Search Results (378)

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

Castanea mollissima Bl. is rich in nutrition and strong in stress resistance, and has nutritional, economic, and ecological values. A protoplast is impactful in somatic fusion and germplasm creation. Here, we propose an effective scheme for the construction of an embryonic suspension cell, protoplast isolation, and fusion. Studies have shown that when 1.0 g yellow loose embryonic callus was inoculated into MS + 1.5 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA + 0.5 mg∙L⁻¹ 2, 4-D liquid medium, a stable suspension cell line can be obtained. After further culturing for 2–4 days, protoplast isolation was performed. First, single-factor screening was conducted on the four enzymes, and then a two-factor random block was further set up to screen the enzyme combinations based on the results. We found that 1.0%cellulase R-10 + 0.5%pectolase Y-23 led to the highest protoplast yield (9.27 × 10⁶/g FW) and the highest activity (92.49%). Furthermore, the protoplast yield could be increased to 9.47 × 10⁶/g FW by adding 0.4 M mannitol and shaking for 8 h. The protoplasts were purified by centrifuging at 40× g for 4 min and then mixed with 30% PEG 6000 at a volume ratio of 1.5:1 for 25 min. The fusion rate could reach 70.00%. This study laid a foundation for the creation of new germplasm by Castanea mollissima Bl. Full article

Chaperonin 60 proteins plays an important role in plant growth and development as well as the response to abiotic stress. As part of the protein homeostasis system, molecular chaperones have attracted increasing attention in recent years due to their involvement in the folding and assembly of key proteins in photosynthesis. However, little is known about the function of maize chaperonin 60 protein. In the study, a gene encoding the chaperonin 60 proteins was cloned from the maize inbred line B73, and named ZmCpn60-3. The gene was 1, 818 bp in length and encoded a protein consisting of 605 amino acids. Phylogenetic analysis showed that ZmCpn60-3 had high similarity with OsCPN60-1, belonging to the β subunits of the chloroplast chaperonin 60 protein family, and it was predicted to be localized in chloroplasts. The ZmCpn60-3 was highly expressed in the stems and tassels of maize, and could be induced by exogenous plant hormones, mycotoxins, and pathogens; Overexpression of ZmCpn60-3 in Arabidopsis improved the resistance to Pst DC3000 by inducing the hypersensitive response and the expression of SA signaling-related genes, and the H₂O₂ and the SA contents of ZmCpn60-3-overexpressing Arabidopsis infected with Pst DC3000 accumulated significantly when compared to the wild-type controls. Experimental data demonstrate that flg22 treatment significantly upregulated transcriptional levels of the PR1 defense gene in ZmCpn60-3-transfected maize protoplasts. Notably, the enhanced resistance phenotype against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in ZmCpn60-3-overexpressing transgenic lines was specifically abolished by pretreatment with ABT, a salicylic acid (SA) biosynthetic inhibitor. Our integrated findings reveal that this chaperonin protein orchestrates plant immune responses through a dual mechanism: triggering a reactive oxygen species (ROS) burst while simultaneously activating SA-mediated signaling cascades, thereby synergistically enhancing host disease resistance. Additionally, yeast two-hybrid assay preliminary data indicated that ZmCpn60-3 might bind to ZmbHLH118 and ZmBURP7, indicating ZmCpn60-3 might be involved in plant abiotic responses. The results provided a reference for comprehensively understanding the resistance mechanism of ZmCpn60-3 in plant responses to abiotic or biotic stress. Full article

LTR retrotransposons are widespread genomic elements that significantly impact genome structure and function. In Arabidopsis thaliana, the EVD LTR retrotransposon encodes a GAG protein essential for retrotransposon particle assembly. Here, we present a comprehensive analysis of the structural features, intracellular localization, and transcriptomic effects of the EVD GAG (evdGAG) protein. Using AlphaFold3, we identified canonical capsid (CA-NTD and CA-CTD) and nucleocapsid (NC) domains, with predicted disordered regions likely facilitating oligomerization. Transient expression of GFP-tagged evdGAG in protoplasts of A. thaliana and distant plant species (Nicotiana benthamiana and Helianthus annuus) revealed the formation of multiple large cytoplasmic aggregates resembling retrosomes, often localized near the nucleus. Stable overexpression of evdGAG in wild-type and ddm1 mutant backgrounds induced significant transcriptomic changes, including up-regulation of stress response and defense-related genes and downregulation of photosynthesis and chloroplast-associated pathways. Importantly, genes linked to stress granule formation were also up-regulated, suggesting a role for evdGAG in modulating cellular stress responses. Our findings provide novel insights into the cellular and molecular properties of plant retrotransposon GAG proteins and their influence on host gene expression. Full article

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10⁶ cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO₄ yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant–fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant–fungal interactions. Full article

Tomato is a widely cultivated vegetable crop worldwide. It is susceptible to various phytopathogens, including fungi, bacteria, viruses, and nematodes. In 2024, an unknown leaf spot disease outbreak, characterized by distinct brown necrotic lesions on leaves, was observed in tomato plants in Yunnan Province, China. Through rigorous pathogen isolation and the fulfillment of Koch’s postulates, it was proved that the fungal isolate could infect tomato leaves and cause typical symptoms. The pathogen isolated from tomato leaves was identified as Nigrospora oryzae based on its morphology and using a multilocus sequence analysis method with the internal transcribed spacer gene (ITS1), beta-tubulin gene (TUB2), and translation elongation factor 1-alpha gene (TEF1-α). This represents the first documented case of N. oryzae infecting tomatoes in the world. Given the damage caused by N. oryzae to tomato plants, we explored biocontrol methods. Through a dual-culture assay on PDA plates, Bacillus velezensis B31 demonstrated significant biocontrol potential, exhibiting strong antagonistic activity toward N. oryzae. In addition, we developed a polyethylene glycol (PEG)-mediated transformation system that successfully introduced pYF11-GFP into the protoplasts of N. oryzae. This achievement provides a foundation for future genetic manipulation studies of N. oryzae. Full article

Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories. Full article