Summary of Progress:

California grape production increasingly faces challenges from drought, soil salinity, micronutrient toxicity, viruses, and nursery production constraints.  Rootstocks are the primary tool growers use to mitigate these stresses, yet the genetic diversity of commercially available rootstocks remains limited.  This project aims to accelerate the development of improved grapevine rootstocks by integrating long-term field trials with modern genomic and high-throughput phenotyping tools.

During the 2025–2026 funding year, we made substantial progress across multiple objectives.  Using vineyard trials established at UC Davis in 2019, 2020, and 2021, we continued collecting phenological and yield component data on advanced rootstock selections derived from decades of breeding efforts.  These trials include material with improved resistance to nematodes, salinity, boron toxicity, phylloxera, fanleaf virus, and drought.  Preliminary analyses reveal clear differences among rootstocks in yield components, vigor, and seasonal development, informing the identification of elite genotypes for future release or further breeding.  We also advanced field evaluations of alternatives to the widely used O39-16 rootstock, evaluating five VR hybrids under both virus-infected and non-infected conditions.

To accelerate long-term breeding progress, we selected 400 advanced rootstocks for a new genomic prediction vineyard.  Grafting is currently underway, with field plantings expected at Davis and Cordelia this summer.  This population will serve as the foundation for developing genomic prediction models that enable earlier and more efficient selection.

Significant advances were also made in understanding salinity and boron tolerance.  We continued genetic analyses of chloride exclusion, identifying a major genomic region controlling this trait and advancing toward gene-level resolution.  In parallel, we published work showing that leaf spectroscopy combined with machine learning can rapidly identify boron-tolerant rootstocks, enabling cost-effective, large-scale screening.

Finally, we continued work on the genetics of graft incompatibility through data analysis and experimental planning using previously established populations.  Together, these efforts strengthen the pipeline for developing rootstocks that are more resilient, easier to propagate, and better adapted to California’s changing production environments.

Because the mission of the breeding program is the development of new grapevine varieties (rootstocks and scions), it is important to recognize that grape breeding is inherently long-term.  Consequently, progress reports and proposals will retain a consistent structure over time, centered on three core activities: (i) evaluation of advanced breeding material, (ii) genetic dissection of key traits, and (iii) the development of high-throughput phenotyping approaches.  The integration of genetics/genomics (ii) with phenomics (iii) is essential for increasing breeding efficiency and accelerating genetic gain.  While specific objectives will evolve as tools and knowledge advance, similarities across proposals reflect the realities of perennial crop breeding rather than a lack of innovation.

Summary of Progress:

Full-length cDNAs of two grapevine cytidine diphosphate diacylglycerol (CDP‐DAG) synthase (CDS) genes VvRBL1-1 (Vitvi10g00174) and VvRBL1-2 (Vitvi19g00570) were cloned from Thompson Seedless leaves. Both VvRBL1-1 and VvRBL1-2 were proposed as genome editing targets for enhancing broad-spectrum disease resistance in grapevine.

For rapid and rough testing of their impact on plant response to different pathogens, we transiently expressed VvRBL1-1/2 in Nicotiana benthamiana and then inoculated grapevine red blotch virus (GRBV), Botrytis cinerea (grey mold), or Pseudomonas syringae DC3000 (bacterial speck).

Transient expression of VvRBL1-2 seems to make plant susceptible to GRBV. Results of B. cinerea and DC3000 inoculations did not generate consistent results, which may be caused by the interference of Agrobacterium and/or high variation of transient VvRBL1-1/2 expression.

We generated stable VvRBL1-1/2 overexpression transformants using N. benthamiana and Arabidopsis, and currently in the process of selecting homozygous single-locus T-DNA insertional lines. Those transgenic lines would again be challenged by different pathogens, without the interference of Agrobacterium or the huge variation of transient gene expression.

I am currently in the process of setting up two research support agreements with UC Davis plant transformation facility to generate VvRBL1-1/2 editing plants and with Fresno State University to hire a part-time technician.

Genome editing is a plant breeding innovation that allows rapid targeted modification of plant genome, like natural mutations, to improve traits such as yield and disease tolerance. Loss-of-function mutations in one or more of ‘Mildew resistance Locus O’ (MLO) genes impart protection to plants from powdery mildew fungi and confer durable broad-spectrum resistance. This mlo-based resistance, detected initially as a natural mutation in barley, has been successfully employed for nearly four decades. Recently, mlo-based resistance was found to be engineered or identified, from natural mutants, in many plant species of economic relevance. Genome editing in grapevine is currently performed through conventional Agrobacterium[1]mediated plasmid delivery, which integrates foreign genes into the genome and is labeled GMO plants. A more recent CRISPR RiboNucleoProtein (RNP) delivery in protoplasts is a DNA-free approach but makes it difficult to regenerate plantlets and identify the gene-edited mutants. This project aims to establish an alternative gene-editing approach for the grapevine model to generate powdery mildew-resistant mutant vines free of foreign genes. We proposed to edit the MLO susceptibility genes through a combination of plasmid- and RNP-delivered CRISPR/Cas9 in microvine to produce DNA-free powdery mildew-resistant plants. The project is currently in the 3rd quarter of the final year, and the first phase of generating mlo mutants through conventional Agrobacterium-mediated gene editing has been concluded. MLOs 3, 4, 13, and 17 in grapevine have been identified as the closest orthologs of MLO genes responsible for powdery mildew resistance in other species. We obtained single mutants of candidate MLOs and the combinations of double and quadruple mlo mutants. The mutant combinations will help identify the appropriate MLO genes for mildew resistance in grapevine. Currently, the genome-edited mlo mutant embryos are at various stages of plantlet regeneration. Preliminary genotyping analyses of the transformants showed mutations in the predicted areas of the MLO genes targeted by the CRISPR system. So far, we have identified over 200 potential mlo mutants. The next phase of the project is the removal of the transgene cassette, integrated into the genome of mlo mutants by employing CRISPR RNP delivery to obtain transgene-free mlo vines. Towards this goal, we concluded the experimental work. We established 1) the methodology to purify the modified Cas9 protein, 2) the RNP complexations and in vitro cleavage tests, 3) the excision of the transgene cassette using in vitro RNPs, 4) the use Cell-Penetrating Peptides (CPP) to facilitate the entry of the CRISPR RNP into regenerable embryogenic cells, and 5) the optimization of the CPP-RNP delivery conditions. The editing of the target genes through the CPP-conjugated RNP delivery into the embryogenic cells was also tested on GFP-expressing embryogenic cell lines. We expect the regenerating mlo-edited plantlets to be ready for genotyping and powdery mildew resistance assays in 3 months, after which the second phase of the project (in vivo excision of the transgenic cassette) will begin to obtain transgene-free powdery mildew resistant vines.

The timing of ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions will affect the ripening process of major cultivars in several growing regions of U.S, which may result in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate effects of climate change is critical, the use of complementary approaches must be investigated. The identification of regulatory genes controlling the timing of ripening initiation is a research avenue to consider for molecular breeding programs in order to identify clones/cultivars more suitable to evolving climatic conditions. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently received scientific attention and its potential for rapid industrial application can be seen as alternate solution to traditional and molecular breeding. Yet, all these translational tools need scientific validation up front to characterize the cause-to-effect relationship between the gene and/or several genes and the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the contribution of a regulatory protein, VitviARF4, to the ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VitviARF4 via genetic engineering and the identification of its potential partners during the ripening process, 2) the identification of ripening-related genes that are targeted by VitviARF4, and 3) the evaluation of fruit composition on genetically engineered berries.

After the establishment of the microvine model at OSU to conduct the genetic engineering experiments, our research efforts were focused this year on several milestones of the project. For the objective 1, we conducted all the microvine transformations (four in total, control, two over-expression, and one knock – down) to either turn on or off the expression of VitviARF4. At least 20 independent transformed plants were selected per transformation event. We also confirmed protein-protein interactions of VitviARF4 with other proteins that play a major role in various physiological process of fruit ripening (sugar, brassinosteroids, ethylene, and epigenetic mechanism). For the objective 2, we demonstrated that we can control the expression of the transgene in transgenic microvines during the development of the plants. This result was critical to the success of the research outcomes of the objective 2. We have generated about 15 to 20 GFP positive transformed lines per construct and few of them were transferred to the greenhouse for being characterized. In parallel, we also conducted a Spray Induced Gene Silencing (SIGS) experiment on pre-véraison berries of V6 microvine. Our preliminary findings seem to support the delaying effect of VitviARF4 on the timing of berry ripening. We observed a faster ripening on berries treated with dsRNA aiming at silencing the expression of the endogenous VitviARF4. However, this experiment is currently repeated to confirm this first results. If confirmed, it might lead to a potential direct application of the SIGS technology in the field to manipulate trait. For the objective 3, we developed a
new analytical method to measure organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). We have built an in-house library of 95 analytes that were tested against berry extracts from pericarp samples collected at different stages of grape berry ripening. We are currently testing the method on mature berries of the microvine and we have identified around 30 analytes covering the major families of compounds existing in grape berry. Dr. Tomasino has optimized the volatile and aroma analyses on mature fruits of regular microvines.