Optimization Pathways and Application Advances in Plant Protoplast Extraction Technology

Protoplasts are plant cells that retain totipotency after removal of the cell wall, and have been widely used in plant research fields such as functional genomics, germplasm innovation through cell engineering, large-scale production of secondary metabolites, as well as analysis of physiological and ecological mechanisms and stress tolerance screening. Although this technology has been standardized in some model plants, non-model plants still require customized protocols based on species-specific characteristics, with certain recalcitrant tissues continuing to face isolation bottlenecks. This review systematically summarizes the optimization pathways and application advances in protoplast extraction technology, and discusses key technical strategies currently employed to improve protoplast yield and viability. In the future, protoplast technology will become deeply integrated with single-cell multi-omics, synthetic biology, and artificial intelligence, progressing toward intelligent, automated, and industrial applications, thereby playing an even more central role in plant science innovation.