Heat stress-induced metabolic responses enhance antioxidant capacity, phenolic and flavonoid accumulation in Bacopa monnieri callus cultures

Heat stress is an important abiotic factor that influences plant growth and metabolism and may function as an effective elicitor for enhancing secondary metabolite production. This study aimed to investigate the effects of heat stress on growth performance, physiological and biochemical responses, and phenolic and flavonoid accumulation in Bacopa monnieri (L.) Wettst. callus cultures under in vitro conditions. Calli were cultured at 25°C (control) and 42°C (heat stress) for 7 days. The results showed that heat stress did not affect survival or response rates (100% in both treatments) but significantly increased callus size. While fresh weight and malondialdehyde content were unaffected by heat stress, dry weight (DW) increased significantly, and electrolyte leakage showed a non-significant increasing trend. In contrast, heat stress significantly increased total phenolic content (TPC) and total flavonoid content (TFC), while reducing the IC₅₀ value from the 2,2-diphenyl-1-picrylhydrazyl assay, indicating enhanced antioxidant capacity. Multivariate analyses (partial least squares discriminant analysis and variable importance in projection scores) identified antioxidant-related parameters (IC₅₀ and TPC) and DW as key variables associated with treatment separation, suggesting that biochemical adjustments contribute importantly to heat-stress responses in callus cultures. Under the conditions tested (42°C for 7 days), heat stress showed potential as an abiotic elicitor, enhancing antioxidant activity and the accumulation of secondary metabolites in B. monnieri callus cultures. These findings provide new insights into temperature-induced physiological and biochemical responses and suggest a potential strategy for enhancing antioxidant-related traits in medicinal plant callus cultures under controlled conditions.