Search Results (96)

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

Sugar beet (Beta vulgaris L.), a biennial sugar crop, provides about 16% of the world’s sucrose production. PEG and Agrobacterium tumefaciens-mediated transformation have been established for sugar beet. However, the traditional transformation of sugar beet is time-consuming, low efficiency, and dependent on tissue regeneration. Recently, the use of Agrobacterium rhizogenes for genetic transformation without tissue culture has become a new possibility. Here, we describe an optimized A. rhizogenes-mediated transformation for the generation of composite sugar beet without tissue culture. By dipping A. rhizogenes K599 colonies onto a wound of hypocotyl and petiole, about 81.7% and 51.1% of shoots and leaves could be induced to produce adventitious roots. Of these, more than 60% of the explants contained transformed adventitious roots. Specifically, we discovered that the transformation efficiency was significantly improved when the MAS promoter was employed instead of the CaMV35S promoter. The transformation in adventitious roots was also validated by qRT-PCR and Western blot at the transcriptional and translational levels. The transformed adventitious roots have great potential for the study of taproot development, sugar accumulation, and resistance to root diseases, which is closely related to sugar beet yield and quality. Full article

Fusarium acuminatum is a major pathogenic fungus causing root rot in alfalfa (Medicago sativa). DNA polymerase theta is known to play a crucial role in repairing DNA double-strand breaks. However, its biological function in F. acuminatum remains unknown. In this study, the POLQ gene was deleted by homologous recombination using Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (with the POLQ gene), the mutants (without the POLQ gene) showed significant phenotypic changes: they produced brown-yellow pigments instead of pink, slowed mycelial growth, and exhibited changes in macroconidia size and shape. The virulence of the mutants was greatly reduced, inducing only mild symptoms in alfalfa. In addition, FITC-WGA staining showed impaired spore germination and hyphal growth. These results suggest that POLQ is a key gene regulating growth and development of F. acuminatum, indicating that DNA repair may play an essential role in the pathogenicity of the pathogen in alfalfa. The POLQ gene could thus be a promising target for limiting F. acuminatum infections in alfalfa. Full article

Soybean (Glycine max L.) is a vital grain and oil crop, serving as a primary source of edible oil, plant-based protein, and livestock feed. Its production is crucial for ensuring global food security. However, soybean yields are severely impacted by various diseases, and the development of disease-resistant cultivars remains the most sustainable strategy for mitigating these losses. While stable genetic transformation is a common approach for studying gene function, virus-induced gene silencing (VIGS) offers a rapid and powerful alternative for functional genomics, enabling efficient screening of candidate genes. Nevertheless, the application of VIGS in soybean has been relatively limited. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for soybean, utilizing Agrobacterium tumefaciens-mediated infection. The TRV vector was delivered through cotyledon nodes, facilitating systemic spread and effective silencing of endogenous genes. Our results demonstrate that this TRV–VIGS system efficiently silences target genes in soybean, inducing significant phenotypic changes with a silencing efficiency ranging from 65% to 95%. Key genes, including phytoene desaturase (GmPDS), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4, were successfully silenced, confirming the system’s robustness. This work establishes a highly efficient TRV–VIGS platform for rapid gene function validation in soybean, providing a valuable tool for future genetic and disease resistance research. Full article

Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for Agrobacterium-mediated transient and stable transformation of Fagopyrum esculentum and F. tartaricum. Leaf and cotyledon tissues were infiltrated with an A. tumefaciens-bearing construct containing eGFP and GUS reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. HvAMIS and HvNMT genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene (eGFP) and selectable marker gene (NptII) was achieved by Agrobacterium-mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy. Full article

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD₆₀₀) of 0.6, an acetosyringone concentration of 100 μmol·L⁻¹ in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L⁻¹, and a selection culture with hygromycin at 9 mg·L⁻¹. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted. Full article

Histone post-translational modifications (HPTMs) can affect gene expression by rearranging chromatin structure. Between these, histone methylation is one of the most studied in filamentous fungi, and different conserved domains coding for methyltransferase were found in Aspergillus spp. genomes. In this work, the role of the histone methyltransferases AcDot1 and AcRmtA in the mycotoxigenic fungus Aspergillus carbonarius was investigated, obtaining knockout or overexpression mutants through Agrobacterium tumefaciens-mediated transformation (ATMT). A. carbonarius is responsible for grape-bunch rot, representing the major source of ochratoxin A (OTA) contamination on grapes. In vivo conditions, the deletion of Acdot1 or AcrmtA resulted in upregulation of growth when the isolates were cultivated on a minimal medium. The influence of Acdot1 on the OTA biosynthesis was differently affected by culture conditions. On rich media, an increase in OTA accumulation was observed, while on minimal medium, lower OTA concentrations were reported. The deletion of AcrmtA always resulted in lower OTA accumulation. However, the expression of OTA biosynthesis genes was regulated by both histone methyltransferases. Of the six analyzed OTA genes, three of them showed altered expression in the knockout mutants, and otaB and otaR1 were common between both mutants. Furthermore, both AcDot1 and AcRmtA play a role in oxidative stress response, induced by 1 mM hydrogen peroxide, by modulating growth, conidiation and OTA biosynthesis. Neither the deletion nor the overexpression of the Acdot1 or AcrmtA affected virulence, while both the sporulation and OTA production were negatively affected in vivo by the deletion of AcrmtA. Full article

Rice false smut, caused by Ustilaginoidea virens, is one of the three major rice diseases in China. It not only seriously affects the rice yield and quality but also endangers human and animal health. Studying the pathogenic mechanism of U. virens has important theoretical significance and application value for clarifying the infection characteristics of the pathogen and cultivating disease-resistant varieties. Plant pathogenic fungi utilize secreted effectors to suppress plant immune responses, which can function in the apoplast or within host cells and are likely glycosylated. However, the posttranslational regulation of these effectors remains unexplored. Deletion of ΔUvALG led to the cessation of secondary infection hyphae growth and a notable decrease in virulence. We observed that ΔUvALG mutants triggered a significant increase in reactive species production within host cells, akin to ALG mutants, which plays a crucial role in halting the growth of infection hyphae in the mutants. ALG functions by sequestering chitin oligosaccharides to prevent their recognition by the rice chitin elicitor, thereby inhibiting the activation of innate immune responses, including reactive species production. Our findings reveal that ALG3 possesses three N-glycosylation sites, and the simultaneous Alg-mediated N-glycosylation of each site is essential for maintaining protein stability and chitin-binding activity, both of which are critical for its effector function. These outcomes underscore the necessity of the Alg-mediated N-glycosylation of ALG to evade host innate immunity. Full article

Monascus, a genus of fungi known for its fermentation capability and production of bioactive compounds, such as Monascus azaphilone pigments and Monacolin K, have received considerable attention because of their potential in biotechnological applications. Understanding the genetic basis of these metabolic pathways is crucial for optimizing the fermentation and enhancing the yield and quality of these products. However, Monascus spp. are not model fungi, and knowledge of their genetics is limited, which is a great challenge in understanding physiological and biochemical phenomena at the genetic level. Since the first application of particle bombardment to explore gene function, it has become feasible to link the phenotypic variation and genomic information on Monascus strains. In recent decades, accurate gene editing assisted by genomic information has provided a solution to analyze the functions of genes involved in the metabolism and development of Monascus spp. at the molecular level. This review summarizes most of the genetic manipulation tools used in Monascus spp. and emphasizes Agrobacterium tumefaciens-mediated transformation and nuclease-guided gene editing, providing comprehensive references for scholars to select suitable genetic manipulation tools to investigate the functions of genes of interest in Monascus spp. Full article

Torenia fournieri L. is a popular ornamental plant in the genus Torenia, widely used in commercial landscaping, especially during the summer. Additionally, Torenia has served as a model ornamental plant in many studies exploring ornamental characteristics and pest control through genetic engineering. To date, no research has been reported on developing insect-resistant Torenia expressing genes from Bacillus thuringiensis (Bt). In this study, a recombinant vector carrying the Cry1Ab gene from Bt, pBI121-Cry1Ab, was constructed and transferred into T. fournieri via Agrobacterium tumefaciens-mediated transformation. A total of 13 shoots survived on the kanamycin selection medium, among which four putative transgenic lines, designated L1, L2, L7, and L11, were molecularly confirmed by PCR and Southern blot analysis, indicating successful integration of the Cry1Ab gene into the genomes of these lines. Quantitative real-time PCR and ELISA results further verified the successful expression of the Cry1Ab gene in the leaves of all four transgenic lines. Insect bioassay results demonstrated that all four transgenic lines showed strong resistance to the insect pest, Mythimna separata, with mortality rates ranging from 59.9% to 100.0%, in contrast to a larval mortality rate of 16.2% in the wild-type Torenia. Additionally, these transgenic lines significantly decreased in larval survival rates compared to those fed on wild-type plants. Furthermore, these transgenic lines activated superoxide dismutase (SOD) activity at 12 and 24 h, and catalase (CAT) activity at 72 h, while suppressing SOD activity at 72 h, and peroxidase (POD) activity over time. Our findings indicate that these transgenic lines exhibit high resistance to the insect pest and provide new insights into controlling insect pests in ornamental plants through genetic approaches. Full article

The Agrobacterium-mediated technique is widely employed for soybean transformation, but the efficiency of this method is still relatively modest, in which multiple factors are involved. Numerous chemical and physiological cues from host plants are needed for A. tumefaciens attraction and subsequent T-DNA integration into the plant genome. Susceptible genotypes may permit this attachment and integration, and the agronomically superior genotypes with susceptibility to A. tumefaciens would play an important role in increasing transformation efficiency. In this study, we aimed to elevate the Agrobacterium-mediated transformation efficiency of soybean by integrating susceptibility alleles from William82 and flavonoids accumulating alleles from LX genotypes in the same soybean line. The crossing was made between LX () and William82 () soybean by hand pollination. Expectedly, the resulting hybrid soybean progenies inherited susceptibility traits and high flavonoid contents (i.e., genistein, genistin, apigenin, naringenin, quercetin, and cinnamic acid) essential for potential plant–pathogen interaction. Furthermore, the progenies and susceptible William82 soybean were subjected to transformation using A. tumefaciens (GV3101) harboring the GmUbi-3XFlag-35S-GFP and reassembled GmUbi3XFlag-35S-GFP: GUS vectors during separate events. Important transformation-related traits like shoot induction and shoot regeneration ability were also significantly improved in progenies. The progenies designated as ZX-3 exhibited superiority over the William82 parental line in all three traits, i.e., shoot induction, regeneration, and Agrobacterium-mediated transformation. The transient transformation efficiency of the ZX-16 line was remarkably higher when half-cotyledon explants were wounded and transformed with A. tumefaciens harboring GUS assembly vector and then co-cultivated on MS medium supplemented with 2 mg/L spermidine, 0.3 g/L GA3, 0.3 mg/L kinetin, and 1.3 mg/L 6-benzylaminopurine. In addition, the shoot elongation was also higher than that of William82 after two weeks of culture on the shoot induction medium. The newly generated soybeans have the potential to serve as a valuable source for high transgene production and represent a promising avenue for future soybean varietal development. Full article

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD₆₀₀ of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila. Full article

Oil palm (Elaeis guineensis Jacq.) is the most efficient oil-producing crop globally, yet progress in its research has been hampered by the lack of effective genetic transformation systems. The EPSPS gene, encoding 5-enolpyruvylshikimate-3-phosphate synthase, has been used as a transgenic selection marker in various crops, including rice and soybean. This study evaluated the EPSPS/glyphosate selection system for oil palm transformation. We constructed a binary expression vector, pCGlyDESCLI-C, containing the TIPS-EiEPSPS selection marker from goosegrass and the mScarlet-I red fluorescent reporter gene. This vector was introduced into oil palm embryonic callus (EC) via Agrobacterium-mediated transformation. After optimizing the transformation steps, positive calli were obtained, and integration of the foreign gene into the oil palm genome was confirmed through molecular analysis. Notably, the selection efficiency of the EPSPS/glyphosate selection system exceeded that of the traditional hpt/hygromycin selection system, demonstrating its advantages. Our findings support the effectiveness of the TIPS-EiEPSPS/glyphosate selection system for oil palm genetic transformation, marking its first application in this species and offering a valuable tool for advancing research on this economically significant crop. Full article

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD_600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa. Full article

Descurainia sophia L. Webb ex Prantl is used in traditional medicine globally. However, the lack of an efficient and reliable genetic transformation system has seriously limited the investigation of gene function and further utilization of D. sophia. In this study, a highly efficient, time-saving, and cost-effective Agrobacterium tumefaciens-mediated genetic transformation system has been developed in D. sophia. In this method, the transformation was accomplished by simply dipping developing D. sophia inflorescences for 45 s into an Agrobacterium suspension (OD₆₀₀ = 0.6) containing 5% sucrose and 0.03% (v/v) Silwet L-77. Treated plants were allowed to set seeds which were then plated on a selective medium with hygromycin B (HygB) to screen transformants. Additionally, the CRISPR/Cas9 genomic editing system was validated by targeting phytoene desaturase (PDS) gene using this floral dip method, and mutant plants with the expected albino phenotype could be obtained in 2.5 months. This genetic transformation and targeted editing system will be a valuable tool for routine investigation of gene function and further exploitation in D. sophia. Full article