DNA-free genome editing in tomato protoplasts using CRISPR/Cas9 ribonucleoprotein delivery

CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly efficient for micro-calli development. G-207.3 liquid medium was more efficient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, five single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis efficiency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials.