Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts’ usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.

Mechanical wounding is a common physical stress affecting Camellia oleifera during cultivation and harvesting; however, its early physiological and transcriptional responses remain poorly understood. In this study, leaves of C. oleifera ‘Huashuo’ were artificially wounded and collected at different time points. Progressive accumulation of H₂O₂ and O₂⁻ was observed following wounding, as indicated by enhanced DAB and NBT staining. The activities of SOD, CAT, POD, GPX, and APX generally increased, with POD showing the greatest increase. GSH content increased overall, whereas AsA content reached its highest level at 6 h. RNA-seq analysis identified 2520 upregulated and 457 downregulated genes in the 1 h versus 0 h comparison, representing the largest number of differentially expressed genes among the examined time-point comparisons. These genes were mainly enriched in plant–pathogen interaction, MAPK signaling pathway–plant, plant hormone signal transduction, phenylpropanoid biosynthesis, and jasmonic acid biosynthesis and signaling pathways. Among the wound-responsive WRKY genes, CoWRKY75 showed a time-dependent increase in expression and reached its highest level at 6 h. RT-qPCR analysis confirmed a trend consistent with the RNA-seq data. CoWRKY75 encodes a 157-amino-acid protein with a typical WRKY domain, and the protein mainly localizes in the nucleus. Heterologous overexpression of CoWRKY75 in Arabidopsis thaliana resulted in a distinct early-flowering phenotype, suggesting that CoWRKY75 may have a role in plant developmental regulation. These results characterize the early physiological and transcriptional responses of C. oleifera leaves to mechanical wounding and identify CoWRKY75 as a candidate WRKY transcription factor associated with the mechanical wounding response.