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…

Exploring aromatic profiles and environmental influences on berry chemistry of V. vinifera Riesling and Vitis sp. L’Acadie blanc in Quebec and Nova Scotia, Canada

Wine quality depends on grape biochemical constituents, including sugars, organic acids, amino acids, and bound and free aroma compounds, which are influenced by vineyard location and environmental factors such as temperature and precipitation [1].

Come proteggere un territorio viticolo: il punto di vista del giurista

La valanga di fango che si è abbattuta nel Salemitano e nell’Avellinese, provocando decine di vittime, è stata causata in larga misura dalle insufficienti opere idrauliche e dalla manca­ta manutenzione di antiquati canali idrici.

Implementing VIS-NIR spectroscopy as a rapid and non-intrusive technique for assessing anthocyanin and phenolic concentrations in Vitis vinifera L. Grenache whole grape berries

Anthocyanins and phenolic compounds play a crucial role in winemaking, contributing to the profile, flavor, color, texture, and stability of wine. Grape clusters, specifically Vitis vinifera L. cv. Grenache, were handpicked from a commercial vineyard sited in Tudelilla, La Rioja, Spain (42°18′ 52.26″, Long. -2°7′ 59.15″, Alt. 582 m) on five distinct dates from veraison to harvest during the 2015 season. Non-contact spectral measurements were conducted on intact grape berries using a VIS-NIR spectrometer operating in the 570 – 1000 nm spectral range under controlled laboratory conditions, positioned at a distance of 25 cm from the berries. The quantification of 16 anthocyanins and phenols in 120 grape clusters was performed using HPLC, established as the reference method for validating the spectral tool.

Mechanization of pre-flowering leaf removal under the temperate-climate conditions of Switzerland

Grapevine leaf removal (LR) in the cluster area is typically done between fruit set and cluster closure to create an unfavorable microclimate for fungal diseases, such as Botrytis cinerea and powdery mildew. Grape growers are now turning their attention to pre-flowering LR, which has additional benefits under certain conditions. When applied before flowering, LR strongly affects fruit set and thus the number of berries per cluster. It is therefore a good yield control tool, replacing time-consuming manual cluster thinning (Poni et al. 2006). It also improves berry structure, that is, skin thickness, skin-to-pulp ratio, and berry composition (total soluble solids, titratable acidity, and polyphenols) (Palliotti et al. 2012; Komm and Moyer 2015). By exacerbating competition for assimilates between reproductive and vegetative organs, pre-flowering LR also poses some risks. Excessive yield loss at the same year’s harvest due to a too low fruit set rate is the main concern: intensive pre-flowering LR (100% of the cluster area) can induce up to 50% yield loss in potted vines (Poni et al. 2005). Other parameters, such as cool climatic conditions during flowering, also affect fruit set rate and make it difficult to predict potential yield at harvest. Repeated and overly intensive preflowering LR can have repercussions over time and induce a decline in bud fruiting and plant vigor (Risco et al. 2014).