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Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates

Summary

Scientists have developed a better method to isolate protoplasts (fungal cells without cell walls) from non-sporulating varieties of gray mold fungus. By optimizing the incubation time, culture container, and enzyme used, they produced more viable protoplasts that can regenerate and be genetically modified. This advancement allows researchers to use CRISPR gene-editing technology to understand and potentially control gray mold, which causes significant crop losses worldwide.

Background

Botrytis cinerea is a necrotrophic fungus causing significant economic losses in commercial crops. Various isolates exhibit different morphological and genetic characteristics, ranging from non-sporogenic to highly virulent forms. Protoplast-mediated transformation has been successfully applied in fungal genetic engineering but requires optimization for different species.

Objective

To develop an improved protoplast production protocol specifically for non-sporulating B. cinerea isolates that produces viable protoplasts with regenerating capacity suitable for CRISPR/Cas9-mediated genetic transformation. The study aimed to optimize three key parameters: incubation time, culture method, and enzymatic treatment.

Results

Two-day incubation of mycelium plugs in petri dishes with 20 mL medium at 40 rpm orbital shaking proved optimal for protoplast isolation. VinoTaste Pro enzyme at 0.2 g concentration in KC buffer produced the highest number of functional protoplasts (2 × 10⁷ per gram of mycelium) with superior regeneration capacity. CRISPR/Cas9-mediated transformation achieved success rates above 90% with 13-19% homokaryotic colonies, significantly outperforming random integration methods.

Conclusion

The optimized protocol successfully produces viable protoplasts from non-sporulating B. cinerea isolates suitable for genetic transformation. VinoTaste Pro serves as an effective, economical replacement for discontinued Glucanex. This method enables biotechnological applications including CRISPR/Cas9-mediated genome editing in previously difficult-to-transform fungal isolates.
  • Published in:Plants (Basel),
  • Study Type:Methods Development Study,
  • Source: PMID: 38999594, DOI: 10.3390/plants13131754
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