• Journal of Plant Biotechnology
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• v.29 no.1
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• pp.45-49
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• 2002

The leaf and petiole segments of Farfugium japonica were cultured to investigate the influence of growth regulators on their callus induction and plant regeneration. The callus induction and growth showed a good response both leaf and petiole on MS media supplemented with 1∼2 mg/L 2,4-D and 1∼2 mg/L BA. Callus induction and growth were more effective in petiole segments than leaf one. The highest percentage of plant regeneration was obtained from 60-day-old calli on MS medium supplemented with 1 mg/L NAA and 2 mg/L BA. When subcultured to the same medium for about 60 days, multiple shoots were developed from regenerating callus. The shoots produced roots after transferring to rooting medium containing 0.5 mg/L IAA. The plantlets over 50 mm in height were successfully acclimatized in vermiculite, and the survival rate was over 95%.

• Korean Journal of Plant Tissue Culture
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• v.28 no.2
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• pp.61-67
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• 2001

Using mature seed-derived callus, optimal conditions for efficient callus growth and plant regeneration, and regeneration efficiency by callus type were investigated in zoysiagrass (Zoysia japonica steud.). Callus induction was highest when the seeds were cultured on MS medium containing 2 mg/L 2,4-D, 0.2 mg/L BAP, 4 mg/L thiamine-HCl and 100 mg/L $\alpha$-ketoglutaric acid. Callus growth was highest when callus were cultured on MS medium containing 0.5 mg/L 2,4-D, 0.05 mg/L BAP, 4 mg/L thiamine-HCl and 100 mg/L $\alpha$-ketoglutaric acid. Plant regeneration was highest when callus was transferred on MS medium containing 3% maltose and 1 mg/L BAP, or 1 mg/L thidiazuron (TDZ). The combinations and concentrations of 2,4-D and BAP were shown to be critical factors for both the frequency and the type of callus. And four morphologically distinct types of callus were induced from the 2,4-D and BAP treatment. Type I,II and III calli produced shoots upon subculture, while the watery callus, type IV produced roots without shoots. Of four types of callus, type I exhibited the maximum frequency (82%) of shoot regeneration and the minimum frequency (4%) of albinism.

• Korean Journal of Plant Tissue Culture
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• v.28 no.2
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• pp.69-74
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• 2001

Calli were induced on MS medium supplemented with 0.5 mg/L 2,4-D by using the leaf explants of haploid which were derived from the diploid and haploid of Nicotiana tabacum cv BY4. These calli were subcultured on MS medium with the combination of 2.0 mg/L 2,4-D, 1.0 mg/L kinetin and 0.1 mg/L BAP. Cell propagation of diploid plants were good in a combination of 2.0 mg/L 2,4-D, 0.1mg/L BAP in vitro conditions, suspension cultures were conducted in equal condition. Homogenized suspension cultured cells were smeared 2.0 mL each on MS medium with 0~100 $\mu$M PFP, to select the resistant colony to PFP, and were examined after 10d, 20d and 30d. Measurment of fresh weight of cells after 30d of culture shows that with more concentration of PFP in medium the fresh weight of the cells decreased. In case of diploid, selected callus was the highest in vitro treated with 5 $\mu$M PFP. It was higher than control until 100 $\mu$M PFP. The active degree of catalase was the highest in vitro with 5 $\mu$M PFP but the lowest in vitro with 10 $\mu$M PFP on the other hand, in case of haploid plant, the active degree of peroxidase and catalase was the highest in vitro treated with 50 $\mu$M PFP. It's sure that enzyme active degree of between diploid and haploid had big differences.

• Journal of Veterinary Clinics
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• v.26 no.6
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• pp.528-533
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• 2009

In the current study, the mesenchymal stem cells (MSCs) isolated and propagated from the human umbilical cord blood (UCB) were tested for their capabilities of differentiation into chondrocytes in vitro. The mesenchymal progenitor cells (MPCs) collected from UCB were cultured in a low glucose DMEM medium with 10% FBS, L-glutamine and antibiotics. The human MSC colonies were positively stained by PAS reaction. When the immunophenotypes of surface antigens on the MSCs were analyzed by fluorescence-activated cell sorter (FACS) analysis, these cells expressed positively MSC-related antigens of CD 29, CD44, CD 90 and CD105, whereas they did not express antigens of CD14, CD31, CD34, CD45, CD133 and HLA-DR. Following induction these MSCs into chondrocytes in the chondrogenic differentiation medium for 3 weeks or more, the cells were stained positively with safranin O. We clearly confirmed that human MSCs were successfully differentiated into chondrocytes by RT-PCR and immunofluorescent stain of type-II collagen protein. These data also indicate that the isolation, proliferation and differentiation of the hUCB-derived MSCs in vitro can be used for elucidating the mechanisms involved in chondrogenesis. Moreover this differentiation technique can be applied to developing cell-based tissue regeneration or repair damaged tissues.

• Journal of Bio-Environment Control
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• v.31 no.2
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• pp.133-141
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• 2022

Bulbophyllum auricomum Lindl. is a rare orchid and has flowers with an attractive fragrance. The present study investigated the tissue culture method for micropropagation. Capsules derived from artificial self-pollination were obtained for the best seed germination in MS basal medium. Plant growth regulators (1.0 mg·L^-1 of BAP and 2.0 mg·L^-1 of NAA) were affected by callus induction from subcultured pseudobulb explants. For the callus subculture, different natural plant extracts were tested in 11 treatment media. Among them, MS medium with 150 mL·L^-1 of coconut water was generally effective in fresh weight (1.75 ± 0.08) and (3.01 ± 0.20) of callus proliferation and PLBs induction at 1 and 2 months, respectively, followed by an MS combination of 30 g·L^-1 of banana and 20 g·L^-1 of potato extract. The results of a comparative study of different MS mediums containing plant growth regulators with a natural extract combination and MS medium supplemented with natural extract only showed that MS medium supplemented with a combination of natural extracts (150 mL·L^-1 of coconut water) and plant growth regulators (2.0 mg·L^-1 of BAP and 1.0 mg·L^-1 of NAA) obtained the highest shoot regeneration (3.37 ± 0.17) and (6.41 ± 0.68) after 1 month and 2 months of culturing, respectively.

• Korean Journal of Veterinary Research
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• v.9 no.1
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• pp.23-62
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• 1969

Throughout the studies the following experimental results were obtained and are summarized: 1. Multiplication of agents in primary cell cultures of both GF classical and CR-64 acute strain of Marek's disease infected chicken kidneys was accompanied by the formation of distinct transformed cell foci. This characteristic nature of cell transformation was passaged regularly by addition of dispersed cell from infected cultures to normal chicken kidney cell cultures, and also transferred was the nature of cell transformation to normal chick-embryo liver and neuroglial cell cultures. No cytopathic changes were noticed in inoculated chick-embryo fibroblast cultures. 2. The same cytopathic effects were noticed in normal kidney cell monolayers after the inoculation of whole blood and huffy coat cells derived from both forms of Marek's disease infected chickens. In these cases, however, the number of transformed cell foci appearing was far less than that of uninoculated monolayers prepared directly from the kidneys of Marek's disease infected chickens. 3. The change in cell culture IS regarded as a specific cell transformation focus induced by an oncogenic virus rather than it plaque in slowly progressing cytopathic effect by non-oncogenic viruses, and it is quite similar to RSV focus in chick-embryo fibroblasts in many respects. 4. The infective agent (cell transformable) were extremely cell-associated and could not be separated in an infective state from cells under the experimental conditions. 5. The focus assay of these agents was valid as shown by the high degree of linear correlation (r=0.97 and 0.99) between the relative infected cell concentration (in inoculum) and the transformed cell foci counted. 6. No differences were observed between the GF classical strain and the CR-64 acute strain of Marek's disease as far as cell culture behavior. 7. Characterization of the isolates by physical and chemical treatments, development of internuclear inclusions in Infected cells, and nucleic acid typing by differential stainings and cytochemical treatments indicated that the natures of these cell transformation agents closely resemble to those described fer the group B herpes viruses. 8. Susceptible chicks inoculated with infected kidney tissue culture cells developed specific lesions of Marek's disease, and in a case of prolonged observation after inoculation (5 weeks) the birds developed clinical symptoms and gross lesions of Marek's disease. Kidney cell cultures prepared from those inoculated birds and sacrificed showed a superior recovery of cell transformation property by formation of distinct foci. 9. Electron microscopic study of infected kidney culture cells (GF agent) by negative staining technique revealed virus particles furnishing the properties of herpes viruses. The particle was measured about $100m{\mu}$ and, so far, no herpes virus envelop has been seen from these preparations. 10. No relationship of both isolates to avian leukosis/sarcoma group viruses and PPLO was observed.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.283-292
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• 2009

Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.

• Journal of Korean Society of Forest Science
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• v.81 no.3
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• pp.273-279
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• 1992

Protoplasts isolated from leaf mesophyll tissues of Populus koreana ${\times}$ P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana ${\times}$ P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of $Ca^{2+}$ in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, $0.45{\mu}M$ 2, 4-D, and $0.5{\mu}M$ BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing $5.0{\mu}M$ zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

• Clinical and Experimental Pediatrics
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• v.50 no.10
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• pp.1011-1017
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• 2007

Purpose : We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. Methods : Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (${\times}1$, ${\times}2$), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. Results : As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). Conclusion : Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.