• Korean Journal of Plant Tissue Culture
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• v.21 no.2
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• pp.99-103
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• 1994

The herbicide bialaphos is a potent inhibitor of glutamine synthetase in higher plants. A bialaphos resistance (bar) gene encoding for an acetyltransferase was isolated from genomic DNA of Pseudomonas syringae pv tabaci. The bar gene was ligated to the binary vector pBI121. Pistils of tobacco plane were heated with the bar gene containing plasmid DNA at various times after pollination. When the treatment was applied at 30 and 40 h after pollination, a number of transgenic plants were obtained. Premary transformation (T$_{0}$ generation) and their progenies (T$_1$T$_2$) were resistant to both bialaphos and kanamycin at a dosage lathal to untransformed control plants. Stable integration of bar gene into chromosomal DNA was proven by Southern blot analysis of genomic DNA isolated from T$_1$progenies. These results show that the bialaphos resistant plane could be obtained by treatment to pistils with the exgenous bar gene through the fertilization cycle of tobacco.o.

• Korean Journal of Plant Tissue Culture
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• v.22 no.2
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• pp.95-99
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• 1995

We have previously established a system for plant regeneration through somatic embryogenesis and Agrobacterium-mediated transformation of Korean ginseng. In this study to produce a fungus-resistant plant, we introduced a bean chitinase gene into ginseng using the transformation system. A binary vector pChi/748 was constructed by introducing the bean basic chitinase gene into EcoRI site of pGA748 which carries the CaMV 35S promoter governing the introduced gene and neomycin phosphotransferase II(NPT-II)gene as a positive selection marker. Cotyledonary explants were cocultured with A. tumefaciens strain LBA4404 harboring the binary vertor pChi/748 for 48 h, and transferred to MS medium supplemented with l mg/L2,4-D,0.1mg/L kinetin, 100 mg/L kanamycin, and 500mg/L carbenicillin. Kanamycin-resistant calli were formed on the cut surface of cotyledonary explants after one month of culture, and subsequently they gave rise to somatic embryos. Upon transfer onto medium containing 1 mg/L each of BA and GA$_3$, most of them converted to plantlets after 5 weeks of culture. The genomic DNA of eight kanamycin-resistant regenerants was subjected to polymerase chain reaction (PCR) using two specific 21-mer oligonucleotides derived from the chitinase gene. PCR-Southern blot analysis confirmed that the chitinase gene was incorporated into six out of the eight regenerants..

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.6
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• pp.826-832
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• 2023

Clostridium perfringens causes diarrhea and other diseases in humans and animals. We investigated the prevalence, toxin gene profiles, and antimicrobial resistance of C. perfringens isolated from commercial jeotgal sample. C. perfringens was isolated from 11 of 22 commercial jeotgals. All C. perfringens strains were positive for the alpha toxin gene, but not for the beta, epsilon, iota, CPE or NetB toxin genes; therefore, all strains were identified as type A C. perfringens. However, the beta2 toxin gene was identified in 54.5% of isolates. Disk diffusion susceptibility tests showed that most isolates were resistant to kanamycin (90.9%), nalidixic acid (72.7%), oxacillin (54.5%), erythromycin (27.3%), ciprofloxacin (9.1%) and clindamycin (9.1%). However, all strains were susceptible to 14 other antimicrobial including amoxicillin, ampicillin, and chloramphenicol. The average minimum inhibitory concentrations against C. perfringens of clindamycin, kanamycin, and nalidixic acid were 128.0, 128.0, and 54.0 ㎍/mL, respectively. These results provide new insight into the necessity for sanitation of commercial jeotgal, and provide evidence to help reduce the risk of contamination with antimicrobial-resistant bacteria.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.267-271
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• 2004

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron $\beta$-glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.327-337
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• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.163-169
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• 1999

This study was conducted to produce herbicide resistant plants by transferring phosphinothricin acetyltransferase (PAT) gene into Populus alba $\times$ Populus glandulosa No .3 using Agrobacterium tumefaciens MP 90/PAT. Leaf segments from in vitro grown shoots of hybrid poplar No. 3 were soaked in a AB medium containing Agrobacterium tumefaciens MP 90/PAT for 10 min and cocultivated for 2 days on MS medium containing 1.0 mg/L 2,4-D and 0.2mg/L kinetin (CIM). Putative transformed calli could be selected after cocultivation of leaf segments on CIM supplemented with 50mg/L kanamycin and 500mg/L cefotaxime for 3 weeks. The selected calli were cultured on CIM supplemented with 50 mg/L kanamycin and 500 mg/L cefotaxime for 5~8 weeks before transfer to WPM containing 1.0mg/L zeatin, 0.1mg/L BAP, 50 mg/L kanamycin and 500mg/L cefotaxime for shoot regeneration. Shoots were regenerated from the callus after 4 week cultivation, and the regenerants were grown on the same medium for 7~l0 weeks. The plants rooted on 1/2 WPM containing 0.2 mg/L IBA and 50 mg/L kanamycin. To confirm the gene insertion into plants, GUS activity was detected by histochemical assay in the transformed plants. Finally, the presence of both NPT II and PAT genes from the transgenic plants were confirmed by PCR amplification with the gene specific primers and subsequent PCR-Southern blot with DIG-labeled PAT gene probe. After acclimatization in pots for 4 weeks, the plants were sprayed by 3 mL/L of Basta to test resistance to the herbicide. The transgenic plants remained green, whereas all the control plants died after one week.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Applied Biological Chemistry
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• v.37 no.4
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• pp.248-254
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• 1994

Bromoxynil is an antidicot herbicide widely used on cereal crops and has a short half life in the soil. A bxn gene, encoding a specific nitrilase that converts bromoxynil to its primary metabolite 3,5-dibromo-4-hydroxybenzoic acid, was inserted in plant binary vector pGA482, and then introduced into tobacco and lettuce plants via Agrobacterium mediated leaf-disc transformation method. Transgenic plants with the bxn gene were selected by kanamycin and regenerated to whole plants. The regenerated transgenic plants were determined level of expression of bxn gene by Northern blot analysis. Leaf-disc analysis and pot-assay confirmed that the transgenic tobacco and lettuce plants were resistant to high doses of bromoxynil.

• Korean Journal of Veterinary Service
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• v.39 no.3
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• pp.175-181
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• 2016

Staphylococcus pseudintermedius is an important opportunistic pathogen of dog and cats. Since 2006 there has been a significant emergence of methicillin-resistant S. pseudintermedius (MRSP) mainly due to clonal spread. The aim of this study was to investigated the prevalence of antibiotic resistance and presence of mecA and femA gene in 91 S. pseudintermedius isolates isolated from dogs and cats associated with various clinic infections. Methicillin resistance was confirmed by oxacillin disc diffusion method. MRSP isolate was detected 19 isolates (20.9%). MRSP and methicillin-resistant S. pseudintermedius (MSSP) isolates were highly resistant to penicillin, kanamycin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, clindamycin, ciprofloxacin, enrofloxacin and choloramphenicol (100~47.3% and 90.3~33.3%, respectively). About 90% of MRSP isolates were multi-drug resistance (resistance to at least five or more antimicrobials), and MSSP isolates was ca 74%. Among the 91 isolates, mecA gene was detected in 25 isolates (27.5%, 19 in MRSP isolates and 6 in MSSP isolates), but none carried the femA gene. Our results indicated MRSA isolates show a strong resistance to antimicrobials commonly used in veterinary medicine. A continuous surveillance and monitoring should be called for to prevent the contamination and spread of MRSP in dogs and cats.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.