Search Results (384)

Efficiency in vitro regeneration is a crucial prerequisite for the application of New Nenomics Techniques (NGTs) in grapevine (Vitis vinifera L.) for improving resistance to biotic and abiotic stresses. This is especially true given that their management must be addressed sustainably, considering the impact of climate change. Unfortunately, in vitro plant regeneration and the establishment of embryogenic calluses are two genotype-dependent processes. Up to now, extensive research has been conducted on major international cultivars, whereas studies on the application of in vitro protocols for autochthonous cultivars remain limited. In this study, protocols for the acquisition of embryogenic calluses were applied on the most relevant Sicilian grapevine cultivars: the red-skinned ‘Frappato’, ‘Nerello mascalese’, and ‘Nero d’Avola’, and the white-skinned ‘Grillo’, ‘Carricante’, and ‘Catarratto’. Stamens and pistils were cultured in two different induction media (PIV and MSII) and at three stages (mother cells in the late premeiotic phase, tetrads, and mature pollen) to induce embryogenic calluses. Five thousand explants per cultivar were cultured, forming calluses in four selected cultivars. Plantlets were successfully generated from calluses of ‘Carricante’, ‘Frappato’, and ‘Nero d’Avola’. Moreover, protoplasts were isolated from ‘Frappato’ and ‘Nero d’Avola’. Our results establish a critical foundation for developing successful regeneration protocols for the future application of NGTs in Sicilian grapevine cultivars. Full article

Apple black rot, a destructive postharvest disease caused by Alternaria alternata, poses significant economic threats during fruit storage and transportation. However, effective biocontrol bacteria to manage this disease remain limited. In this study, Leuconostoc mesenteroides strain GY-2, isolated from healthy apple fruit surfaces, had a remarkable biocontrol ability on apple black rot. While GY-2 exhibited no direct inhibitory effects in confrontation assays, volatile organic compounds (VOCs) emitted by the strain suppressed colony diameter of A. alternata by 70.8% in dual plate assays, indicating potent fungistatic activity. Notably, these VOCs produced by L. mesenteroides displayed broad-spectrum antifungal properties against multiple apple fungal pathogens. Microscopic analysis revealed that VOC exposure induced structural anomalies in A. alternata hyphae, including surface perforations and protoplast leakage, suggesting membrane integrity disruption. The VOCs produced by strain GY-2 were identified; four compounds had antifungal activities, among them, isoamylol exhibited the highest antifungal activity. Applying bacterial suspensions of strain GY-2 on apple fruit significantly reduced 91.4% of lesion areas of black rot. The strain exhibited robust colonization capacity on fruit surfaces, maintaining viable populations for over 15 days post-application, guaranteeing a sustained disease prevention. Furthermore, GY-2 treatment enhanced systemic resistance in apple fruit, as evidenced by upregulated antioxidant enzymes and defense-related enzymes. Importantly, application of GY-2 did not adversely affect key parameters of fruit quality, including firmness, soluble solids content, or acidity. These findings showed that the bacterial L. mesenteroides GY-2 was a promising biocontrol agent for managing postharvest black rot of apple fruit. Full article

Mitoviral-derived sequences are frequently detected in plant genomes, encoding an RNA-dependent RNA polymerase (RdRp). These sequences share many similarities with mitoviruses that are known to commonly infect plant mitochondria. However, the functional characterization of nuclear-encoded mitoviral-RdRp remains unclear. This study elucidates the critical role of mRdRp (AT2G07749) in maintaining mitochondrial homeostasis and embryo viability, highlighting the dual role of viral-derived genes in plant development and stress response. Phylogenetic analysis reveals that mRdRp shares 96.8% identity with the mitoviral RdRp encoded by mitochondrial-genomes, suggesting that this nuclear mRdRp gene originated from horizontal transfer events following ancestral plant-mitovirus infections. To dissect mRdRp function, we generated a mRdRp knockout mutant via CRISPR-Cas9 or knockdown mutant by RNA interference (RNAi). These mRdRp mutants exhibited severe developmental defects, including dwarfism, embryo lethality, and sterility. Phenotypic assays further showed that mRdRp mutants displayed heightened susceptibility to ABA and rotenone, indicating impaired adaptive capacity to both hormonal and metabolic stress. Loss of mRdRp led to fragmented mitochondrial networks and a significant reduction in mitochondrial abundance in both leaf protoplasts and root meristematic cells. Additionally, mitochondrial-derived small RNA (sRNA) aberrantly accumulated in mRdRp mutants, which potentially disrupts endogenous RNA-silencing pathways that rely on sRNA-mediated gene regulation. Collectively, these results provide mechanistic insights into the function integration of a virus-derived gene into plant cellular networks, advancing our understanding of host–virus coevolution and the role of horizontally transferred viral genes in shaping plant physiology. Full article

Macrophomina phaseolina is a widely distributed soilborne phytopathogenic fungus that causes destructive diseases such as charcoal rot and stem canker, posing serious threats to crop yield and quality. In recent years, mycoviruses have gained attention as potential biological control agents. In this study, a novel double-stranded RNA (dsRNA) virus was identified from M. phaseolina strain 22C-8, isolated from sesame (Sesamum indicum L.) charcoal rot samples in Fuyang, Anhui Province, China. The viral genome comprised four dsRNA segments, each encoding a single open reading frame (ORF) predicted to encode RNA-dependent RNA polymerase (RdRp), coat protein (CP), and two hypothetical proteins. Phylogenetic analysis classified the virus as a new member of the genus Betachrysovirus in the family Chrysoviridae, and it was designated Macrophomina phaseolina chrysovirus 2 (MpChrV2). Pathogenicity assays in sesame seedlings revealed that MpChrV2 infection significantly reduced the virulence of M. phaseolina strain 22C-8. In contrast, virus-free derivatives (22C-8-VF18), obtained via protoplast regeneration, caused more severe symptoms and exhibited enhanced growth rates, indicating that MpChrV2 alters fungal physiology and pathogenicity. These findings suggest that MpChrV2 possesses a typical hypovirulence phenotype and holds promise as a biocontrol agent for sesame charcoal rot. Full article

Stropharia rugosoannulata is a cultivated edible mushroom characterized by its nutritional composition and efficient cellulolytic enzymatic systems. However, the lack of genetic tools has significantly impeded the investigation of its molecular mechanisms, severely constraining the study of functional genomic and precision breeding in S. rugosoannulata. It was demonstrated in this study that the Agrobacterium-tumefaciens-mediated genetic transformation (ATMT) system is applicable for the transformation of S. rugosoannulata protoplasts. Through this proposal, we successfully achieved the expression of exogenous genes (mCherry gene encoding red fluorescent protein, hph gene encoding hygromycin B phosphotransferase, and GUS gene encoding β-glucuronidase) and the endogenous mutant gene SDI encoding the iron-sulfur protein subunit of succinate dehydrogenase in S. rugosoannulata. Furthermore, this study employed endogenous promoters of GPD encoding glyceraldehyde-3-phosphate dehydrogenase and SDI to enhance transformation efficiency and drive target gene expression. This study establishes the feasibility of ATMT in S. rugosoannulata systems, while achieving stable expression of a panel of selectable marker genes and reporter genes critical for genetic research in S. rugosoannulata. Full article

Sclerotinia sclerotiorum, a devastating phytopathogenic fungus with global distribution, exhibits a broad host range encompassing over 700 plant species. Sclerotinia stem rot caused by this pathogen poses a significant threat to sustainable oilseed rape production. Coniothyrium minitans, a mycoparasite of S. sclerotiorum, is a promising biological control agent against this devastating disease. C. minitans-based formulations have been commercially developed for field application. A transcriptomic analysis revealed significant upregulation of the chitinase-encoding gene CmCH1 in C. minitans during interaction with S. sclerotiorum. Knockout of either CmCH1 or another chitinase-encoding gene CmCH10 in C. minitans did not markedly affect the mycelial growth, development, and parasitism of S. sclerotiorum. However, knockout CmCH1 and CmCH10 simultaneously resulted in reduced growth rate, impaired protoplast release, enhanced cell wall integrity, and diminished mycoparasitic capability. These results indicate that CmCH1 and CmCH10 collectively influence remodeling of the cell wall in C. minitans and its mycoparasitic activity. Full article

Protoplasts offer a promising alternative to embryogenic cell suspensions (ECS) for gene editing in banana, potentially overcoming several limitations associated with ECS-based transformation systems. This study aimed to optimize protoplast isolation and regeneration in Cavendish banana (cv. Williams) and to assess their suitability for transient gene expression. Enzymatic digestion of ECS using cellulase and macerozyme consistently yielded approximately 3 × 10⁶ protoplasts per milliliter of settled cell volume. Protoplast yield was further enhanced, by approximately threefold, through the addition of an antioxidant mixture (ascorbic acid, citric acid and L-cysteine) combined with 0.01% bovine serum albumin. Polyethylene glycol-mediated transfection with a green fluorescent protein reporter gene yielded transient expression in approximately 0.75% of protoplasts five days post-transfection. While phenotypically normal plants were regenerated from untransfected protoplasts after 12 weeks in agarose bead culture with conditioned liquid medium, no regeneration was observed from transfected cells. These findings establish a reproducible protocol for protoplast isolation and plant regeneration in Cavendish banana and provide insight into the barriers limiting successful regeneration following transfection. Full article

Efficient protoplast isolation and gene transfection remain significant challenges in gymnosperms, particularly in Pinus species, where stable transformation is highly limited. Conventional pine protoplast preparation methods have resulted in extremely low transfection efficiencies, hindering functional genomic studies. This study presents an optimized method for isolating high-yield, viable protoplasts from Pinus densiflora (Korean red pine), providing a robust system for transient gene expression assays. Splitting one-month-old cotyledons produced the highest mesophyll protoplast yield (5.0 × 10⁶ cells/g FW), which further increased to 1.2 × 10⁷ cells/g FW after optimizing the enzyme mixture (4.5% cellulase, 0.7% pectinase, 3% hemicellulase), maintaining viability above 86%. Developing xylem and whole-stem protoplasts were also successfully isolated by mitigating resin leakage and debris contamination, with a 17% sucrose gradient yielding 7.4 × 10⁴ cells/g FW at 81.9% viability. Overcoming prior inefficiencies, this protocol significantly enhances gene transfection efficiency, achieving 94.1% GFP transformation with 82.9% viability. Furthermore, transient activation assays confirmed strong activation of pine-derived reporters by native effectors, underscoring the assay’s suitability for studying gymnosperm-specific gene regulation. Given the limited stable transformation strategies available for Pinus species, this optimized protoplast transient gene expression system provides a practical and reliable platform for transient gene expression analysis, offering valuable opportunities for studying gene function and regulation in gymnosperms. Full article

Welsh onion (Allium fistulosum L.), a globally significant vegetable, flavoring agent, and phytomedicine resource, has remained unavailable with established transient expression platforms for functional genomic investigations. To address this critical methodological limitation, we present systematically optimized protocols for both Agrobacterium-mediated hairy root transformation and protoplast transient expression systems, achieving significant advances in transformation efficiency for this species. Through systematic optimization of key parameters, including Agrobacterium rhizogenes (A. rhizogenes) strain selection (with Ar.Qual demonstrating superior performance), explant type efficacy, bacterial suspension optical density (OD₆₀₀ = 0.3), and acetosyringone induction concentration (100 μM), we established a highly efficient stem disc infection methodology, achieving 88.75% hairy root induction efficiency. Subsequent optimization of protoplast isolation protocols identified the optimal enzymatic digestion conditions: 6-h dark digestion of young leaves using 1.0% (w/v) Cellulase R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M mannitol, yielding 3.3 × 10⁶ viable protoplasts g⁻¹ FW with 90% viability. System functionality validation through PEG-mediated transient transformation demonstrated successful green fluorescent protein (GFP) reporter gene expression, confirmed by fluorescence microscopy. As the first documented transient expression platforms for Welsh onion, these protocols enable essential molecular investigations, including in planta promoter activity profiling, subcellular protein localization, and CRISPR-based genome-editing validation. This methodological breakthrough overcomes previous technical constraints in Welsh onion molecular biology, providing critical tools for accelerated gene functional characterization in this agriculturally important species. Full article

Volvariella volvacea, a fungal species of Volvariella within the Pluteaceae family, is predominantly cultivated in southern China. Polysaccharides, the primary bioactive constituents of V. volvacea, exhibit diverse pharmacological activities. However, current cultivation practices face challenges due to the genetic heterogeneity of strains, leading to inconsistent content and compositional variability of polysaccharides and other functional components. ARTP, denoting atmospheric and room-temperature plasma, is a technology capable of generating plasma jets at ambient pressure with temperatures ranging from 25 to 40 °C. These jets feature high concentrations of highly reactive species, including but not limited to excited-state helium atoms, oxygen atoms, nitrogen atoms, and OH radicals. This study aims to develop high-yielding exopolysaccharide (EPS) strains through integrated ARTP mutagenesis and genome shuffling, thereby overcoming current cultivation bottlenecks. ARTP mutagenesis and genome shuffling significantly boosted EPS production in V. volvacea. ARTP generated nine stable mutants with >20% higher EPS yields. Subsequent genome shuffling (three rounds of protoplast fusion) produced the hybrid strain SL212, which achieved 46.85 g/L of EPS, an 111.67% increase over that of the parent strain under identical conditions. Metabolomics and transcriptomics analyses revealed that differential metabolites and genes were mainly enriched in galactose metabolism, ABC transporter pathways, and the tricarboxylic acid cycle. These pathways enhance monosaccharide biosynthesis and generate ATP, providing both precursors and energy for polysaccharide polymerization, thereby driving EPS overproduction. Preliminary mechanistic analysis identified the key contributing factors driving the elevated polysaccharide biosynthesis. Full article

Gastrodia elata Blume is an important medicinal orchid, yet its large-scale cultivation is increasingly threatened by fungal diseases. The 4-coumarate–CoA ligase (4CL) gene family directs a key step in phenylpropanoid metabolism and plant defence, but its composition and function in G. elata have not been investigated. We mined the G. elata genome for 4CL homologues, mapped their chromosomal locations, and analysed their gene structures, conserved motifs, phylogenetic relationships, promoter cis-elements and codon usage bias. Publicly available transcriptomes were used to examine tissue-specific expression and responses to fungal infection. Subcellular localisation of selected proteins was verified by transient expression in Arabidopsis protoplasts. Fourteen Ge4CL genes were identified and grouped into three clades. Two members, Ge4CL2 and Ge4CL5, were strongly upregulated in tubers challenged with fungal pathogens. Ge4CL2 localised to the nucleus, whereas Ge4CL5 localised to both the nucleus and the cytoplasm. Codon usage analysis suggested that Escherichia coli and Oryza sativa are suitable heterologous hosts for Ge4CL expression. This study provides the first genome-wide catalogue of 4CL genes in G. elata and suggests that Ge4CL2 and Ge4CL5 may participate in antifungal defence, although functional confirmation is still required. The dataset furnishes a foundation for functional characterisation and the molecular breeding of disease-resistant G. elata cultivars. Full article

SnRK kinases, central regulators of plant stress response, remain uncharacterized in Taxus—an ancient gymnosperm valued for paclitaxel production. This study aimed to identify the Taxus SnRK family and elucidate its functional roles. Specifically, we identified SnRK genes through genomic analysis and assessed tissue-specific expression via transcriptomics, while regulatory networks were deciphered using WGCNA. To overcome experimental constraints, a PEG-mediated protoplast transient expression system was developed using calli, followed by dual-luciferase assays. Consequently, 19 SnRK genes (2 SnRK1, 4 SnRK2, 13 SnRK3) were identified, with tissue-specific expression revealing TaSnRK1.2 upregulation under methyl jasmonate (MeJA) and in stress-resilient tissues (bark/root). Subsequently, WGCNA uncovered a bark/root-specific module containing TaSnRK1.2 with predicted TF interactions (TaGRAS/TaERF). Critically, homologous dual-luciferase assays demonstrated TaSnRK1.2 activates TaGRAS and TaERF promoters (4.34-fold and 3.11-fold induction, respectively). This study establishes the Taxus SnRK family and identifies TaSnRK1.2 as a hub integrating stress signals (e.g., MeJA) to modulate downstream TF networks, while the novel protoplast system enables future functional studies in this medicinal plant. Full article

Chloroplasts, as the organelles primarily responsible for photosynthesis, require a substantial supply of iron ions. Conversely, due to Fe toxicity, the homeostasis of these ions is subject to tight regulation. Permease in chloroplast 1 (PIC1) has been identified as the primary iron importer into chloroplasts. However, previous studies suggested the existence of a distinct pathway for Fe transfer to chloroplasts, likely involving mitoferrin-like 1 (MFL1) protein. In this work, Arabidopsis MFL1 (AtMFL1) and its cucumber homolog (CsMFL1) were characterized using, among others, Arabidopsis protoplasts as well as both yeast and Arabidopsis mutants. Localization of both proteins in chloroplasts has been shown to be mediated via an N-terminal transit peptide. At the gene level, MFL1 expression profiles differed between the model plant and the crop plant under varying Fe availability. The expression of other genes involved in chloroplast Fe homeostasis, including iron acquisition, trafficking, and storage, was affected to some extent in both AtMFL1 knockout and overexpressing plants. Moreover, root growth and photosynthetic parameters changed unfavorably in the mutant lines. The obtained results imply that AtMFL1 and CsMFL1, as putative chloroplast iron transporters, play a role in both iron management and the proper functioning of the plant. Full article

The type V CRISPR/Cas12f system, with its broad PAM recognition range, small size, and ease of delivery, has significantly contributed to the gene editing toolbox. In this study, enOsCas12f1 activity was detected during transient expression in rice protoplasts. The results showed that enOsCas12f1 exhibited DNA cleavage activity when it recognized TTN PAMs. Subsequently, we examined the gene editing efficiency of enOsCas12f1 in stably transformed rice plants, and the results showed that enOsCas12f1 could identify the TTT and TTC PAM sequences of the OsPDS gene, resulting in gene mutations and an albino phenotype. The editing efficiencies of TTT and TTC PAMs were 6.21% and 44.21%, respectively. Furthermore, all mutations were base deletions, ranging in size from 7 to 29 base pairs. Then, we used enOsCas12f1 to edit the promoter and 5′ UTR of the OsDREB1C gene, demonstrating that enOsCas12f1 could stably produce base deletion, mutant rice plants. Additionally, we fused the transcriptional activation domain TV with the dead enOsCas12f1 to enhance the expression of the target gene OsIPA1. Our study demonstrates that enOsCas12f1 can be utilized for rice gene modification, thereby expanding the toolbox for rice gene editing. Full article

Rice (Oryza sativa L.), a staple food for over half of the global population, plays a pivotal role in food security. Among its two primary groups, japonica and indica, temperate japonica varieties are particularly valued for their high-quality grain and culinary uses. Although some of these varieties are adapted to cooler climates, they often suffer from reduced productivity or increased disease susceptibility when cultivated in warmer productive environments. These limitations underscore the need for breeding programs to incorporate biotechnological tools that can enhance the adaptability and resilience of the plants. However, New Genomic Techniques (NGTs), including CRISPR-Cas9, require robust in vitro systems, which are still underdeveloped for temperate japonica genotypes. In this study, we developed a reproducible and adaptable protocol for protoplast isolation and regeneration from the temperate japonica cultivars ‘Ónix’ and ‘Platino’ using somatic embryos as the starting tissue. Protoplasts were isolated via enzymatic digestion (1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10) in 0.6 M AA medium over 18–20 h at 28 °C. Regeneration was achieved through encapsulation in alginate beads and coculture with feeder extracts in 2N6 medium, leading to embryogenic callus formation within 35 days. Seedlings were regenerated in N6R and N6F media and acclimatized under greenhouse conditions within three months. The isolated protoplast quality displayed viability rates of 70–99% within 48 h and supported transient PEG-mediated transfection with GFP. Additionally, the transient expression of a gene editing CRISPR-Cas9 construct targeting the DROUGHT AND SALT TOLERANCE (OsDST) gene confirmed genome editing capability. This protocol offers a scalable and genotype-adaptable system for protoplast-based regeneration and gene editing in temperate japonica rice, supporting the application of NGTs in the breeding of cold-adapted cultivars. Full article