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.