• Journal of The Korean Society of Grassland and Forage Science
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• v.21 no.3
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• pp.151-156
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• 2001

This study was conducted to obtain the transformed alfalfa (Medicago sativa L.) plants with thermotolerance gene (BcHSP17.6) using Agrobacterium tumefaciens LBA4404 and we confirmed the transformed gene from the regenerated alfalfa plants. The expression vector, pBKH4, harboring BcHSP17.6 gene was used for production of transgenic alfalfa plants. In a process for transformation, the callus of alfalfa was cocultivated with Agrobacterium tumefaciens and transformed calli were selected on kanamycin-containing SH-3-kc medium to regenerate into into the plant. The complete transgenic alfalfa plants were produced by cultivation for about 4 months on several regeneration media, SH-nk-c, SH-l lb-c, SH-sp-c, and SH-IBA. The transgenic alfalfa plants were analyzed by isolation of genomic DNA and PCR/Southem blot.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• KSBB Journal
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• v.27 no.6
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• pp.341-346
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• 2012

Indican (indoxyl-${\beta}$-D-glucoside) is a colorless natural compound and can be used as a precursor for the production of indigo. This production step only require an enzyme, ${\beta}$-glucosidase, that readily screened from microbial resource by using selective media supplemented with indican as a sole carbon source. Agrobacterium tumefaciens was well grown in this media and thus presumed to produce a related enzyme. The corresponding gene, encoding a protein with a calculated molecular mass of 51 kDa, was cloned and overexpressed as MBP fusion proteins. The purified enzyme was determined to be a dimer and showed the maximum activity for indican at pH 7.0 and $40^{\circ}C$. The kinetic parameters for indican, Km and Vmax, were determined to be 1.4 mM and 373.8 ${\mu}M/min/mg$, respectively. The conversion yield of indican into indigo using this enzyme was about 1.7-1.8 folds higher than that of previously isolated enzyme from Sinorhizobium meliloti. Additionally, this enzyme was able to hydrolyze various ${\beta}$-1,4 glycoside substrates.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.66-75
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• 2016

Agrobacterium-mediated gene transfer has recently been developed to improve rice transformation. In this study, 3 different transformation methods were tested including soaking, co-cultivation, and vacuum infiltration. Agrobacterium tumefaciens GV3101 harboring the binary vector pGreen:: LeGSNOR was used in this experiment. This study aimed to identify the most appropriate method for transferring LeGSNOR into rice. Vacuum infiltration of the embryonic calli for 5 min in Ilpum resulted in high transformation efficiency based on confirmation by PCR, RT-PCR, and qRT-PCR analyses. In conclusion, we described the development of an efficient transformation protocol for the stable integration of foreign genes into rice; furthermore, the study results confirmed that PCR is suitable for efficient detection of the integrated gene. The vacuum infiltration system is a potentially useful tool for future studies focusing on transferring important genes into rice seed calli, and may help reduce time and effort.

• Horticultural Science & Technology
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• v.18 no.6
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• pp.797-801
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• 2000

The present study was conducted to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in hot pepper. Both regeneration ratio and transformation frequency after the cocultivation with A. tumefaciens were affected by inoculation time and artificial wounding. Transformation frequency was increased over 50% by combining artificial wounding with 120 s of inoculation treatment. Confirmation for the transformation of regenerated shoots was carried out by histochemical ${\beta}$-glucuronidase assay and polymerase chain reaction analysis using npt II primer.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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• pp.415-418
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• 2004

The effects of sonication and vacuum infiltration on transformation efficiency was investigated by using immature embryos of Korean wheat as explants. Two Agrobacterium tumefaciens strains, KYRT1 and EHA105, carrying pCAMBIA 1305.1 were used. Transformation efficiency was demonstrated by the detection of $\beta-glucu-ronidase$ (GUS) activity. GUS expression showed clear difference among Korean wheat cultivars. Geurumil showed higher GUS expression efficiency $79.1\%$ compared with other cultivars. The effects of the duration of vacuum infiltration and sonication treatment showed a tendency high GUS expression efficiency by their combination. In comparison with other Agrobacterium strains, KYRT1 showed high efficiency in most Korean cultivars.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1034-1036
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• 2005

Chinese cabbage (Brassica campestris ssp. napus var. pekinensis Makino) seeds/seedlings were transformed via vacuum-infiltration with recombinant Agrobacterium tumefaciens LBA4404 cells. The agroinfiltration method was determined to be unsuccessful for Chinese cabbage transformation during the analysis of hepatitis B surface antigen expression by ELISA. However, treatment of sodium hypochlorite solution, prior to agroinfiltration, to pregerminated or germinating 1 day- or 2 days-old seeds was proven effectively to enhance transformation efficiency, suggesting that chemical wounding caused by sodium hypochlorite reaction might facilitate Agrobacterium infection and, therefore, transient gene expression in Chinese cabbage sprouts.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.115-121
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• 2003

In this study, plant regeneration and Agrobacterium tumefaciens-mediated transformation Kentucky bluegrass(Poa pratensis L.) were evaluated. Three different types of calli were produced depending on the combinations of growth regulators. They were non-friable brown or gray-colored callus (type I), compact, friable and yellow or white-colored callus (typeII), and soft, watery translucent callus with differentiated structure (typeIII). The highest regenerable organogenic callus (typeII) was obtained on the medium containing 1mg/L, 2,4-D and 0.1mg/L BA. Additionally, the production of typeII calli increased significantly when AgNO$_3$ was added to the callus induction and growth medium. The highest frequency of multiple shout formation from typeII callus was obtained on MS medium containing 1mg/L BA and 1mg/L Thidiazuron(TDZ). The organogenic calli(typeII) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pIG121Hm with $\beta$-glucuronidase gene, and various factors were found to influence the transfer-DNA delivery efficiency. The highest transient GUS activity was observed on typeIIcallus. In the present work, we reported the first transient GUS activity of Kentucky bluegrass mediated by Agrobacterium tumefaciens. Our system may contribute to genetic improvement for breed-recalcirtrant grass species, Kentucky bluegrass.

• Journal of Plant Biology
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• v.35 no.2
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• pp.155-163
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• 1992

To elucidate the regulatory mechanism of virE operon in Agrobacterium tumefaciens pTiA6 plasmid at the molecular level, the regulatory site of virE promoter was determined using truncated virE recombinant plasmids obtained by 5' deletion analysis of virE promoter. The size of deleted nucleotides of p]S201, a functional recombinant plasmid, was found to be about 130 nucleotides from 5'-end of virE promoter. On the other hand the size of deleted nucleotides of p]S301, nonfunctional recombinant plasmid, was identified 263 nucleotides by DNA sequencing. Hence it was thought that the essential site of virE promoter was located between about 130th nucleotide and 263th nucleotide. Since the inverted repeat sequence (AACTTTGCGCTATAGGCAMGTT) is included in this essential site of virE promoter, it could be the first recognition site of the RNA polymerase in virE promoter.omoter.

• Applied Biological Chemistry
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• v.28 no.2
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• pp.92-97
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• 1985

UV irradiation method was applied to Agrobacterium tumefaciens A 136 to obtain arginine auxotrophic mutant which is applicable as a host of Ti-plasmid. When the bacterial growth was measured at 600 nm, it showed the exponential phase between 7 and 16 hours after 2% inoculation (v/v) in TY medium and the generation time of 4.8 hours. Survival rate of $1{\sim}0.1%$ was reserved when irradiated at the intensity of $800\;{\mu}w/cm^2$ for $30{\sim}50sec$. Fifteen mutants were selected among 5,000 colonies after UV irradiation. Two of them were identified as arginine auxotrophs, three of them as asparagine auxotrophs, ana the other not as arginine, asparagine, glycine nor cysteine auxotrophs.