Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.