Efficient protoplast isolation and transfection for CRISPR/Cas9-based genome editing in pea (Pisum sativum L.)

Pea (Pisum sativum L.) is an important protein-rich legume crop that fixes nitrogen. However, the cultivation of pea worldwide is facing challenges due to multiple constraints such as climate change, biotic and abiotic stresses, and the increasing demand for plant-based protein, which have necessitated the genetic trait improvement of pea. CRISPR/Cas gene editing has emerged as a promising genetic tool for precise trait improvement in plants. Nevertheless, its application is often hindered by the tedious and time-consuming plant transformation process. Therefore, protoplast-based transfection offers a valuable platform for validating the efficiency of CRISPR/Cas editing reagents prior to proceeding with stable transformation methods. The study aimed to optimise parameters for efficient protoplast isolation and precise genome editing in pea via CRISPR/Cas9 system. The polyethylene glycol (PEG) mediated transfection was validated in pea protoplasts using green fluorescent protein (GFP) as a reporter gene. Subsequently, pea phytoene desaturase (PsPDS) gene was edited in the protoplasts. The best protoplast transfection efficiency (59 ± 2.64%) was obtained using 20% PEG, with 20 µg plasmid DNA, and 15 min of incubation. Further, two gRNAs were designed for targeting PsPDS gene and their efficiency was validated by in-vitro cleavage assay. Multiplexed gene construct carrying the combination of gRNA1 and gRNA2 was used for transfection of pea protoplasts that showed up to 97% of targeted mutagenesis and confirmed the accuracy of gene editing. This study established a high-throughput platform for in-vivo testing of CRISPR reagents in pea protoplasts, offering a valuable tool for developing genetically improved, transgene-free pea plants. Using a single-cell system with protoplasts helps eliminate chimerism, enabling precise and reliable assessment of gene editing outcomes. The present research emphasizes the potential of pea protoplast platform as an effective system for in-vivo functional genomics and genome editing applications aimed to develop genetically improved pea crop.