• Asian Journal of Turfgrass Science
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• v.18 no.3
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• pp.141-148
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• 2004

In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.289-296
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• 2006

In order to establish highly efficient gene transfer condition at early stage of soybean transformation, various experiments were performed and compared their efficiencies by transient GUS analysis; those conditions are genotype determination of Korean soybean cultivars for amenability to Agro-infection, appropriate agar and selective agent concentration, orientation of explant placement, hormone pre-culture, and liquid selection condition. In the genotype screen of Korean soybean varieties, 14 amenable genotypes were selected. For efficient Agrobacterium washing, cefotaxime was chosen and hygromycin at the concentration of 10 and 15 ppm was used as selection agent in the media. Agar concentration was slightly better in 0.6% and 0.8% for both shoot and callus formation, and explant placement with adaxial side down showed high frequency of GUS expression. For wounding treatment, oriental needle was efficient than scalpel for shoot formation and gene transfer. To increase the frequency of gene transfer, hormone pre-treatment was applied. BA at the concentration of 5 and 10 ppm resulted in better survival at the late stage of selection in shoot elongation media. Selection in liquid media after hormone pre-treatment seemed to be effective to remove the escaped non-transformants at early stage of procedure. Considering the results obtained, Eunhakong could be the first choice as a material for soybean transformation among Korean soybean genotypes.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1034-1036
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• 2005

Chinese cabbage (Brassica campestris ssp. napus var. pekinensis Makino) seeds/seedlings were transformed via vacuum-infiltration with recombinant Agrobacterium tumefaciens LBA4404 cells. The agroinfiltration method was determined to be unsuccessful for Chinese cabbage transformation during the analysis of hepatitis B surface antigen expression by ELISA. However, treatment of sodium hypochlorite solution, prior to agroinfiltration, to pregerminated or germinating 1 day- or 2 days-old seeds was proven effectively to enhance transformation efficiency, suggesting that chemical wounding caused by sodium hypochlorite reaction might facilitate Agrobacterium infection and, therefore, transient gene expression in Chinese cabbage sprouts.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.2
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• pp.103-108
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• 2000

To produce of transgenic orchardgrass, the seed-derived calli of orchardgrass (Dactylis glomerata L.) co-cultivated with Agrobacterium turnefaciens EHAlOl harboring binary vector pIG121-Hm were selected with hygromycin and then transferred onto N6 regeneration medium containing 1 rngl l of NAA, 5 rngl l of kinetin, 250 rngl l of carbenicillin and 50 mg/ l of hygromycin. The efficiency of transformation was differed on cultivars, that is, 'Potomac' appeared 12% of transformation efficiency while 'Amba' did 5.5%. The addition of acetosyringone during co-cultivation was a key to successhl transformation of orchardgrass. Transgene fragments were identified by PCR analysis and the constitutive expression of GUS gene was confirmed by Northern blot analysis. (Key words : Acetosyringone, Agrobacterium tumefaciens, Orchardgrass (Dactylis glomerata L.), Transformation)

• Journal of Plant Biotechnology
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• v.5 no.2
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• pp.87-93
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• 2003

Brachiaria (Poaceae) is the most important forage genus for cattle production in Brazil. The genetic breeding of this genus is limited by the incompatibility among species, differences in ploidy level and the natural cloning of plants by apomixis (Valle and Miles 1992). However, plant regeneration via tissue culture methods and genetic engineering provide an opportunity to introduce new characteristics in plants of this genus. We have developed methods for the 'genetic modification of Brachiaria brizantha cv. Marandu via biolistic transformation. A higher number of shoots was obtained with 4 mg/L 2.4-diclorophenoxyacetic acid and 0.2 mg/L benzylaminopurine in calli induction medium and 0.1 mg/L naphtaleneacetic acid and 4.0 mg/L kinetin in shoot regeneration medium. A selection curve for mannose was determined to use phospho mannose isomerase (PMI) gene of Escherichia coli as a selection marker. Calli formation was inhibited from 5 g/L mannose, even in the presence of sucrose while calli that were formed in the presence of mannose failed to develop embryos showing that PMI gene can be used for selection of transformants of this grass. Different promoters were tested to evaluate the efficiency based on the detection of the GUS gene expression (Jefferson et al. 1987). The monocot promoters, act1-D and ubi-1, resulted in higher expression levels than dicot promoters, ubi-3 and act-2, or the CaMV35S and CVMV promoters.

• Molecules and Cells
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• v.25 no.3
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• pp.368-375
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• 2008

Approximately 120 UDP-glycosyltransferases (UGTs), which are classified into 14 distinct groups (A to N), have been annotated in the Arabidopsis genome. UGTs catalyze the transfer of sugars to various acceptor molecules including flavonoids. Previously, UGT71C1 was shown to glycosylate the 3-OH of hydroxycinnamates and flavonoids in vitro. Such secondary metabolites are known to play important roles in plant growth and development. To help define the role of UGT71C1 in planta, we investigated its expression patterns, and isolated and characterized a loss-of-function mutation in the UGT71C1 gene (named ugt71c1-1). Our analyses by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), microarray data mining, and histochemical detection of GUS activity driven by the UGT71C1 promoter region, revealed the tissue-specific expression patterns of UGT71C1 with highest expression in roots. Interestingly, upon treatment with methyl viologen (MV, paraquat), ugt71c1-1 plants displayed enhanced resistance to oxidative stress, and ROS scavenging activity was higher than normal. Metabolite profiling revealed that the levels of two major glycosides of quercetin and kaempferol were reduced in ugt71c1-1 plants. In addition, when exposed to MV-induced oxidative stress, eight representative ROS response genes were expressed at lower levels in ugt71c1-1 plants, indicating that ugt71c1-1 probably has higher non-enzymatic antioxidant activity. Taken together, our results indicate that ugt71c1-1 has increased resistance to oxidative stress, suggesting that UGT71C1 plays a role in some glycosylation pathways affecting secondary metabolites such as flavonoids in response to oxidative stress.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.52-52
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• 2018

Development of herbicide resistant transgenic legume plants through Agrobacterium-mediated transformation has been worked in many previous studied. Plant regeneration after infection is the important step to obtain successful transgenic plants. Many attempts try to find the optimum media condition for plant regeneration after infection. However, the transformation efficiency of legume plants is still low. In this study, regeneration of some Korean legume species including two soybean cultivars (Dawon and Pungsan) and pea have been done with organogenesis which is used various kind of explants such as cotyledonary-nodes in soybean and bud-containing tissue in pea. We developed the optimum media condition for plant regeneration regulators under Agrobacterium-mediated transformation using different kind and various concentration of plant growth. As the results, B5 medium containing 2 mg/L of 6-benzylaminopurine was selected in this study for the optimum plant regeneration media. The segments were inoculated with Agrobacterium suspension harbored an IG2 vector containing bar gene which confers resistance to phosphinotricin (PPT) in 3, 5 and 7 days. The transformation efficiency was achieved in Dawon 3.03 % and pea 1.46 % with co-cultivation period of 7 days which is showed a high number of GUS positive expression period.

• The Plant Pathology Journal
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• v.15 no.6
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• pp.313-318
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• 1999

Cloning of the 5'untranslated region (5' UTR) and Nterminus of the glycoprotein precursor (G2G1) open reading frame of tomato spotted wilt virus has been problematic, possibly because of the toxicity of a signal peptide at the beginning of th G2G1 protein precursor. The toxicity of the signal peptide to bacterial growth and the reason for the expression of the peptide gene in Escherichia coli were investigated by cloning the 5' UTR and the signal peptide sequence separately. Cells transformed with the plasmid containing both the first 30 amino acids of the glycoprotein and the 5' UTR showed a severe growth inhibition whereas transformants harboring either the plasmid with the signal sequence or the 5'UTR alone did not show any ingibition. An E. coli promoter-like sequence was found in the 5'UTR and tis promoter acivity was confirmed with a promoter-less GUS gene cloned downstream of the 5'UTR. In the cloning of the Tomato spotted wilt virus (TSWV) glycoprotein G2G1 open reading frame all the recovered plasmids contained stop codons in the signal sequence region. However, clones containing no stop codon were recovered when the signal sequence and the 5'UTR were cloned separately.

• Plant Resources
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• v.7 no.1
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• pp.39-46
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• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Journal of Life Science
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• v.14 no.4
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• pp.644-649
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• 2004

During plant development, active gibberellins (GAs) control many aspects of plant growth and development including seed germination, stem elongation, flower induction, anther development and seed growth. To understand the biosynthesis and functional role of active GAs in high plants, this study investigated GA 3$\beta$-hydroxylase gene en-coding $GA_1$ and$GA_4$ catalizing last step in GA biosynthetic pathway. The antisense GA 3$\beta$-hydroxylase gene was inserted into expression vector, pIG121-Hm. Calli derived from mature seeds of rice (Oryza satiiva L. cv. Donjinbyeo) were co-cultivated with Agrohacterium tumefaciens EHA101 earring a pIG121-Hm containing hygromycin resistance ($Hyg^r$) and antisense GA 3$\beta$-hydroxylase gene. Seventeen transgenic plants obtained inhibiting GA 3$\beta$-hydroxylase. Transgenic plants had shorter plant height more than that of the Dongjinbyeo. Stable integration of antisense GA 3$\beta$-hydroxylase gene was confirmed by polymerase chain reaction of genomic DNA isolated from the leaf organs of the $T_o$ generation.