• Journal of Plant Biotechnology
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• v.43 no.2
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• pp.240-247
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• 2016

In this study, routinely used transformation method, which includes transferring explants onto co-cultivation medium after inoculating them with bacterial solution for a while, was compared with 3 different inoculation methods. In every 3 methods, hypocotyl explants excised from 7-day-old sterile flax seedlings having cotyledon leaves and no root system dried under air flow in sterile cabin for 35 min were inoculated with different volumes of bacterial solution at different inoculation periods. GV2260 line of Agrobacterium tumefaciens having 'pBIN 19' plasmid containing npt II (neomycin phosphotransferase II) gene and GUS reporter gene was used in transformation studies. After inoculation, hypocotyl segments of seedlings (0.5 cm in length) - were excised and left to co-cultivation for 2 days. Then, explants were transferred to regeneration medium supplemented with different antibiotics. The presence of npt-II and GUS genes in transformants was confirmed by PCR and GUS analysis. The highest results in all characters examined in all cultivars were obtained from the 2 inoculation method in which hypocotyls excised from seedlings inoculated with $500{\mu}l$ of bacterial solution after drying in sterile cabin for 35 min were used.

• Journal of The Korean Society of Grassland and Forage Science
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• v.13 no.3
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• pp.177-183
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• 1993

This study was conducted to obtain the transformed tobacco plants with S. cerevisiae Acid phosphatase gene(PH05) using Agrobacterium tumefaciens and th confirm plant transformation and gene expression. the results obtained were summarized as follows: APase activity of Saccharomyces cereviase NA 87-11A was remarkably showed up as deep red color when assayed by Tohe and Oshima(1974). PH05 fragment, Apase gene, was obtained from pVC727G and the graphically estimated size was about 1.5kb by agarose gel electrophoresis. The sequencing results of 5'end and 3'end of PH05 using dideoxy chain termination method were coinsided with the full length nucleotide already. pBKJ I vector was constructed by isolation of PH05 fragment from pVC727-1 and pBKSI-1 digesred with Sma I and Xba I. Isolated plasmid from transformed A. tumefaciens with constructed pBKJ I when it was electrophoresed with agarose gel. The dosc of tobacco leaf was cocultivated 재소 transformed Agronacterium tumefaciens. Transformed shoots were selected on kanamtcin-containing MS-n/B medium and they were regenerated. The transgenic tobacco plants were elucidated by isolation of genomic DNA and genomic southern hybridization using ${\alpha}-^{32}P$ labelled PH05 fragments. The PH05 in transformed tobacco plants was expressed in leaf, stem and root, and its APase activity was estimated as deep red color by Tohe method.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.37-43
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• 1998

The highest transformation frequency was observed when cotyledonary and hypocotyl explants of flowering cabbage (Brassica oleracea var. acephala DC) 'Eunbae' were cultured on shoot induction medium without kanamycin for 1 day, then cocultured with Agrobacterium tumefaciens LBA4404;;pGA1036 harboring tomato inhibitor II promoter and $\beta$-glucuronidae (GUS) fusion gene for 3 days. These explants were transferred to MS medium containing 20 mg/L kanamycin, 500 mg/L carbenicillin, and 1 mg/L BA. The explants were subsequently subcultured every 2 weeks. Incorporation of the GUS gene into flowering cabbage was confirmed by PCR analysis of DNA. Southern blot analysis showed that ECL-labeled GUS gene was hybridized to the expected amplified genomic DNA fragment of about 366 bp from transgenic flowering cabbage. Histochemical analysis based on the enzymatic activity of the GUS protein indicated that PI-II promoter activity was sysmatically associated with vascular tissue in wonded as well as in non-wounded leaves, petioles and stems, but not in roots. Partial wounding with razor blade showed not systemic induction but partial induction.

• Applied Biological Chemistry
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• v.30 no.4
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• pp.293-299
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• 1987

Bradyrhizobium japonicum spheroplasts were prepared by culturing cells in the presence of glycine, follwed by treatment with lysozyme. The cells were examined by electron microscopy during the formation of spheroplast. Then Ti plasmid from Agrobacterium tumefaciens 15955 was introduced into Bradyrhizobium japonicum by glycine-lysozyme induced spheroplast transformation. After cell wall regeneration, transformants were selected by the ability of utilization of octopine. Transformation were received at a frequency of $1{\times}10^{-7}$. The transformants obtained from spheroplast transformation harbored the introduced Ti plasmid, which was identified by agarose gel electrophoresis. Furthermore, the differences in their gene products were observed between the transformant and the recipient cell by two-dimensional polyacrylamide gel electrophoresis. The transformants which still possessed the same ability nodulate soybean (Glycine max.) as that of the original host strain, acquired the ability to induce tumor on Petunia hybrida like Agrobacterium, but formed the small crown galls in size compared to those of Agrobacterium tumefaciens.

• Korean Journal of Plant Tissue Culture
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• v.25 no.4
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• pp.267-271
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• 1998

Cotyledon explants of lettuce were cocultured with Agrobacterium tumefaciens LBA4404::pBKS-GR1 harboring glutathione reductase(GR) gene in MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 2ip for 48 hr. These explants were transferred to MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 2ip, 50 mg/L kanamycin, and 500 mg/L carbenicillin. After 4 weeks of subculture, kanamycin-resistant shoots were obtained on selection medium. Leaves of putative transformants survived on selection medium containing 100 mg/L kanamycin. Incoporation of the GR gene into lettuce was confirmed by PCR analysis of genomic DNA. Southern blot analysis showed that ECL-labeled GR gene was hybridized to the expected amplified genomic DNA fragment of about 1.8 kb from transgenic lettuce. The presence of mRNA in transgenic lettuce was confirmed by RT-PCR with total RNA of transgenic lettuce. In progeny test of transformants, R$_1$ seeds were resistant to kanamycin (200mg/L) on MS medium.

• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.215-219
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• 2003

This study was conducted to find optimum bacterial density for improving the efficiency of transformation mediated by Agrobacterium tumefaciens in apples. Regeneration(15％) and transformation frequency(10％) were increased in resuspension-culture density $A_{600}$ 1.3 from preculture density $A_{600}$ 0.7 of Agrobacterium tumefaciens in ′Fuji′. In ′Gala′, 20% regeneration and 16% transformation frequency were observed at optimum bacterial density $A_{600}$ 0.7 form preculture density $A_{600}$ 1.3. ′Mclntosh as well as "Gala" were 25％regeneration and 10％ transformation frequency. Hence a frequency optimum condition of bacterial density for the efficient transformation of apple could be depend on apple genotypes.

• BMB Reports
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• v.41 no.6
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• pp.472-478
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• 2008

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.62-72
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• 2001

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 has been successfully cloned and expressed in Escherichia coli. Expression of D-carbamoylase gene under the 17 promoter in different host strains showed that the optimal expression was achieved in E. coli JM109 (DE3) with a 9-fold increase in enzyme production compared to the wild-type strain. The co-expression of the GroEL/ES protein with D-carbamoylase protein caused an in vivo solubilization of D-carbamoylase in an active form. The synergistic effect of GroEL/ES at 28$^{\circ}C$ led to 60 ％ solubilization of the total expressed target protein with a 6.2-fold increase in enzyme activity in comparison to that expressed without GroEL/ES and 43-fold increase in enzyme activity compared to A. tumefaciens AM 10. Attempts to express D-carbamoylase in an altered redox cytoplasmic milieu did not improve the enzyme production in an active form. The Histidyl-tagged D-carbamoylase was purified in a single step by Nickel-affinity chromatography and was found to have a specific activity of 9.5 U/mg protein.

• KSBB Journal
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• v.24 no.1
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• pp.75-79
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• 2009

The effect of inorganic, organic nitrogen sources and amino acids on the coenzyme $Q_{10}$ production and coenzyme $Q_{10}$ component ratio was investigated. Among the nine organic nitrogen sources, CSP showed a remarkable enhancing effect on the production of coenzyme $Q_{10}$. But this enhancement was not observed in medium containing Bacto peptone, tryptone, casamino acid and soybean meal. These differences on the production of coenzyme $Q_{10}$ may be due to differences in kinds and amounts of component amino acids and peptides in the various organic nitrogen sources tested. In the addition of inorganic nitrogens, only $(NH_4)_2SO_4$ increase the coenzyme $Q_{10}$ production by 2.0 times compare to the control group. The addition of L-tyrosine to the medium containing Bacto tryptone, was also determined to be crucial for the coenzyme $Q_{10}$ production. But phenylalanin and tryptophan, other aromatic amino acids, had no stimulatory effect on the coenzyme $Q_{10}$ production. These results show that the production and components ratio of coenzyme $Q_{10}$ was greatly affected by the kinds and the concentration of inorganic, organic nitrogen sources as well as amino acids.