Isolation and Transfection of Protoplasts From Maize Mesophyll Cells.

Protoplast systems are widely used in plant research as versatile platforms for studying cellular processes and validating gene editing tools. In maize, they are particularly valuable because stable transformation in immature embryos is slow and labor-intensive, often requiring months to regenerate plants. However, existing protocols often yield inconsistent results in protoplast recovery, transfection efficiency, and viability. We present an optimized protocol for maize mesophyll protoplast isolation and PEG-mediated transfection. Two-week-old etiolated seedlings are processed using vertical cutting, improving the yield and viability of protoplasts. Protoplasts are then immediately transformed with a CRISPR/Cas9 construct after isolation, via PEG4000 with only 10 μg of plasmid DNA, reducing the resource demands of standard methods. Modified washing and storage conditions extend transformed protoplast viability to seven days, enabling longer-term monitoring and expanded downstream analyses. Editing outcomes are quantified by sequencing target sites and calculating efficiency with Cas-Analyzer. This protocol provides a rapid, efficient, and reproducible method for the rapid evaluation of gene editing in maize. This protocol offers a methodology to accelerate agricultural crop studies and broader plant molecular biology. Key features • Optimized maize mesophyll protoplast isolation using vertical cutting to improve yield, consistency, and viability. • Efficient PEG4000-mediated transformation requiring only 10 μg of plasmid DNA for CRISPR delivery. • Extended protoplast viability up to seven days through modified washing and storage conditions, enabling longer monitoring and analysis. • Rapid and reproducible gene-editing evaluation via targeted sequencing and Cas-Analyzer quantification.