Efficient DNA‐Free Protoplast Gene Editing of Elite Winegrape Cultivars for the Generation of Clones With Reduced Downy Mildew Susceptibility

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Background and Aims The grape and wine sector in Australia and around the world is under increasing pressure from climate change, disease threats and sustainability concerns, necessitating the development of fast and innovative solutions that can also preserve the sought‐after quality traits of elite cultivars. Methods and Results A simplified and cost‐effective method for DNA‐free protoplast gene editing and plant regeneration was assessed for its broad applicability using the two most common red and white winegrape cultivars commercially grown in Australia. Plant regeneration and editing efficiencies were investigated with downy mildew disease and associated susceptibility genes, Vvi DMR6-1 and Vvi DMR6-2, as the trait and gene‐editing targets. High rates of edited plant regenerants displaying normal growth and development were recorded for all four cultivars within 4–5 months, and flow cytometry assays were employed to assess the ploidy status of regenerants and filter out nondiploid plants. Fungal assays with glasshouse‐grown, edited Chardonnay plants suggested a previously unreported role for Vvi DMR6- 2 and jasmonic acid in grapevine susceptibility to downy mildew. Conclusions This study has demonstrated that a relatively simple and robust protoplast isolation, DNA‐free protoplast transfection and plant regeneration workflow can be used to efficiently produce nontransgenic, diploid, edited clones with desired phenotypes of four elite winegrape cultivars, including the highly recalcitrant Cabernet Sauvignon. Significance of the Study The DNA‐free editing workflow reported in this study is a major step towards addressing many of the challenges that the winegrape sector is facing by enabling fast and targeted genetic improvements of elite cultivars. Resulting improved clones could be a key component of typicity‐preserving strategies to counteract major threats to the sector as demonstrated by the increased resistance of edited Chardonnay plants to downy mildew infection.