Agroinfiltration is a newly developed plant transient gene expression technique, which is simple, rapid and reproducible. It has been widely used in analyses of foreign gene expression, hypersensitive reaction, gene silencing, promoter activity and identification of new disease-resistance genes. In this paper, we describe the principle and the operation procedure of Agroinfiltration and its application in diverse aspects of plant molecular biology research. Our experiences in modification of the Agroinfiltration technique are also provided.
Agrobacterium tumefaciens ; genetics ; Genetic Vectors ; genetics ; Plants ; genetics ; Plants, Genetically Modified ; Research Design

Chromosomal virulence genes acvB, abvA, chvA of Agrobacterium tumefaciens were cloned with the technique of transposon 5 insertion. The chromosome genes are necessary for Agrobacterium tumfaciens absorbing to cell ular surface of plant, the adherence reaction can't be executed and result in losing the toxicity if mutations are occurred in some chromosome genes. The chromosome toxicity gene is inactivated due to transposon Tn5 be inserted and the accept ant cell infected with Agrobacterium tumefaciens can't cause tumor ultimately. This article briefly introduces the research way of thinking and strategy of this technique and the important roles of every gene, which are taken of in the process of T-DNA's form, transfer, integration, and expression etc. This article also gives a presumption to T-DNA's transport: The plant cell wall's porin may be T-DNA's natural channel.
Agrobacterium tumefaciens ; genetics ; metabolism ; pathogenicity ; DNA Transposable Elements ; genetics ; physiology ; Virulence ; genetics

The main aim of this study was to transform the enhanced green fluorescent protein gene (egfp) into biocontrol fungus Paecilomyces lilacinus strain 9410. We constructed the expression vector pUPNGT of the fusion gene nptII-egfp using pcDNA3.1(-) as a helper plasmid. The egfp gene was then transformed into P. lilacinus strain 9410 via Agrobacterium tumefaciens-mediated transformation. PCR and Southern blotting analysis showed that the egfp gene was integrated into the genomes of the tested transformants and the integration manner was single-copy. The transformants could generate green fluorescence when they were excited by 488 nm blue laser. These results indicated that the egfp gene had been successfully transformed into P. lilacinus 9410 and expressed in the tested transformants. Our work may provide a new approach to assess environmental safety and practical biocontrol efficacy ofP. lilacinus under different conditions.
Agrobacterium tumefaciens ; genetics ; Green Fluorescent Proteins ; genetics ; Paecilomyces ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Transformation, Genetic

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1 x 10(5) spores when 1 x 10(6) spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600 = 0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 microg/mL and selection medium contained 100 microg/mL of hygromycin B and 200 microg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.
Agrobacterium tumefaciens ; genetics ; Ascomycota ; genetics ; Cucumis melo ; microbiology ; Plant Diseases ; microbiology ; Plants, Genetically Modified ; Transformation, Genetic

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.
Panax/genetics* ; Promoter Regions, Genetic ; Agrobacterium tumefaciens/genetics* ; Computational Biology ; Cloning, Molecular

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.
Agrobacterium tumefaciens ; metabolism ; Breeding ; Cucumis sativus ; genetics ; microbiology ; Plants, Genetically Modified ; microbiology ; Research ; Transformation, Genetic

Candida glycerinogenes WL2002-5 (C.g) is an important industrial strain for glycerol production. To further improve glycerol production, we reconstructed a binary vector pCAM3300-zeocin-CgGPD1, introduced it to Agrobacterium tumefaciens LBA4404 by electroporation, and then transformed the T-DNA harboring the CgGPD1 to Candida glycerinogenes by Agrobacterium tumefaciens-mediated transformation (ATMT). After 96 h fermentation with glucose as the substrate, we screened a transformant named C.g-G8 with high glycerol production. Compared with the wild strain, the glucose consumption rate of C.g-G8 and the glycerol production were 12.97% and 18.06% higher, respectively. During the fermentation, the activity of glycerol-3-phosphate dehydrogenase of C.g-G8 was 27.55% higher than that of the wild strain. The recombinant Candida glycerinogenes with high glycerol production was successful constructed by ATMT method.
Agrobacterium tumefaciens ; enzymology ; genetics ; Candida ; genetics ; metabolism ; Electroporation ; Fermentation ; Glycerol ; analysis ; metabolism ; Glycerolphosphate Dehydrogenase ; genetics ; Recombination, Genetic ; Transformation, Genetic

It was clearly demonstrated that T-DNA of Agrobacterium tumefeciens Ti plasmid was integrated into the cells of crown gall in our experiment. This paper reported the influences of some kinds of physical-chemistry factors on the growth of crown gall of Panax quinquefolium and the production of its main active compounds--ginsenoside Re and ginsensoside Rg1. The results showed that White medium was the best one for ginsensoside Rg1 accumulation (0.095%) among the six media, but ginsensoside Re accumulation (0.194%) was the highest on the MS medium; The highest contents of ginsensoside Re (0.147%) and ginsensoside Rg1 (0.061%) were on the culture 36d and 32d after innoculum respectively; The optimum pH was 5.6 for ginsensoside Rg1 synthesis(0.054%), and 5.8 for ginsensoside Re synthesis(0. 184% ); The contents of ginsensoside Re and ginsensoside Rg1 was the highest in the inoculum of 4 g and 2 g/flask (FW) respectively. The result also indicated that the concentration of inositol in 0.05 g/L could obviously promote ginsensoside Re synthesis (0.182%), and in 0.30 g/L for ginsensoside Rg1 (0.055%).
Agrobacterium tumefaciens ; genetics ; physiology ; Cell Culture Techniques ; Ginsenosides ; biosynthesis ; Hydrogen-Ion Concentration ; Panax ; growth & development ; metabolism ; microbiology ; Plant Tumors ; microbiology

In order to induce male sterility of Brassica campestris L. subsp. chinensis Makino var. parachinensis, we introduced the chimeric pTA29-barnase gene into it by Agrobacteriumtume faciens transformation. We obtained the transgenic plants, and determined them by PCR, Southern blotting and RT-PCR analysis. Results indicated that the RNase (barnase) gene had been transferred into genome of plant, and its expression level was different among transformation plants. All transgenic plants were male sterile; there was no vigor or a little pollen without fertility in the anther of transgenic plants. The transgenic plants failed to produce seeds under the condition self-control pollination, but hybrid seeds set were obtained when these transgenic plants were cross-pollinated artificially with normal pollen from untransformed plants. Progeny from cross-pollinated maintainer line with transgenic plants segregated in the 1:1 for male sterility and male fertility, and these phenotypes corresponded directly to the presence or absence of the chimeri TA29-barnase gene. The male fertile plants of co-separated progenies could die by spraying 10 mg/L PPT in cotyledon seedling stage. The hybrid F1 between male sterility and other varieties showed heterosis in yield and growth. All these show that it is an efficient method to induce male sterility in Brassica campestris L. subsp. chinensis Makino var. parachinensis by TA29-barnase ene, there is potential on heterosis breeding of Brassica campestris L. subsp. chinensis Makino var. parachinensis.
Agrobacterium tumefaciens ; genetics ; Brassica ; genetics ; growth & development ; Gene Transfer Techniques ; Genes, Plant ; genetics ; Plant Infertility ; genetics ; Plants, Genetically Modified ; genetics ; Ribonucleases ; genetics ; Transformation, Genetic