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Article|13 Dec 2023|OPEN
An efficient protoplast-based genome editing protocol for Vitis species
David M. Tricoli1 , and Juan M. Debernardi,1
1Plant Transformation Facility, University of California, Davis, CA 95616, USA
*Corresponding author. E-mail: dmtricoli@ucdavis.edu

Horticulture Research 11,
Article number: uhad266 (2024)
doi: https://doi.org/10.1093/hr/uhad266
Views: 24

Received: 27 Jun 2023
Accepted: 01 Dec 2023
Published online: 13 Dec 2023

Abstract

CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants. However, poor regeneration of plants from protoplasts has hindered their implementation for genome editing. Here, we report an efficient protocol for regenerating plants from protoplasts from multiple grape varieties. By encapsulating the protoplasts in calcium alginate beads and co-culturing them with feeder cultures, the protoplasts divide to form callus colonies that regenerate into embryos and ultimately plants. This protocol worked successfully in wine and table grape (Vitis vinifera) varieties, as well as grape rootstocks and the grapevine wild relative Vitis arizonica. Moreover, by transfecting protoplasts with CRISPR-plasmid or ribonucleoprotein (RNP) complexes, we regenerated albino plants with edits in VvPHYTOENE DESATURASE gene in three varieties and in V. arizonica. The results reveal the potential of this platform to facilitate genome editing in Vitis species.