Tiny but powerful: protoplast isolation in Solanum melongena L. and RNPs mediated genome editing

Traditional genetic transformation approaches relying on Agrobacterium tumefaciens for the delivery of CRISPR/Cas9 reagents usually provide plants that stably integrate the gene construct in their genome. To meet the EU commission's proposal for a new legislation on plants obtained by new genomic techniques (NGTs), it is important to develop new protocols that produce transgene-free genome edited plants (NGT category 1). Protoplasts are a promising platform, since delivery of CRISPR/Cas9 reagents as ribonucleoproteins (RNPs) is effective in cells lacking their wall. This allows genetic modifications from a transient application, leaving no traces in the recipient genome apart from the desired targeted mutations. With the aim of implementing transgene-free editing of eggplant (Solanum melongena L.), we adapted and improved a protocol previously established in potato and tomato for the isolation of protoplasts from cotyledonary leaves and subsequent CRISPR/Cas9 reagents delivery. Isolated protoplasts were subjected to in vitro culture and regeneration, and the first shoots were regenerated from calli approximately 4-5 months after isolation. Alongside, two transfection protocols were tested for the delivery of RNPs into eggplant protoplasts, one using polyethylene glycol (PEG) in two concentrations (25% and 40%) and one exploiting two formulations of lipofectamines (Lipofectamine CRISPRMAX™ and Lipofectamine™ 3000), all targeting SmChl_H gene, whose inactivation can cause a chlorotic phenotype. Efficient callus regeneration from transfected protoplasts was obtained and the editing efficiency (calculated as the percentage of edited calli on the total of calli that underwent sequencing) was evaluated. 25% PEG treatment provided the highest editing efficiency, and fully edited biallelic calli were retrieved, showing the expected chlorotic phenotype. Even if the efficiency of in vitro regeneration of plants from calli still needs improvement, edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.