• 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)

• Microbiology and Biotechnology Letters
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• v.27 no.4
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• pp.339-343
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• 1999

Pasteurella haemolytica is well known to cause severe pneumonia, consolidation and oedema of the lung, and fibrinous pleurisy under the stress and infection of virus in the cattle. In the course of our screening for antimicrobial agents against P.haemloytica, compound 51086 has been isolated from the fermentation broth of Streptimyces sp. 51086. The compound 51086 was purified by SiO2, Sephadex LH-20 and ODS column chromatographies and HPLC, subsequently. The structure of compound 51086 was determined as hygromycin A by combination of 1H NMR, 13C NMR, HMBC, and ESI-MS. This compound showed significant antibacterial activity against P.haemolytica and P.multocida.

• Asian Journal of Turfgrass Science
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• v.18 no.4
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• pp.211-219
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• 2004

Transgenic zoysiagrass (Zoysia japonica cv. Zenith) plants have been obtained by particle bombardment of embryogenic callus with the plasmid pSMABuba, which contains hygromycin resistance (hpt) and bialaphos resistance (bar) genes. Parameters on DNA delivery efficiency of the particle bombardment were partially optimized using transient expression assay of a chimeric $\beta-glucuronidase$(gusA) gene driven by the CaMV 35S promoter. Stably transfarmed zoysiagrass plants were recovered with a selection scheme using hygromycin. Transgenic zoysiagrass plants were confirmed by PCR analysis with specific primer for bar gene. Expression of the transgene in transformed zoysiagrass plants was demonstrated by Reverse transcriptase (RT)-PCR analysis. All the tested transgenic plants showed herbicide BastaR resistance at the field application rate of $0.1\%-0.3\%$.

• ALGAE
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• v.28 no.4
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• pp.379-387
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• 2013

The purpose of this study was to express bovine lactoferrin N-lobe in Chlorella vulgaris, a green microalga, using the pCAMBIA1304 vector. Chlorella-codon-optimized bovine lactoferrin N-lobe (Lfb-N gene) was cloned in the expression vector pCAMBIA1304, creating the plasmid pCAMLfb-N. pCAMLfb-N was then introduced into C. vulgaris by electro-transformation. Transformants were separated from BG-11 plates containing 20 ${\mu}g\;mL^{-1}$ hygromycin. Polymerase chain reaction was used to screen transformants harboring Lfb-N gene. Finally, total soluble protein was extracted from the transformants, and the expression of Lfb-N protein was detected using western blotting. Using this method, we successfully expressed bovine lactoferrin in C. vulgaris. Therefore, our results suggested that recombinant lactoferrin N-lobe, which has many uses in the biomedical and pharmaceutical industries, can be produced economically.

• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.233-239
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• 2003

We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.603-614
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• 2002

Human gingival fibroblasts have proven to useful as a species specific cell culture system in various system on periodontal disease and regeneration. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary human gingival fibroblasts by the E6 and E7 genes of the Human papilloma virus(HPV) 16. The full length of HPV 16 E6 and E7 was cloned from the pBR322 into BamHl and Sal I of a pBabe vector including hygromycin B resistance. Before pBabeE6/E7 plasmid transfection, peak 8 GFP including G418 resistance was transfected into primary GF to check the transfection efficency. PBabe E6/E7 plasmid was transfected using Lipofectamine plus following manufacter's instruction into primary normal human gingival fibroblasts in 60mm dishes with FBS free DMEM. After 2 days of transfection, the cells were treated with hygromycin for 2 weeks until the transfected control cells died. The resulting hygromycin resistant colonies were pooled, and clonned, and sucessful　transfection was established for immortalized gingival fibroblast cell lines. Immoralized GF cells showed stellate shape, that is similar to that of orange grains, and more rapid growth and higher proliferation than that of primary gingival fibroblasts. This cell lines overcame crisis and could be cultured over 30 subcultured, could be use for three dimentional culture, epithelial-mesenchymal interaction study.

• Journal of Microbiology
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• v.44 no.4
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• pp.383-395
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• 2006

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 $transformants/{\mu}g$ DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 $transformants/l0^7$ spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transform ants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

• The Korean Journal of Mycology
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• v.38 no.1
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• pp.48-53
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• 2010

Agrobacterium-mediated transformation was conducted in order to generate DNA insertional mutants of Flammulina velutipes. Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into gill tissues of Flammulina velutipes strain KACC42777. The transformants resistant on hygromycine ($30\;{\mu}g/ml$) were confirmed by PCR. The targeted insertional sites were amplified by inverse PCR and sequenced. To find the phenotype variation of all generated transformants, bottle cultivation which followed by the standard cultivation protocol were conducted. Color variation was observed on the cultivated fruiting bodies. Furthermore, the transformant pool will be used as a good genetic resources for studying gene function.

• Applied Biological Chemistry
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• v.39 no.1
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• pp.25-31
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• 1996

Transgenic tobacco plants expressing calmodulin derivative($lys{\rightarrow}ile$ 115 calmodulin) and hygromycin resistance genes or hygromycin resistance gene alone(control) were generated by Agrobacterium-mediated DNA transfer. Seeds obtained from the transgenic plants($F_o$) were tested for resistance to hygromycin and the expected 3 : 1 ratio was observed. The expression of calmodulin derivative in the tobacco plants was identified by a combined method of Western blot and Chemiluminescence. The effects of surface sterilizers on the germiation of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacoo plants expressing the calmodulin derivative showed no fungi contamination with normal germination by treating with sterilized water alone or sodium hypochlorite(2% effective chlorine). In contrast, seeds from the control transgenic tobacco plants showed severe contamination with fungi by treating with sterilized water alone and showed no contamination with normal germination by treating with sodium hypochlorite(2% chlorine). The effects of calcium concentration on the germination of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacco plants expressing the calmodulin derivative showed better germination frequency than that of the control transgenic tobacco seeds in the medium containing 30 mM $CaCl_2$. The data raise the possibility that the expression of calmodulin derivative gene in tobacco plants could increase adaptability of the seeds to environmental stresses.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.175-182
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• 1995

Rice is one of the most successful monocot in regenerating fertile and genetically stable transgenic plants. However there is no report of a rice line developed in Korea that can be used for regeneration of fertile and genetically stable transformants. In this paper we first demonstrate that a Korean variety Nakdongbyeo, is suitable to obtain transgenic rice plants. Protoplasts from embryogenic suspension cultures were co-transformed with HPT (hygromycin phosphotransferase) and GUS ($\beta$-glucuronidase) genes in separate plasmids in the presence of PEG (polyethylene glycol). In 5 independent experiment, the average frequency of calli showing hygromycin resistance were 1.73%. Plantlets were regenerated from the Hy $g^{R}$ calli. The average efficiency of plantlet regeneration was apprbximately 27%. Based on the GUS activities of hygromycin resistant calli, ca.35% of the resistant calli carried active GUS genes. The R0 transgenic plantlets were grown to maturity and Rl seeds were obtained. By examining the in siぉ activity of GUS in Rl seeds and seedlings, we confirmed that the GUS transgene driven by a CaMV 35S (cauliflower mosaic virus) promoter showed proper expression patterns. We also confirmed Mendelian segregation of the HPT transgene in the Rl generation.n.