Abstract
BACKGROUND: Tissue culture is one of the most important methods for propagating orchids. Notably, many orchid seedlings exhibit autonomous flowering during the cultivation process. To explore the underlying mechanism, Anoectochilus roxburghii (Wall.) Lindl., an orchid that spontaneously forms in vitro flowers, was analyzed in this study. METHODS: Bud samples at the early, middle, and fully open stages were collected for transcriptome sequencing, followed by differential expression, trend, enrichment and protein-protein interaction (PPI) network analyses. RESULTS: Differential gene expression analysis identified 2364, 4137, and 6522 differentially expressed genes (DEGs) in the early vs. middle, middle vs. open, and early vs. open comparisons, respectively. These DEGs were significantly enriched in various metabolic and biosynthetic pathways, particularly in ko01100 (metabolic pathways). PPI network analysis further identified hub genes, including MCM3, MCM4, and MCM7, which are associated with DNA replication, and CURL3, which is linked to plant hormone signal transduction pathways. CONCLUSION: Our findings provide novel insights into the molecular mechanisms driving in vitro flowering in A. roxburghii, highlighting the importance of metabolic and biosynthetic process signaling in this unique developmental transition. These results provide valuable resources for future studies on orchid propagation and floral development.