• Journal of Plant Biotechnology
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• 제30권3호
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• pp.215-219
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• 2003

This study was conducted to find optimum bacterial density for improving the efficiency of transformation mediated by Agrobacterium tumefaciens in apples. Regeneration(15％) and transformation frequency(10％) were increased in resuspension-culture density $A_{600}$ 1.3 from preculture density $A_{600}$ 0.7 of Agrobacterium tumefaciens in ′Fuji′. In ′Gala′, 20% regeneration and 16% transformation frequency were observed at optimum bacterial density $A_{600}$ 0.7 form preculture density $A_{600}$ 1.3. ′Mclntosh as well as "Gala" were 25％regeneration and 10％ transformation frequency. Hence a frequency optimum condition of bacterial density for the efficient transformation of apple could be depend on apple genotypes.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• The Journal of Advanced Prosthodontics
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• 제11권2호
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• pp.81-87
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• 2019

PURPOSE. The purpose of this study was to evaluate the effects of various protocols and systems for finishing and polishing monolithic zirconia on surface topography, phase transformation, and bacterial adhesion. MATERIALS AND METHODS. Three hundred monolithic zirconia specimens were fabricated and then treated with three finishing and polishing systems (Jota [JO], Meisinger [ME], and Edenta [ED]) using four surface treatment protocols: coarse finishing alone (C); coarse finishing and medium polishing (CM); coarse finishing and fine polishing (CF); and coarse finishing, medium polishing, and fine polishing (CMF). Surface roughness, crystal phase transformation, and bacterial adhesion were evaluated using atomic force microscopy, X-ray diffraction, and streptococcal biofilm formation assay, respectively. One-way and two-way analysis of variance with Tukey post hoc tests were used to analyze the results (${\alpha}=.05$). RESULTS. In this study, the surface treatment protocols and systems had significant effects on the resulting roughness. The CMF protocol produced the lowest roughness values, followed by CM and CF. Use of the JO system produced the lowest roughness values and the smallest biofilm mass, while the ME system produced the smallest partial transformation ratio. The ED group exhibited the highest roughness values, biofilm mass, and partial transformation ratio. CONCLUSION. Stepwise surface treatment of monolithic zirconia, combined with careful polishing system selection, is essential to obtaining optimal microstructural and biological surface results.

• Journal of Microbiology and Biotechnology
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• 제21권4호
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• pp.333-340
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• 2011

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P<0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.

• 한국식품영양학회지
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• 제5권2호
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• pp.77-83
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• 1992

The optimum conditions and mechanisms for the plasmid-mediated genetic transformation of intact cells of Bacillus brevis Pl76-2, an extracellular protein producing bacterium by electroporation were investigated. It was found that pUB110 Plasmid DNA can be introduced into intact bacterial cells by electroporation. The frequency of transformation by this electroporation system depended upon the initial electric field strength, the capacity of the electric discharge capacitor, growth stage, number of successive pulses and composition of electroporation buffer. It was effective for transformation that cells were harvested, washed and resuspended with HSM [7M HEPES(PH 7.4), 272mM sucrose, 1 mM MgCl2] electroporation buffer when cell growth was attained to 1.2 at OD660. A maximum frequency of transformation of 2.40$\times$104 transformants per$\mu$g plasmid DNA was obtained by two succesive Pulses with an initial electric field strength of 12.5kV/cm and with a capacitance of 7.3uF.

• KSBB Journal
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• 제6권3호
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• pp.223-230
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• 1991

Using the shuttle vector pECCGl between Escherichia coli and Corynebacterium glutamicum and C. glutamicum strain JS231 grown in the medium supplemented with penicillin-G, which inhibits the formation of cross-links in the peptidoglycan of bacterial cell wall, various parameters involved in electroporation system including resistance, electric field strength, capacitance, DNA concentration, and cell density were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Using cells grown with 0.3U/ml of penicillin-G and harvested at A600 of 0.7-0.8, transformation efficiencies of 107-l08 transformants/$\mu\textrm{g}$ of DNA with Corynebcctertum glutamicum strain JS231 and wild type ATCC13032 were achieved under conditions of 12.5kV/cm of electric field strength, 400 ohms of resistance, $25\mu$F of capacitance, 3$\times$108 cells per transformation(1.2$\times$1010 cells/ml) and 100ng of plasmid DNA per transformation.

• 식물조직배양학회지
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• 제27권5호
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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.

• Mycobiology
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• 제29권3호
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.

• 한국어류학회지
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• 제19권2호
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• pp.83-87
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• 2007

어류 에드워드감염증에 대한 예방 재조합 ghost 백신을 개발하기 위한 연구의 일환으로 어류 병원성 세균인 Edwardsiella trada를 대상으로 플라스미드 형질전환을 실시하고 형질전환 안정성을 평가하였으며, 형질 도입된 재조합 ghost 세균의 E-lysis 유전자 발현을 분석하였다. E. tarda를 대상으로 한 ghost 유도는 대장균에 비해 상대적으로 장시간의 반응 시간이 요구되며 lysis의 개시가 지연된 점을 고려 시 발현된 E protein의 용해 능력 또는 E-gene의 전사발현 양이 E. tarda에서 다소 약화되는 것으로 나타났다. 그러나 대장균에 비해 ghost 유도 속도가 다소 낮음에도 불구하고 반응이 완성되었을 시점에서의 E. tarda의 ghost 효율은 대장균과 전혀 차이가 없이 99.99% 이상의 유도효율을 나타내었다.

• The Plant Pathology Journal
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• 제38권3호
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• pp.239-247
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• 2022

Bacterial wilt caused by the pathogen Ralstonia solanacearum is a devastating disease of potato crops. Harmonizing immunity to pathogens and crop yield is a balance between productive, economic, and environmental interests. In this work, the agronomic performance of two events of potato cultivar INIA Iporá expressing the Arabidopsis thaliana EFR gene (Iporá EFR 3 and Iporá EFR 12) previously selected for their high resistance to bacterial wilt was evaluated under pathogen-free conditions. During two cultivation cycles, the evaluated phenotypic characteristics were emergence, beginning of flowering, vigor, growth, leaf morphology, yield, number and size of tubers, analyzed under biosecurity standards. The phenotypic characteristics evaluated did not show differences, except in the morphology of the leaf with a more globose appearance and a shortening of the rachis in the transformation events with respect to untransformed Iporá. The Iporá EFR 3 genotype showed a ~40% yield decrease in reference to untransformed Iporá in the two trials, while Iporá EFR 12 did not differ statistically from untransformed Iporá. Iporá EFR 12 shows performance stability in the absence of the pathogen, compared to the untransformed control, positioning it as an interesting candidate for regions where the presence of the pathogen is endemic and bacterial wilt has a high economic impact.