Abstract

Orchidaceae is a highly valuable horticultural and medicinal plant family worldwide; however, large-scale propagation and conservation remain severely limited. Orchid seeds depend on symbiotic fungi for germination, and pre-symbiotic communication is essential for establishing a successful association, a process that remains poorly understood. In this study, exudates were collected from the germination-promoting fungus Ceratobasidium sp. GS2 and applied to seeds of the terrestrial orchid Gymnadenia conopsea to investigate downstream responses. Multi-omics approaches, including RNA-seq, metabolomics, and phylogenetic analysis, combined with biological validation, revealed that the exudates elicited transcriptional and physiological responses in G. conopsea seeds, potentially promoting dormancy release. Exposure to fungal exudates increased the levels of brassinosteroids, cytokinins, and fatty acids in seeds. Exogenous hormone application confirmed that brassinosteroids and cytokinins promote fungal colonization and facilitate symbiotic seed germination. Phylogenetic analysis revealed the conservation of symbiotic genes in partially mycoheterotrophic orchids, and functional characterization confirmed the role of GcRAM2 in G. conopsea . These findings provide new insights into the mechanisms underlying orchid mycorrhizal symbiosis.