Data Sheet 1_A novel application of Raman spectroscopy to monitor cell wall removal during protoplasting in grapevine.pdf

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.</p>Results<p>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.</p>Discussion<p>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.</p>