IVES Conference Series

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

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.