A novel application of Raman spectroscopy to monitor cell wall removal during protoplasting in grapevine

Introduction Grapevine is one of the most widely cultivated and economically important perennial fruit crops worldwide. However, its genetic improvement is severely constrained by a long juvenile phase and a high degree of recalcitrance in in vitro regeneration and biotechnology applications. Recently, the feasibility of DNA-free genome editing through a protoplast-based platform has been demonstrated in grapevine. However, the successful application of this approach requires the optimization of complex and genotype-dependent protocols, ranging from tissue culture establishment and protoplast isolation to cell regeneration and whole-plant recovery. Methods Here, we investigated Raman spectroscopy (RS) as a noninvasive and label-free approach to monitor cell wall degradation during protoplast isolation in two grapevine cultivars, Vitis vinifera cv. Syrah and cv. Thompson Seedless. The RS-based assessment was integrated into a protoplast-to-plant workflow, which was subsequently evaluated through protoplast cultivation and whole-plant regeneration. Results For both cultivars, RS revealed specific cellulose-, xyloglucan-, and lignin-related spectral features in embryogenic calli that were absent or reduced in isolated protoplasts following enzymatic digestion. These spectral changes were supported by Fluorescent Brightener 28 staining and successful polyethylene glycol PEG-mediated GFP transfection, confirming that cell wall degradation was functionally sufficient for exogenous material delivery and downstream applications. The isolated protoplasts retained developmental competence, as demonstrated by somatic embryogenesis and whole-plant regeneration in both cultivars. Discussion These findings indicate that RS provides a standardized, label-free quality-control step for monitoring the protoplasting process in grapevine without compromising cell integrity. By providing detailed molecular profiling before and after enzymatic digestion, RS allows objective validation of cell wall removal. Successful plant regeneration serves as a biological downstream validation, confirming that the isolated protoplasts, in which cell wall digestion was demonstrated by RS, preserved developmental competence and totipotency. Integrated into protoplast workflows, RS enhances reproducibility and efficiency for downstream biotechnological applications, including DNA-free genome editing in recalcitrant woody species.