Open GPB

IVES 9 Tag: Open GPB ( Page 5 )

Optimization of in vitro establishment of grapevine varieties for fast micropropagation 

Micropropagation is an important alternative to conventional methods of plant propagation. The objective of this study was to optimize a protocol for in vitro micropropagation of selected grapevine hybrids (H19 and H20) that are included in our breeding program. For the sprouting initiation experiment, nodal cuttings with only one axillary bud from two hybrids were separated, disinfected, and cultivated in 50% Murashige Skoog nutrient medium (½ MS) and Woody Plant Medium (WPM), adding 4.4 µM benzyladenine (BA) in both mediums.

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Optimizing protocol for a rapid and cost effective DNA isolation for Marker Assisted Selection pipeline

Grapevine is a plant that holds significant socioeconomic importance due to its production of grapes for fresh consumption, wines, and juices. However, climate changes and susceptibility to diseases pose a threat to the quality and yield of these products. The breeding of new genotypes that are resistant/tolerant to biotic and abiotic stresses is essential to overcome the impact of climate changes. In this regard, Marker-assisted selection (MAS), which uses DNA markers, is a crucial tool in breeding programs. The efficiency and economy of this method depend on finding rapid DNA isolation methods.

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In vitro tissue culture as a tool for Croatian grapevine germplasm management

In vitro culture makes it possible to carry out specific studies that would not be possible with whole plants grown in the field or in a greenhouse. Cryopreservation allows long-term preservation without metabolic changes in the plant material and cryotherapy can be efficient in virus elimination, which is a major scientific challenge.
The preculture media of cryopreservation protocols were evaluated on three Croatian grape varieties with different antioxidants (salicylic acid, ascorbic acid and glutathione). The highest growth in vitro was achieved on the medium with the addition of glutathione and the lowest with the addition of salicylic acid.

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Effects of Silver Thiosulphate and Salicylic Acid on the long-term maintenance of the embryogenic callus of Vitis vinifera

New Plant Breeding Techniques (NPBTs) have the potential to revolutionize the genetic improvement of grapevine. However, the practical application of these techniques is limited by several challenges, such as the difficulty in generating embryogenic calluses, maintaining their competence during in vitro cultivation, and regenerating plants without defects. To overcome these challenges, we conducted a study to test the effect of two treatments on callus cultures derived from different grapevine varieties, with and without embryogenic competence. The tested substances were Silver Thiosulphate (STS) an ethylene inhibitor, and Salicylic Acid (SA), an elicitor with different effects depending on the concentration of use beyond the ethylene inhibitor activity.

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In vitro regeneration of grapevine cv. Aglianico via somatic embryogenesis: preliminary studies for next genome editing applications  

Italy is a rich hub of viticultural biodiversity harboring hundreds of indigenous grape varieties that have adapted over centuries to the diverse climatic and geographic conditions of its regions. Preserving this biodiversity is essential for maintaining a diversified genetic pool, crucial for addressing future challenges such as climate change and emerging plant diseases. Rising temperatures, precipitation pattern variations, and extreme weather events can affect grape ripening, crop quality, and contribute to disease development. Integrated disease management necessitates exploration of novel strategies. Biotechnologies emerge as a significant player in tackling modern viticulture challenges.

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From protein-centered to gene-centered approaches to investigate DNA-protein interactions in grapevine

DNA-binding proteins play a pivotal role in critical cellular processes such as DNA replication, transcription, recombination, repair, and other essential activities. Consequently, investigating the interactions between DNA and proteins is of paramount importance to gain insights into these fundamental cellular mechanisms. Several methodologies have been devised to uncover DNA-protein interactions, which can be broadly categorized into two approaches. The “protein-centered” approach focuses on identifying the DNA sequences bound by a specific transcription factor or a set of TFs. Techniques falling within this category include chromatin immunoprecipitation, and protein-binding microarrays.

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Mobilizing endogenous transposable elements for grapevine improvement: a genomic and epigenomic approach in New Zealand Sauvignon Blanc

Efforts to improve the New Zealand wine industry’s climate resilience and sustainability through grapevine improvement are limited by germplasm availability and a reliance on Sauvignon Blanc exports. To address this, we are working to generate a population of 12,000 individuals with unique genetic traits, from which to select future clones for major export varieties.

Sauvignon Blanc plantlets are being regenerated from embryogenic callus, using an approach designed to mobilise endogenous transposable elements as mutagens.

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

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.

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DNA-Free genome editing confers disease resistance in grapevine

Grapevine (Vitis Vinifera L.), one of the most important cultivated fruit crops, is facing significant challenges due to climate change. Specifically, increasing temperatures negatively impact the physiological traits and disrupt plant phenology. Additionally, increased virulence in pathogen attacks and pests leads to significant yield loss, requiring widespread application of plant protection products. Traditional agronomic practices offer only partial mitigation, requiring the development of precise and effective intervention strategies. The economic worth of viticulture has prompted continuous efforts in grapevine genetic improvement programs, traditionally involving conventional breeding and clonal selection that, however, are complex and time-consuming approaches.

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Functional characterization of grapevine MLO genes to define their roles in Powdery mildew susceptibility by CRISPR/Cas9 genome editing

Successful powdery mildew (PM) infection in plants relies on Mildew Resistance Locus O (MLO) genes, which encode susceptibility factors essential for fungal penetration. In Arabidopsis, loss-of-function mutations in three clade-V MLOs, AtMLO2, 6, and 12 confer complete resistance to PM infection. Since then, efforts are on to discover MLO genes contributing to PM susceptibility in many species to introduce mlo-based PM-resistance. Earlier studies in tomato and grapevine, using the RNAi approach, attributed PM susceptibility to SlMLO1, 5, and 8 and VvMLO3, 13, and 17, respectively indicating likely functional redundancy among MLOs.

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Development and application of CRISPR/Cas in grapevine

The development and application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) technologies have revolutionized genome editing in plants due to its simplicity, high efficiency, and versatility. As an economically important fruit crop worldwide, grapevine genome editing using CRISPR/Cas technologies has also been reported these years. Here we introduce the development briefly of the most popular CRISPR/Cas9 system and also the state-of-the-art CRISPR technologies developed so far. Moreover, we summarize CRISPR/Cas9-mediated applications for gene functional study and trait improvement in grapevine.

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Exploring the impact of NPR3 gene silencing on the interaction between grapevine and mycorrhizal fungi through genome editing

One of the main plant defence mechanisms is the Systemic Acquired Resistance (SAR) mediated by Salicylic Acid (SA). This is a heightened and broad-spectrum immune response initiated by the exposure to pathogens, inducing resistance not only in the infected site, but also throughout the entire plant. It was demonstrated that plant immune system can be regulated by two classes of SA receptors: NONEXPRESSOR OF PR GENES 1 (NPR1) and NPR1-LIKE PROTEIN 3 and 4 (NPR3/NPR4). While NPR1 is required for SA-induction followed by the expression of pathogenesis-related (PR) protein and resistance against pathogens, NPR3/NPR4 serve as transcriptional co-repressors of SA-responsive genes.

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

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.

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Deciphering the function and regulation of VviEPFL9 paralogs to modulate stomatal density in grapevine through New Genomic Techniques

Stomata are microscopic pores mainly located in leaf epidermis, allowing gas exchanges between plants and atmosphere. Stomatal initiation relies on the transcription factor SPEECHLESS which is mainly regulated by the MAP kinase cascade, in turn controlled by small signaling peptides, the Epidermal Patterning Factors (EPF and EPF-Like), namely EPF1, EPF2 and EPFL9. While EPF1 and EPF2 induce the inhibition of SPEECHLESS, their antagonist, EPFL9, stabilizes it, leading to stomatal formation. In grapevine, there are two paralogs for EPFL9, VviEPFL9-1 and VviEPFL9-2. Despite their structural similarity, it remains unclear whether they are differentially regulated and have distinct roles.

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Exploring the inner secrets of grapevine: a journey through plant-microbe interactions

Throughout centuries of anthropocentric breeding, plants have been selectively bred to enhance their quality traits and yield, often overlooking the importance of neglected attributes, like those involved in the interactions with beneficial microorganisms. This phenomenon led to an alteration in the distribution of photosynthetic products, shifting from defence mechanisms to growth, commonly described as ‘domestication syndrome’. Addressing the losses stemming from this condition is imperative just as unravelling the concealed communication between grapevines and beneficial microorganisms.

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Exploring diversity of grapevine responses to Flavescence dorée infection

Flavescence dorée, a serious threat to grapevine cultivation in several European Countries, is caused by phytoplasmas in the 16Sr-V ribosomal group, classified as quarantine organisms in the EU and transmitted mainly by the insect vector Scaphoideus titanus. The disease is controlled only by indirect and preventive measures, with important economic and environmental concerns. Genetic resources from the great variety of Vitis vinifera germplasm together with application of new genomic techniques could be applied to produce resistant/tolerant plants, once the genetic bases of susceptibility are elucidated. In a current Italian project (BIORES*) we are evaluating different international and local grapevine cvs. as well as microvine plants for their response to FD transmission and multiplication in controlled conditions.

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Physiological means to curb the enthusiasm of viruses from infecting grapevines  

The two most deadly viruses infecting and threatening the productivity of grapevines worldwide are leafroll and red blotch viruses. There is no cure for viral diseases other than roguing the symptomatic vines and replacing them with certified vines derived from clean, virus-tested stocks.
Given that phloem plays a central role in virus infection, this study aimed to purge the virus by girdling the phloem of leafroll-infected vines at different phenological stages of infected grapevines. Phloem-girdling was performed on canes at veraison to varying regions between the proximal and distal clusters.

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Selection of beneficial endophytes from Sicilian grapevine germplasm 

The recent expansion of arid areas due to climate change is putting grapevine and the other traditional productions at risk in all Mediterranean countries with a limited availability of fundamental resources such as water. It is possible to improve the resilience of vineyards by developing sustainable agricultural practices based on biological and natural resources such as endophytic microorganisms that colonize inner plant tissues, and which can potentially increase the tolerance to abiotic stresses. A selection of grapevine endophytes was conducted from 2021 to 2023 as part of the PRIMA project PROSIT.

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