A highly efficient protocol for tomato protoplast isolation and transfection via seedling etiolation

Protoplast-based transient expression systems serve as indispensable platforms for functional genomics, genome editing, and synthetic biology. While tomato ( Solanum lycopersicum ) is a premier model crop for genetic research, its utility in protoplast-based assays has been constrained by suboptimal isolation yields and low transfection efficiencies. In this study, we report that etiolation treatment of tomato seedlings (cv. Micro-Tom) significantly enhances both protoplast recovery and PEG-mediated transfection. Cotyledons subjected to an 8-day dark treatment yielded an average of 1.47 × 10 7 protoplasts/g FW, representing a 12-fold increase compared with non-etiolated controls (1.22 × 10 6 protoplasts/g FW). Furthermore, etiolated protoplasts exhibited superior viability at 90.8%, compared to 53.4% in light-grown counterparts. Most notably, the transfection efficiency using a GFP-reporter plasmid reached 58.2% in etiolated protoplasts, a 2.2-fold improvement over the 25.8% observed in non-etiolated cotyledons. Our results suggest that etiolation optimizes cellular physiology and enhances structural stability for exogenous DNA delivery. This study establishes a robust and streamlined protocol for high-efficiency protoplast isolation and transfection, providing a powerful tool to accelerate precision breeding and functional genomics in tomato.