Cisgenesis and genome editing: powerful tools for fruit breeding

Over the past few years, the new plant breeding technologies emerged as outstanding tools in the scenario of the green biotechnologies all over the world. Cisgenesis and genome editing opened up a new breedomics era, allowing to introduce or to disrupt specific genes in the plant genome in shorter time compared to traditional breeding and with minimal and precise genetic modifications. While the application of these technologies in model plant and herbaceous crops underwent great and rapid development in the last few years, in fruit trees only few studies have been published so far. Many challenges in fact still hamper the gene transfer techniques in grapevine and apple resulting in a low efficiency of the process even in the presence of a selectable marker gene. Thus, in order to obtain cisgenic fruit crops, methods based on site-specific recombination for the removal of the selectable marker gene after the selection phase appeared to be more feasible and effective, if compared to approaches that avoid their use and rely the discrimination of transformed event on demanding and labor intensive screening analysis. At FEM, we compared different induction strategies for the excision of the undesired cassette in transformed grapevine and proved the high efficiency of an heat-shock inducible system. Furthermore, experiments are in progress to apply the CRISPR system in grapevine and apple for the inactivation of genes playing key role in susceptibility to powdery mildew and fire-blight respectively. Our method relies on a binary vector carrying the specific sgRNA and Cas9 delivered into plant cell via Agrobacterium tumefaciens. With the aim of adopting a “clean” genome editing approach and exploiting the knowledge deriving from our studies on cisgenesis, we designed the construct to allow the removal of Cas9/sgRNA expression cassette after the targeted mutation has been obtained.