Generation of mildew-resistant grapevine clones via genome editing

Cultivated grapevine (Vitis vinifera) is highly susceptible to several fungal and fungal-like diseases such as powdery mildew (PM) and downy mildew (DM), which require large use of pesticides for their containment. In other crops, knock-out mutations of host genes controlling susceptibility to such diseases have proven a successful strategy to obtain resistant plants. Recent works demonstrated the importance of the MLO family as susceptibility genes for PM resistance also in grapevine, whereas DMR6 has been identified as DM susceptibility gene in Arabidopsis thaliana, and its involvement in DM susceptibility is yet to be demonstrated in grapevine. Today, emerging new breeding technologies such as CRISPR/Cas9, recently applied also to grapevine, allow to generate knock-out mutants in specific DNA targets while maintaining the varietal and clonal identity as by traditional breeding. In this work we aim to knock-out specific grapevine susceptibility genes, such as MLO and DMR6 genes via CRISPR/Cas9 technology, to generate PM and DM resistant clones of grapevine. Embryogenic callus of Chardonnay and microvine varieties were transformed via Agrobacterium tumefaciens with CRISPR/Cas9 vectors designed to specifically edit these genes. In vitro plants regenerated form these embryogenic calli were sequenced confirming the presence of small insertion and deletions or single nucleotide polymorphisms at the target sites