• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.13-18
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• 2003

This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.

• Horticultural Science & Technology
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• v.28 no.3
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• pp.449-455
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• 2010

'Moulinrouge' was selected as the best regenerating cultivar among 18 different spray-type chrysanthemum cultivars bred in the Gyeongnam Flowers Breeding Research Institute. When the leaf explants from standard- and spray-type chrysanthemum 'Jinba' and 'Moulinrouge' were incubated on MS basal medium supplemented with $0.5mg{\cdot}L^{-1}$ BA and $1.0mg{\cdot}L^{-1}$ NAA, both 'Jinba' and 'Moulinrouge' induced adventitious shoots that can be regenerated into plantlets. Based on these regeneration conditions, we developed an efficient $Agrobacterium$-mediated chrysanthemum 'Moulinrouge' transformation method by using sequential selection of shoots from low ($10mg{\cdot}L^{-1}$) to high ($30mg{\cdot}L^{-1}$) concentrations of kanamycin after co-cultivation of leaf explants with $Agrobacterium$ for 10 days and induction of shoots. All kanamycin resistant plants investigated with genomic PCR analysis carried the report gene, $AtSICKLE$, in their genome. Although expression levels of the report gene in the transgenic plants investigated with RT-PCR were relatively low because of inefficiency of CaMV 35S promoter in chrysanthemum, transgenic lines expressing $AtSICKLE$ efficiently showed leaf epinasty phenotype. We expect that our results will provide a useful method that can perform a high-throughput investigation of genes isolated and studied well in model plants for molecular breeding of chrysanthemum.

• The Journal of Korean Society for Radiation Therapy
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• v.26 no.1
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• pp.137-147
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• 2014

Purpose : The purpose of this study is to verify the accuracy of dose delivery according to the patient's breathing cycle in Gated Volumetric Modulated Arc Therapy Materials and Methods : TrueBeam STxTM(Varian Medical System, Palo Alto, CA) was used in this experiment. The Computed tomography(CT) images that were acquired with RANDO Phantom(Alderson Research Laboratories Inc. Stamford. CT, USA), using Computerized treatment planning system(Eclipse 10.0, Varian, USA), were used to create VMAT plans using 10MV FFF with 1500 cGy/fx (case 1, 2, 3) and 220 cGy/fx(case 4, 5, 6) of doserate of 1200 MU/min. The regular respiratory period of 1.5, 2.5, 3.5 and 4.5 sec and the patients respiratory period of 2.2 and 3.5 sec were reproduced with the $QUASAR^{TM}$ Respiratory Motion Phantom(Modus Medical Devices Inc), and it was set up to deliver radiation at the phase mode between the ranges of 30 to 70%. The results were measured at respective respiratory conditions by a 2-Dimensional ion chamber array detector(I'mRT Matrixx, IBA Dosimetry, Germany) and a MultiCube Phantom(IBA Dosimetry, Germany), and the Gamma pass rate(3 mm, 3%) were compared by the IMRT analysis program(OmniPro I'mRT system software Version 1.7b, IBA Dosimetry, Germany) Results : The gamma pass rates of Case 1, 2, 3, 4, 5 and 6 were the results of 100.0, 97.6, 98.1, 96.3, 93.0, 94.8% at a regular respiratory period of 1.5 sec and 98.8, 99.5, 97.5, 99.5, 98.3, 99.6% at 2.5 sec, 99.6, 96.6, 97.5, 99.2, 97.8, 99.1% at 3.5 sec and 99.4, 96.3, 97.2, 99.0, 98.0, 99.3% at 4.5 sec, respectively. When a patient's respiration was reproduced, 97.7, 95.4, 96.2, 98.9, 96.2, 98.4% at average respiratory period of 2.2 sec, and 97.3, 97.5, 96.8, 100.0, 99.3, 99.8% at 3.5 sec, respectively. Conclusion : The experiment showed clinically reliable results of a Gamma pass rate of 95% or more when 2.5 sec or more of a regular breathing period and the patient's breathing were reproduced. While it showed the results of 93.0% and 94.8% at a regular breathing period of 1.5 sec of Case 5 and 6, it could be confirmed that the accurate dose delivery could be possible on the most respiratory conditions because based on the results of 100 patients's respiratory period analysis as no one sustained a respiration of 1.5 sec. But, pretreatment dose verification should be precede because we can't exclude the possibility of error occurrence due to extremely short respiratory period, also a training at the simulation and careful monitoring are necessary for a patient to maintain stable breathing. Consequently, more reliable and accurate treatments can be administered.

• The Journal of Korean Society for Radiation Therapy
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• v.29 no.2
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• pp.43-51
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• 2017

Purpose: The purpose of this study is to evaluate the dosimetric effects of couch attenuation and air gaps using 3D phantom for prone breast radiation therapy. Materials and method: A 3D printer(Builder Extreme 1000) and computed tomography (CT) images of a breast cancer patient were used to manufacture the customized breast phantom. Eclipse External Beam Planning 13.6 (Varian Medical Systems Palo Alto, CA, USA) was used to create the treatment plan with a dose of 200 cGy per fraction with 6 MV energy. The Optically Stimulated Luminescence Detector(OSLD) was used to measure the skin dose at four points (Med 1, Med 2, Lat 1, Lat 2) on the 3D phantom and ion-chamber (FC65-G) were used to perform the in-vivo dosimetry at the two points (Anterior, Posterior). The Skin dose and in-vivo dosimetry were measured with reference air gap (3 cm) and increased air gaps (1, 2, 3, 4, 5, 6 cm) from reference distance between the couch and 3D phantom. Results: As a result, measurement for the skin dose at lateral point showed a similar value within ${\pm}4%$ compared to the plan. While the air gap increased, skin dose at medial 1 was reduced. And it was also reduced over 7 % when the air gap was more than 3 cm compared to radiation therapy plan. At medial 2 it was reduced over 4 % as well. The changes of dose from variety of the air gap showed similar value within ${\pm}1%$ at posterior. As the air gap was increased, the dose at anterior was also increased and it was increased by 1 % from the air gap distance more than 3 cm. Conclusion: Dosimetrical measurement using 3D phantom is very useful to evaluate the dosimetric effects of couch attenuation and air gaps for prone breast radiation therapy. And it is possible to reduce the skin dose and increase the accuracy of the radiation dose delivery by appling the optimized air gap.

• Journal of Plant Biotechnology
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• v.44 no.1
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• pp.82-88
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• 2017

Transgenic lily plants have been obtained after particle bombardment, using PDS-1000/He system and scale explants of lilies, followed by PPT (D-L-phosphinothricin) selection. In this study, scales of the lily plants cv. 'red flame' were bombarded with a plasmid containing the bar gene as a selectable marker, and the AtSIZ gene as a gene of interest, showing salt tolerance and drought tolerance respectively, and both being driven by the CaMV 35S promoter. For optimization of a protocol, factors which optimized and showed a high transformation efficiency under following conditions, were considered: a bombardment pressure of 1100 psi, a target distance of 6 cm and $1.0{\mu}m$ of gold particle, and 24-h pre-culture and post-culture on MS medium containing 0.2 M sorbitol and 0.2 M mannitol as osmoticum agents. After bombardment, all the bombarded scales of lily were transferred to MS medium without selective agents, for a week. Subsequently, these bombarded scales were transferred to a selection MS medium containing 10 mg/l PPT, and incubated for a month for further selection, after which they were cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. After transferring into hormone-free MS medium, the PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets. PCR analysis revealed that the surviving putatively transformed plantlets indicated the presence of both the bar gene and the AtSIZ gene. In conclusion, when 100 scales of lily cv. Red flame are bombarded, this study produced approximately 17-18 transgenic plantlets with an optimized bombardment protocol. The protocol described here can contribute to the breeding program of lilies.

• Horticultural Science & Technology
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• v.18 no.5
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• pp.594-598
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• 2000

This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.13-19
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• 1998

We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.63-69
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• 1997

Erythropoietin (EPO) is a glycoprotein that mediates the growth and differentiation of erythroid progenitors. In order to produce recombinant human erythropoietin in tobacco plant, the EPO genomic DNA (5.4 kb) was cloned into plant expression vectors, pBI$\Delta$GUS121, pBD$\Delta$GUS121 and pPEV-1, and introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated transformation. After selection on MS media containing kanamycin (Km), 10 Km-resistant plants were obtained per each construct. The correct integration of EPO genomic DNA in the genome of transgenic plant was confirmed by polymerase chain reaction (PCR). Northern blot showed that transcripts of 1.8 kb length were produced in leaves of the plants, but there was no difference of mRNA amount according to promoter number and 5'-untranslated sequence (UTS). The proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbit anti-human EPO polyclonal antibody. The expressed protein appeared as smaller band of apparent mass of 30 kDa as compared to the EPO protein from human urine (37 kDa), suggesting that the modification (glycosylation) system in tobacco plant might be different from that of mammalian cells.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.285-293
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• 2004

FMDV is a viral pathogen that caused foot-and-mouth disease in animals. VP1 is a major capsid protein of FMDV. It is known as one of best materials for the FMDV diagnosis and for the development of protein vaccine. In this study, 633 bp of VP1 gene was modified for the expression of VP1 in plant, based on the VP1 DNA sequence from FMDV taiwan O type and from FMDV isolated vietnam. The. deduced DNA fragment was artificially synthesized using the multiple fragment extension with long-nucleotides. A new plant transgenic vector system, pCAMBIA139011 was constructed on the basis of pBI12l and pCAMBIA1390. Using this vector system and GFP gene or modified VP1 gene, each target gene was introduced into Nicotiana tabacum. The insertion of whole target gene was successfully confirmed in each transgenic plant named GFP-A7 and VP1-4, respectively. The expression level of each gene was estimated by RT-PCR and Real-Time PCR using VP1, GFP specific primers.

• The Korean Journal of Mycology
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• v.47 no.4
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• pp.359-371
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• 2019

To optimize the expression and secretion of ferritin protein associated with ion storage in the mushroom, Pleurotus eryngii, a recombinant secretion vector, harboring the ferritin gene, was constructed using a pPEVPR1b vector under the control of the CaMV 35S promoter and signal sequence of pathogen related protein (PR1b). The ferritin gene was isolated from the T-Fer vector following digestion with EcoRI and HindIII. The gene was then introduced into the pPEVPR1b secretion vector, and it was then named pPEVPR1b-Fer. The recombinant vector was transferred into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformants were selected on MCM medium supplemented with kanamycin and its expression was confirmed by SDS-PAGE and western blotting. Expression of ferritin protein was optimized by modifying the culture conditions such as incubation time and temperature in batch and 20 L airlift type fermenter. The optimal conditions for ferritin production were achieved at 25℃ and after incubating for 8 days on MCM medium. The amount of ferritin protein was 2.4 mg/g mycelia, as measured by a quantitative protein assay. However, the signal sequence of PR1b (32 amino acids) seems to be correctly processed by peptidase and ferritin protein may be targeted in the apoplast region of mycelia, and it might not be secreted in the culture medium. The iron binding activity was confirmed by Perls' staining in a 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in P. eryngii mycelia. Mycelium powder containing ferritin was tested as a feed additive in broilers. The addition of ferritin powder stimulated the growth of young broilers and improved their feed efficiency and production index.