Immersion Frequency Optimisation and Species-Specific Metabolic Profiles of Colchicum autumnale and Colchicum bivonae in Temporary Immersion Systems

Temporary immersion systems (TISs) are an advanced biotechnological platform for the large-scale cultivation of medicinal plants and the consistent production of high-value secondary metabolites. In this study, we evaluated three immersion regimes with stand-by periods of 4, 8, or 12 h, each paired with a 15-minute immersion period, to optimise shoot growth and colchicine accumulation in Colchicum autumnale L. and Colchicum bivonae Guss. The 4 h stand-by/15 min immersion regime yielded the highest growth index (C. autumnale: 0.75 ± 0.08; C. bivonae: 1.25 ± 0.03) and maximum colchicine content (C. autumnale: 0.19 ± 0.01 mg/g dry biomass; C. bivonae: 0.25 ± 0.02 mg/g dry biomass). Using gas chromatography-mass spectrometry (GC-MS), detailed metabolic profiling of cultures grown under this optimised regime was performed, resulting in the identification of 46 metabolites, including amino acids, organic acids, sugars, sugar alcohols, phenolic, and fatty acids. Volcano plot analysis revealed 11 upregulated and 5 downregulated metabolites in C. autumnale relative to C. bivonae. Significance analysis of metabolomics (SAM) identified 34 metabolites with statistically significant differences between two species. Hierarchical clustering and partial least squares discriminant analysis (PLS-DA) confirmed clear species separation, with Component 1 explaining 68.8% of the total metabolic variance. Glucose-6-phosphate (VIP = 2.01), citric acid (VIP = 1.85), asparagine (VIP = 1.67), and γ-aminobutyric acid (GABA; VIP = 1.52) were the primary biomarkers differentiating the species. These findings confirm that TISs provide an optimised environment for biomass accumulation and stable alkaloid biosynthesis in the Colchicum genus, with C. bivonae emerging as a promising candidate for biotechnological exploitation.