Grapevine (Vitis vinifera L.), a globally significant crop, is highly susceptible to Botrytis cinerea, the causative agent of gray mold disease. This study investigates transcriptomic responses to B. cinerea in tolerant and susceptible grapevine genotypes using RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were identified at three time points (T1, T2, T3), highlighting both genotype-independent and genotype-specific responses. Early-stage infection (T1) revealed rapid and robust activation of defense pathways in both genotypes, though the tolerant genotype showed enhanced modulation of metabolic processes by T2, prioritizing secondary metabolism and stress adaptation over growth. In contrast, the susceptible genotype exhibited less coordinated metabolic reprogramming, with delayed or weaker activation of key defense mechanisms. Gene Ontology and KEGG analyses identified critical pathways, including phenylpropanoid biosynthesis-like lignin metabolism, MAPK signaling, as well as candidate genes such as WRKY transcription factors and enzymes involved in cell wall fortification and antifungal compound biosynthesis. Genotype-specific responses emphasized metabolic flexibility as a determinant of resistance, with the tolerant genotype exhibiting superior resource allocation to defense pathways. These findings provide insights into the molecular basis of grapevine resistance to B. cinerea, offering potential targets for breeding or genetic engineering to enhance resilience and reduce fungicide dependency.

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

Vitis vinifera is an important fruit crop which is mainly consumed fresh or used for the production of wine. Genetic improvement programs through New Genomic Techniques (NGTs) aim to develop grapevine varieties resistant to biotic and abiotic stresses or enhancing nutraceutical properties. In order to apply NGTs, maintaining embryogenic calluses from flower tissues is critical. Optimizing culture conditions—pH, gelling agents, temperature, light, growth regulators, and gas composition—is essential for inducing efficient embryogenic responses tailored to each genotype/explant. Ethylene, a pivotal gaseous plant hormone, significantly influences tissue culture by affecting organogenesis and embryogenesis processes in several plants. Modulating ethylene levels shows promise for improving tissue culture vitality. This study evaluates in Vitis vinifera the effects of silver thiosulfate (STS) and salicylic acid (SA) on embryogenic callus growth, specifically investigating their roles in maintaining and inducing embryogenic competence. STS, particularly at 40 µM and 60 µM concentrations, effectively preserved embryogenic competence in Italia and Red Globe calluses, while high SA concentrations showed varied and occasionally adverse effects. At the same time, STS markedly suppressed the non-embryogenic callus growth in recalcitrant variety Italia, potentially increasing the ratio between embryogenic to non-embryogenic calluses development.

While resistance or tolerance is a highly sought-after trait in new grape varieties, producing such grapes does not guarantee acceptance in the market. The objective of this study was to explore consumers’ appreciation of a novel tolerant table grape variety by integrating chemical, textural, and sensory analyses with near-infrared (NIR) spectroscopy. To achieve this, we developed multivariate prediction models using artificial neural networks (ANNs) that were applied to the spectra of samples subjected to sensory analysis, allowing us to predict their composition. This approach offers a non-destructive way to conduct sensory analysis, as a single NIR spectrum can assess consumer appreciation and identify the chemical and physical characteristics of each berry. For the grape variety tested, we observed a significant favorable consensus among consumers. Therefore, instead of identifying differences, we focused on determining the optimal maturity ranges that enhance consumer appreciation for this variety. Our findings represent a step toward non-destructive sensory analysis; however, further research is still needed to refine this approach.

This work evaluates how the vineyard-level application of selected indigenous non-Saccharomyces yeasts influences the chemical and aromatic profile of Primitivo wines, focusing on fermentation-derived metabolites and phenolic composition. The traditional method for making red wine relies on the use of sulfur dioxide (SO₂) to ensure wine preservation, prevent oxidation, and produce microbiologically stable products. Applying beneficial non-Saccharomyces yeasts to grape surfaces shortly before harvest is a novel winemaking biotechnology aimed at producing wines with reduced SO₂ inputs. This approach can influence fermentation outcomes, modulate wine chemistry, and reinforce regional identity. The produced wines were analyzed for conventional oenological parameters, as well as for their phenolic and aromatic profiles. Results showed that field applications of our strains of L. thermotolerans and St. bacillaris positively modulate both the phenolic and aromatic profile of young Primitivo wine produced with reduced amounts of SO₂.

DNA-free genome editing by the direct delivery of CRISPR-associated nucleases has emerged as a promising technology due to its precision and reduced risk of off-target effects. However, existing purification protocols for native Cas proteins require the use of complex instrumentation, which limits their application. Here, we present a simplified protocol for the purification of native Cas9, Cas12RR and dCas9-VP64 nucleases optimized for DNA-free genome editing. Our approach leverages a streamlined affinity and ion exchange chromatography coupled with minimal downstream processing, ensuring a good yield and activity of the purified proteins. The in vitro analysis of the purified ribonucleoprotein complex demonstrated a good efficiency of DNA target cleavage. This simplified protocol increases the opportunity to adopt CRISPR technology, and enables broader access to DNA-free genome editing tools also for laboratories that are not specifically equipped for protein purification.