The overall objective of this research project is to develop and use a genome editing technology for trait improvement of elite grape cultivars. In our 2018-2019 work, we successfully demonstrated the feasibility of editing a grape color gene, VvMybA1, by delivering a VvMybA1editing construct, p201H-MybA1Double1(1176/1180), into V. vinifera ‘Chardonnay’ embryogenic callus through stable Agrobacterium transformation. Subsequently in this project year, we focused on editing the grape color gene using a non-transgenic approach. Our non-transgenic approach is based on the facts that genome editing components (i.e. Cas9 and gRNA) on the T-DNA of a construct in Agrobacterium can transiently be expressed in target cells before degraded in or stably integrated into the host cell genome and the transiently expressed gene editing components, when taking place at a right time with appropriate strength, can result in successful editing of a gene in the host cell. By using GUS as a reporter gene, we observed that the transient expression of GUS in ‘Chardonnay’ embryogenic callus reached the peak after 2-3 days of co-incubation with Agrobacterium carrying the GUS construct. Based on the GUS observations, we co-incubated ‘Chardonnay’ embryogenic callus with Agrobacterium carrying the same construct p201H-MybA1Double1(1176/1180) we used in the 2018-2019 stable transformation for editing the VvMyb1A color gene. The co-incubation experiments were conducted for 2 and 3 days, respectively. High throughput sequencing revealed that editing took place in both 2- and 3-day incubations. Various types of editing were found, including deletions, insertions, and substitutions. Among the deletion events, single bp deletion was most frequent, as expected. Deletions involving multiple bps (2-15 bps) were also observed. Many of these events were likely resulted from transient expression of the CRISPR-Cas9 and VvMyb1A gRNAs. While the overall editing frequencies were very low (<0.015%), we demonstrated the success of editing a grape gene through transient Agrobacterium transformation. Now the key issue for grape gene editing is not so much about whether we can edit a grape gene in a non-transgenic manner, but about how to select the edited events from millions of non-edited cells. Solving this issue will be the focus of the proposed 2020-2021 project.
As a part of this continuing project, we induced some transgenic vines from the embryogenic callus transformed with the p201H-MybA1Double1(1176/1180) construct in the previous project year 2018-2019. Unfortunately, the induced vines showed extremely low vigor in tissue boxes, likely due to some undesirable clonal variation in the batch of ‘Chardonnay’ callus used. New ‘Chardonnay’ callus transformed with the same construct was produced in this project year and transgenic vines from the callus will be induced. The purpose of generating stable transgenic vines from the experiment is to demonstrate the phenotypic effect after the transposon Gret1 is removed from the VvMybA1 gene in ‘Chardonnay’.