Osmotic stress modulates physio-biochemical traits and the expression of key genes involved in taxane biosynthesis in Taxus baccata L. cell cultures

• Osmotic stress enhanced the biosynthesis of taxanes in Taxus baccata cell cultures. • All treatments except polyethylene glycol reduced cell viability and biomass from day 7 onward. • Sorbitol (2%) raised taxol yield 32.12-fold on day 7 vs. control. • Osmotic stress enhanced extracellular taxanes accumulation over intracellular levels • Sorbitol induced the highest expression levels of the studied genes, whereas mannitol had the lowest effect. Induced environmental stresses are recognized as an effective strategy to enhance the production of specialized metabolites in in vitro cultures. This study investigated the effects of osmotic agents including mannitol (3%), polyethylene glycol 6000 (3%), sucrose (6%), and sorbitol (2%) at varying exposure times on the growth, physiological, biochemical, and metabolic responses of Taxus baccata L. cells during a 28-day batch culture. Additionally, the expression patterns of four key genes ( txs, t13αoh, bapt , and dbtnbt ), encoding enzymes involved in the taxane biosynthetic pathway, were analyzed. All treatments, except polyethylene glycol, led to a significant reduction in cell viability, fresh weight, and dry weight starting from the 7th day of culture. Increased levels of hydrogen peroxide and membrane lipid peroxidation were observed, accompanied by elevated activity of antioxidant enzymes such as catalase and ascorbate peroxidase, as well as a rise in free proline content within the cells. All treatments significantly enhanced the production of taxanes, with extracellular levels exceeding intracellular levels. The most pronounced effect was observed with 2% sorbitol on the 7th day of culture, which resulted in a 32.12-fold increase in taxol production compared to the control. A strong positive correlation was found between the expression patterns of the studied genes and the levels of taxane production. Among the osmotic agents, sorbitol had the greatest impact on gene expression, while mannitol had the lowest. These findings provide valuable insights for metabolic modeling aimed at optimizing large-scale T. baccata cell cultures to enhance taxane production.