Location: Crop Improvement and Genetics Research
Project Number: 2030-21220-003-014-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2024
End Date: Aug 31, 2026
Objective:
Novel technologies that enhance plant transformation/regeneration and the efficiency of genome editing/genetic engineering of grapevine will be developed. The following cooperative research objectives will be pursued:
1. Development, optimization and use of a grape Red-Blotch virus-based replicon to efficiently produce transgene-free CRISPR-edited Microvine plants.
2. Use of Developmental Regulator (DR) genes, GRF-GIF and VitviIPT7 and other candidate genes for enhancing transformation and shoot regeneration in Microvine.
Approach:
Developing an efficient CRISPR editing technology that produces transgene-free grapevine is paramount for the grape industry. Several approaches are currently being pursued in grapevine that primarily use biolistic, protoplast transfection, or nanomaterial delivery of editing components to grapevine cells. However, inherent limitations of these technologies suggest a need for additional editing toolkits are needed for efficiently generating transgene-free edited plants. A DNA-based delivery strategy of a self-replicating viral system introduced to the intact plant regenerable cells will be examined (Olivares et al. 2021). A novel Grape Red Blotch Virus-based construct is being developed and optimized as an editing delivery system, which includes an Agrobacterium and peptide-mediated delivery of the GRBV self-replicon system in embryogenic cells of microvine V4. Microvine V4 will be transformed with constructs carrying the GRBV replicon, a fluorescent reporter gene, and CRISPR editing components for targeted gene editing. Initially, model grapevine genes (Phytoene Desaturase, others etc.) will be targeted to identify the optimal constructs and tissue culture/transformation conditions for generating edited Microvine explants. When a functional and efficient system is developed for producing edited Microvine plants, it will be utilized to develop knock-outs of candidate genes (identified by our ARS collaborators) potentially involved in response to smoke exposure.
