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

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, VviDMR6-1 and VviDMR6-2, 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 VviDMR6-2 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.