terclim by ICS banner
IVES 9 IVES Conference Series 9 Open-GPB 9 Open-GPB-2024 9 Flash - New biotechnological tools 9 Enhancing hydric stress tolerance by editing the VviMYB60 promoter with CRISPR/Cas9 

Enhancing hydric stress tolerance by editing the VviMYB60 promoter with CRISPR/Cas9 

Abstract

Climate change presents increasing challenges to viticulture, particularly with rising water stress contributing significantly to yield losses and damages. The identification of the MYB60 transcription factor, which regulates stomatal opening and closing in Arabidopsis thaliana and Vitis vinifera, offers potential solutions. Notably, knockout studies in Arabidopsis have shown reduced stomatal opening and increased drought tolerance in myb60 mutants. Additionally, the grapevine ortholog, VviMYB60, can restore the wild-type phenotype of Arabidopsis myb60 mutants. Further investigation of the Arabidopsis promoter region has revealed that mutations in DOF motifs lead to reduced expression of AtMYB60.

Utilizing the advancements in CRISPR/Cas9 genome editing, this project aims to modify the VviMYB60 promoter region to lower gene expression, thereby reducing stomatal opening in grapevines. Binary vectors for genome editing were constructed to target two specific regions of the VviMYB60 promoter. Agrobacterium-mediated transformation was performed on Chardonnay embryogenic calli, resulting in the successful regeneration of plants under selection conditions. Sanger sequencing analysis of the targeted region confirmed the occurrence of genetic edits in four of the six lines analyzed so far.

To further characterize the edited lines, next-generation sequencing will be utilized, providing a more comprehensive understanding of the mutations, as well as gene expression of VviMYB60 will be evaluated to confirm that the editing reduces its expression. Morphological and physiological parameters will be measured after acclimatation in greenhouse and finally these edited lines will undergo drought tolerance testing to assess their performance.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Article

Authors

Manuela Campa1*, Loredana Moffa2, Luca Nerva2, Walter Chitarra2, Johan Burger1

1 Genetics Department, Faculty of Agrisciences, Stellenbosch University, 7600 Stellenbosch, South Africa
2 Research Centre for Viticulture and Enology, Council for Agricultural Research and Economics (CREA-VE), Via XXVIII Aprile 26, 31015 Conegliano (Italy)

Contact the author*

Keywords

CRISPR/Cas9, VviMYB60, promoter, drought tolerance, stomatal regulation

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

Aromatic profile evolution of corvina, corvinone and rondinella grapes during withering

AIM AND METHODS: Grape withering is one of the key steps in the production of the most renowned red wines of the Valpolicella area, namely Amarone and Recioto. This practice, which was already used since Roman times, entails important modifications in grape composition and in the chemical and sensorial characteristics of the corresponding wines, especially in terms of aromatic profile. The aim of this research is evaluating the aromatic evolution during grape withering of the three main varieties used in Valpolicella wines: Corvina, Corvinone and Rondinella.Samples of the three varieties were analyzed at harvest and at different stages of withering, namely10%, 20% and 30% of weight loss. Free and glycosidically bound compounds were extracted and analyzed using Gas Chromatography- Mass Spectrometry (GC-MS). RESULTS: For all the samples the data were normalized to eliminate the effect of concentration due to grape dehydration. Terpene content and evolution varied considerably in relationship to grape variety. Corvinone was richer in cyclic terpenes (including phellandrene, limonene, and cymene) and they decreased during withering.

Soave beyond the zonation

In a previous zoning program (1998-2002), climatic and pedological factors were able to distinguish 14 terroir within the Soave DOC area where wine characteristics are well recognizable. Nevertheless, in the past vinegrowers identified several vineyards where a better quality of the grapes and wines could be obtained. So, « beyond the zonation » will aim to suggest a new methodology to characterise the Cru, starting with 15 vineyards that were selected in the Soave Classico DOC area. In the year 2005, a meteorological station was positioned in each vineyard and temperature data were collected; because of the limited area of investigation, only 3 rain sensors were set up.

Determinazione della frazione aromatica dei vini, quale strumento per-la valorizzazione del territorio viticolo

La caratterizzazione della frazione volatile aromatica dei vini attraverso l’analisi quali­quantitativa dei diversi composti, ha portato corne primo risultato la netta differenziazione delle annate in prova.

Characterization of winegrape berries’ composition on sorting tables using hyperspectral imaging and AI

Comprehensive evaluation of grape composition at winery receiving areas often requires multiple measurements to ensure representativeness, as well as the use of analytical techniques that are time-consuming and involve sample preparation.

Development of FTIR partial least squares models for polyphenol quantification in red wine during fermentation

Polyphenolic compounds are considered to have a major impact on the quality of red wines. Sensory impact, such as astringency and bitterness, stems directly from tannin composition. Thenceforth, quick analytical measurement of phenolic compounds appears to be a real challenge for winemaking monitoring and process control. Many methods were developed to analyzed polyphenols in wine, but they are time-consuming and require chemistry skills and equipment, not suitable for a rapid routine analysis. A reliable and rapid method to obtain this kind of measurement is Fourier Transform Infrared (FTIR) spectroscopy.