• 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.45 no.1
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• pp.63-70
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• 2018

Brazzein is the smallest sweet protein and was isolated from the fruit pulp of Pentadiplandra brazzeana Baillon, native to tropical Africa. From ancient times, the indigenous people used this fruit in their diet to add sweetness to their daily food. Brazzein is 500 to 2000 times sweeter than sucrose on a weight basis and 9500 times sweeter on a molar basis. This unique property has led to increasing interest in this protein. However, it is expensive and difficult to produce brazzein other than in its native growing conditions which limits its availability for use as a food additive. In this study, we report high production yields of, brazzein protein in transgenic rice plants. An ORF region encoding brazzein and driven by the $2{\times}CaMV\;35S$ promoter was introduced into rice genome (Oryza sativa Japonica) via Agrobacterium-mediated transformation. After transformation, 17 regenerated plant lines were obtained and these transgene-containing plants were confirmed by PCR analysis. In addition, the selected plant lines were analyzed by Taqman PCR and results showed that 9 T0 lines were found to have a single copy out of 17 transgenic plants. Moreover, high and genetically stable expression of brazzein was confirmed by western blot analysis. These results demonstrate that recombinant brazzein was efficiently expressed in transgenic rice plants, and that we have developed a new rice variety with a natural sweetener.

• Journal of Korean Society of Forest Science
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• v.86 no.3
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• pp.261-269
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• 1997

Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.375-380
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• 2007

This study was conducted to develop herbicide-resistance domestic maize plants by introducing the CP4 5-enol-pyruvylshikimate-3-phosphate synthase (CP4 EPSPS) gene using Agrobacterium tumefaciens-mediated immature embryo transformation. Immature embryos of five genotypes (HW1, KL103, HW3, HW4, HW7) were co-cultivated with strains Agrobacterium tumefaciens (strain C58C1) containing the binary vector (pCAMBIA2300) carrying Ubiquitin promoter-CP4 EPSPS gene and Cauliflower mosaic virus 35S (CaMV35S) promoter-nptll gene conferring resistance to paromomycin as a selective agent. The presence and expression of CP4 EPSPS transgene were confirmed by PCR, RT-PCR and Northern blot analysis, respectively. Also, the resistance to glyphosate in the transgenic maize ($T_1$) was analyzed by shikimate accumulation assay. The frequency (%) of paromomycin-resistance callus was 0.37, 0.03, 2.20, 2.37, and 0.81% in pure lines HW1, KL103, HW3, HW4 and HW7, respectively. EPSP transgene sequences were amplified in putative transgenic plants that regenerated from paromomycin-resistance calli of two inbred lines (HW3, HW4). Of them, RT-PCR and Northern blot analyses revealed that the transgene was only expressed in two transgenic events (M266, M104) of HW4 inbred line, and a mild glyphosate resistance of transgenic event (M266) was confirmed by the lower shikimate accumulation in leaf segments. These results demonstrate that transgenic maize with herbicide-resistance traits in Korean genotype can be genetically obtained.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.323-329
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• 2007

Transgene expression was analyzed in tomato plants. Four lines of neomycin phosphotransferase II gene (NPTII) and the trehalose biosynthetic fusion gene (TPSP) transformed $T_0$ plants showed kanamycin resistance on selection medium. However, the analysis of phenotype (kanamycin resistance) and mRNA expression in $T_1$ plants indicated that the expression of the NPTII and TPSP transgenes was down-regulated to an undetectable level in two independent lines 1 and 11. Southern analysis demonstrated that the lines 1 and 11 had multicopies of the transgenes, whereas the typical transgenic lines 2 and 10 had 1 or 2 copies. DNA methylation analysis using methylation sensitive enzyme detected accumulated CpG DNA methylation on TPSP coding region and CaMV35S promoter region in the line 11, but not the typical transgenic line 2. These results suggest that multicopy transgene in plants is attributed to down-regulation of the transgene expression via transcriptional gene silencing.

• Journal of The Korean Society of Grassland and Forage Science
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• v.29 no.4
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• pp.299-306
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• 2009

Oxidative stress is the main limiting factor in crop productivity. To solve global environmental problems using the plant biotechnology, we have developed on the oxidative stress-tolerant transgenic tall fescue plants via Agrobacterium-mediated genetic transformation method. In order to develop transgenic tall fescue (Festuca arundinacea Schreb.) plants with enhanced tolerance to multiple environmental stresses, nucleotide diphosphate kinase gene under the control of CaMV35S promoter were introduced into genome of tall fescue plants. Proteomic analysis revealed that transgenic tall fescue not only accumulated NDP kinase 2 protein in their cells, but also induced several other antioxindative enzyme-related proteins. When leaf discs of transgenic plants were subjected to cold stress, they showed approximately 30% less damage than wild-type plants. In addition, transgenic tall fescue plants showed normal growth when transgenic plants were subjected to $4^{\circ}C$ for 3 days treatments. These results suggest that transgene is important in ROS scavenging by induction of antioxidative proteins, and could improve abiotic stress tolerance in transgenic tall fescue plants.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.305-311
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• 1997

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

• Journal of Life Science
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• v.12 no.1
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• pp.96-105
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• 2002

To investigate expression of the artificial gene encoding a repeated tripeptide lysyl-glutamyl-tryptophan in tobacco plant, the plant binary vector, pART404 has been constructed, which contains the duplicated CaMV 35S promoter, an artificial gene coding for repetitive polymer (Lys-Glu-Trp)$_{64}$, and nopaline synthase (nos) terminator. The recombinant expression vector was introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated trans-formation. The transgenic calli selected by kanamycin containing medium were then regenerated to whole plants. Southern blot analysis indicated that five transgenic plants (No. 1, 7, 9, 43, 45) showed the hybridizing signals at 1.1 kb of the expected size on EcoRI digestion and each of the transgenic plants contained 1 or 3 copies of the artificial gene inserted into its genome. By northern blot analysis, the size of the hybridized total RNA was estimated to be approximately 1.2 kb and the RNA appeared generally to have the integrity. Western blot indicated that the protein was detected at the position of 33 kDa and the expression level of the polypeptide in the transgenic plant (No. 45) was measured to approximately 0.1% of the total protein.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.75-80
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• 2009

In order to develop transgenic sweetpotato plants [Ipomoea batatas (L.) Lam. cv. Yulmi] with enhanced tolerance to oxidative stress, we constructed transformation vectors expressing 2-Cys peroxiredoxin (Prx) gene under the control of the stress-inducible SWPA2 or enhanced 35S promoter (named as SP or EP). Transgenic sweetpotato plants were attempted to generate from embryogenic calli using an Agrobacterium-mediated transformation system. Embryogenic calli gave rise to somatic embryos and then converted into plantlets on MS medium containing 100 mg/L kanamycin. Transgenic plants were regenerated in the same medium. Southern blot analysis confirmed that the Prx gene was inserted into the genome of the plants. To further study we selected the transgenic plant lines with enhanced tolerance against methyl viologen (MV). When sweetpotato leaf discs were subjected to methyl MV at $20{\mu}M$, transgenic plants showed about 40% higher tolerance than non-transgenic or empty vector-transformed plants.

• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.93-97
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• 2006

The calmodulin as a Ca$^{2+}$-binding protein mediates cellular Ca$^{2+}$ signals in response to a wide array of stimuli in higher eukaryotes. Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca$^{2+}$ signals. SCaM-5 is a soybean calmodulin that is involved in plant defense signaling. Here, we constructed a SCaM-5 CDNA under control of CaMV 35S promoter and transformed it into tomato (Lycopersicon esculentum). The constitutive over-expression of SCaM-5 in tomato plants exhibited a high levels of pathogenesis-related (PR) gene expression, and conferred an enhanced resistance to two fungal pathogen (Phytophthora capsici, Fusarium oxysporum), and a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Thus, this results collectively suggest that SCaM-5 plays an important role in plant defense of tomato.