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Somatic embryogenesis (SE) is a developmental process related to the regeneration of tissue-cultured plants, which serves as a useful technique for crop breeding and improvement. However, SE in cotton is difficult and elusive due to the lack of precise cellular level information on the reprogramming of gene expression patterns involved in somatic embryogenesis. Here, we investigate the spatial and single-cell expression profiles of key genes and the metabolic patterns of key metabolites by integrated single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics (ST), and spatial metabolomics (SM). To evaluate the results of these analyses, we functionally characterized the potential roles of two representative marker genes, AATP1 and DOX2, in the regulation of cotton somatic embryo development. A publicly available web-based resource database ( https://cotton.cricaas.com.cn/somaticembryo/ ) in this study provides convenience for future studies of the expression patterns of marker genes at specific developmental stages during the process of SE in cotton.