Insights into the developmental trajectories of zygotic embryo, embryogenic callus and somatic embryo in coconut by single-cell transcriptomic analysis

Comprehensive utilization of coconut tissue culture in propagation and transformation is limited by the low efficiency of somatic embryogenesis. Understanding the potential mechanisms and developmental regulators (DRs) will facilitate the process. Here, we performed single-cell RNA sequencing (scRNA-seq) using protoplasts isolated from the zygotic embryo (ZE), embryogenic callus (EC) and somatic embryo (SE) of 'Hainan Tall' coconut. Cell types were identified with the assistance of known cell marker genes and further validated by RNA in situ hybridization. Differentiation trajectories from the ZE to SE transition were then constructed and identified potential DRs in the process. Notably, developmental initiation cells for both ZE and SE were identified as quiescent center-like cells (QC-L), while it was the dividing cell (DC) in EC. CnGRF12 (Growth Regulating Factor) was preferentially expressed in these developmental initiation cells, and its co-expression with CnGIF1 (GRF-interacting Factor) by transient transformation suggested that CnGRF12 has potential as a DR to accelerate transformation. Our comprehensive cell atlas provides single-cell-level insights into the developmental trajectories of ZE, EC and SE cells in coconut, and potential DRs investigated in this study could be useful for the future genotype-independent transformation and gene editing of coconut.