Transient gene expression in mesophyll protoplasts is a valuable approach for investigating gene function, plant physiological processes, and molecular mechanisms. Rubber dandelion (Taraxacum kok-saghyz, TKS) is an ideal model for studying rubber biosynthesis and serves as a promising source of natural rubber and inulin. However, developing efficient protoplast-based systems for TKS remains challenging. In this study, we established a robust method for isolating mesophyll protoplasts from TKS by optimizing enzymatic conditions for cell wall digestion. We subjected the protoplasts to PEG/calcium-mediated transfection and evaluated promoter activities, expressed and detected target proteins, confirmed the subcellular localization of the target proteins, and examined transcription factor-DNA interactions under physiological conditions. We also developed a rapid assessment strategy for genome-editing tools in TKS protoplasts using multiple reporter systems. We evaluated these optimized tools in a tissue-culture-free hairy root transformation system, establishing a dual-platform toolkit for functional genomics in TKS. This work provides an efficient approach for TKS protoplast preparation, facilitating studies of gene function and advancing biotechnological research in this rubber-producing crop.
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