• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.329-333
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• 1998

$\beta$-Glucuronidase (GUS) gene of Escherichia coli was introduced into sweet potato (Ipomoea batatas (L.) Lam.) cells by particle bombardment and expressed in the regenerated plants. Microprojectiles coated with DNA of a binary vector pBI121 carrying CaMV35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker were bombarded on embryogenic calli which originated from shoot apical meristem-derived callus and transferred to Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid and 100 mg/L kanamycin. Bombarded calli were subcultured at 4 week intervals for six months. Kanamycin-resistant calli transferred to MS medium supplemented with 0.03 mg/L 2iP, 0.03 mg/L ABA, and 50 mg/L kanamycin gave rise to somatic embryos. Upon transfer to MS basal medium without kanamycin, they developed into plantlets. PCR and northern analyses of six regenerants transplanted to potting soil confirmed that the GUS gene was inserted into the genome of the six regenerated plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the vascular bundle and the epidermal layer of leaf, petiole, and tuberous root.

• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.13-18
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• 2003

This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.

• Journal of Life Science
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• v.20 no.9
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• pp.1314-1323
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• 2010

In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.

• Journal of Plant Biotechnology
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• v.1 no.2
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• pp.85-90
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• 1999

In order to protect fungal diseases, leaf disc explants of Solanum tuberosum cultivar, Belchip, was infected with an Agrobacterium MP90 strain containing chimeric gene construct, consisting of antibiotic resistance and chitinase gene driven by the CaMV 35S promoter, for transformation. Regenerated multiple shoots were selected on a medium containing kanamycin and carbenicillin after exposure to Agrobacterium. The presence and integration of the npt II and chitinase gene were confirmed by polymerase chain reaction(PCR). Northern blot analysis indicated that the genes coding for the enzyme could be expressed in potato plants. The chitinase activity of transgenic potato plants was higher than the control potato.

• Journal of Plant Biotechnology
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• v.2 no.3
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• pp.143-149
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• 2000

An Efficient in vitro regeneration system and an optimized Agrobacterium mediated transformation protocol are described, based on the use of young seedling cotyledons of Capsicum annuum L. Optimal regeneration efficiency can be obtained by cultivating cotyledon explants on media containing 4 mg/L benzyladenine and 0.1 mg/L indolacetic acid. The effect of antibiotics used to eliminate Agrobacteria, as well as the toxic level of some generally used selection agents (kanamycin, geneticin, hygromycin, phosphinotricin and methotrexate) in regenerating pepper tissues were determined. To enable the comparison of different selection markers in identical vector background, a set of binary vectors containing the marker genes for NPTII, HPT, DHFR and BAR respectively, as well as the CaMV 35S promoter/enhancer-GUS chimaeric gene was constructed and introduced into four different Agrobacterium host strains.

• Asian Journal of Turfgrass Science
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• v.15 no.1
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• pp.9-14
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• 2001

Callus formation and plant regeneration from the seeds of zoysiagrass cv. Zenith was tested on MS basal medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D) and of several cytokinins. A concentration of 1mg/L 2,4-D on medium stimulated callus formation. In the presence of 5mg/L 2,4-D, addition of 1mg/L kinetin significantly enhanced callus formation and plant regeneration over 2,4-D alone. To transfer foreign DNA into turfgrass, parameters for the bombardment of embryogenic callus with the particle bombardment were partially optimized using transient expression assay of a $chimeric \beta$-glucuronidase(GUS) gene driven by the CaMV 35S promoter. GUS gene was strongly expressed at helium pressure 1,100 psi and 6~9cm target distance.

• Plant Resources
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• v.4 no.3
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• pp.181-188
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• 2001

We tried to introduce two forsythia genes related in lignan biosynthesis, dirigent protein and pinoresinol/lariciresinol (Ph) reductase, into potatoes for accumulation of lignans in transgenic potatoes. We made binary vectors overexpressing dirigent protein gene and P/L reductase gene driven by a CaMV35S promoter and transformed into potatoes via Agrobacterium mediated transformation. And in order to control the metabolic flux of lignan biosynthesis pathway, we tried to inhibit chalcone synthase genes of potatoes by antisense inhibition technique also. We tried to use PCR screening method for selection of transgenic plants of different vectors. We tried to determine and compare lignan contents from different transgenic potato lines.

• BMB Reports
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• v.41 no.7
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• pp.542-547
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• 2008

D-type cyclins control the onset of cell division and the response to extracellular signals during the G1 phase. In this study, we transformed a D-type cyclin gene, Nicta;CycD3;4, from Nicotiana tabacum using an Agrobacterium-mediated method. A predicted 1.1 kb cyclin gene was present in all of the transgenic plants, but not in wild-type. Northern analyses showed that the expression level of the Nicta;CycD3;4 gene in all of the transgenic plants was strong when compared to the wild-type plants, suggesting that Nicta;CycD3;4 gene driven by the CaMV 35S promoter was being overexpressed. Our results revealed that transgenic plants overexpressing Nicta;CycD3;4 had an accelerated growth rate when compared to wild-type plants, and that the transgenic plants exhibited a smaller cell size and a decreased cell population in young leaves when compared to wild-type plants.

• Asian Journal of Turfgrass Science
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• v.18 no.4
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• pp.211-219
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• 2004

Transgenic zoysiagrass (Zoysia japonica cv. Zenith) plants have been obtained by particle bombardment of embryogenic callus with the plasmid pSMABuba, which contains hygromycin resistance (hpt) and bialaphos resistance (bar) genes. Parameters on DNA delivery efficiency of the particle bombardment were partially optimized using transient expression assay of a chimeric $\beta-glucuronidase$(gusA) gene driven by the CaMV 35S promoter. Stably transfarmed zoysiagrass plants were recovered with a selection scheme using hygromycin. Transgenic zoysiagrass plants were confirmed by PCR analysis with specific primer for bar gene. Expression of the transgene in transformed zoysiagrass plants was demonstrated by Reverse transcriptase (RT)-PCR analysis. All the tested transgenic plants showed herbicide BastaR resistance at the field application rate of $0.1\%-0.3\%$.

• KSBB Journal
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• v.16 no.5
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• pp.480-485
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• 2001

Genetic transformation in dandelion(Taraxacum mongolicum Hand). was studied. We used for transformation by Agrobacterium tumefaciens strian LBA4404 harboring a binary vector pBI121 carrying the CaMV 35S promoter-GUS gene fusion used as a reporter gene and NOS promoter-NPTII gene as a positive selection marker. To obtain transformed plants, leaf explants of dandelion were cocultured with Agrobacterium tumefaciens LBA4404 for 10 mins, then transferred to MS medium containing 1 $\mu$M IAA, 1$\mu$M BA, 100$\mu$g/ML carbenicillin and 50 $\mu$g/ML kanarmycin sulfate. After two weeks of subculture of the explants, Kanamycin-resistant shoots were formed on explants survived. When subjected to GUS histochemical assay, all of the regenerants showed the GUS-positive responses. Plantlets were be be transformed to soil for further growth.