In vitro induced osmotic stress changes morphological, physiological, biochemical and phytochemical characteristics of Centella asiatica (L.) Urban

Centella asiatica (L.) Urban is an important medicinal plant valued for its centellosides, a group of bioactive pentacyclic triterpenoids with extensive pharmaceutical and cosmetic applications. In vitro osmotic stress has been established as a promising strategy for enhancing specialized metabolite production; however, comparative information on the responses of C. asiatica to commonly used osmotic treatments remains limited. This study investigated the morphological, stomatal, physiological, biochemical, and phytochemical responses of C. asiatica plantlets to representative in vitro osmotic treatments using: polyethylene glycol (PEG 4000; 1, 2, and 4%), sucrose (45, 60, and 80 g/L), and sorbitol (20, 30, and 40 g/L). Most morphological traits, including biomass accumulation, plant height, and leaf number, were not significantly affected by the applied treatments. In contrast, leaf area index and root length responded significantly, with the highest leaf area index observed under 45 g/L sucrose and the greatest root elongation under 4% PEG. Among the treatments examined by SEM, osmotic treatments reduced stomatal density and altered stomatal dimensions on both leaf surfaces. Photosynthetic pigment contents varied among treatments, with particularly low chlorophyll and carotenoid contents under 60 g/L sucrose and 40 g/L sorbitol. Total soluble protein content increased under high sucrose concentrations, whereas antioxidant enzyme responses differed among treatments. Catalase and guaiacol peroxidase activities were highest under 1% PEG and 45 g/L sucrose, respectively, whereas ascorbate peroxidase and superoxide dismutase activities were highest under 40 g/L sorbitol. Total phenolic content remained unchanged, whereas total flavonoid content was highest under 40 g/L sorbitol and 80 g/L sucrose. Among the treatments evaluated, 40 g/L sorbitol was associated with the highest total centelloside accumulation, primarily through increased madecassoside and asiaticoside contents, whereas 4% PEG was associated with greater accumulation of madecassic acid and asiatic acid. Among the treatments tested, 40 g/L sorbitol was associated with the highest total centelloside accumulation, with no significant reduction in fresh weight, dry weight, or plant height. However, leaf area index was significantly reduced under 20 g/L sorbitol, and photosynthetic pigment contents were reduced particularly under 40 g/L sorbitol. These findings suggest a potential growth–metabolite trade-off and should be interpreted as a comparison among representative osmotic treatment regimes rather than as evidence of the intrinsic superiority of any individual osmotic agent. Not applicable.