• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.257-262
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• 2005

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic melon. Cotyledonary-node explants of melon (Cucumis melo L. cv. Super VIP) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing the binary vector (pPTN289) carrying with CaMV 35S promoter-gus gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selective agent, and the binary vector (pPTN290) carrying with Ubiquitin promoter-GUS gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent, respectively. The maximum transformation efficiency (0.12%) was only obtained from the cotyledonary-node explants co-cultivated with EHA101 strain (pPTN289) on selection medium with 5 mg/L glufosinate and not produced a transgenic melon from the cotyledon or cotyledonary-node co-cultivated with other strains. Finally, five plants transformed showed the resistance in glufosinate antibiotic and the GUS positive response in leaf ($T_0$), flower ($T_0$), seeds ($T_1$) and plantlet ($T_1$). Southern blot analysis revealed that the gus gene integrated into each genome of transgenic melon.

• Journal of Applied Biological Chemistry
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• v.54 no.1
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• pp.26-32
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• 2011

To improve genetic transformation of Brassica napus winter cultivar 'Youngsan', factors influencing shoot regeneration and transformation from cotyledonary petioles were investigated. Shoot induction was enhanced in the combination of 0.5 mg/L NAA and 2~4 mg/L kinetin. Silver nitrate was essential for successful shoot regeneration, ranging from 5 to 9 mg/L. The addition of $GA_3$ promoted plant regeneration. Among the tested Agrobacterium strains, co-cultivation times, and antibiotic selection regimes, choice of appropriate Agrobacterium strain was the most critical factor for efficient transformation of B. napus cv. 'Youngsan'. The EHA105 succinamopine strain was the most efficient and the maximum transformation efficiency was 26.8%. Transgenic shoots were selected on 10 mg/L phosphinothricin (PPT) containing media. The transgenic plants expressing bar and gus genes were resistant for commercial herbicide "Basta" and stained with X-Gluc. Southern blot hybridization indicated that the presence of one to three gus gene copies per genome and inheritance of the gus gene into the $T_1$ generation.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.75-80
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• 2009

In order to develop transgenic sweetpotato plants [Ipomoea batatas (L.) Lam. cv. Yulmi] with enhanced tolerance to oxidative stress, we constructed transformation vectors expressing 2-Cys peroxiredoxin (Prx) gene under the control of the stress-inducible SWPA2 or enhanced 35S promoter (named as SP or EP). Transgenic sweetpotato plants were attempted to generate from embryogenic calli using an Agrobacterium-mediated transformation system. Embryogenic calli gave rise to somatic embryos and then converted into plantlets on MS medium containing 100 mg/L kanamycin. Transgenic plants were regenerated in the same medium. Southern blot analysis confirmed that the Prx gene was inserted into the genome of the plants. To further study we selected the transgenic plant lines with enhanced tolerance against methyl viologen (MV). When sweetpotato leaf discs were subjected to methyl MV at $20{\mu}M$, transgenic plants showed about 40% higher tolerance than non-transgenic or empty vector-transformed plants.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.69-74
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• 2008

This study aims to establish the efficient soybean transformation system and develop soybean [Glycine max (L.) Merill] transformants using cotyledonary node explants. The cotyledonary node of soybean were co-cultivated with Agrobacterium tumefaciens strains (KYRT1, EHA105). These strains contain the binary vector pCAMBIA3301 which carries a herbicide-resistant far gene. Korean cultivars (Danbaekkong, Eunhakong) and foreign cultivars (Jack, Peking) were the most efficient in regenerating cotyledonary node. Therefore, they were chosen for the transformation. Results showed that the T-DNA transfer reached up to 60% and transformation efficiency reached up to 3% in the cotyledonary node explants from Jack cultivar, co-cultivated with EHA105 strain. Histochemical GUS evaluation showed that 12 individual lines, transformed with the 현 gene, have positive response. The transformed soybeans have been confirmed in the $T_0$ generation through phenotypic assay using herbicide $Basta^{(R)}$ and Southern blot analysis.

• Korean Journal of Plant Tissue Culture
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• v.22 no.2
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• pp.95-99
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• 1995

We have previously established a system for plant regeneration through somatic embryogenesis and Agrobacterium-mediated transformation of Korean ginseng. In this study to produce a fungus-resistant plant, we introduced a bean chitinase gene into ginseng using the transformation system. A binary vector pChi/748 was constructed by introducing the bean basic chitinase gene into EcoRI site of pGA748 which carries the CaMV 35S promoter governing the introduced gene and neomycin phosphotransferase II(NPT-II)gene as a positive selection marker. Cotyledonary explants were cocultured with A. tumefaciens strain LBA4404 harboring the binary vertor pChi/748 for 48 h, and transferred to MS medium supplemented with l mg/L2,4-D,0.1mg/L kinetin, 100 mg/L kanamycin, and 500mg/L carbenicillin. Kanamycin-resistant calli were formed on the cut surface of cotyledonary explants after one month of culture, and subsequently they gave rise to somatic embryos. Upon transfer onto medium containing 1 mg/L each of BA and GA$_3$, most of them converted to plantlets after 5 weeks of culture. The genomic DNA of eight kanamycin-resistant regenerants was subjected to polymerase chain reaction (PCR) using two specific 21-mer oligonucleotides derived from the chitinase gene. PCR-Southern blot analysis confirmed that the chitinase gene was incorporated into six out of the eight regenerants..

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.329-334
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• 1995

Transformation system of rolC gene, dwarf gene in Lycium chinenese Mill. established by using system. Pin-punctured leaves induced numerous adventious buds in abaxial side when cultured on 3/2 MS medium containing 2.0 mg/L zeatin. Survival rate and shoot regeneration frequency of leaf explants decreased as kanamycin sulfate level increased. Shoot buds were not regenerated on 3/2 MS medium containing 10 mg/L kanamycin sulfate and 2.0 mg/L zeaein. Of the level tested, 10 mg/L of kanamycin sulfate was optimum in selection of kanamycin sulfate resistant plant. Co-culture time of bacteria and leaf explants was affected at the frequency of shoot regeneration and survival of leaf explants. Leaf explants co-cultivated during above 48hr severely decreased survival rate and shooting rate. Best result on survival rate and shooting rate were obtained when exposed for 24 h. 80 explants of 105 leaf explants survived on 3/2 MS medium containing 2.0 mg/L zeatin and 10 mg/L kanamycin sulfate, and 15 shoots was regenerated on the same medium. To select kanamycin sulfate resistant plant, regenerate as cultured on 3/2 MS medium containing 10 mg/L kanamycin sulfate, and obtained 5 kanamycin resistant plants. Southern blot analysis conformed that the rolC gene was incorporated into the genomic DNA of kanamycin resistant plants.

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.85-92
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• 2002

This study has been focused on improving transformation efficiency of rice using Agrobacterium tumefaciens. We have demonstrated the effect of this system when the GPAT gene related to the cold-resistance was transferred by Agrobacterium tumefaciens in rice. Transformation conditions were modified using intron $\beta$-glucuronidase (GUS) expression as a reporter gene in the rice. In this study, mature seed-derived calli of rice (Oruza sativa L. cv. Dongjin) were pre-cultured for 3 days and then infected with Agrobacterium. When this infected calli were cultured in the dark for 10 days on co-cu]lure medium containing 50 mg/L of CaCl$_2$, 30 mg/L of acetosyringone, 2 mg/L of 2,4-D, 120 mg/L of betaine, high GUS expression was observed. In the present transformation system, the efficiency of transformation of GPAT gene was about 54%. Stable integration of GPAT gene into chromosomal DNA was proven by southern blot analysis of genomic DNA isolated from T$_{0}$ progenies. The progenies (T1 generation) derived from primary transformant of 5 lines were segregated with a 3 (resistant) : 1 (sensitive ratio) in medium containing hygromycin. This high frequency transformation system can be used as a useful tool in transformation of another monocotyledon.n.

• 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.

• Korean Journal of Breeding Science
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• v.41 no.4
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• pp.482-487
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• 2009

The apple rootstock M.26 (Malus pumila) is very popular apple rootstock with semi-dwarf habit and the trees on M.26 produce high quality fruit at a young age. Since it is prone to poor prop ability to soil, however, young trees require staking in windy locations. The rolC gene was introduced into M.26 by Agrobacterium tumefaciens LBA4404 harboring pBI121 to obtain its transformants with dwarfism and enhanced rooting ability. One regenerated transgenic line was confirmed by polymerase chain reaction (PCR) analysis and Southern blot analysis of genomic DNA for the existence of rolC gene. The characteristics of transgenic line in vitro were not significantly different from non-transgenic line except for the active root formation and lateral root number. The rolC transgenic line showed reduced stem length and increased root number in vitro. Rooting ability was examined in the isolated greenhouse after mound layering. Compared to non-transgenic M.26, rolC transgenic line showed significantly higher rooting ability. The transgenic line did not show any other observable variation in shoot phenotype compared with non-transgenic line excepting increased branching

• Biomedical Science Letters
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• v.2 no.2
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• pp.223-229
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• 1996

HLA-B27 gene, one of the HLA-class I molecule, is strongly associated with ankylosing spondylitis. It has been most frequently used as a disease-correlated HLA gene by clinicians. In most laboratories, conventional HLA-B27 typing is still performed by cell cytotoxicity tests or fluorescence serology with specific antibodies. In this study, DNA typing method for HLA-B27 was developed by using group specific Polymerase Chain Reaction (PCR). Four HLA-B27 cell lines (HOM-2, JESTHOM, WT24 and BTB) and fifty six B27 Korean individuals defined by serology were used. The results of control cell and B-27 positive individual samples were correlated well with the data which was performed by serological method. All of B27 positive PCR products gave positive signals on Southern blot hybridization with B27 specific probe. This study shows that the HLA-B27 DNA typing is a relatively simple, fast and practical tool for the determination of the HLA-B27 gene in routine clinical laboratory work.