PVP-40 mediated enhancement of mesophyll protoplast yield and viability for transient gene expression in black huckleberry
Bibliographic record
Abstract
Abstract Background 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. Results 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. Conclusions 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".