• Korean Journal of Microbiology
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• v.32 no.1
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• pp.53-59
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• 1994

Agrobacterium tumefaciens KU12 contains pTiKU12 (240kb) of the octopine type Ti plamsid and pTi12 (45 kb) of the cryptic plasmid. To make the avirulent A. tumefaciens, the octopine type Ti plasmid, pTiKU12, was cured with elevated temperature (37${\circ}C$) and ethidium bromide (EtBr), respectively. Also the cryptic plasmid, pTi12, was cured by the introduction of recombinant plasmid, pYWXP, made by pTi12 replication origin and pUC19. pYWXP was cured by elevated temperature (37${\circ}C$) and EtBr simultaneously.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.260-267
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• 2006

The Liquid Culture Assay using a recombinant Agrobacterium tumefaciens strain has been developed as a means for quorum sensing autoinducer screening. However, the low aqueous solubility of marine natural product extracts used as potential autoinducers has been a hindrance in the screening process. Although the addition of organic solvents and/or surfactants could increase aqueous solubility, errors in data interpretation including false positive results could be a serious problem. Therefore, determining the best possible solvent and surfactant at the optimum concentration is crucial. Evaluating methanol, ethanol, 1-propanol, DMSO and DMF at concentration ranges of 0~10% revealed < 2% methanol to be most favorable when tested for ${\beta}$-gal activity and growth inhibition of the recombinant A. tumefaciens strain. On the other hand, among surfactants tested, Triton X-100 was similarly effective in increasing the delivery of autoinducers for activity at less than 0.05% concentration.

• Journal of Yeungnam Medical Science
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• v.2 no.1
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• pp.175-181
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• 1985

Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called Ti plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow Ti plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of Ti plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts and Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high $Ca^{2+}$ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high $Ca^{2+}$ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observations suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.292-299
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• 1993

Agrobacterium tumefaciens KU12 isolated from Korea is able to induce tumors on various plants and catabolize octopine as a sole carbon and nitrogen source. A, tumefaciens KU12 contains three plasmids. Their sizes are 45.5 kb. 240 kb. and > 240 kb. respectively. For the purpose of identification of octopine type Ti plasmid, avirulent A, tumefacients A136 is transformed with plasmids isolated from KU12 by direct transformation. Transformants containing Ti plasmid were grown on AB medium containing octopine as a sole nitrogen source. The isolated strain, named KU911, contains only 240 kb plasmid. As a result of induction of crown gall and Southern hybridization with other octopine Ti plasmid pTiAch5, 240 kb plasmid named pTiKU12 was Ti plasmid.

• Journal of Applied Biological Chemistry
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• v.51 no.3
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• pp.117-122
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• 2008

Inactivation of Agrobacterium tumefaciens was evaluated on the inoculated fresh Radix Ginseng by electron beam irradiation or aqueous chlorine dioxide ($ClO_2$) treatment. Two groups of fresh ginsengs were prepared and inoculated with A. tumefaciens. One group was then irradiated at 0, 2, and 4 kGy using an electron beam accelerator, and the other group was treated with 0, 50, and 100 ppm of aqueous $ClO_2$. Microbiological data indicated that populations of A. tumefaciens significantly decreased with increasing irradiation dose or aqueous $ClO_2$ concentration. In particular, A. tumefaciens was eliminated by irradiation at 4 kGy, and 100 ppm $ClO_2$ treatment reduced the populations of A. tumefaciens by 1.44 log CFU/g. These results suggest that electron beam irradiation or aqueous $ClO_2$ treatment can be useful in improving the microbial safety of fresh ginsengs during storage.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.13-17
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• 2004

To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.91-95
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• 2005

Agrobacterium tumefaciens-mediated immature embryo transformation was used to produce transgenic maize. Immature embryo of Hi II genotype were co-cultivated with strains Agrobacterium tumefaciens (C58C1) containing the binary vectors (pPTN290) carrying with Ubiquitin promoter-GUS gene as reporter gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent. Seven embryogenic callus lines transformed showed the resistance in paromomycin antibiotics. Histochemical GUS assay showed that 7 individual lines transformed with the GUS gene were positive response among the transformants. Southern blot analysis revealed that the nptll gene segregated and expressed in their progeny.

• The Plant Pathology Journal
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• v.39 no.2
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• pp.220-227
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• 2023

Rose crown gall caused by Agrobacterium tumefaciens is a major disease that damages the production of cutroses in Korea. The effective prevention methods for this disease include the use of resistant varieties. This study was conducted to evaluate the resistance of 58 Korean cultivars and six foreign cultivars to crown gall disease with nodal explants in vitro. Among 180 A. tumefaciens strains, pathogenic strain RC12 was selected as an inoculant strain. The strain RC12 was identified based on characteristics of some selective media, pathogenicity test, and polymerase chain reaction analysis. Forty rose cultivars formed tumors on explants inoculated with A. tumefaciens RC12. However, 24 cultivars, including 22 Korean cultivars and 2 foreign cultivars, showed resistance to A. tumefaciens RC12 without forming any tumors. Six cultivars with tumor formation rates of over 30% formed initial tumors within 23 days after inoculation. Six cultivars with low tumor formation rates of around 5% formed initial tumors after 28 days of inoculation. It was found that gall formation rate was highly correlated with the initial gall formation period. Thus, the relationship between the period of gall formation and the rate of gall formation could be useful for assessing resistance to crown gall disease. In vitro inoculation methods could be used to evaluate resistance of cut-rose cultivars to crown gall diseases.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.2
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• pp.103-108
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• 2000

To produce of transgenic orchardgrass, the seed-derived calli of orchardgrass (Dactylis glomerata L.) co-cultivated with Agrobacterium turnefaciens EHAlOl harboring binary vector pIG121-Hm were selected with hygromycin and then transferred onto N6 regeneration medium containing 1 rngl l of NAA, 5 rngl l of kinetin, 250 rngl l of carbenicillin and 50 mg/ l of hygromycin. The efficiency of transformation was differed on cultivars, that is, 'Potomac' appeared 12% of transformation efficiency while 'Amba' did 5.5%. The addition of acetosyringone during co-cultivation was a key to successhl transformation of orchardgrass. Transgene fragments were identified by PCR analysis and the constitutive expression of GUS gene was confirmed by Northern blot analysis. (Key words : Acetosyringone, Agrobacterium tumefaciens, Orchardgrass (Dactylis glomerata L.), Transformation)

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.387-393
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• 2000

The process by which Agrobacterium tumefaciens genetically transforms plants involves a complex series of reactions communicated between the pathogen and the plants. To identify plant factors involved in agrobacterium-mediated plant transformation, a large number of T-DNA inserted Arabidopsis thaliana mutant lines were investigated for susceptibility to Agrobacterium infection by using an in vitro root inoculation assay. Based on the phenotype of tumorigenesis, twelve T-DNA inserted Arabidopsis mutants(rat) that were resistant to Agrobacterium transformation were found. Three mutants, rat1, rat3, and rat4 were characterized in detail. They showed low transient GUS activity and very low stable transformation efficiency compared to the wild-type plant. The resistance phenotype of rat1 and rats resulted from decreased attachment of Agrobacterium tumefaciens to inoculated root explants. They may be deficient in plant actors that are necessary for bacterial attachment to plant cells. The disrupted genes in rat1, rat3, and rat4 mutants were coding a arabinogalactan protein, a likely cell wall protein and a cellulose synthase-like protein, respectively.