Development of an efficient protoplast isolation and transfection protocol for olive

In the context of climate change, the development of novel breeding strategies is imperative to enhance plant resilience. Within the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system emerges as a valuable tool for targeted gene editing. The absence of a cell wall in protoplasts makes them a versatile cell-based model system for investigating gene and protein functions. Moreover, protoplasts represent an opportunity for DNA-free genome editing, through transfection with ribonucleoproteins (RNPs), which are preassembled complexes comprising purified Cas9 protein and guide RNAs. This study outlines an optimized protocol for isolating olive protoplasts, with a specific focus on maximizing yield, ensuring viability and promoting callus differentiation. Protoplasts were obtained from both leaves and calli induced in different media. Three distinct enzymatic solutions for cell-wall digestion were investigated. Following isolation, protoplasts were transfected using a PEG/Ca2+ solution and the pAVA393:GFP plasmid. The efficiency of protoplast transfection, evaluated 48 h post-transfection, showed value higher than 50% of transfected protoplasts. Culturing protoplasts in a liquid medium for callus induction revealed the formation of microcolonies. Ongoing experiments are exploring callus development as well as de novo shoot organogenesis and/or somatic embryogenesis. The results underscore the potential of olive protoplasts as a valuable tool to be applied for DNA-free genome editing.