New plant breeding technologies towards a more sustainable viticulture

European grapevine cultivars are highly susceptible to many pathogens that are managed through large pesticide use. Nevertheless, the European policies promote pesticide use reduction and new environmentally friendly methods for a more sustainable agriculture. In this framework, grapevine genetic improvement could benefit from New Plant Breeding Technologies. In order to reduce fungal susceptibility, we will produce knock-out plants from embryogenic calli using CRISPR/Cas9 technology. Studies in barley reported the acquisition of powdery mildew resistance by knocking out susceptibility genes belonging to the MLO (Mildew Locus O) family. In this study, our approach takes advantage from CRISPR/Cas9 technology to perform a multiple knockout of MLO genes. Among the 17 VvMLOs reported in grapevine we designed constructs to target VvMLO6 and VvMLO7. Golden Gate assembly was used to produce three different constructs (containing two guideRNAs for each gene) to knocking-out the targets singularly or by producing a double mutant. Usually, the genetic engineering techniques, mediated by A. tumefaciens, involve the insertion of exogenous selectable marker genes. These markers are required for selection of transgenic plants, but they are undesirable to be retained in commercial transgenic plants due to possible toxicity or allergenicity to humans and potential environmental hazard. To overcome these limits, we opted for a “clean” editing strategy developing an inducible excision system. This approach is based on a recombinase technology involving the Cre-loxP system from the P1 bacteriophage under a heat-shock inducible promoter to be activated once the editing event(s) will be confirmed. Obtainment of embryogenic calli is one of the main bottlenecks for application of CRISPR/Cas9: for two seasons, we collected inflorescences from Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese cultivars and two rootstocks, 110 Richter and SO4, cultured and maintained in vitro up to embryo development and then used to perform Agrobacterium tumefaciens GV3101 mediated transformation.