• The Korean Journal of Pesticide Science
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• v.8 no.1
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• pp.22-29
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• 2004

To evaluate mutagenicity of Phosphinotricin Acetyltransferase(PAT) which is expressed by the glufosinate-resistance gene pat, in vitro reverse mutation test using Salmonella typhimurium, chromosome aberration test using chinese hamster lung(CHL) cells and in vivo micronucleus test of mice were performed. In the reverse mutation, the PAT did not induce mutagenicity in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation at $5000{\mu}g/plate$. In the chromosome aberration test, the results showed no incidence of increased structural and numerical chromosome aberrations at any doses tested(100, 10, $1{\mu}g/mL$). In micronucleus test, the ratio of micronuclei was measured in polychromatic erythrocytes of bone marrow of male ICR mice intraperitoneally administrated with PAT(1250, 625, and 313 mg/kg), the results showed no incidence of increased micronucleated polychromatic erythrocytes (MNPCE). These results indicate that PAT might not have mutagenic potential in vitro and vivo systems.

• Horticultural Science & Technology
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• v.19 no.1
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• pp.17-21
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• 2001

To develop a selection system for regenerating plants from transformed tissues, effects of four antibiotics (kanamycin, hygromycin, carbenicillin, cefotaxime) and herbicide (phosphinotricin) on shoot regeneration from cotyledon and hypocotyl explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis) were studied. For cotyledon, shoot induction was not significantly affected by kanamycin at $1mg{\cdot}L^{-1}$, but the number of shoots formed was significantly reduced at $2mg{\cdot}L^{-1}$, and no shoots were regenerated from any explants at $6mg{\cdot}L^{-1}$ or higher. Hypocotyl explants showed similar result as cotyledon. Kanamycin at $7mg{\cdot}L^{-1}$ may be adequate for selecting Chinese cabbage transformants. Hygromycin at $4mg{\cdot}L^{-1}$ or higher completely inhibited the growth and shoot regeneration of Chinese cabbage explants. Therefore, resistance gene to hygromycin may also be used as a selective marker for Chinese cabbage transformation. Carbenicillin and cefotaxime, the cephalosporin type of antibiotics, had little effect on shoot regeneration of Chinese cabbage explants. Since carbenicillin and cefotaxime have low toxicity to Chinese cabbage, they are suitable for use in tissue culture to eliminate Agrobacterium in transformation experiments after co-cultivation. Shoot regeneration from cotyledon and hypocotyl explants was significantly reduced in presence of $1mg{\cdot}L^{-1}$ phosphinotricin (PPT) and completely inhibited by $2mg{\cdot}L^{-1}$ or higher. PPT, same as antibiotics, may also be used to select transformed cells. Since Chinese cabbage is known to be recalcitrant to in vitro shoot regeneration compared to other Brassica species, even though lower levels of selectable markers result in more transformants but simultaneously allow more untransformed escapes to develop, lower levels of antibiotics and herbicides could be successfully used as a selectable marker to reduce selection pressure.

• 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.

• 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.

• 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.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.52-52
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• 2018

Development of herbicide resistant transgenic legume plants through Agrobacterium-mediated transformation has been worked in many previous studied. Plant regeneration after infection is the important step to obtain successful transgenic plants. Many attempts try to find the optimum media condition for plant regeneration after infection. However, the transformation efficiency of legume plants is still low. In this study, regeneration of some Korean legume species including two soybean cultivars (Dawon and Pungsan) and pea have been done with organogenesis which is used various kind of explants such as cotyledonary-nodes in soybean and bud-containing tissue in pea. We developed the optimum media condition for plant regeneration regulators under Agrobacterium-mediated transformation using different kind and various concentration of plant growth. As the results, B5 medium containing 2 mg/L of 6-benzylaminopurine was selected in this study for the optimum plant regeneration media. The segments were inoculated with Agrobacterium suspension harbored an IG2 vector containing bar gene which confers resistance to phosphinotricin (PPT) in 3, 5 and 7 days. The transformation efficiency was achieved in Dawon 3.03 % and pea 1.46 % with co-cultivation period of 7 days which is showed a high number of GUS positive expression period.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.36-36
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• 2018

Soybean is the important crop in Asian countries as protein source, oil production and animal feed. Improving soybean using genetic transformation is the principal tool in nowadays. Developing herbicide resistant transgenic soybean plants through Agrobacterium-mediated transformation has been worked in many previous studied. However, the transformation efficiency is still low. Many attempts try to find the optimum media condition for plant regeneration after infection. After transformation, the plant regeneration is very important condition to promote growth of transgenic plant. In this study, we optimized a regeneration condition for two Korean soybean cultivar, Dawonkong and Pungsannamulkong using cotyledon, cotyledonary nodes and hypocotyl as explant. The results showed that shoot regeneration of cotyledonary nodes on B5 medium containing 2 mg/L 6-benzylaminopurine showed the highest percentage of regeneration in Dawonkong (75.8%) while Pungsannamulkong presented high number of shoots 2.12 shoots per explant. For transformation condition, co-cultivation in 7 days showed a high number of GUS positive expression. Most of explants can survived under media including 5 mg/L of glufocinate which refers phosphinotricin for 2-week selection. Washing with 400 mg/L of cefotaxime in several times and selection in plant regeneration media with 400 mg/L of cefotaxime can prevent bacteria growth, effectively.

• Plant Resources
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• v.7 no.1
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• pp.39-46
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• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.1
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• pp.61-68
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• 2004

A reproducible transformation system via optimized regeneration media for Korean rice cultivars was established using Agrobacterium tumefeciens LBA4404 (pSBM-PPGN; gusA and bar). Although japonica rice genotypes were easier to produce transgenic plants compared to Tongil type cultivars, transformation efficiencies were not always correlated with regeneration efficiencies of non-transgenic callus on the control medium. Regeneration efficiencies of Donganbyeo, Ilmibyeo, and Manchubyeo were over 50% in non-transgenic control, however, transformation efficiencies were significantly low when only sucrose was added to the media as a carbon source. However, the medium, MSRK5SS-Pr (or MSRK5SM-Pr), that contains $5\textrm{mgL}^{-1}$ kinetin, $0.5\textrm{mgL}^{-1}$ NAA, 2 % sucrose (or maltose), 3% sorbitol, and $500\textrm{mgL}^{-1}$ proline, was the most efficient not only for regeneration of non-transgenic callus but also for regeneration of transgenic callus in the presence of L-phosphinotricin (PPT). Average transformation efficiencies of 16 Korean rice cultivars were significantly enhanced by using the optimized medium from 1.5% to 5.8% in independent callus lines and from 2.9% to 19.4% in tromsgenic plants obained. Approximately 98.9% (876 out of 885) transgenic plants obtained on optimized media showed basta resistance. Stable integration, inheritance and expression of gusA and bar genes were continued by GUS assay and PCR and Southern analysis of the bar gene. With Pst1 digestion of genomic DNA of transgenic plants, one to five copies of T-DNA segment were observed; however, 76% (19 out of 25 transgenic plants) has low copy number of T-DNA. The transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.293-300
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• 2011

This study is conducted to develop an efficient transformation system via particle bombardment with PLBs (Protocorm-like bodies) in Cymbidium. For this, pCAMBIA3301 vector which carries a herbicide-resistant bar gene and gus gene as a reporter gene was used for transformation with Cymbidium cultivars 'Youngflower ${\times}$ masako' line. To select transformants, proper concentration of herbicide, PPT (phosphinotricin), should be determined. As a result, 5 mg/l of PPT was selected as a proper concentration. Further, proper conditions for particle bombardment were determined to obtain a high frequency of transformation. Results showed that 1.0 ${\mu}g$ of DNA concentration, 1,100 and 1,350 psi for helium gas pressure, 1.0 ${\mu}m$ of gold particle and 6 cm of target distance showed the best result for the particle bombardment experiment. Also, pre-treatment with combination 0.2 M sorbitol and 0.2 M mannitol for 4 hrs prior to genetic transformation increased the transformation efficiency up to 2.5 times. Using transformation system developed in this study, 3.2 ~ 4.0 transgenic cymbidium plants can be produced from 100 bombarded PLBs on average. Putative transgenic plants produced in this system confirmed the presence of the bar gene by PCR analysis. Also, leaves from randomely selected five transgenic lines were applied for Basta solution (0.5% v/v) to check the resistance to the PPT herbicide. As a result, three of them showed resistance and one of them showed the strongest resistance with the maintenance of green color as non-transformed plants showed. Using this established transformation system, more genes of interests can be introduced into Cymbidium plants by genetic transformation in the future.