New Plant Breeding Techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses, satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. Applying genome editing via CRISPR/Cas9 in grapevine, we focused our attention on susceptibility genes: to control powdery mildew we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family VvMLO7 and VvMLO6. In parallel we also focused our attention on abiotic stresses, in particular drought stress, performing a knock-out of two different gene classes: I) two different isoforms of GST (Glutathione S-Transferase) that are involved in increasing of ABA (abscisic acid), lateral root branching and osmolytes accumulation; II) two different isoforms of PME (Pectin Methyl Esterase) involved in regulation of woody hydraulic proprieties modifying pit membrane thickness and porosity.
In parallel to genome editing, we used cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important grape cultivars. This locus is formed by two different genes, TNL2A and TNL2B, that were inserted together (with native promoters and terminators).
To avoid one of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation, hence the insertion of unrelated transgene, we exploited an inducible excision system based on a Cre-Lox recombinase technology. The system is controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confirmed allowing the removal of CRISPR/Cas and selection markers both in genome editing and cisgenic approach.
