Location: Grape Genetics Research Unit (GGRU)
Project Number: 8060-21220-008-007-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2022
End Date: Sep 14, 2025
Objective:
Gene editing technologies have successfully been applied to grapevine gene and trait modification, but most of the work were done through transgenic methods. In heterozygous, clonally-propagated crops with strong cultivar recognition like grapevine, non-transgenic editing is highly desirable. Several non-transgenic editing approaches have actively been pursued by others, including in vitro ribonucleoprotein (RNP) transfection, transient expression of transgenes without DNA integration, uses of nanotechnologies and virus-like particles and employment of bacterial secretory systems for Cas9/gRNA delivery. None of them have been proven working effectively in grapevines. In this proposed collaboration, ARS plans to evaluate some of these approaches and explore some new ones for development of an efficient, non-transgenic method for editing genes in grapevine.
Approach:
ARS will collaboratively pursue development of several non-transgenic editing methods in this study. They include: 1. Cas9/gRNA RNP delivery to intact, regenerable cells (e.g., calli induced from immature florescence) through bombardment; 2. Cas9/gRNA RNP transfection of embryogenic cells whose walls are completely or partially removed using electroporation; and 3. Transient expression of new Cas9/gRNA plasmids containing morphological genes [e.g., BABY BOOM (BBM), WUSCHEL (WUS), and other MADS-box genes] for regeneration promotion through Agrobacterium and/or bombardment transformation; Vitis vinifera cv. ‘Chardonnay’, ‘Thompson Seedless’ or ‘Scarlet Royal’ will be used as the plant material for the research. Trait genes of interest such as FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and marker genes such as GREEN FLOURESCENT PROTEIN (GFP) and ß-GLUCURONIDASE gene (gusA) will be used as target genes for editing.
