IVES 9 IVES Conference Series 9 Open-GPB 9 Open-GPB-2024 9 Category: Poster - New biotechnological tools

Poster – New biotechnological tools

IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

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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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

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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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

A versatile genome editing platform for grapevine: improving biotic and abiotic stress resilience 

New Plant Breeding Techniques (NPBTs) have arisen with the objective of surmounting the constraints inherent in conventional breeding methodologies, thereby enhancing plant resilience against both biotic and abiotic stresses. To date the application of genome editing in grapevine is still limited by the necessity to overcome recalcitrance to produce embryogenic calli and to regenerate plants. In our studies, we developed a smart and versatile genetic transformation system carrying all the most promising features of different genome editing approaches. In specific, we joined the GRF-GIF expression to improve regeneration, the systemic movement of the editing transcripts through tRNA-like sequences (TLS) and the cisgenic-like approach to remove transgenes.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

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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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Enhancing plant defense: carbon dots for efficient spray-induced gene silencing 

Ectopic RNA application for plant defense faces challenges in tree crops, including size, diffusion, and stability of active compounds such as ribonucleoproteins and nucleic acids. While existing strategies involve expressing dsRNA in transgenic plants targeting pathogens, our research strives to develop a transient RNAi system based on Spray-Induced Gene Silencing (SIGS). This approach aims to circumvent legal barriers and public concerns associated with genetically modified organisms (GMOs). Our strategy integrates SIGS with branched polyethyleneimine-functionalized Carbon Dots (bPEI-CDs) as nanocarriers, effectively addressing unique delivery challenges in plant defense as RNA stability and uptake enhancement

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Exogenous dsRNA applications to identify novel candidate susceptibility genes to downy mildew

One of the major threats to viticulture is represented by fungal pathogens. Plasmopara viticola, an oomycete causing grapevine downy mildew, is one of the principal causes of grape production losses. The most efficient management strategies are represented by a combination of agronomical practices, fungicides’ applications, and use of resistant varieties. Plant resistance is conferred by the presence of resistance (R) genes. Opposed to them, susceptibility (S) genes are encoded by plants and exploited by pathogens to promote infection. Loss or mutation of S genes can limit the ability of pathogens to infect the host. By exploiting post-transcriptional gene silencing, known as RNA intereference (RNAi), it is possible to knock-down the expression of S genes, promoting plant resistance.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

New breeding frontiers: application of the CRISPR-cas9 system in grapevine (V. vinifera L.) and improvements in plant regeneration

Nowadays, many policies are being adopted for direct agriculture towards more sustainable approaches. To continue to maintain a high production using fewer fertilizers, pesticides and water resources, agronomic techniques must be combined with biotechnological approaches. In grapevine, the breeding programs are restricted by the fact that it has a highly heterozygous genome, therefore, if on the one hand, we try to improve the characteristics, on the other hand it is necessary to preserve the original genome of the varieties. CRISPR-cas9 system is one of the smartest tools to carry out highly precise genetic modifications leaving the genetic background unchanged.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Novel approaches and promising perspectives for enhancing grapevine editing and regeneration

Grapevine (Vitis vinifera L.) is a challenging plant species to transform and regenerate due to its complex genome and biological characteristics. This limits the development of cisgenic and gene-edited varieties. One hurdle is selecting the best starting tissue for the transformation process, much like isolating suitable tissue for protoplasts. One promising method involves delivering CRISPR/Cas components to protoplasts isolated from embryogenic calli, which are then induced to regenerate. However, this process is inefficient, time-consuming, and only applicable to a few genotypes. To enhance grapevine regeneration efficiency, the expression of developmental and plant growth regulators shows promise in escaping the recalcitrance encountered in traditional tissue culture methods.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

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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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Obtaining high-quality RNA from grape tissues, including berry pulp, berry skins, stems, rachis, or roots, is challenging due to their composition, which includes polysaccharides, phenolic compounds, sugars, and organic acids that can negatively affect RNA extraction. For instance, polyphenols and other secondary metabolites can bind to RNA, making it difficult to extract a pure sample. Additionally, RNA can co-precipitate with polysaccharides, leading to lower extraction yield. Also, sugars and organic acids can interfere with the pH and ionic properties of the extraction buffer. To address these challenges, we optimized a protocol for RNA isolation from grape tissues.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

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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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Plant regeneration via somatic embryogenesis and preliminary trials for the application of the DNA-free genome editing in grapevine cv. Corvina veronese

Grapevine (Vitis spp.) is a globally significant fruit crop, and enhancing its agronomic and oenological traits is crucial to meet changing agricultural conditions and consumer demands. Conventional breeding has played a key role in domesticating grapevine varieties, but it is a time-consuming process to develop new cultivars with desirable traits for cultivation.
New plant breeding techniques (NpBTs) offer a potential revolution in grapevine cultivation, and genome editing has shown promise for targeted mutagenesis. The success of these biotechnological approaches relies on efficient in vitro regeneration protocols, particularly through somatic embryogenesis (SE).

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Cis-acting regulatory elements are DNA sequences that can be bound by transcription factors to regulate the expression of genes in a condition-dependent and tissue-specific way. It is nowadays possible to search for DNA motives and sequences that a given transcription factor is binding or at least can, but it is still hard to have a glance at all the transcription factors that are contemporaneously located at the same locus. Inspired by an existing technique that uses the CRISPR-Cas system in mammal cells, we are trying to develop a protocol to study such regulation in Vitis vinifera. Using the highly sequence-specific binding capacity of a catalytically inactive Cas9 protein (dCas9), our idea is to set up a system to target a desired sequence and precipitate all the crosslinked proteins and distantly interacting chromatin at this locus and analyze them.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Somatic embryogenesis and polyploidy in grapevine: morphological shoot and leaf traits variations

Somatic embryogenesis (SE) has been used in a variety of biotechnology applications such as virus elimination, cryopreservation, induced mutagenesis and genetic transformation. The SE induction process may cause DNA alterations and ploidy changes, which may provide a source of genetic variability useful for the improvement of agronomic characteristics of plants. This research aims at investigating the spontaneous alterations of the genome in grapevine plants regenerated through SE. Regenerants obtained from different embryogenic events from three different grapevine genotypes (Catarratto, Frappato and Nero d’Avola) were analysed.

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IVES Conference SeriesOpen-GPBOpen-GPB-2024Poster - New biotechnological tools

Using nanopore skim-sequencing to characterise regional epigenetic variability in New Zealand Sauvignon Blanc

Recent advancements in genomic sequencing technologies have enabled more detailed and direct studies of DNA methylation, which can help characterise epigenetic variations in plants. The Grapevine Improvement team at the Bragato Research Institute is studying the use of Oxford Nanopore sequencing to identify epigenetic changes associated with environmental differences among clonally-propagated grapevines.

This study involved sequencing DNA from the same Sauvignon Blanc clone, sourced from diverse New Zealand viticultural regions, using the PromethION platform.

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