体细胞胚胎发生是植物细胞全能性的体现,在种质资源保存、林木育种及基因编辑等领域具有重要应用价值。由于缺乏通用技术体系,林木体胚发生效率种间差异显著,亟须突破外植体脱分化效率低与体胚成熟率不高的瓶颈。深入解析体胚发生的分子机制是优化体系、推动体胚技术产业化的关键。单细胞测序技术可以在单细胞水平解析转录组异质性与表观遗传调控动态信息,揭示林木体胚发生过程中分子调控机制和细胞命运转换轨迹,为林木遗传育种提供新视角。然而,木本植物细胞特有的细胞壁结构与组织的复杂性严重制约了该技术的应用。本研究系统综述单细胞测序技术的发展历程、木本植物组织样本制备策略及生物信息分析流程,通过列举龙眼(Dimocarpus longan)、椰子(Cocos nucifera)等典型案例,分析其在林木体胚发生研究中的瓶颈与策略,探讨该技术在细胞命运轨迹解析、激素响应机制及多组学系统研究中的应用潜力,并展望未来技术优化与跨物种数据库建设的路径,为林木体胚发生机制研究提供理论支撑。

Somatic embryogenesis (SE), a manifestation of plant cellular totipotency, holds significant application value in plant germplasm resource conservation, elite tree breeding and industrial utilization, cultivar and gene-editing applications. However, substantial variations in embryogenic capacity exist among tree species, coupled with the absence of universally applicable protocols, highlighting an urgent need to improve dedifferentiation efficiency and somatic embryo induction rates. Elucidating the molecular mechanisms underlying somatic embryogenesis is essential for further enhancing embryogenesis efficiency, optimizing somatic embryo induction systems, and expanding industrial production. Single-cell sequencing (scRNA-seq) has emerged as a transformative tool for dissecting transcriptional heterogeneity, epigenetic dynamics, and cell fate transitions at single-cell resolution, offering novel insights into the regulatory networks underlying SE. However, the application of scRNA-seq in woody species is constrained by lignified cell walls, vascular complexity, and limited species-specific databases. This review systematically outlines the technological evolution of scRNA-seq, strategies for preparing woody plant tissues (e.g., protoplast isolation, nuclei extraction), and bioinformatic workflows for data analysis. By integrating case studies in Dimocarpus longan and Cocos nucifera, we highlight the utility of scRNA-seq in reconstructing developmental trajectories, unraveling hormone signaling crosstalk, and enabling multi-omics integration during SE. For instance, in D. longan, scRNA-seq revealed 12 cell clusters in embryogenic callus, with pseudotime analysis identifying histone deacetylation as a key regulator of early embryogenesis. Similarly, coconut studies demonstrated distinct transcriptional landscapes among zygotic embryos, callus, and somatic embryos, pinpointing CnGRF12 as a critical transcription factor in cell fate determination. Challenges in woody plant scRNA-seq include spatial information loss during cell dissociation, technical noise from protoplast preparation, and the scarcity of cross-species marker gene databases. To address these, we propose strategies such as combining snRNA-seq with spatial transcriptomics, optimizing enzymatic digestion protocols, and establishing unified annotation frameworks. Furthermore, advancements in multi-omics platforms (e.g., scATAC-seq, CITE-seq) and computational tools (e.g., Monocle for trajectory inference) are discussed as avenues to enhance resolution and biological relevance. Future directions emphasize the need for large-scale single-cell atlases, standardized protocols for recalcitrant species, and collaborative databases to bridge knowledge gaps between model and non-model plants. By leveraging these advancements, scRNA-seq holds immense potential to accelerate mechanistic studies of SE, optimize regeneration systems, and advance precision breeding in forestry.