified as the optimal consensus treatments, as they ranked in the highest statistical groups across the majority of measured traits. Fresh weight and dry weight reached their highest values under 1 mM MJ + 2 mM SA, with increases of approximately 47% and 64% over the control, respectively. MSI improved by ~50% under both optimal treatments. Protein content increased by ~2.9-fold, while activities of SOD, CAT, and GPX rose approximately 3-fold compared to the control. Anthocyanin accumulation peaked (~2.5-fold over control) under 2 mM MJ + 3 mM SA, with 1 mM MJ + 3 mM SA placing in the same statistical group. Essential oil yield increased approximately 2-fold under the optimal combined treatments. All four TIA pathway genes (TDC, STR, SGD, and DAT) were upregulated by 2.4- to 3.8-fold, with STR showing the greatest induction (~3.8-fold) and SGD the least (~2.2–2.4-fold). Higher concentrations of SA (3 mM) in combination with lower MJ levels, or certain high-dose combinations, produced diminished responses for some traits, indicating concentration-dependent optima.

Discussion: These findings indicate that simultaneous MJ and SA elicitation activates coordinated physiological and transcriptional responses, with 1 mM MJ + 3 mM SA and 2 mM MJ + 2 mM SA as consensus optima. Enhanced antioxidant capacity, membrane stability, essential oil yield, and TDC/STR/SGD/DAT expression support a synergistic, concentration-dependent effect that may improve anticancer alkaloid production. Future field-scale validation and direct vinblastine/vincristine quantification are needed.

Conclusion: Co‑application of MJ and SA induces a strong combined effect, enhancing antioxidant capacity, essential oil accumulation, and transcriptional activation of TIA biosynthetic genes. This effect offers a possible approach to boost the production of anticancer alkaloids in C. roseus. Future field‑scale validation and direct quantification of vinblastine/vincristine are recommended.