Simulated microgravity enhances cell growth, callus formation, antioxidant responses, and lignan biosynthesis in Phyllanthus amarus

Gravity is a fundamental environmental signal that regulates plant growth, development, and metabolism. In medicinal plants, altered gravity conditions may serve as a useful abiotic cue for modulating secondary metabolite biosynthesis. In this study, we investigated the effects of simulated microgravity generated by a 2D clinostat on growth, cellular morphology, antioxidant enzyme activity, regeneration capacity, and lignan accumulation in single cells of Phyllanthus amarus , an important medicinal species rich in hepatoprotective lignans. Cell suspension cultures were established from callus tissues and early logarithmic-phase single cells (20-day-old) were selected for treatment. Based on screening experiments, 5 rpm for 10 days produced the best response, resulting in the highest cell growth while maintaining relatively stable morphology. Simulated microgravity altered intracellular organization, including a more uniform distribution of statoliths, and significantly increased antioxidant enzyme activities, including superoxide dismutase, catalase, and ascorbate peroxidase. Under clinostat treatment, phyllanthin, which was undetectable under normal gravity, accumulated to 3.5 µg/g, whereas hypophyllanthin content increased approximately 3.1-fold relative to the control. After transfer back to normal gravity, previously treated cells showed markedly improved regeneration, with callus formation increasing from 38% in control to 90% in the simulated microgravity group. These findings indicate that simulated microgravity may be an effective physical elicitor for improving both regenerative competence and lignan biosynthesis in P. amarus cell cultures, with potential applications in medicinal plant biotechnology.