• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.41-45
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• 2008

Laccase (EC 1.10.3.2) is a small group of enzymes that catalyze the oxidation of a broad range of phenolic compounds including hazardous and recalcitrant pollutants in the environment. This study attempted to develop an efficient system for production of a recombinant laccase by chloroplast genetic transformation of tobacco. Chloroplast transformation vector was constructed and introduced into the tobacco chloroplast genome using particle bombardment. Chloroplast-transformed plants were subsequently regenerated. PCR and southern blot analyses confirmed stable integration of the laccase gene into the chloroplast genome. Northern blot analysis revealed that mRNA of the laccase gene was highly expressed in chloroplast-transformed plants.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.71-80
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• 2005

Effects of low temperature ($8^{\circ}C$) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber; Cucumis sativus L.) and a chilling-resistant (figleaf gourd; Cucurbita ficifolia Bouche) crop have been measured at the levels of whole root systems (root hydraulic conductivity, $Lp_r$) and of individual cortical cells (cell hydraulic conductivity, Lp). In figleaf gourd, there was a reduction only in hydrostatic $Lp_r$ but not in osmotic $Lp_r$ suggesting that the activity of water channels was not much affected by low root temperature (LRT)treatment in this species. Changes in cell Lp in response to chilling and recovery were similar asroot level, although they were more intense at the root level. Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (both at the root and cell level) often resulted in Lp and $Lp_r$ values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger forosmotic (representing the cell-to-cell passage of water) than for hydrostatic $Lp_r$. After a short term (1 d) exposure to $8\;^{\circ}C$ followed by 1 d at $20\;^{\circ}C$, hydrostatic $Lp_r$ of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. On the contrary, osmotic $Lp_r$ and cell Lp in both species remained high by a factor of 3 as compared to the control, possibly due to an increased activity of water channels. After pre-conditioning of roots at LRT, increased hydraulic conductivitywas completely inhibited by $HgCl_2$ at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins and alterations of root anatomy determine the water uptake in both species. To better understand the aquaporin function in plants under various stress conditions, we examined the transgenic Arabidopsisand tobacco plants that constitutively overexpress ArabidopsisPIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates were found between the transgenic and wild-type plants under favorable growth conditions. By contrast, overexpression of PIP1;4 or PIP2;5 had a negative effect on seed germination and seedling growth under drought stress, whereas it had a positive effect under cold stress and no effect under salt stress. Measurement of water transport by cell pressure probe revealed that these observed phenotypes under different stress conditions were closely correlated with the ability of water transport by each aquaporin in the transgenic plants. Together, our results demonstrate that PIP-type aquaporins play roles in seed germination, seedling growth, and stress response of Arabidopsis and tobacco plants under various stress conditions, and emphasize the importance of a single aquaporin-mediated water transport in these cellular processes.

• 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.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.

• Plant Biotechnology Reports
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• v.2 no.3
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• pp.199-206
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• 2008

We have developed an efficient system of assessing the ability of a gene silencing cassette to silence transcripts from recalcitrant or poorly studied plant species by using a model plant as a host for the gene of interest. Tobacco plants transgenic for Lachrymatory Factor Synthase (LFS) enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. In this work, we have demonstrated that the RNAi construct is a suitable candidate for the development of a non-lachrymatory onion. Our model plant RNAi system has wide-reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in the pharmacological, food and process industries.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.256-262
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• 2003

To enhance the production of heavy chain immunoglobulin G (HC IgG) in the suspension cultures of transgenic tobacco cells (Nicotiana tabacum), the effects of adding various cytokinins (i.e., growth regulators) and organic nitrogen sources to culture media were investigated. Four different cytokinins including kinetin, isopentenyladenine (IPA), 6-benzylaminepurine (BA), and zeatin were tested with or without dichlorophenoxyacetic acid (2,4-D), which is a typical growth regulator supplemented in the standard Murashige and Skoog (MS) medium. The productivity of intracellular HC IgG was increased by 36 and $42\%$, compared to the control, especially when IPA (2 mg/l) or BA (0.2 mg/l) was added to the media in the presence of 2,4-D, respectively. In the study of organic nitrogen sources, addition of each casein hydrolysate and tryptone to the culture media at a final concentration of 0.01 and 1 g/l, respectively. increased the productivity or he IgG as much as 68 and $67\%$, respectively, in comparison with the control, which was is MS medium without supplementation of any organic nitrogen sources. This study shows that the optimization of media composition could offer significant improvements in the production of foreign proteins in the suspension cultures of transgenic plants.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.135-140
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• 2013

A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (mCry1Ac) in the transgenic Bt rice plants which were made via conventional Agrobacterium-mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.121.2-122
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• 2003

Since the demonstration that the transgenic plants expressing tobacco mosaic virus(TMV) coat protein(CP) gene showed resistance to TMV infection, there have been numerous attempts to produce virus-resistant plant by introducing of a part of or modified viral genome. This study was conducted to investigate the characterization and variability of disease outbreak of transgenic potato(T-potato) with the CP gene of potato leaf roll virus(PLRV) in an isolated field from 2000 to 2002. In the field inspection, incidence of PLRV on T-potato showed only 3.5％, while non-transgenic potato(N-potato) revealed 13.4％. Infection rate of PLRV was considerably low on T-potato with 4.2％ compared to 15.4％ of N-potato in ELISA tests. Those of potato virus M, potato virus Y and potato virus X on both potatoes were not statistically different. Infection of potato virus A was not observed on both potatoes. Incidence of potato late blight caused by Phytopkhora infestans on T-potato and N-potato did not differ each other with 52.7％, and 50.8％, respectively, Mating type of the causal fungus isolated from both potatoes was all Al types. Results indicates that the CP gene of PLRV affects specifically to the virus in the transgenic potato.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• Journal of Plant Biology
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• v.36 no.3
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• pp.267-273
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• 1993

Promoters of the chlorophyll a/b bidning protein genes, cab1, and cab2, of Arabidopsis thaliana were studied for their functions in differential expression during greening of etiolated shoots. The etiolated shoots were derived from leaves of transgenic tobacco plants with the cab-CAT (chloramphenicol acetyltransferase) translational fusions, and CAT activity was measured to monitor the activities of the cab promoters. Cab1 promoter activity increased rapidly and showed saturation after about 24 hours of greening, but that of cab2 increased with about 2 day-lag period and showed saturation after 6 days. Cab1 promoter activity was more sensitive to levulinic acid (LA) compared with cab2 activity. Cab2 promoter activity was inhibited more sensitively by chloramphynicol (CAP) than by inhibitors of Chl formation. Cab1 promoter activity was, however, inhibited less sensitively by CAP than by LA. The treatment of abscisic acid (ABA) did not block Chl synthesis so significantly as LA treatment did, and cab2 promoter activity was much less sensitive to ABA compared with that of cab1. These results suggest that cab1 expression is strongly related with Chl formation, possibly with $\delta$-aminolevulinic acid accumulation, and cab2 expression is suppressed more by the blockage of translation of Chl a-apoproteins than by the blockage of Chl a accumulation.