Protoplast technology in woody plants: current advances and potential applications in genetic improvement

Abstract Woody plants have garnered significant attention in recent years for their essential ecological and economic contributions. Protoplasts, isolated from plant cells, have exhibited remarkable totipotency and offered immense potential in a broad array of biological and biotechnological fields. These include, but are not limited to, protein gene expression regulation, functional gene analysis, subcellular localization, interaction studies, gene editing and single-cell sequencing. This review offers a comprehensive overview of protoplast isolation methods, key influencing factors, purification techniques and viability assessment. It further explores the use of protoplast transient expression systems for gene function characterization, while highlighting the diverse applications of protoplast-based technologies, such as fusion, regeneration, genome editing and single-cell sequencing. With technological advancements, future breakthroughs in these areas will be poised to create new avenues for research, genetic improvement and biotechnological innovations in woody plants.