• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.323-327
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• 1998

The flavonoid biosynthetic pathway has been studied as a genetic model system, particularly in Petunia hybrida. In order to study the flavonoid biosynthetic pathway, we constructed a fusion gene system between Cauliflower Mosaic Virus (CaMV) 35S promoter and eggplant flavonoid 3', 5'-hydroxylase in pBI 121 plasmid. An optimal condition for plant regeneration was observed when internode explants were cultured on MS medium supplemented with IAA 0.2 mg/L plus BA 3 mg/L. For plant transformation internode explants of Petunia hybrida were precultured on BM medium supplemented with IAA 0.2 mg/L plus BA 3 mg/L. Putative transgenic plants were selected on medium containing kanamycin 50 mg/L plus cefotaxim 300 mg/L. Putative selected transformants were confirmed by amplification of selectable marker gene (nptII) by polymerase chain reaction (PCR) and Southern hybridization of flavonoid 3',5'-hydroxylase gene.

• Plant Resources
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• v.6 no.2
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• pp.108-113
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• 2003

This study was carried out to obtain basic information for possibility of oral vaccine in carrot using Agrobacteruim -mediated transformation system. The epitope region of porcine epidemic diarrhea virus (PEDV) spike gene which is classified as a member of the Coronaviridae and causes an acute enteritis in pigs was successfully expressed in carrot (Daucus carota) using the Agrobacterium-mediated transformation system. Hypocotyl segments of in vitro germinated plantlets were infected with Agrobacteriun tumefaciens LBA 4404 harboring PEDV spike gene. Embryogenic callus (EC) was induced on MS selection medium with 1 mg/L 2,4-D, 50 mg/L kanamycin and 300 mg/L cefotaxime after 45 days of culture. Subcultured ECs on MS selection medium without 2,4-D were converted to somatic embryos (SE) of various stage; globular, heart and torpedo stage. Putative transgenic embryos were selected on MS medium with 50 mg/L kanamycin and 300 mg/L cefotaxime. Regenerated plantlets from transformed SE were induced on MS medium containing 50 mg/L kanamycin after 30 days of culture. Genomic PCR confirmed the integration of PEDV spike gene into nuclear genome of carrot and northern blot analysis demonstrated the expression of PEDV spike gene in transgenic carrot.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.255-259
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• 2004

Agrobacterium tumefaciens-mediated cotyledonary node transformation was used to produce transgenic soybean. Cotyledonary node explants of three cultivars and one genotype were co-cultivated with strains Agrobacterium (LBA4404, GV3101, EHA101, C58) containing the binary vectors (pCAMBIA3301 and pPTN289) carrying with CaMV 35S promoter-GUS gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selectable marker. There was a significant difference in the transformation frequency depend on bacteria strain. The EHA101 strain of the bacterial strains employed gave the maximum efficiency (3.6%). One hundred-six lines transformed showed the resistance in glufosinate. Histochemical GUS assay showed that at least 11 plants transformed with the GUS gene were positive response. The soybean transformants were obtained from the Thorne (5 plants), 1049 (5 plants) and Bakun (1 plant), respectively. Southern blot analysis and leaf painting assay revealed that the GUS and bar gene segregated and expressed in their progeny.

• 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.

• Journal of Plant Biotechnology
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• v.38 no.3
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• pp.198-202
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• 2011

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

• Journal of the Korean Society of Tobacco Science
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• v.25 no.1
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• pp.12-19
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• 2003

Transgenic tobacco plants were selected by using the transformation of putrescine N-methyltransferase(PMT) gene, the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine. PMT was fused in reverse orientation to the CaMV 35S promoter of the plant expression vector pBTEX(pPAB3) to produce tobacco plants of low nicotine content. To compare nicotine content, only pBTEX vector and PMT gene which was fused in forward orientation to the CaMV 35S promoter(pPAB2) were also transformed to the leaf tobacco plants(Nicotiana tabacum cv. NC82 and N. tabacum cv. Br2l). The presence of sense- and antisense-PMT gene, and pBTEX vector in the transgenic plant was confirmed by genomic PCR.

• KSBB Journal
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• v.6 no.4
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• pp.385-388
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• 1991

Embryogenic cell suspension cultures of B. striata were established as transferred selected embryogenic callus into liquid medium. Protoplasts isolated from embryogenic cell suspensions were electroporated in buffered solutions containing plasmid DNA of pBI121. Transient GUS (beta-glucuronidase) activity measurement and selection for kanamycin resistent showed that expression of foreign genes and stable transformation were achieved. GUS transient gene expression was increased by increasing DNA concentration of pBI121 plasmid and affected by the level of the applied voltage. An optimal level of GUS activity was obtained after electroporation with a pulse of 200-300 voltage/1180 uF. Protoplast viability was up to the 80% at the optimal voltage.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.322-328
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• 2003

The Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5'and 3' gene targeting sequences. In this study, we examined the minimum size of specific hooks required for a single-copy gene isolation and compared the utility of different TAR vectors, radial and unique vectors, by cloning the same single-copy gene. The efficiency of TAR cloning of the hHPRT gene was same using hooks varying from 750 to 63 bp. The number of transformants decreased approximately 20-fold when the TAR vector contained two unique hooks versus using a radial vector, but the percentage of positive recombinants increased over 2-fold when a unique TAR vector was used. Therefore, we suggest that the two-unique TAR vector is suitable for general TAR cloning given its high selectivity, and the radial TAR vector is more suitable when genomic DNA is in limited quantity, for example, DNA isolated from pathological specimens. Moreover, we confirm the minimal length of a unique sequence in a TAR vector is approximately 60 bp for a single-copy gene isolation.

• 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..

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.135-141
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• 2002

Agrobacterium-mediated transformation has been unsuccessful for monocot plants except for a few important crops such as barley, rice, maize and wheat. We discussed here that a successful transformation of monocots demands certain critical conditions. The requirements for an efficient transformation are a selection of target tissues competent for plant regeneration and Agrobacterium-infection, and various factors promoting Agrobacterium-infection. The factors were divided into two to activate Agrobacterium and to increase plant cell's susceptibility against Agrobacterium. Optimization of these factors significantly increased transformation efficiency of zoysia grass and rice plants. A technical improvement in transformation system for monocots will promote improvement of the breed as well as a study of gene functions in monocots.