• Korean Journal of Microbiology
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• v.50 no.4
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• pp.372-375
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• 2014

This experiment was undertaken to investigate proper conditions for protoplast isolation and genetic transformation of the white rot fungi, Polyporus brumalis. The protoplasts were formed from mycelia at a frequency of $1{\times}10^7/ml$ with 0.5% Usukizyme. The transformation vector (pHYgpt) was constructed using hygromycin resistance gene (hph) for the selectable maker. The yield was 100-160 transformants/${\mu}g$ DNA in a transformation mediated by 40% polyethylene glycol solution with aurintricarboxylic acid, heparin and supermidine. The genomic integration of the pHYgpt was confirmed by hph-specific PCR and the expected amplified band appeared only in the transformants. These results could be an efficient tool in gene engineering of the genus polyporus.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.657-662
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• 2008

To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, the recombinant DgHSP17.2 gene was introduced into orchardgrass plants using the Agrobacterium-mediated transformation method and expressed constitutively under the control of the CaMV 35S promoter. The results of genomic DNA PCR and Southern analysis showed a DNA band and hybridization signal on agarose gel and X-ray film in transgenic orchardgrass plants harboring the recombinant DgHSP17.2 gene, but a DNA band and hybridization signal were not observed in the wild type and empty vector control plants. The same result was also obtained in RT-PCR and Southern blot analysis, and these transgenic orchardgrass plants did not show any morphological aberration both in the culture bottle and soil mixture. When leaf discs cut from transgenic orchardgrass plants with recombinant DgHsp17.2 gene were exposed to lethal temperature (heat treatment at $60^{\circ}C$ for 50 min), 60-80% of the leaf discs showed only damage symptoms, but non-transgenic leaf discs showed a lethal condition. These results indicate that the DgHsp17.2 gene may act as a protector from heat stress in plants.

• Journal of Life Science
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• v.12 no.1
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• pp.67-76
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• 2002

The effect of transgene inactivation in T2, T3 and F2 generations was analyzed in progeny seedlings which had been generated by Agrobacterium (LBA4404/pBI121)-mediated transformation in Arabidopsis thaliana. In a system which investigated in the expression of $\beta$-glucuronidase(GUS)gene in kanamycin-resistant (ke $n^{R}$)seedlings, GUS inactivated seedlings were observed in 5 of 12 tested lines of T2 generation and the frequency of GUS inactivation was approximately 2.3%. Lines with multi-copies of T-DNA exhibited severe GUS gene inactivation with the frequency of 5.8% in T2 generation. In T3 generation lines exhibited GUS gene inactivation with the frequency of 1.3%. In contrast, inactivation increased dramatically up to 12.6% in multi-copy T-DNA line. A similar phenomenon was also found in F2 progeny from a transgenic line which had been crossed with wild-type Arabidopsis plant, WS-O (GUS gene inactivation frequency 9.9%). These results indicate that the foreign gene introduced into the plant was inactivated progressively in its transmission during subsequent generations and the transgenic line with multi-copies of T-DNA tended to show more increased inactivation.

• Korean Journal of Plant Tissue Culture
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• v.27 no.1
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• pp.7-12
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• 2000

Explants of lettuce (Lactuca sativa L.) were co-cultivated with Agrobacterium tumifacience GV 3101 strain containing nptII gene and cold regulated gene (BN115) from Brassica napus for transformation. Multiple shoots were obtained from the explants in the selection medium (MS basal medium supplemented with 100 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5 mg/L kinetin) after 3 to 4 weeks of co-culture. The putative transgenic shoots were transferred to rooting medium (1/2 MS basal medium supplemented with 100 mg/L kanamycin and 250 mg/L carbenicillin). The selected shoots were tested with PCR analysis using nptll, BN115 primers whether cold-regulated gene was introduced to genome of the plants. The vir G primers were particularly used to check contamination of Agrobacterium during PCR analysis. The nptII and BN115 primers produced the specific PCR bands in the putative transgenic lines but the vir G primers did not. These results confirmed that the PCR products were not the result of contamination with Agrobacterium. Additionally the Southern analysis of the PCR products and RT-PCR analysis proved that the cold-regulated gene was successfully integrated and transcribed in the putative transgenic lettuce plants.

• 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.

• Journal of Plant Biology
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• v.39 no.2
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• pp.93-98
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• 1996

In order to enhance the system of potato transformation and further regeneration, potato was transformed using the Agrobacterium tumefaciens harboring $\beta$-glucuronidase (GUS) gene. We found that a series fo modified medium ttained 100% shoot regeneration within 5 weeks after the preincubated explants on stage I medium were infected with Agrobacterium. Callus appeared at the cut edges of stem segments on stage II medium, mainly at the basal parts. Some explants started to form shoots after two to three weeks on stage III medium containing kanamycin (50 mg/L). When transferred to MS medium containing 200 mg/L kanamycin, 81% of the transformed shoots formed roots at the cut edge of the plantlets. In contrast, untrasformed shoots never rooted and became yellowish after few weeks under the same conditions. Southern and northern analysis indicated in vitro shoot regeneration on the callus derived from the potato explants, which were incubated with Agrobacteria. The regeneration cycle was shortened after the transformatin and finally the transformation efficiency was highly enhanced.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.869-872
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• 2009

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplast-regenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA-producing T. niveum.

• Korean Journal of Plant Tissue Culture
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• v.28 no.6
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• pp.353-357
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• 2001

Transformation of ginseng plants was achieved by biolistic system with cotyledon explants and callus using phosphinothricin acetyl-transferase (PAT) gene resisting to a herbicide of Bialaphos. The binary vector for transformation was constructed with disarmed Ti-plasmid and with double 355 promoter. The introduced NPT II and PAT genes of the transgenic ginseng plants were successfully identified by the PCR, and the survival test on the medium with basta. The transgenic ginseng plants were propagated using repetitive secondary embryogenesis. The transgenic ginseng plantlets had normal structures of roots and shoots, and dormant buds for new year sprouting. We transferred the transgenic plants to greenhouse and observed the continuing growth until a new year.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.235-238
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• 2011

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant($hyg^r$) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of $hyg^r$ gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.274-276
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• 1997

Transformation of Coprinus congregatus with a linearized plasmid has been successfully carried out using phosphinothricin resistance gene as a dominant selectable marker. The transforming frequency was about 500 transformants per microgram of DNA using the protoplast-$CaCl_2$ method. The transforming vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from a transformant by Southern hybridization.