A Stable and Rapid Protoplast-Based CRISPR/Cas9-Ribonucleoprotein Complex Screening System for Octoploid Strawberry...

Area of Science:

• Plant Biotechnology
• Genomics
• Horticultural Science

Background:

• The CRISPR/Cas9 system offers powerful gene editing capabilities for crop improvement.
• Cultivated octoploid strawberry (Fragaria× ananassa) presents genome editing challenges due to its complex polyploid nature.
• Efficient genome editing is crucial for advancing functional genomics and breeding in strawberries.

Purpose of the Study:

• To establish a DNA-free, protoplast-based genome editing system for cultivated octoploid strawberry.
• To optimize protoplast isolation and transient transfection for efficient gene editing.
• To demonstrate the application of CRISPR/Cas9 for targeted gene modification in strawberry.

Main Methods:

• Optimized protoplast isolation from strawberry leaves using specific culture media and enzyme combinations (Viscozyme, Celluclast, Pectinex).
• Optimized transient transfection protocols using polyethylene glycol (PEG) for high expression efficiency.
• Delivered Cas9 ribonucleoproteins (RNPs) targeting FaPDS and FaPG1 genes via optimized transfection.

Main Results:

• Achieved efficient protoplast isolation with high yields using optimized Murashige and Skoog medium and enzyme treatments.
• Determined optimal transient transfection conditions (40% PEG 4000, 20 min incubation) for high expression efficiency (up to 52.5%).
• Successfully performed DNA-free CRISPR/Cas9 genome editing targeting specific genes in octoploid strawberry.

Conclusions:

• Established a robust protoplast isolation and DNA-free CRISPR/Cas9 genome editing protocol for cultivated strawberry.
• The optimized system provides a valuable platform for functional genomics research in octoploid strawberries.
• This approach facilitates molecular breeding and crop improvement in complex polyploid species.