• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.165-171
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• 2001

New visible and fast assay system have been developed for tobacco transformant introduced with adenosine deaminase (ADA) marker gene, which converts cytotoxic adenosine analogues to non-toxic inosine analogues and ammonia. Ammonia was changed to blue color in the solution of phenol-nitoprusside and alkaline-hypochlorite. It was possible to detect activity of ADA visibly on the holes of 96 well plate using tiny explant of transgenic tobacco leaves within 1 hour incubation time. As substrates of ADA enzyme from transgenic plant on the plate, a number of adenosine analogues such as 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine were possible for detection of ADA activity. Optimal condition of substrate for ADA enzyme was each 10 mM and pH 7.5 in adenosine solution. Especially, transgenic plant did not convert adenosine to inosine and ammonia in the presence of ADA inhibitor deoxycoformycin, which means that ammonia produced from transgenic plant is due to expression of ADA gene. Now, we show that this detection system can be easily, sensitively, fast and cheaply as well as visibly assayed in vitro as GUS gene system with very small size of transformant explant.

• Korean Journal of Plant Resources
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• v.27 no.3
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• pp.242-248
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• 2014

This study was conducted to establish an efficient transformation system by using somatic embryogenesis in an important Korean native conifer, Korean fir (Abies koreana). Embryogenic masses were induced from mature zygotic embryos of the Korean fir on Schenk and Hildebrandt medium, which was supplemented with thidiazuron. For genetic transformation, the embryogenic masses were co-cultivated with a disarmed Agrobacterium tumefaciens strain C58/pMP90 containing the plasmid vector pBIV10 or LBA4404 containing the plasmid vector MP90. Both vectors contain the kanamycin resistance and beta-glucuronidase (GUS) reporter genes. A total of 48 lines of embryogenic masses were selected on mLV medium containing $50{\mu}g/mL$ of kanamycin after 4 weeks of culture, following 3 days of co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 (none of the lines was cultivated with strain LBA4404 carrying MP90). Quantitative real-time PCR was performed, and high levels of GUS transcripts were observed in the 48 putative transgenic lines; however, the control (non-transgenic line) showed negative results. Results of histochemical staining showed that the expression of the GUS reporter gene was observed in somatic embryos that developed from the embryogenic masses of all 48 lines. Stably transformed cultures were successfully produced by co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 in Korean fir. Here, we have reported an Agrobacterium-mediated gene transfer protocol via somatic embryogenesis that may be helpful in developing breeding and conservation strategies for the Korean fir.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.1
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)

• The Plant Pathology Journal
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• v.25 no.4
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• pp.333-343
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• 2009

Here, we show that an endophytic bacterial strain, Enterobacter asburiae B1 exhibits the ability to elicit ISR in cucumber, tobacco and Arabidopsis thaliana. This indicates that strain B1 has a widespread ability to elicit ISR on various host plants. In this study, E. asburiae strain B1 did not show antifungal activity against tested major fungal pathogens, Colletotrichum orbiculare, Botrytis cinerea, Phytophthora capsici, Rhizoctonia solani, and Fusarium oxysporum. Moreover, the siderophore production by E. asburiae strain B1 was observed under in vitro condition. In greenhouse experiments, the root treatment of strain B1 significantly reduced disease severity of cucumber anthracnose caused by fungal pathogen C. orbiculare compared to nontreated control plants. By root treatment of strain B1 more than 50% disease control against anthracnose on cucumber was observed in all greenhouse experiments. Simultaneously, under the greenhouse condition, the soil drench of strain B1 and a chemical inducer benzothiadiazole (BTH) to tobacco plants induced GUS activity which is linked with activation of PR promoter gene. Furthermore, in Arabidopsis thaliana plants the soil drench of strain B1 induced the defense gene expression of PR1 and PDF1.2 related to salicylic acid and jasmonic acid/ethylene signaling pathways, respectively. In this study, for the main focus on root colonization by strain B1 associated with defense responses, bacterial cells of strain B1 was tagged with the gfp gene encoding the green fluorescent protein in order to determine the colonization pattern of strain B1 in cucumber. The gfp-tagged B1 cells were found on root surface and internal colonization in root, stem, and leaf. In addition to this, the scanning electron microscopy observation showed that E. asburiae strain B1 was able to colonized cucumber root surface.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.475-478
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• 2000

The cytosolic glutathione reductase(GR) gene of chinese cabbage was introduced into Lettuce (Lactuca sativa L.) with particle bombardment method. When cotyledon explants were treated with osmoticum-conditioning medium (0.6 M sorbitol/mannitol) 4 hours prior to and 16 hours after bombardment, it was identified by GUS assay that this condition was the most efficient one for introduction of foreign genes into cotyledon tissue of lettuce with particle bombardment. The stable integration of a GR gene was confirmed by the PCR analysis. 0.3, 0.6, 1.5 kbp PCR fragments of transgenes were obtained by three types of primers designed on the basis of GR sequence. To know whether the expression of the GR gene of pBKs-GR 1 can be stably maintained in the next generation, T$_2$selfing seeds obtained from the transformed mother plants were sowed on MS medium supplemented with 200 mg/L kanamycin sulfate. 70% of seedlings showed resistance to kanamycin.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.319-326
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• 2010

An interest in the production of seed-oil based fuel and raw materials, which comes from renewable plant sources, has been intrigued by the phenomenon of global warming and shortage of fossil fuels. Rapeseed (Brassica napus) is the most important oilseed crop, which produces seeds with 40% oil. It is desirable to develop genetically modified rapeseed producing oils, which can be easily converted to biodiesel. As an initial step for development of genetically modified rapeseed for the production of biofuels or bio-based materials, Korean rapeseed cultivars, Naehan, Youngsan, Tammi and Halla, were analyzed. Four Korean rapeseed cultivars produce 32 to 40% oil of seed dry weight, which is rich in oleic acid (more than 60 mole%). The cotyledonary petioles of rapeseed cultivar, Halla, were transformed using Agrobacterium tumefaciens strain GV3101, carrying the uidA gene encoding $\beta$-glucuronidase (GUS) as a reporter gene and the phosphinothricin acetyltransferase (PAT) gene as a selectable marker. The stable integration of PAT gene in the genome of transgenic rapeseeds was confirmed by PCR analysis. Expression of uidA gene in various rapeseed organs was determined by fluorometric assay and histochemical staining. Transformation efficiency of a Korean rapeseed Halla cultivar was 10.4%. Genetic inheritance of transgenes was confirmed in $T_2$ generation.

• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.123-129
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• 2001

Electroporation of microcalli and embryonic axes of a Brazilian Indica rice cultivar was performed. Some parameters influencing the recovery of transformed callus have been defined through transient npt II expression. Such parameters included the presence of light during incubation of microcalli used as target for electroporation, heat shock at 45$^{\circ}C$, macerozyme pre-digestion of target tissues and the number of pulses during electroporation. Transgenic calli were obtained from embryonic axes after electroporation with plasmid pDM302, which encodes the gene phosphinotricin acetyl transferase (bar) under the control of Act-1 promoter. Integration of the introduced gene into the genome was demonstrated by Southern blot hybridization.

• Journal of Life Science
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• v.14 no.4
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• pp.657-663
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• 2004

Tobacco transformants harboring cinnamic acid 4-hydroxylase gene (C4H) fused with susquiterpene cyclase promoter was developed in order to regulate biosynthesis of phenolic compounds by the expression of the introduced gene. Twenty transformants for each specific promoter were used to analyze the incorporation of the chimeric genes by PCR and Southern blot analysis. PCR products of NPTII(neomycin phosphotransferase) gene (553bp) were detected in the transgenic tobacco plants. The incorporation of the chimeric gene was confirmed in the Southern blot analysis. C4H activity in the transgenic plants was elevated by UV-irradiation and its level was higher compared to that of control plants.

• Journal of Plant Biotechnology
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• v.7 no.4
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• pp.219-223
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• 2005

We investigated chemical-wounding effect on Agrobacterium-mediated Chinese cabbage transformation via vacuum infiltration. Pre-germinated or germinating Chinese cabbage seeds were infiltrated with Agrobacterium tumefaciens LBA4404 cells carrying either GUS gene (pBI121) or hepatitis B virus surface antigen DNA (pBIHBsAg). Prior to agroinfiltration process, the seeds were soaked in sodium hydrosulfite (SHS) solution or just in sterile water as a control. Comparative transformation efficiency was determined by both of histochemistry and ELISA. We could demonstrate that SHS solution treatment especially to 1-day or 2-days old germinating seeds efficiently improved transformation process, and therefore, transient expression level. This strongly indicated that Agrobacterium infection could be facilitated indeed by SHS-causing wounds on Chinese cabbage seeds.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.278-284
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• 2011

The CCR4-CAF1-NOT complex-mediated degradation of mRNA is a fundamental aspect of gene regulation in eukaryotes. We herein examined the role of AtCAF1 in the innate immune and wound responses of plants. Our results showed that overexpression of AtCAF1 significantly downregulated the transcript level of EFR but not FLS2 and BRI1, as well as abolished up-regulated expression pattern of EFR in response to wounding. Consistently, Agrobacteriummediated transient expression of GUS was highly enhanced in the transgenic plants overexpressing AtCAF. Furthermore, JA responsive genes were down-regulated by overexpression of AtCAF, causing the transgenic plants overexpressing AtCAF more susceptible to necrotrophic fungal pathogen, Botrytis cinerea. These results suggest that The CCR4-CAF1-NOT complex-mediated degradation of mRNA negatively regulates wounding-mediated disease resistance in Arabidopsis thaliana.