• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.13-17
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• 2004

To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.673-677
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• 2006

A perfusion culture of transgenic Nicotiana tabacum cell suspensions, transformed to express recombinant glucuronidase (GUS), was successfully performed in a 5-1 stirred tank bioreactor. With 0.1 $day^{-1}$ of perfusion rate, the maximum dry cell weight (DCW) reached to 29.5 g/l in 16 days, which was 2.1-fold higher than the obtained in batch culture (14.3 g/l). In terms of the production of GUS, the volumetric activity could be increased up to 12.8 U/ml by using perfusion, compared with 4.9 U/ml in batch culture. The specific GUS activities in both perfusion and batch cultures were maintained at similar levels, 200-400 U/g DCW. Consequently, a perfusion culture could be a good strategy for the enhanced production of recombinant proteins in a plant cell culture system.

• Journal of Korean Society of Forest Science
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• v.86 no.3
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• pp.261-269
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• 1997

Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.267-271
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• 2004

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron $\beta$-glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

• Korean Journal of Plant Tissue Culture
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• v.21 no.1
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• pp.55-58
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• 1994

Agrobacterium tumefaciens LABA4404 harboring plant binary vector, pBI121, was used for genetic transformation of lettuce (Lactuca sativa t.). Cotyledon segments were infected with A. tumefaciens LBA4404 by cocultivation method and regenerated. Regenerated letture was subject to molecular analyses for integration into plant nuclear genome and expression of ${\beta}$-glucumnidase (GUS) gene. Southern and Northern blot analyses demonstrated that GUS gene was integrated into plant nuclear genome and expressed into its mRNA. The expression of GUS gene into its protein was confirmed by specetrophotometric assay of GUS activity.

• Journal of Plant Biology
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• v.37 no.1
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• pp.17-25
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• 1994

Two potato (Solanum tuberosum L.) cultivars were transformed by Agrobacterium tumefaciens harboring cauliflower mosaic virus (CaMV) 35S promoter and $\beta$-glucuronidase (GUS) gene. Expression patterns of the CaMV 35S promoter according to tissue types and developmental stages, and genetic stability of GUS gene were investigated in the clonal progenies of transgenic potatoes. Kanamycin-resistant shoot emerged from tuber disc after 4 weeks of culture, and root was induced 6 weeks after culture on the selection medium. Shooting frequency of cvs. Superior and Dejima were 43% and 27%, respectively. Mature transformants and their clonal progenies showed no phenotypical abnormality. GUS activity was expressed primarily at parenchymatous cells of phloem tissue around the vascular cambium in the stem and root, and higher activity was found at the apical meristem of shoot, root and adventious shoot bud. GUS activity was higher at tubers of young explants than at stored tubers. These facts indicate that expression level of the CaMV 35S promoter differed according to tissue types and developmental stages of the organs. The GUS gene was stably inherited to each clonal progeny and normally expressed.

• Fisheries and Aquatic Sciences
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• v.9 no.4
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• pp.135-139
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• 2006

We optimized the biological and physical parameters for DNA delivery into thalli of the red alga Porphyra yezoensis using a particle bombardment device. The efficiency of transformation was determined using the ${\beta}-glucuronidase$ (GUS) assay. The optimal helium pressure, distance of tungsten particle flight, and ratio of DNA to tungsten particles were $23kgf/cm^2$, 8 cm, and $5{\mu}g/mg$ tungsten, respectively. During bombardment, osmotic treatment with a mixture of 0.6 M mannitol and sorbitol increased the efficiency of GUS transformation. After 2 days, the blue color indicating GUS activity was observed using a histochemical assay.

• 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.

• The Plant Pathology Journal
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• v.21 no.3
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• pp.288-292
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• 2005

Pathogenesis-related protein-1a (PR-1a) is strongly induced in tobacco plants by pathogen attack, exogenous salicylic acid (SA) application and by other developmental processes. In order to develop a rapid screening system for the selection of plant defense-inducing compounds originated from various sources, we have transformed tobacco Samsun NN plants with a chimeric construct consisting of GUS $(\beta-glucuronidase)$. In the $T_1$ generation, three transgenic lines having stable GUS expression were selected for further promoter analysis. Using GUS histochemical assay, we observed strong GUS induction driven by PR-1a promoter in PR1a-GUS transgenic tobacco leaves in response to the exogenous application of SA or benzol (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH), a SA­derivative compound. In addition, GUS expression was maintained locally or systemically in PR1a-GUS transgenic line $\#5\;T_2$ generation) until after 3 days when they were treated with same chemicals. Our results suggested that the PR1a-GUS reporter gene system in tobacco plants may be applicable for the large-scale screening of defense-inducing substances.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.176-181
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• 2005

To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to $\beta-glucuronidase$ (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5'-flanking region of RCaM gene regulates wound-inducible expression.