• Proceedings of the Korean Society of Crop Science Conference
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• 2005.10b
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• pp.288-289
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• 2005

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.230-234
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• 2019

Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplast-mediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1178-1187
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• 2024

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

• Journal of The Korean Society of Grassland and Forage Science
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• v.29 no.3
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• pp.165-170
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• 2009

A system for the production of transgenic plants has been developed for perennial ryegrass (Lolium perenne L.) via Agrobacterium-mediated transformation. Included in this study were two factors which may affect the gene transfer efficiency: concentrations of acetosyringone (AS, 0 to 300 ${\mu}M$), and co-culture period (1 to 7 days). Both factors were very important to achieve high efficiency gene transformation in the perennial ryegrass. The highest transformation efficiency was obtained when embryogenic calli were inoculated with Agrobacterium in the presence of 100 ${\mu}M$ AS with the culture medium for 5 days. Phosphinothricin resistant calli were developed with into complete plants. GUS histochemical assay, polymerase chain reaction (PCR) and Northern blot analysis of transgenic plants demonstrated that transgenes were integrated into the genome of perennial ryegrass. Using this protocol, it was possible to obtain transformants efficiently for further study.

• The Plant Pathology Journal
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• v.21 no.1
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• pp.39-46
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• 2005

The promoter region flanking the 5’ CAZFP1 coding region was isolated from the genomic DNA of Capsicum annuum. To identify the upstream region of the CAZFP1 gene required for promoter activity, a series of CAZFP1 promoter deletion derivatives was created. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in tobacco leaves after infection by Pseudomonas syringae pv. tabaci, or treatment with methyl jasmonate (MeJA), ethylene, abscisic acid (ABA), salicylic acid (SA), cold and wounding. Promoter fragments of 685 bp or longer showed 7-fold or greater induction after P. s. pv. tabaci infection and MeJA treatment. The CAZFP1 full-length promoter (-999 bp) also showed 6-fold induction in response to ethylene. The transiently transformed tobacco leaves with the CAZFP1 full length promoter fused-GUS gene showed more than 5-fold induction in response to SA, ABA and cold. These results suggest that the CAZFP1 promoter contains responsive elements for pathogen, MeJA, ethylene, SA, ABA and cold.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.35-41
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• 2000

Transgenic cabbage (Brassica oleracea ssp. capitata) plants resistant to the commercial herbicide Bast $a^{R}$ were obtained by Agrobacterium tumefaciens - mediated transformation. Hypocotyl segments of in vitro grown plants were infected with Agrobacterium tumefaciens LBA 4404 harboring plasmid pMOG6-Bar which contains hpt and bar genes. Explants were cultured on callus induction medium (MS basal medium + 1 mg/L NAA + 2 mg/L BA + 2 mg/L AgN $O_3$+ 100 mg/L carbenicillin + 250 mg/L cefotaxime) supplemented with 15 mg/L hygromycin. Hygromycin resistant calluses were transferred to shoot regeneration medium (MS basal medium + 0.1 mg/L NAA + 2 mg/L BA + 3% sucrose + 2 mg/L AgN $O_3$+ 15 mg/L hygromycin + 250 mg/L cefotaxime + 100 mg/L carbenicillin). In order to induce roots, elongated shoots were placed on the MS medium without plant growth regulators and hygromycin. Southern blot analysis of several putative transgenic plants indicated that one to five intact copies of Apt and bar genes were incorporated into the genome. Expression of bar gene was confirmed by Northern blot analysis and by herbicide resistant phenotype. Seed progeny from self-pollinated transformants expressed the herbicide resistance and showed Mendelian segregation of the introduced gene.e.

• 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.31 no.4
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• pp.267-271
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• 2004

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron $\beta$-glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.489-492
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• 2006

For molecular breeding purpose, genetic transformation of Brassica napus cultivars has been extensively performed using Agrobacterium method. B. napus cv. napus, one of major oil crops, can be transformed via Agrobacterium-based method. We demonstrated that Agrobacterium-mediated transformation via vacuum infiltration slightly worked for the seedlings of B. napus cv. napus according to fluorometric GUS enzyme analysis. In contrast, transformation efficiency was highly enhanced when the seedlings, prior to agroinfiltration, were treated with sodium hydrosulfite solution as a chemical wounding agent. GUS gene expression in transformed seedlings that was confirmed by RT-PCR suggests their usefulness for the development of transient expression system.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1018-1022
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• 2004

Optimized conditions for Agrobacterium-mediated lisianthus pollen transformation were adjusted using various factors such as temperature, pH and sucrose concentration. Pollen tube growth was successfully achieved in a medium (pollen germination medium; PGM) containing $7-15\%$ sucrose with pH in the range of 5.5-7.0 at temperature of $20-27^{\circ}C$. Lisianthus pollen was vacuum-infiltrated with Agrobacterium cell suspension for 20 min, and transformed pollen was confirmed by GUS histochemistry and Southern hybridization following RT-PCR. Transgenic pollen system may be utilized for establishing an area of plant transient expression systems based on the convenient pollen transformation procedure presented in here.