Somatic embryogenesis-induced epigenetic changes promoting catechin accumulation in Vaccinium vitis-idaea L

Somatic embryogenesis triggers epigenetic changes that influence catechin accumulation in lingonberry, helping to improve phytochemical quality in micropropagated plants. Vaccinium vitis-idaea L. (lingonberry), a boreal superfruit valued for its high antioxidant content, exhibits high potential for regeneration via somatic embryogenesis (SE). However, in-vitro culture often induces epigenetic variations that can influence phytochemical stability. To find out whether DNA methylation plays a role during SE, we compared DNA methylation levels in the regenerants of two lingonberry genotypes, Erntedank (Cv1) and a hybrid designated as "H1" (identity undisclosed). We demonstrate that SE in lingonberries induces epigenetic modifications, particularly epigenetic memory and biological imprinting that contribute to enhanced catechin accumulation, as revealed by highly sensitive DNA methylation profiling using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) and catechin analysis via proton nuclear magnetic resonance (¹H NMR) across four experimental groups: in-vitro-derived shoots, in-vitro and ex-vitro SE-regenerated plants, and controls. The optimized UHPLC protocol enabled robust quantification of 5-methylcytosine (5mC), revealing significantly elevated DNA methylation in in-vitro-derived shoots (Cv1-TC: 84.02%, H1-TC: 87.30%) compared to ex-vitro SE-regenerated plants (Cv1-GC: 53.79%, H1-GC: 60.27%). Parallel ¹H NMR analysis confirmed the presence of catechins, with ex-vitro SE plants showing the strongest signals across aromatic (6.93-6.06 ppm), sugar (4.57-4.01 ppm), and aliphatic (2.93-2.54 ppm) regions. In-vitro-derived shoots exhibited high methylation but minimal catechin signals, suggesting epigenetic suppression of flavonoid biosynthesis under culture-induced stress. This negative relation indicates that the methylation status influences secondary metabolite profiles. The findings underscore the importance of monitoring epigenetic and biochemical integrity during plant regeneration. This dual-platform strategy provides a valuable tool for optimizing micropropagation systems, monitoring epigenetic changes, and improving phytochemical consistency in lingonberry.