Search Results (97)

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

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

Loquat (Eriobotrya japonica Lindl.) is one of the most important subtropical evergreen fruit trees. However, due to the lack of widely applicable genetic transformation platforms, the research about gene functional characterization and molecular mechanisms is largely confined. In this study, the efficient protocol of protoplast isolation (the enzyme solution composed of 2.4% macerozyme R-10, 4.8% cellulase RS, dissolved in a 0.6 M mannitol solution) and the method of protoplast purification (CPW solution containing 5% sucrose and 11% mannitol) have been achieved with protoplast yields of 12.6 × 10⁶/g·FW, reaching a viability rate of up to 91%. A protoplast transient gene expression system has been established with an efficiency of approximately 40% using GFP reporter gene. Using this reliable and efficient system, the protein localization characteristics of transcription factor EjDELLA, EjbHLH79, and marker gene OsPHT4 were also utilized for further analysis. To our knowledge, this is the first report on establishing an efficient system for protoplast isolation, purification, and transformation of loquat mesophyll. The system reported here will definitely promote rapid progress in breeding, genetic transformation, and molecular research. Full article

Climate change profoundly impacts the health, productivity, and resilience of forest ecosystems and threatens the sustainability of forest products and wood-based industries. Innovations to enhance tree growth, development, and adaptation offer unprecedented opportunities to strengthen ecosystem resilience and mitigate the effects of climate change. Here, we established a method for protoplast isolation, purification, and CRISPR-Cas ribonucleoprotein (RNP) delivery in Pinus taeda and Abies fraseri as a step towards accelerating the genetic improvement of these coniferous tree species. In this system, purified protoplasts could be isolated from somatic embryos with up to 2 × 10⁶ protoplasts/g of tissue and transfected with proteins and nucleotides, achieving delivery efficiencies up to 13.5%. The delivery of functional RNPs targeting phenylalanine ammonia lyase in P. taeda and phytoene desaturase in A. fraseri yielded gene editing efficiencies that reached 2.1% and 0.3%, respectively. This demonstration of RNP delivery for DNA-free genome editing in the protoplasts of P. taeda and A. fraseri illustrates the potential of CRISPR-Cas to enhance the traits of value in ecologically and economically important tree species. The editing system provides a foundation for future efforts to regenerate genome-edited forest trees to improve ecosystem health and natural resource sustainability. Full article

Menaquinone-7 (MK-7) plays a crucial role in preventing fractures and certain cardiovascular diseases and is one of the essential vitamins in the human body. In this study, a strain of Bacillus subtilis that produces MK-7 was isolated from commercially available natto fermentation agents, with an MK-7 titer of 75 mg/L. It was named L-5. Firstly, by employing Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology and protoplast fusion techniques, mutants resistant to 1-hydroxy-2-naphthoic acid (HNA) and diphenylamine (DPA) were obtained, with the titer of MK-7 reaching 196 mg/L. It was named R-8. Based on whole-genome sequencing technology, four mutants involved in the MK-7 synthesis pathway of strain L-5 were identified: 2-succinyl-5-enol-pyruvate-6-hydroxy-3-cyclohexen-1-carboxylic acid, MenD (S249L); (1,4)-dihydroxy-2-naphthalic acid-heptaisoprenyltransferase, MenA (S196L); 1-deoxy-D-xylose-5-phosphate synthetase, Dxs (N60D, Q185H); and hydroxy acid reductive isomerase, Dxr (Q351K). The overexpression of these mutants led to increases in MK-7 production of 19 mg/L, 20 mg/L, 17 mg/L, and 16 mg/L, respectively, compared to the unmutated genes. These mutations have been shown to be effective. To further enhance the production of MK-7, the mutants menD (S249L), menA (S196L), Dxs (N60D, Q185H), and Dxr (Q351K) were co-expressed. The final titer of MK-7 reached 239 mg/L. This study provides theoretical support for the future genetic modification of key enzymes in the MK-7 biosynthetic pathway. Full article

Areca palm (Areca catechu L.) is an economically significant crop in tropical and subtropical regions. However, an efficient transformation and gene editing system for genetic improvement has still not been established. In this study, protoplasts were isolated from juvenile leaves, followed by PEG-mediated transformation and gene editing targeting the areca palm AcPDS via the CRISPR/Cas9 system. High yield (9.08 × 10⁶ cells/g FW) and viability (91.57%) protoplasts were isolated successfully by digestion for 5 h in an enzyme solution. Transformation efficiency (11.85%) was obtained through PEG-mediated transformation (incubation for 30 min in the mixture containing 40% PEG-4000, 400 mM CaCl₂, 30 µg of plasmid DNA, and 100 µL of protoplasts). Furthermore, subcellular localization was established by the cotransformation of GFP and pNLS-mCherry in the protoplasts. Moreover, the editing efficiency (2.82%) of AcPDS using the CRISPR/Cas9 system was detected by Hi-TOM sequencing. This study established an efficient transformation and gene editing system based on protoplasts in areca palm, which will be beneficial for gene function verification and genetic improvement in areca palm and other tropical palm crops. Full article

Phytophthora cinnamomi is a major plant pathogen that affects economically important crops and natural ecosystems, posing a threat to global biodiversity. While gene editing has emerged as a powerful tool for functional genomics in various Phytophthora species, its application in P. cinnamomi remains underexplored. To address this gap, our study investigated the challenges of implementing CRISPR/Cas9-mediated gene editing in P. cinnamomi, with the insights gained applicable to other gene editing platforms. We designed guide RNAs (gRNAs) targeting β-cinnamomin, a highly basic elicitin expressed by the pathogen during early infection stages, known for its role in sterol recruitment. Using an “all-in-one” plasmid containing the gRNA, Cas9, and an antibiotic resistance gene as a selectable marker, we transformed P. cinnamomi protoplasts via PEG/CaCl₂-mediated methods. The successful integration of the nptII gene, which confers geneticin (G418) resistance, was confirmed in heterokaryotic regenerants. To isolate pure mutants and eliminate wild-type dominance, we derived homokaryotic colonies from nptII-positive transformants. Mutation screening was performed using mismatch detection assays, T7 endonuclease 1 (T7E1), and restriction fragment length polymorphism (RFLP), followed by Sanger sequencing. Despite the integration of the nptII gene, the β-cinnamomin sequence in the transformants remained identical to the wild-type sequence, indicating challenges in detecting and achieving targeted gene disruption. This study identifies critical steps for optimising mutagenesis in P. cinnamomi, highlighting the importance of thorough preliminary screening, effective separation of heterokaryotic populations, and the isolation of homokaryotic colonies. Full article

The Brassica oleracea L. species embrace important horticultural crops, such as broccoli, cauliflower, and cabbage, which are highly valued for their beneficial nutritional effects. However, the complexity of flower emasculation in these species has forced breeders to adopt biotechnological approaches such as somatic hybridization to ease hybrid seed production. Protoplasts entail a versatile tool in plant biotechnology, supporting breeding strategies that involve genome editing and hybridization. This review discusses the use of somatic hybridization in B. oleracea L. as a biotechnological method for developing fusion products with desirable agronomic traits, particularly cytoplasmic male sterile (CMS) condition. These CMS lines are critical for implementing a cost-effective, efficient, and reliable system for producing F1 hybrids. We present recent studies on CMS systems in B. oleracea L. crops, providing an overview of established models that explain the mechanisms of CMS and fertility restoration. Additionally, we emphasize key insights gained from protoplast fusion applied to B. oleracea L. breeding. Key steps including pre-treatments of donor plants, the main tissues used as sources of parental protoplasts, methods for obtaining somatic hybrids and cybrids, and the importance of establishing a reliable plant regeneration method are discussed. Finally, the review explores the incorporation of genome editing technologies, such as CRISPR-Cas9, to introduce multiple agronomic traits in Brassica species. This combination of advanced biotechnological tools holds significant promise for enhancing B. oleracea breeding programs in the actual climate change context. Full article

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts. Full article

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction. Full article

Advances in cell fusion technology have propelled breeding into the realm of somatic hybridization, enabling the transfer of genetic material independent of sexual reproduction. This has facilitated genome recombination both within and between species. Despite its use in plant breeding for over fifty years, somatic hybridization has been limited by cumbersome procedures, such as protoplast isolation, hybridized-cell selection and cultivation, and regeneration, particularly in woody perennial species that are difficult to regenerate. This review summarizes the development of somatic hybridization, explores the challenges and solutions associated with cell fusion technology in woody perennials, and outlines the process of protoplast regeneration. Recent advancements in genome editing and plant cell regeneration present new opportunities for applying somatic hybridization in breeding. We offer a perspective on integrating these emerging technologies to enhance somatic hybridization in woody perennial plants. Full article

The sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and foods commonly used in China, Japan, Korea, and other Asian countries. To provide a high-quality reference genome and deepen our understanding of the genome of W. hoelen to elucidate various biological phenomena. In this study, we assembled three genomes of W. hoelen using a combination of Nanopore and Illumina sequencing strategies. The fifteen-chromosome genome L7 of W. hoelen was assembled with two-sided telomere and rDNA sequences for the first time. The chromosome count was subsequently confirmed through collinearity analysis, correcting the previous belief that W. hoelen had only fourteen chromosomes. Moreover, the aneuploid genome was discovered in W. hoelen for the first time through sequencing depth analysis of different chromosomes, and only some strains of W. hoelen exhibit aneuploid genomes. According to the genome analysis of homokaryotic offspring and protoplast-isolated strains, a potential variation in chromosome allocation patterns was revealed. Moreover, the gene function enrichment analysis of genes on reduplicated chromosomes demonstrated that aneuploidy in the genome may be the result of environmental adaptation for W. hoelen. The discovery of an aneuploid genome also provides new ideas for genetic improvement of W. hoelen. Full article

The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in Phelipanche aegyptiaca. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for P. aegyptiaca. Even the parasitic mechanisms of P. aegyptiaca and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in Phelipanche aegyptiaca haustorium. The optimal protoplast yield and activity were 6.2 × 10⁶ protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 μg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts’ isolation and transient transformation system of Phelipanche aegyptiaca haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants. Full article