Location: Crop Improvement and Genetics Research
Project Number: 2030-21220-003-024-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2025
End Date: Aug 31, 2026
Objective:
Technologies that improve the speed and efficiency of grapevine gene editing, transformation, and regeneration need further development. To enable these methodological refinements the following cooperative research objectives will be pursued:
1. Increasing the reliability of cell penetrating peptides to deliver genome engineering reagents to a greater variety of grape tissues.
2. Improving the rate and efficiencies of grapevine regeneration by expanding the catalog of developmental regulatory genes and implementing alternate methods for their expression.
Approach:
The ability to generate transgene-free, CRISPR-edited plants is paramount for the grape industry. Previous gene editing approaches have been pursued in grapevine, primarily using biolistic, protoplast transfection, or nanomaterial to deliver the components to cells. However, limitations of these technologies suggest a need to develop additional editing techniques to efficiently generate transgene-free edited plants. The application of CPPs has shown to successfully deliver multiple self-replicating DNA-based geminiviral vectors (GVRs) to grape cells (Olivares et al., 2021), including a newly developed Grape Red Blotch Virus (GRBV). To take advantage of this methodology, CPPs will be used for delivery of GVRs and gene editing reagents across an array of tissue types that are challenging to access with current techniques (e.g. regenerating tissues in culture, in planta shoot meristems, etc.).
To further take advantage of these delivery methods, we will expand beyond gene editing reagents and deliver developmental regulatory genes (DRs) looking to overcome the bottleneck of poor grapevine regeneration after genetic transformation. In Microvine V4 grapes, efforts using the DR miR--GRF-GIF (Debernardi et al., 2020) have demonstrated a noticeable but limited boost to tissue regeneration. Testing additional DRs, including but not limited to VitviIPT, controlled by different expression systems (e.g. constitutive overexpression, inducible expression, etc.) is expected to further enable improvements to regeneration speed and efficiency. Using transcriptional information from multiple grape developmental stages (e.g. globular, heart, torpedo and the meristematic center), candidate genes will be isolated for application towards tissue regeneration. With these improvements to the Microvine editing platform, this methodology will promote a more high-throughput generation of candidate gene knock-outs (identified by our ARS collaborators) to define their involvement in response to smoke exposure.