Breeding, Genetics, and Germplasm Evaluation
The overall objective of this research project is to develop and apply a genome editing tool for trait improvement in grape cultivars. During 2018-2019, we built a CRISPR-Cas9 construct for modifying the color gene VvMybA1 and transformed it into V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium transformation. We successfully demonstrated the efficacy of the editing machinery components in the construct and obtained transgenic callus containing cells with edited color gene VvMybA1. A 10-kb-long Gret1 transposon fragment in the promoter region of VvMybA1 in ‘Chardonnay’ was removed in the edited cells. Further analysis revealed 20-60 bp deletions at or near the target sites. The overall frequencies of these deletions/insertions were generally low, with the maximum being about 0.5%. From this work, we were the first to demonstrate the feasibility of removing a large piece of DNA from a locus in grapevine. We also demonstrated the technical feasibility for modifying the color gene, an important fruit quality trait. In parallel, we tried two new delivery methods for editing VvMybA1 and VvPDS genes in callus: a) bombardment of plasmid DNA for transiently expressing both Cas9 and gRNA components in grape embryogenic callus; and b) bombardment of in vitro preassembled Cas9–gRNA ribonucleoproteins into grape embryogenic callus. Our results showed that the direct delivery methods didn’t worked well, and the successful editing rate was too low to have any practical utility. This is not a surprise as no successful edited vines have been produced by direct delivery methods due to tremendous technical challenges. We will propose new approaches for improving the direct delivery methods in the project year 2019-2020.