Rouppe van der Voort, J. | bioRxiv Science

bioRxiv2022

Vitis vinifera plants edited in DMR6 genes show improved resistance to downy mildew.

The production and cultivation of vines (Vitis vinifera) tolerant or resistant to diseases such as downy mildew (DM) is a promising strategy to reduce fungicides and help viticulture sustainability. In many crops, generation of knock-out mutants in host genes controlling susceptibility to DM, such as Downy Mildew Resistant 6 (DMR6) is a strategy of proven success to obtain resistant plants, while the effect of mutations in DMR6 genes has yet to be demonstrated in grapevine. In addition, small mutations in genes governing important traits can be obtained by gene-editing while maintaining the genetic background of commercially important clones. Moreover, very recent advances in the technology of gene-editing allowed to produce non-transgenic grapevine mutants, by regeneration of protoplasts previously edited with the CRISPR-Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge on the targets, and an extensive evaluation of the impact of their mutation on plant phenotype and fitness. In this work we generated single and double knock-out mutants in DMR6 susceptibility (S) genes in multiple grapevine cultivars with improved resistance to DM.

plant biology↗

bioRxiv2021

Regeneration of Plants from DNA-free Edited Grapevine Protoplasts

CRISPR-Cas technology has widely extended the application fields of genome editing in plant breeding, making possible specific and minimal mutations within a genetic pool. With respect to standard genome editing technologies, CRISPR-Cas machinery can be introduced in the form of ribonucleoproteins (RNPs), thus avoiding the introduction of exogenous DNA into cells. The interest on the application of DNA-free delivery into plant cells is constantly increasing, especially in the case of valuable woody plants elite varieties where CRISPR-Cas9 technology would preserve their genotype, while still resulting into targeted genetic modifications. The use of single cells fits well the requirements of New Breeding Technologies, by ensuring both the CRISPR-Cas DNA-free delivery as RNPs and, since every plant will be regenerated from a single edited cell, the absence of chimerism. However, the use of protoplasts cell culture from woody plants is generally hampered by low editing efficiencies and an unsuccessful regenerative process. We here describe a successful DNA-free methodology to obtain fully edited grapevine plants, regenerated from protoplasts obtained from V. vinifera cv. Crimson seedless L. embryogenic callus. The transfected protoplasts were edited on the Downy Mildew susceptibility gene VvDMR6-2. The regenerated edited plants exhibited homozygous deletions of 1bp or 2bp, and homozygous insertion of 1bp.

plant biology↗