• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.10a
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• pp.14-14
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• 2010

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed two different kinds of transgenic potato plants (Solanumtuberosum L. cv. Taedong Valley) overexpressing strawberry GalUR and mouse GLoase gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the these genes in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid(AsA) levels in transgenic tubers were determined by high-performance liquid chromatography(HPLC). The over-expression of these genes resulted in 2-4 folds increase in AsA intransgenic potato and the levels of AsA were positively correlated with increased geneactivity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen(MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of these gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. We have also investigated the mechanism of the abiotic stress tolerance upon enhancing the level of the ascorbate in transgenic potato. The transgenic potato plants overexpressing GalUR gene with enhanced accumulation of ascorbate were investigated to analyze the antioxidants activity of enzymes involved in the ascorbate-glutathione cycle and their tolerance mechanism against different abiotic stresses under invitro conditions. Transformed potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed significant increase in the activities of superoxide dismutase(SOD, EC 1.15.1.1), catalase, enzymes of ascorbate-glutathione cycle enzymes such as ascorbate peroxidase(APX, EC 1.11.1.11), dehydroascorbate reductase(DHAR, EC 1.8.5.1), and glutathione reductase(GR, EC 1.8.1.7) as well as the levels of ascorbate, GSH and proline when compared to the untransformed tubers. The increased enzyme activities correlated with their mRNA transcript accumulation in the stressed transgenic tubers. Pronounced differences in redox status were also observed in stressed transgenic potato tubers that showed more tolerance to abiotic stresses when compared to untransformed tubers. From the present study, it is evident that improved to lerance against abiotic stresses in transgenic tubers is due to the increased activity of enzymes involved in the antioxidant system together with enhanced ascorbate accumulated in transformed tubers when compared to untransformed tubers. At moment we also investigating the role of enhanced reduced glutathione level for the maintenance of the methylglyoxal level as it is evident that methylglyoxal is a potent cytotoxic compound produced under the abiotic stress and the maintenance of the methylglyoxal level is important to survive the plant under stress conditions.

• Journal of Applied Biological Chemistry
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• v.58 no.4
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• pp.325-332
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• 2015

Rapeseed (Brassica napus) is now the second largest oilseed crop after soybean. Cold temperature tolerance is an important agronomic trait in winter rapeseed that determines the plant's ability to control below freezing temperatures. To improve cold tolerance of rapeseed plants, an expression vector containing an Barley Ice recrystallization inhibition protein (HvIRIP) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into rapeseed plants. Transgenic expression of HvIRIP was proved by southern- and northern-blot analyses. The level of freezing tolerance of transgenic $T_3$ plants was found to be significantly greater than that of wild-type rapeseed plants by freezing assay. Proline accumulation during cold stress was also highly induced in the transgenic rapeseed plants. The transgenic plants exhibited considerable tolerance against oxidative damage induced by cold stress. Our results indicated that heterologous HvIRIP expression in transgenic rapeseed plants may induce several oxidative-stress responsive genes to protect from cold stress.

• Korean Journal of Environmental Agriculture
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• v.32 no.2
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• pp.148-154
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• 2013

BACKGROUND: Genetically modified(GM) trigonal cactus(Hylocereus trigonus Saff.) contained a coat protein gene of cactus virus X (CVX), which conferred resistance to the virus, phosphinothricin acetyltransferase (bar) gene, which conferred herbicide resistance, and a cauliflower mosaic virus 35S promoter (CaMV 35S). This study was conducted to evaluate the possible impact of GM trigonal cactus cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere of GM and non-GM trigonal cactus cultivation soils. The total numbers of bacteria, and actinomycete in the rhizosphere soils cultivated GM and non-GM trigonal cactus were similar to each other, and there was no significant difference. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM trigonal cactus were Proteobacteria, Uncultured archaeon, and Uncultured bacterium. The denaturing gradient gel electrophoresis (DGGE) profiles show a similar patterns, significant difference was not observed in each other. DNA was isolated from soil cultivated GM and non-GM trigonal cactus, we analyzed the persistence of the inserted gene by PCR. Amplification of the inserted genes was not observed in the soil DNA, which was collected after harvest. CONCLUSION(S): This result suggests that the GM trigonal cactus cultivation does not change significantly the microbial community.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Journal of The Korean Society of Grassland and Forage Science
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• v.19 no.3
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• pp.281-290
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• 1999

The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with 35S promoter of cauliflower mosaic virus (CaMV) and introduced into tobacco plants (Nicotiana tabacum L. cv. Samsun) via Agrobacterium-mediated transformation. As expected, ipt gene was constitutively expressed in all tissues of transgenic plants. Several primary transgenic plants were obtained that expressed different level of transcripts for ipt gene. Three of transgenic plants with different expression level of ipt gene were selected and selfed to obtain homozygous line for further analysis. A number of interesting phenotypic changes such as viviparous leaves, delayed senescence, larger axillary shoots, an abundance of tiny shoots at the apex and a release of lateral buds, were observed in transgenic plants. Chlorophyll content was 1.5- t.o 4-fold higher in transgenic plants as compared with non-transformed plants. These results indicate that the cytokinin synthesized in transgenic plants could improve forage crop yield by delay of leaf senescence and increase of leaf number.

• 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.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.251-257
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• 2011

Rice is the most important crop as a model plant for functional genomics of monocotyledons. Rice is usually transformed using Agrobacterium tumefaciens. However, the transformation efficiency using previous method is still low. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the inoculation and co-cultivation step. We directly inoculated Agrobacterium containing a CIPK15 gene under the control of CaMV 35S promoter and NOS terminator in the pCAM1300 vector into the pre-soaked seeds in N6D media for 24 hours. After 7 days of culture at $25^{\circ}C$, calli were formed on seeds cultured on the co-cultivation medium containing an antioxidant compound (1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (3 mg/L silver nitrate). We obtained 35 and 22 transgenic plants in rice cultivars, Gopumbyeo and Ilpumbyeo, with increase of transformation efficiency by 30.4% and 22.6%, respectively compared to the general transformation method. The new method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

• Korean Journal of Plant Resources
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• v.19 no.5
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• pp.634-639
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• 2006

In order to develop a disease-resistant root stock for the growth of watermelon, an efficient regeneration system of the gourd(Lagenaria leucantha Duch.) inbred line GO701-2 via organogenesis was established in this experiment. Using proximal parts of cotyledon explant excised from germinated seedling in vitro, maximum adventitious shoot formation (39%) was achieved on MS medium where cytokinin (BA) and auxin (IAA) were added at a concentration of 3mg/L and 0.1mg/L, respectively. Roots of the elongated shoots were successfully formed on MS medium without adding any plant growth regulators. The cucumber CsGolS1 gene known as a resistance gene against biotic and abiotic stresses, was constructed into the binary vector pBI121 under the control of CaMV 35S promoter. When the gene was introduced into the genome of gourd by Agrobacterium-mediated transformation, putative transgenic plants were obtained with the transformation efficiency of approximately 20 percent.

• 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.

• 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.