Development of a highly efficient and widely applicable protoplast isolation and transformation system for strawberry

Protoplasts are a valuable biotechnological tool for plant genetic improvement, yet efficient isolation and transformation systems with broad applicability across strawberry ( Fragaria vesca/ananassa ) species remain underdeveloped. Here, we developed an optimized protocol encompassing callus induction, suspension cell culture, and protoplast isolation and transformation derived from suspension cells, mesophyll cells and petals. Our results revealed maximal callus induction efficacy on MS medium supplemented with 1.0 mg · L -1 6-BA and 0.1 mg · L -1 NAA, while optimal suspension cell growth was achieved by inoculating 1 g of yellowish, loose embryogenic callus into 40 mL of liquid medium. Peak protoplast yield and viability were achieved following enzymatic digestion (1.5% Cellulase R-10, 0.75% Macerozyme R-10, 0.5% Snailase, and 0.4 mol · L -1 D-mannitol) for 8 h (leaves and petals) and 10 h (suspension cells). Notably, we developed the universally applicable protoplast isolation system for strawberries spanning various ploidy levels and varieties. Transient transformation efficiency was optimized using 20 μg plasmid DNA and 40% PEG4000 for 20 min, enabling successful application in subcellular localization, protein-protein interaction, protein detection and assessment of U6 promoter-driven genome editing efficiency. This efficient, versatile, and time-saving protoplast-based system provides a powerful platform for diverse strawberry research applications, including somatic cell fusion, genome editing, functional genomics, and multi-omics analyses.