• Korean Journal of Plant Tissue Culture
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• v.21 no.2
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• pp.99-103
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• 1994

The herbicide bialaphos is a potent inhibitor of glutamine synthetase in higher plants. A bialaphos resistance (bar) gene encoding for an acetyltransferase was isolated from genomic DNA of Pseudomonas syringae pv tabaci. The bar gene was ligated to the binary vector pBI121. Pistils of tobacco plane were heated with the bar gene containing plasmid DNA at various times after pollination. When the treatment was applied at 30 and 40 h after pollination, a number of transgenic plants were obtained. Premary transformation (T$_{0}$ generation) and their progenies (T$_1$T$_2$) were resistant to both bialaphos and kanamycin at a dosage lathal to untransformed control plants. Stable integration of bar gene into chromosomal DNA was proven by Southern blot analysis of genomic DNA isolated from T$_1$progenies. These results show that the bialaphos resistant plane could be obtained by treatment to pistils with the exgenous bar gene through the fertilization cycle of tobacco.o.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.261-266
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• 2004

This experiment was carried out to confirm the stability of bar gene introduced into petunia plant through Agrobacterium-mediated transformation, in successive generation, or after crossing or back-crossing. Some of different 25 transgenic plants were used in crossing and back-crossing to wild type, or repeated-selfing to T$_4$ generation. On the processing of experiment, it was found that some lines lost their resistant ability to herbicide basta, or showed non-Mendelian segregation mode: produced much more susceptible segregants than resistant plants. Even though there are exceptional cases, which was off from expected, the genetic stability of bar gene introduced could be confirmed strongly, because in almost case, the segregation of resistant and susceptible plants to basta was done under Mendelian-law according to single gene dominant model.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.

• 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.

• Journal of Life Science
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• v.14 no.2
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• pp.368-373
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• 2004

We have obtained fertile transgenic rice plants resistant to the broad spectrum herbicide Bast $a^{(R)}$ (active ingredient phosphinothricin, PPT) by PEG-mediated transformation procedure. The plasmid pCaMV35S::Bar was used to deliver the bar gene into embryogenic suspension culture-derived protoplasts of rice (Oryza sativa L.). Transformed plants were regenerated and selected on medium containing 15 mg/l of phosphinothricin. Stable integration and expression of the bar gene in transgenic rice plants was confirmed by Southern and Northern blot analysis. Transgenic $R_1$ plants were also confirmed by assays for phosphinothricin acetyltransferase (PAT) activity. The bar gene was expressed in the primary transgenic rice plants and in the next generation progeny, in which it showed a 3 : 1 Mendelian inheritance pattern. Transgenic $R_1$ and $R_2$ plants were resistant to the herbicide Bast $a^{(R)}$ when sprayed at rates used in field practice.ice.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.13-20
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• 2008

Potato (Solanum tuberosum L.), one of the most important food crops, is susceptible to a number of devastating fungal pathogens in addition to bacterial and other pathogens. Producing disease-resistant cultivars has been an effective and useful strategy to combat the attack of pathogens. Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus strain HUT 6037 and bialaphos resistance (bar) genes in a binary plasmid vector, pEKH1. Polymerase chain reaction (PCR) analysis revealed that the ChiC and bar genes are integrated into the genome of transgenic plants. Different insertion sites of the transgenes (one to six sites for ChiC and three to seven for bar) were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of the ChiC gene at the messenger RNA (mRNA) level was confirmed by Northern blot analysis and that of the bar gene by herbicide resistance assay. The results obviously confirmed that the ChiC and bar genes are successfully integrated and expressed into the genome, resulting in the production of bialaphos-resistant transgenic plants. Disease-resistance assay of the in vitro and greenhouse-grown transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight).

• BMB Reports
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• v.38 no.1
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• pp.41-48
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• 2005

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1130-1134
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• 2005

To monitor the possibility of horizontal gene transfer between transgenic potato and bacteria in the environment, the gene flow from glufosinate-tolerant potato to bacteria in soils was investigated. The soil samples treated with the leaf tissue of either glufosinate-tolerant or glufosinate-sensitive potato were subjected to PCR and Southern hybridization to determine possible occurrence of glufosinate-resistant soil bacteria and to detect the bar (phosphinothricin acetyltransferase) gene, conferring tolerance to glufosinate. The bar gene was not detected from genomic DNAs extracted at different time intervals from the soil samples, which had been treated with the leaf tissue of either transgenic or non-transgenic potato for 2 to 8 weeks. In addition, the level of glufosinate-resistant bacteria isolated from the soil samples treated with the leaf tissue of transgenic potato was similar to that of the samples treated with non-transgenic potato after 4 months of incubation at $25^{\circ}C$. The bar gene was not detected in the genomic DNAs extracted from colonies growing on the plate containing glufosinate, indicating that the bacteria could acquire the resistant phenotype to glufosinate by another mechanism without the uptake of the bar gene from glufosinate-tolerant potato.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.143-149
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• 2003

This experiment was carried out to confirm the stability of bar gene introduced into petunia plant through Agrobacerium-mediated transformation. Twenty-five transgenic plants T$_{0}$ plants, back cross (BC$_1$) populations to wild type and F$_1$plants between different T$_{0}$ plants were prepared, and polymerase chain reaction(PCR), PCR-Southern blot analysis, and field test with 0.1% Basta treatment were done. The results of PCR, PCR-Southern blot hybridization, and field test indicated that NPTII and bar gene introduced into the genome of petuina plants were stably transmitted to their progenies, and conferred the plants resistance to herbicide, Basta.sta.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.255-259
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• 2004

Agrobacterium tumefaciens-mediated cotyledonary node transformation was used to produce transgenic soybean. Cotyledonary node explants of three cultivars and one genotype were co-cultivated with strains Agrobacterium (LBA4404, GV3101, EHA101, C58) containing the binary vectors (pCAMBIA3301 and pPTN289) carrying with CaMV 35S promoter-GUS gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selectable marker. There was a significant difference in the transformation frequency depend on bacteria strain. The EHA101 strain of the bacterial strains employed gave the maximum efficiency (3.6%). One hundred-six lines transformed showed the resistance in glufosinate. Histochemical GUS assay showed that at least 11 plants transformed with the GUS gene were positive response. The soybean transformants were obtained from the Thorne (5 plants), 1049 (5 plants) and Bakun (1 plant), respectively. Southern blot analysis and leaf painting assay revealed that the GUS and bar gene segregated and expressed in their progeny.