Biotic and Abiotic Elicitation Enhances Silymarin Biosynthesis in Silybum marianum Callus Cultures: Integrating HPLC Profiling and Gene Expression Analysis.

<b>Background and Objective:</b> <i>Silybum marianum</i> (milk thistle) is a valuable medicinal plant that produces silymarin, a complex of flavonolignans with well-documented hepatoprotective, antioxidant and anti-inflammatory properties. However, silymarin production from field-grown plants is limited by low yield, environmental variability and inconsistent metabolite accumulation. This study aimed to evaluate the effects of biotic and abiotic elicitors on biomass growth, silymarin biosynthesis and the expression of key biosynthetic genes in <i>S. marianum</i> callus cultures. <b>Materials and Methods:</b> Callus cultures were established from sterile explants and treated with biotic elicitors (yeast extract and chitosan) and abiotic elicitors (methyl jasmonate and silver nanoparticles) at different concentrations under optimized <i>in vitro</i> conditions. Biomass accumulation, total flavonolignan content and the expression of the phenylpropanoid pathway genes PAL, CHS and TAT were analyzed. High-Performance Liquid Chromatography (HPLC) was employed to quantify the major silymarin constituents, including silybin A, silybin B, silychristin, silydianin and isosilybin. <b>Results:</b> Elicitor treatments significantly enhanced silymarin biosynthesis compared with the untreated control. The combined treatment of methyl jasmonate (100 μM) and chitosan (50 mg/L) produced the highest response, resulting in a 10.4-fold increase in total silymarin content (14.08 mg/g DW) and an 18.42-fold upregulation of CHS expression. The HPLC analysis confirmed increased accumulation of the major silymarin flavonolignans, demonstrating a synergistic effect of combined biotic and abiotic elicitation on secondary metabolite production. <b>Conclusion:</b> Combined biotic and abiotic elicitation effectively enhanced silymarin biosynthesis in <i>S. marianum</i> callus cultures, with increased expression of selected phenylpropanoid-associated genes and higher flavonolignan accumulation. The findings support combined elicitation as a promising controlled <i>in vitro</i> approach, but validation at larger culture scales and across broader molecular targets is required before industrial application.