Somatic embryogenesis is pivotal for conifer propagation, yet its molecular basis remains poorly understood in gymnosperms. Single-nucleus RNA-seq offers unprecedented resolution for deciphering cell-type-specific transcriptional dynamics during gymnosperm embryogenesis. Here, we constructed a high-resolution transcriptomic atlas of Norway spruce (Picea abies) using high-efficiency and non/low-efficiency proembryogenic masses (HEMs and LEMs) collected before and after somatic embryo induction. Analysis of 55,635 nuclei identified 11 clusters, including somatic embryogenic cells (SCs) responsible for somatic embryo formation. Developmental trajectory analysis revealed a polarity-associated state S6 enriched in SCs, which was abundant in HEMs but nearly absent in LEMs. Key polarity genes, such as ROP-Guanine Exchange Factors, a homolog of PIN-FORMED 1, and PaWOX2, were enriched in the S6 state. Strikingly, overexpression of P. abies AGAMOUS-like 104 (PaAGL104), an S6-enriched transcription factor, induced excessive suspensor growth and apical-basal defects in both HEMs and LEMs embryos. TUNEL assays showed impaired suspensor elimination via suppressed programmed cell death in PaAGL104 overexpressors. Spatial expression analysis using a PaAGL104pro::NLS-mCherry reporter line revealed dynamic expression from ubiquitous to basal-enriched in the embryo proper. This region potentially contains the "tube cell", a gymnosperm-specific stem cell crucial for suspensor formation and embryonic polarization. Our study identifies embryonic polarization as a key developmental checkpoint in conifer somatic embryogenesis and provides a high-resolution transcriptomic atlas of gymnosperm callus. These findings advance our understanding of totipotency in spruce and offer practical insights for improving clonal propagation in forestry.
Zhu et al. (2026) studied this question.