Comparative Metabolomics Reveals Enhanced TCA Cycle and Suppressed Secondary Metabolism as Metabolic Hallmarks of Embryogenic Calli in Picea mongolica

Somatic embryogenesis (SE) plays a pivotal role in the propagation and genetic improvement of coniferous trees; however, its efficiency is frequently limited by the reduced embryogenic potential of callus cultures. Here, we investigated the metabolic determinants underlying this phenomenon in Picea mongolica by conducting a comparative metabolomic analysis of embryogenic calli (EC) and non-embryogenic calli (NEC). We observed significant metabolic differences between EC and NEC using an integrated approach combining morphological observations and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. EC exhibited increased central carbon metabolism, characterized by enhanced citrate cycle (TCA) flux, with significantly increased levels of the key TCA intermediates, citric acid and L-malic acid—18.8- and 3.6-fold higher, respectively, than those in NEC. Conversely, NEC displayed a divergent metabolic state, characterized by the accumulation of various amino acids and the activation of secondary metabolic pathways, especially alkaloid biosynthesis. These results indicate that embryogenic competence in P. mongolica is supported by a distinct metabolic program that prioritizes energy generation and efficient carbon-nitrogen allocation for biosynthetic processes. Conversely, the non-embryogenic state arises from a shift in metabolic resources toward secondary metabolism. These findings provide key metabolic insights and a theoretical basis for enhancing conifer SE systems.