Abstract
Protoplasts are single cells enclosed by the plasma membrane after cell wall removal. They are widely used in various biotechnological applications, including gene functional analysis, verification of genome editing reagents, and plant regeneration. Recent advances in genome editing have enabled the production of non-chimeric and transgene-free genome-edited plants using protoplasts. This process involves protoplast isolation, transformation, and regeneration, requiring advanced technical skills. Challenges in isolation and regeneration have limited their use in genome editing. In grapevines, however, very few studies have reported the use of protoplasts isolated from leaves. Efficient isolation and transformation protocols for Chardonnay remain lacking and require cultivar-specific optimization. In this study, we established a reliable and efficient protoplast isolation and transformation system by optimizing conditions for protoplast isolation and PEG-mediated transformation in Chardonnay cultivar. The yield of viable protoplasts was approximately 75 × 10(6) per gram of leaf material, with a viability of 91%. A transformation efficiency of 87% was achieved under the optimized conditions. To evaluate the regeneration ability of mesophyll protoplast, transformed and untransformed protoplasts were cultured on solid and liquid MS media supplemented with 2 mg/L 2,4-D and 0.5 mg/L BA to facilitate microcalli formation. Microcalli formed on the feeder layer and developed into calli when transferred to liquid MS culture with 2 mg/L 2,4-D and 0.5 mg/L BA. However, the calli were unable to regenerate into roots or shoots. These findings provide a foundation for further optimization of protoplast-based regeneration systems in grapevines, with the potential to enhance genome editing applications in this species.