terclim by ICS banner
IVES 9 IVES Conference Series 9 DNA-free editing to improve stress resilience of wine grape genotypes recalcitrant-to-regeneration

DNA-free editing to improve stress resilience of wine grape genotypes recalcitrant-to-regeneration

Abstract

Wine viticulture, being firmly linked to the vine-terroir relationship, has always encountered significant bottlenecks to genetic innovation. Nonetheless, the development of new breeding strategies leading to the selection of stress resilient genotypes is urgent, especially in viticulture, where it would allow reducing the use of chemical treatments adopted to control fungal diseases. Genome editing represents an extremely promising breeding technique. Unfortunately, the well-known recalcitrance of several wine grape cultivars to in vitro regeneration strongly limits the exploitation of this approach, which to our knowledge has so far been developed on table grape genotypes with high regeneration potential. By targeting the phytoene desaturase gene as visual editing proof, we developed a genome editing and regeneration protocol to produce transgene-free grapevine plants exploiting the lipofectamine–mediated delivery of CRISPR–Cas9 ribonucleoproteins into protoplasts. We regenerated edited grapevines of Vitis vinifera‘Nebbiolo’, a cultivar extremely recalcitrant to in vitro regeneration and at the basis of outstanding quality wines, such as ‘Barolo’ and ‘Barbaresco’. Successful editing was confirmed by a combination of approaches: HRM, Sanger and amplicon deep sequencing, phenotype visualization. We then exploited the method to silence two micro(mi)RNAs involved in biotic stress responses: vv-miR482, which is conserved in different species, and the grapevine-specific vv-miR3623. Since NBS-LRR disease-resistance genes are the targets of those miRNAs, the objective is to regenerate vines with a broad-spectrum level of plant tolerance/resistance to different pathogens. The developed strategy could be extended to other important wine grape varieties and recalcitrant woody species.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Article

Authors

Giorgio Gambino1, Floriana Nuzzo1, Amedeo Moine1, Walter Chitarra1,2, Chiara Pagliarani1, Annalisa Petrelli3, Paolo Boccacci1, Andrea Delliri1, Riccardo Velasco2, Luca Nerva1,2, Irene Perrone1*

1 Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Strada delle Cacce 73, 10135 Torino (Italy)
2 Research Centre for Viticulture and Enology, Council for Agricultural Research and Economics (CREA-VE), Via XXVIII Aprile 26, 31015 Conegliano (Italy)
3 Open Lab – Department of Veterinary Sciences, University of Turin (DSV-UNITO), Largo Paolo Braccini 2, 10095 Grugliasco, Italy

Contact the author*

Keywords

genome editing, protoplast regeneration, lipofectamines, microRNAs, biotic stress

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

Evaluation of biodegradable mulch for weed control with focus on vineyard performance

Context and Purpose of the Study. The use of herbicides and mechanical soil tillage, particularly on steep slopes, poses significant ecological challenges, including soil compaction and erosion.

Hyperspectral imaging for the appraisal of varietal aroma composition along maturation in intact Vitis vinifera L. Tempranillo Blanco berries

The knowledge of the grape aromatic composition during ripening provides very important information for winegrowers, who may carry out different viticultural practices, or determine the harvest date more accurately. However, there are currently no tools that allow this measurement to be carried out in a non-invasive and rapid way. For this reason, the aim of this work was to design a non-invasive methodology, based on hyperspectral imaging to estimate the aromatic composition and total soluble solids (TSS) of Tempranillo Blanco berries during ripening.

Effect of ozone application for low-input postharvest dehydration of wine grapes 

The postharvest dehydration of grapes is a traditional practice to obtain wines with unique traits (e.g. sweet, dry/reinforced). The modern facilities (dehydrating rooms) used for this purpose are equipped with systems for artificially controlling the inside environment parameters, to obtain the desired dehydration kinetic and preserve the grapes from grey mold (Botrytis cinerea) infection, However, the conditioning systems are extremely energy-demanding and the identification and practical applications of solutions effective in controlling/reducing the postharvest decay would reduce the costs of the operation of the dehydration facilities. To this end, we explored the potential of ozone-based treatments on harvested grapes and preliminarily tested if the treatment could impact the normal behavior and metabolism of grapes during the traditionally slow dehydration practice.

Differentiating and grouping of oltrepo’ pavese environments according to grape maturation

The maturation patterns process has been very studied. In particular the modelization of the sugars and titratable acidity during the ripening period was an important approach, in particular for the prediction of harvest date (Barillere et al., 1988; Jourion et al.,1987; Maujean et al., 1983; Scienza, 1989). In Oltrepò Pavese, the widest viticultural district of Lombardy – Northern Italy – (about 15000 hectares), grape maturation trends shows high variability, due to the large variation in environmental characteristics of vineyards (altitude, exposure, soil type, mesoclimate) and to “cultivar x environment” interaction.

Inhibition of Oenococcus oeni during alcoholic fermentation by a selected Lactiplantibacillus plantarum strain

The use of selected cultures of the species Lactiplantibacillus plantarum in Oenology has grown in prominence in recent years. While initial applications of this species centred very much around malolactic fermentation (MLF), there is strong evidence to show that certain strains can be harnessed for their bio-protective effects. Unwanted spontaneous MLF during alcoholic fermentation (AF), driven by rogue Oenococcus oeni, is a winemaking deviation that is very difficult to manage when it occurs. This work set out to determine the efficacy of one particular strain of Lactiplantibacillus plantarum(Viniflora® NoVA™ Protect), against this problem in Cabernet Sauvignon must. The work was carried out at commercial scale and in a winery environment and compared the bio-protective culture with the more traditional approach of reducing must pH by the addition of tartaric acid. The combination of both was also investigated. The concentration of both Oenococcus oeni and Lactiplantibacillus plantarum was determined using qPCR. The adventitious Oenococcus oeni showed the most growth during AF in the control wine, whereas in the wines treated with Lactiplantibacillus plantarum a bacteriostatic effect against this species was observed. This effect was comparable to the wines treated with tartaric acid. This has particular commercial relevance for controlling the flora in musts with high pH, or when the addition of tartaric acid is either not permitted or is prohibitive for other reasons.