Agrobacterium tumefaciens can harm various fruit trees, leading to significant economic losses in agricultural production. It is urgent to develop new pesticides to effectively treat this bacterial disease. In this study, four new sesquiterpene derivatives, trichoderenes A-D (1-4), along with six known compounds (5-10), were obtained from the marine-derived fungus Trichoderma effusum. The structures of 1-4 were elucidated by extensive spectroscopic analyses, and the calculated ECD, ORD, and NMR methods. Structurally, the hydrogen bond formed between the 1-OH group and the methoxy group enabled 1 to adopt a structure resembling that of resorcylic acid lactones, thereby producing the ECD cotton effect. Compound 3 represents the first example of C12 nor-sesquiterpene skeleton. Compounds 1-10 were tested for their antimicrobial activity against A. tumefactions. Among them, compounds 1-3 and 8-10 exhibited inhibitory activity against A. tumefactions with MIC values of 3.1, 12.5, 12.5, 6.2, 25.0, and 12.5 μg/mL, respectively.

Agrobacterium-mediated transient expression is a flexible and efficient technique for introducing genes into plants, allowing for rapid and temporary gene expression. Agroinfiltration of Arabidopsis seedlings is a newly developed Agrobacterium-based transient expression system. The expression of target genes and the localization of relevant proteins can be observed within 3 days using this method. In this chapter, we present the detailed protocol for transient transformation in Arabidopsis thaliana seedlings utilizing vacuum infiltration of Agrobacterium. This procedure enables rapid and temporary gene expression by introducing exogenous DNA into Arabidopsis seedlings, particularly in easily accessible tissues such as cotyledons. This protocol provides a detailed description of experimental procedures, including Arabidopsis seedlings cultivation, the preparation of Agrobacterium suspensions, and subsequent steps leading to confocal microscope observation. Through this protocol, researchers can efficiently investigate gene function and subcellular localization in Arabidopsis cotyledons within 8 days in total.

Promoters are the genomic regions upstream of genes that RNA polymerase binds in order to initiate gene transcription. Understanding the regulation of gene expression depends on being able to identify promoters, because they are the most important component of gene expression. Agrobacterium tumefaciens (A. tumefaciens) strain C58 was the subject of this study with the goal of creating a machine learning-based model to predict promoters. In this study, nucleotide density (ND), k-mer, and one-hot were used to encode the promoter sequence. Support vector machine (SVM) on fivefold cross-validation with incremental feature selection (IFS) was used to optimize the generated features. These improved characteristics were then used to distinguish promoter sequences by feeding them into the random forest (RF) classifier. Tenfold cross-validation (CV) analysis revealed that the projected model has the ability to produce an accuracy of 84.22%.

This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of β-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

CONCLUSIONS: We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics studies of both hemp-type and drug-type cannabis. Cannabis (Cannabis sativa L.) holds great promise in the medical and food industries due to its diverse chemical composition, including specialized cannabinoids. However, the study of key genes involved in various biological processes, including secondary metabolite biosynthesis, has been hampered by the lack of efficient in vivo functional analysis methods. Here, we present a novel, short-cycle, high-efficiency transformation method for cannabis seedlings using Agrobacterium tumefaciens. We used the RUBY reporter system to monitor transformation results without the need for chemical treatments or specialized equipment. Four strains of A. tumefaciens (GV3101, EHA105, LBA4404, and AGL1) were evaluated for transformation efficiency, with LBA4404 and AGL1 showing superior performance. The versatility of the system was further demonstrated by successful transformation with GFP and GUS reporter genes. In addition, syringe infiltration was explored as an alternative to vacuum infiltration, offering simplicity and efficiency for high-throughput applications. Our method allows rapid and efficient in vivo transformation of cannabis seedlings, facilitating large-scale protein expression and high-throughput characterization studies.

Sunflower is one of the four major oil crops in the world. \'Zaoaidatou\' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

A CRISPR/Cas12i.3-based gene editing platform is established in broomcorn millet (Panicum miliaceum) and used to create new elite germplasm for this ancient crop.

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

(1) Background: Sanghuangporus baumii, a valuable medicinal fungus, has limited studies on its gene function due to the lack of a genetic transformation system. (2) Methods: This study aimed to establish an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for S. baumii. This study involved cloning the promoter (glyceraldehyde-3-phosphate dehydrogenase, gpd) of S. baumii, reconstructing the transformation vector, optimizing the treatment of receptor tissues, and inventing a new method for screening positive transformants. (3) Results: The established ATMT system involved replacing the CaMV35S promoter of pCAMBIA-1301 with the gpd promoter of S. baumii to construct the pCAMBIA-SH-gpd transformation vector. The vectors were then transferred to A. tumefaciens (EHA105) for infection. This study found that the transformation efficiency was higher in the infection using pCAMBIA-SH-gpd vectors than using pCAMBIA-1301 vectors. The mycelia of S. baumii were homogenized for 20 s and collected as the genetic transformation receptor. After 20 min of co-culture and 48 h of incubation in 15 mL PDL medium at 25 °C, new colonies grew. (4) Conclusions: These colonies were transferred to PDA medium (hygromycin 4 μg/mL, cefotaxime 300 μg/mL), and the transformation efficiency was determined to be 33.7% using PCR.

CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.