Efficient Protoplast Isolation and PEG-mediated Transformation protocols for blueberry Vaccinium corymbosum

• An efficient system for isolating and transforming blueberry callus protoplasts was established. • A transformation efficiency of 40.4 % was achieved when the protoplasts were treated with 45 % PEG4000 and a high concentration Ca 2+ . • This system provides a valuable platform for the exploration of gene function, genome editing and protoplast regeneration. The blueberry is an important commercial fruit tree with high nutritional value in its fruit. Due to the complexity of its ploidy, genetic improvement in blueberry faces challenges such as difficult genetic transformation and low efficiency. Protoplasts can be used as efficient recipients for transient transformation and serve as important tools for gene function and editing. However, no one has reported a protoplast transformation system in blueberries. In this study, we established an efficient system for the isolation and transient transformation of blueberry protoplasts. The 30-day culture of callus from leaf discs was selected as the optimum callus for the isolation of protoplasts with the highest yield and viability. The callus was then incubated in an enzymolysis solution containing 1.2 % (w/v) Cellulase R-10, 0.8 % (w/v) Macerozyme R-10, and 0.5 M d -mannitol under dark for 5 h, and the protoplast yield obtained was 2.95×10 6 g −1 (FW) with 90.4 % viability. Furthermore, by optimizing various transformation conditions, a transformation efficiency of 40.4 % was achieved when 35–40 μg plasmids were mixed with 100 μL protoplasts and incubated with 45 % (w/v) PEG in the dark for 35 min. Additionally, the results of VcHKT1;1 subcellular localization further verified the reliability of this system. Overall, our study reports a highly efficient system for isolation and transient transformation of blueberry protoplasts and provides crucial technical support for the exploration of blueberry physiology and molecular mechanism, as well as promising prospects for blueberry breeding.