• Plant Biotechnology Reports
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• v.5 no.2
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• pp.157-167
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• 2011

In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacteriummediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of $T_0$, $T_1$ and $T_2$ peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.47 to 1.83% depending on the genotype, which was higher than that obtained by CIT without the GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led to successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for conducting the genetic transformation process of pepper. In addition, a co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues of paprika was monitored to select the right transformant.

• KSBB Journal
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• v.5 no.3
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• pp.247-253
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• 1990

These studies were carried out to obtain the transformant from carrot cells by using binary vector pGA472 with NPT II gene to confer kanamycin resistance in the plant cells. The binary vector pGA472 was mobilized from E. coli MC1000 into A. tumefaciens strains isolated in the Korea, C23-1. K29-1, and disarmed Ti-plasmid PC2760, and A28l using a tri-parental mating method with E. coli HB101/pRK2013. Transconjugants, C23-1/pGA472, K29-1/pGA472, PC2 760/pGA472 and A28l/pGA472 were obtaind on the minimum AB media containing tetracycline and kanamycin, were comfirmed to hold the Ti-plasmid and pGA472 binary vector on the 0.7% agarose gel. Transformed carrot calli were initiated on the MS media supplemented with l00$\mu\textrm{g}$/ml kanamycin and 250$\mu\textrm{g}$/ml carbenicillin after co-cultivation of carrot explant and transconjugant Agrobacteria. Selected callus was grown vigouousley for subculture on the medium containing 100$\mu\textrm{g}$/ml kanamycin, thus indication that the selected callus was transformed with NPT II gene.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.567-573
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• 2010

To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.41-46
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• 2008

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in $T_1$ and $T_2$ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. $T_2$ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.65.2-65
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• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• Korean Journal of Breeding Science
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• v.40 no.3
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• pp.278-285
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• 2008

An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a ${\beta}-glucuronidase$ (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l $N_6-benzylaminopurine$ (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.

• Journal of Korean Society of Forest Science
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• v.87 no.3
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• pp.334-340
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• 1998

This research was conducted to identify plant regulatory regions by gene tagging method. A promoterless GUS coding sequence was introduced to Populus nigra ${\times}$ P. maximowiczii via Agrobacterium strains(LBA4404/EHA101), and putative transgenic poplars were selected by culturing on medium containing G418($60mg/{\ell}$) and by GUS assay. Among them one positive plant was to amplify the native sequences flanking to the introduced GUS gene in plant genome by inverse PCR method and from this 730 by DNA product was obtained. After subcloning and sequencing, it has 88% homology to the Eucalyptus gunnii CAD(cinnamyl alcohol dehydrogenase) gene. The GUS gene fused with the putative promoter reinserted into poplar leaves by particle bombardment method to test the funtional promoter activity. Upon staining with X-gluc, many blue spots appeared on the leaf segments bombarded by the chimeric gene 2-3 days, thus the isolated DNA fragment contain some possible coding region as well as a putative regulatory sequences of poplar CAD gene.

• Korean Journal of Soil Science and Fertilizer
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• v.35 no.2
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• pp.118-126
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• 2002

To evaluate the correlations of microbial populations with soil healthiness and crop production and establish the criteria for microbial population of soil types. We analyzed the microbial community structure of 13 soils which were different in physical and chemical properties and cultivation methods. According to the analysis of microbial population suing the dilution plate method, the large differences of the microbial population structures among soil types were shown: aerobic bacteria $2-27{\times}10^6$, fluorescent Pseudomonas $1-1,364{\times}10^5$, Gram negative bacteria $1-126{\times}10^4$, and mesophilic Bacillus $1-110{\times}10^5$. The density of Gram negative bacteria was highest on red pepper cultivating soils (sample no. 4 and 6) of Umsung and Gesan, Chungbuk, and the density of the fluorescent Pseudomonas was highest on greenhouse soil (sample no. 7) of Jinju, Kyungnam. The crop productivity of three soils was high as compared with those of other soils. It was supposed that the density of fluorescent Pseudomonas and mesophilic Bacillus were correlated with the incresed crop production. By MIDI analysis, 579 strains isolated from 13 soils composed of a variety of microbes including 102 isolates of Agrobacterium, 112 isolates of Bacillus, 32 isolates of Pseudomonas, 44 isolates of Kocuria, and 34 isolates of Pseudomonas. Among the 624 isolates of Gram negative bacteria, Pseudomonas including P. putida and p. fluorescens occupied the highest density (51%), and Stenotrophomonas maltophilia and Burkholderia cepacia also appeared at high density. From RAPD analysis, the fluorescent Pseudomonas strains isolated from 13 soil types showed a high level of strain diversities and were grouped into 2 - 14 patterns according to soil types. Many of unknown bacteria were recovered from the paddy soil, and needed to be further characterized on the molecular basis.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.540-545
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• 2006

T-DNA-mediated transformation is a common method for generating transgenic plants with insertional mutagenesis. In order to identify important genes involved in shoot development, a system of promoter trap insertional mutagenesis was employed in Arabidopsis thaliana. For this system, an efficient promoter trap vector, pFGL561 was developed. The pFGL561 includes a basta-resistant gene, an intron with multiple splicing donor and acceptor sites, and a promoter-less GFP reporter gene. Using floral-dipping method, we made total 300 $T_1$ promoter-trap lines which were screened for GFP expression. GFP signals in the $T_1$ plants were detected with high frequency, 26.7%, and the signals were reconfirmed in $T_2$ plants. To isolate the genes that are involved in shoot development, phenotypes were analyzed in $T_2$ plants of the 19 $T_1$ lines that had GFP signals in shoot apex, and 6 $T_1$ lines were selected that had abnormal shoot development. These lines will be very useful for the investigation of shoot development.

• Journal of Plant Biotechnology
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• v.44 no.1
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• pp.89-96
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• 2017

The CRISPR/Cas9 is a core technology that can result in a paradigm for breeding new varieties. This study describes in detail the sgRNA design, vector construction, and the development of a transgenic plant and its molecular analysis, and demonstrates how gene editing technology through the CRISPR/Cas9 system can be applied easily and accurately. CRISPR/Cas9 facilitates targeted gene editing through RNA-guided DNA cleavage, followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. It also allows the generation of heritable-targeted gene mutations and corrections. Here, we present detailed procedures involved in the CRISPR/Cas9 system to acquire faster, easier and more cost-efficient gene edited transgenic rice. The protocol described here establishes the strategies and steps for the selection of targets, design of sgRNA, vector construction, and analysis of the transgenic lines. The same principles can be used to customize the versatile CRISPR/Cas9 system, for application to other plant species.