• 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)

• Applied Biological Chemistry
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• v.47 no.4
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• pp.430-433
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• 2004

Lilium cv. Casablanca pollen grains stored at $-70^{\circ}C$ were grown in pollen germination medium with Agrobacterium tumefaciens LBA4404 cells harboring pBI121 for 18 hr at $27^{\circ}C$. Following this, cefotaxime (250 mg/L) was treated for 6 hr to eradicate the bacterial cells. Histochemical GUS analysis revealed that the transgenic pollen displayed deep blue color mostly from 12 hr after the co-cultivation. Presence of $200\;{\mu}M$ acetosyringone was determined not to be more effective for GUS transformation than its absence. GUS DNA integration in the transgenic pollen genomic DNA was clearly demonstrated by Southern blot analysis.

• Journal of The Korean Society of Grassland and Forage Science
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• v.21 no.3
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• pp.145-150
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• 2001

This study was conducted to obtain the transformed birdsfoot trefoil (Lotus corniculatus L.) plants with BcHSP17.6 gene using Agrobacterium turnefaciens LBA4404 and we confirmed transformed gene from the regenerated birdsfoot trefoil plants. The expression vector, pBKH4 vector, harboring BcHSP17.6 gene was used for production of transgenic birdsfoot trefoil plants. The callus of birdsfoot trefoil was cocultivated with Agrobacteriurn turnefaciens and transformed calli were selected on kanamycin-containing SH-kc medium to regenerate into plants. The transformed birdsfoot trefoil plants were produced 4 momths after cultivation on BOi2Y medium. The transgenic birdsfoot trefoil plants were analyzed by isolation of genomic DNA and genomic Southern hybridization using a -32P labelled BcHSPl7.6 fragments. (Key words : Birdsfoot trefoil, Transgenic plant. BcHSP17.6 gene, Callus induction, Plant regeneration)

• Korean Journal of Plant Tissue Culture
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• v.25 no.4
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• pp.283-288
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• 1998

The bialaphos is a potent inhibitor of glutamine synthease in higher plants and is used as a non-selective herbicide. We have used the bialaphos resistant gene(Bar) encoding for an acetyltransferase isolated from Streptomyces hygroscopicus SF1293. Callus derived from mature seeds of rice(Oryza sativa L. cv. Dong Jin) were co-cultivated with Agrobacterium tumefaciens EHA101 carring a plasmid pGPTV-HB containing genes for hygromycin resistance (HygR) and Bar. Transgenic plants showing in vitro resistance to 50 mg/L hygromycin and 10 mg/L bialaphos were obtained by using a two-step selection/regeneration procedure. Transformation efficiency of rice was about 30% which was as high as reported in other dicotyledons. Progenies ($\textrm{T}_{1}$ generation) derived from primary transformant of 17 lines were segregated with a 3 resistant : 1 sensitive ratio in medium containing hygromycin and bialaphos. Stable integration of Bar gene into chromosomal DNA was proven by Southern blot analysis of genomic DNA isolated from $\textrm{T}_{2}$ progenies. Transgenic plants ($\textrm{T}_{3}$) grown in the field were resistant to bialaphos (Basta) at a dosage lethal to wild type plants.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.138-147
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• 2024

Agrobacterium-mediated transformation (AMT) is a method that allows for the stable integration of DNA fragments into the plant genome. Transgenic plants generated through AMT typically exhibit a lower copy number of the transgene compared to those induced by particle bombardment. Furthermore, AMT offers a straightforward and efficient approach for generating transgenic plants. While the transformation efficiency of wheat is comparatively lower than that of other monocot plants such as Rice (Oryza sativa L.) and Maize (Zea mays L.), the cultivars 'Bobwhites' and 'Fielder' are commonly employed for wheat transformation. To date, there have been no reported instances of successful development of transgenic plants using Korean wheat varieties through AMT. This study aims to assess the transformation efficiency of 43 Korean wheat cultivars using the GUS assay, with the goal of identifying suitable Korean wheat cultivars for AMT. The pCAMBIA1301 vector, carrying the β-glucuronidase (GUS) gene, was incorporated into Agrobacterium strain EH105. Following the inoculation of Agrobacterium into immature embryos, GUS assays were conducted 'Saeol', 'Jopum', and 'Jonong' showed 100% (the number of embryos showing GUS spots/the number of embryos used for AMT) among 43 cultivars. In addition, cultivars with more than 70% were 'Saekeumgang', 'Jojung', 'Tapdong', 'Anbaek', 'Dabun', 'Sugang', 'Keumgang', 'Jeokjung', 'Seodun', 'Joeun', 'Dajung', and 'Baekjung'. It seems that the 15 cultivars above showed the possibility of using AMT. On the other hand, 'Yeonbaek', 'Goso', 'Baekgang', and 'Johan' showed less than 20% and GUS spots were not observed in 'Gru', 'Gobun', 'Milseong', and 'Shinmichal-1'. This study explores transient GUS expression in Korean wheat cultivars seven days after AMT. The observed initial high efficiency of transient transformation suggests the potential for subsequent stable transformation efficiency. Korean wheat cultivars demonstrating elevated transient transformation efficiency could serve as promising candidates for the development of stable transgenic wheat.

• 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.

• Korean Journal of Agricultural Science
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• v.20 no.2
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• pp.133-144
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• 1993

Calli were induced on microtuber disks of potato(S.tuberosum) infected with three binary vectors transconjugated with C58, A281 and LBA 4404 of Agrobacterium tumefaciens and pBI121. The frequency inducing callus was the highest by infection of C121 carrying pC58 and pBI121, and shoots were differentiated on the calli without any hormonal application. Transformed calli were selected by their resistance to kanamycin and identified by GUS activity. The frequency of callus formation by infection of binary vector strain was affected according to the hormonal application.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.281-287
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• 1999

Codonopsis lanceolata ("Deoduck" in Korea) is a perennial herb, and belongs to family, Campanulaceae. Its taproot is used a good source of a wild vegetable as well as an herbaceous medicine. In this study, to develop a bialaphos-resistant transgenic Codonopsis, seed germination mechanism and somatic embryogenesis of the plant were investigated, and Agrobacterium-mediated transformation with bar gene encoding phosphinothricin acetyltransferase (PAT) was performed. Attempt were made to regenerate plant from cells via somatic embryogenesis. When the cotyledons, nodes and leaf disks were cultured on MS medium containing 2,4-D and zeatin, embryogenic calli were induced. Upon transferring the somatic embryos to N6 solid medium without plant growth regulators, they developed into plantlets under continuous illumination. All plants were dead on MS basal medium containing 10 mg/L phosphinothricin (PPT) and Basta, respectively. The explants did not produce calli in the medium containing 200 mg/L kanamycin. The explants were cocultured with Agrobacterium tumefaciens for 2 days, and transformants were selected in MS basal medium containing 1.0 mg/L 2,4-D, 100 mg/L kanamycin and 500 mg/L carbenicillin. After the selection, embryogenic calli were induced and then somatic embryos were produced by subsequent subculturing. The somatic embryos were germiated on N6 basal medium containing 200 mg/L kanamycin and 500 mg/L carbenicillin. PCR analysis showed that nptII and bar genes were introduced in the Deoduck transformants. After the confirmation of bar gene expression in RNA and protein level, the transgenic Deoduck will be used to study the genetics of filial generation with the herbicide control gene, bar.gene, bar.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.37-41
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• 1997

The in vitro regeneration and genetic transformation systems in hot pepper(Capsicum annuum L.) have not been routinely available, which has been a major limiting factor in the application of new genetic manipulations. An efficient procedure to regenerate whole pepper plants and to generate transgenic plants expressing a foreign gene was established. A relatively high frequency of plant regeneration was observed when hypocotyl and cotyledon explants were cultured on MS medium supplemented with NAA 0.1 mg/L plus zeatin 2.0 mg/L or IBA 10.0 mg/L plus BAP 1.0 mg/L. Addition of AgNO$_3$5 $\mu$M to these media improved the regeneration frequency up to 8%. For plant transformation, hypocotyl and cotyledon explants of hot pepper were precultured on shoot induction media without kanamycin added for 2 days, and then cocultured with Agrobacterium tumefaciens pDY183 for 2 days. Putative transformants were obtained from selection media containing 100 mg/L kanamycin sulfate and 500 mg/L carbenicillin. Putatively selected transformants were confirmed by amplification of selectable marker genes (ADA and NPT II) by polymerase chain reacion. Successful transcripts of ADA gene were detected by Northern blot analysis. Enzyme activity of ADA was also examined by spectrophotometric analysis, and expression of ADA gene in hot pepper suggests the potential application of ADA gene as a selectable marker in plants.

• Korean Journal of Medicinal Crop Science
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• v.13 no.1
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• pp.1-5
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• 2005

While the rusty-colored root is common in ginsengs culture and, often results in a severe economic loss, the major factors have not been found. This study was focused on the determination of a potential relationship between rusty root and endophytic bacteria. The number of endophytes was $9.6\;{\times}\;10^1{\sim}1.5\;{\times}\;10^2\;cfu/g$ fw in normal ginseng roots compared to $3.7\;{\times}\;10^6{\sim}5.1\;{\times}\;10^7\;cfu/g$ fw in rusty ones. Of 31 isolates from rusty ginseng roots, twenty-four isolates repeatedly induced severe to moderate rust on root while seven isolates induced slight rust. The bacteria responsible for rusty ginseng roots were mainly Gram negative aerobic. Rust inducing bacteria were identified as Agrobacterium tumefaciens, A. rhizogenes, Burkholderia phenazinium, Ensifer adharens, Lysobacter gummosus, Microbacterium luteolum, M. oxydans, Pseudomonas marginalis, P. veronii, Pseudomonas sp., Rhizobium leguminosarum, R. tropica, Rhodococcus erythropolis, Rh. globerulus, Variovorax paradoxus on the basis of bacteriological characters and 16S rDNA sequences analysis. The results in this study strongly suggested that the rusty ginseng roots were produced by infection and growth of endophytic bacteria.