PVP-40 mediated enhancement of mesophyll protoplast yield and viability for transient gene expression in black...

Black huckleberry (Vaccinium membranaceum) is a native fruit species of high nutritional, medicinal, ecological, and economic value. The black huckleberries, abundant in bioactive compounds, offer significant antioxidants and anti-inflammatory effects and play a key role in maintaining wildlife and forest ecosystems. Despite its importance, protoplast isolation and gene editing have not been reported in this species. These techniques are essential for functional genomics and crop improvement, but the recalcitrant nature of this species, complex genome, and variable ploidy present significant challenges for cellular and molecular manipulation. This study aimed to establish a reliable protocol for efficient mesophyll protoplast isolation and transient gene expression in V. membranaceum using in vitro-grown leaves.

A systematic optimization of enzyme composition, osmotic concentration, antioxidant supplementation, and pH was undertaken to enhance protoplast yield and viability in V. membranaceum. The optimized enzymatic combination of 2% cellulase R-10, 1% hemicellulase, 1% Macerozyme R-10, and 1.5% pectinase facilitated efficient cell wall degradation while maintaining structural integrity. The inclusion of 0.6 M mannitol ensured osmotic stability, and 1% PVP-40 effectively suppressed phenolic oxidation, significantly improving protoplast viability. A near-neutral pH of 5.8 supported optimal enzyme activity without inducing cellular damage. Under these optimized conditions, 14 h enzymatic digestion produced 7.20 × 10⁶ protoplasts g⁻¹ FW with 95.1% viability. Subsequent optimization of PEG-mediated transformation identified 40% PEG-4000 with 30 µg plasmid DNA as the most effective combination, achieving 75.1% transient expression efficiency. Nuclear localization of GFP-tagged proteins, confirmed by DAPI staining, validated the robustness of the optimized system.

This study presents a standardized, PVP-40-enhanced protocol for mesophyll protoplast isolation with notable yield and viability in V. membranaceum, supporting efficient transient gene expression. The method provides a robust platform for functional genomics, gene editing, and biotechnological applications in this underutilized species and other related plant species.