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Plant embryogenic calli (ECs) can undergo somatic embryogenesis to regenerate plants. This process is mediated by regulatory factors, such as transcription factors and specifically expressed genes, but the precise molecular mechanisms underlying somatic embryogenesis at the single-cell level remain unclear. In this study, we performed high-resolution single-cell RNA sequencing analysis to determine the cellular changes in the EC of the woody plant species Dimocarpus longan (longan) and clarify the continuous cell differentiation trajectories at the transcriptome level. The highly heterogeneous cells in the EC were divided into 12 putative clusters (e.g., proliferating, meristematic, vascular, and epidermal cell clusters). We determined cluster-enriched expression marker genes and found that overexpression of the epidermal cell marker gene GDSL ESTERASE/LIPASE-1 inhibited the hydrolysis of triacylglycerol. In addition, the stability of autophagy was critical for the somatic embryogenesis of longan. The pseudo-timeline analysis elucidated the continuous cell differentiation trajectories from early embryonic cell division to vascular and epidermal cell differentiation during the somatic embryogenesis of longan. Moreover, key transcriptional regulators associated with cell fates were revealed. We found that ETHYLENE RESPONSIVE FACTOR 6 was characterized as a heat-sensitive factor that negatively regulates longan somatic embryogenesis under high-temperature stress conditions. The results of this study provide new spatiotemporal insights into cell division and differentiation during longan somatic embryogenesis at single-cell resolution., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)