• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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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.

• 생명과학회지
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• 제12권1호
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• pp.67-76
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• 2002

Agrobacterium(LBA4404/pBI1121)을 이용하여 형질전환된 애기장대 (Arabidopsis thaliana)를 대상으로 T2, T3, F3세대에서의 도입된 외래 유전자의 비활성화 현상을 조사하였다. Kanamaycin저항성 개체들의 GUS유전자 발현을 분석한 결과, T2세대에서 조사된 12계통 중 5계통에서 GUS 비활성 개체가 관찰되었다 (GUS유전자 비활성율 2.3%). Multi copy T-DNA 계통을 조사한 결과, GUS 비활성 정도가 더욱 심해짐이 관찰되었다 (5.8%). T3 세대에서 single copy T-DNA 계통들은 1.3%의 GUS 비활성율을 보인 반면, multi-copy T-DNA 계통에서의 비활성율은 12.6%로 급격히 증가하였다. 유사한 현상이 형질전환 식물체와 정상개체를 교배하여 생산된 F2 계통에서도 관찰되었다 (비활성율 9.9%). 본 실험으로 식물체에 도입된 외래 유전자가 후대에서의 전이과정동안 점진적으로 비활성화되고, 이 현상은 multi copy T-DNA 계통에서 훨씬 심각함이 밝혀졌다.

• 식물조직배양학회지
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• 제27권1호
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• pp.7-12
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• 2000

저온관련 유전자인 BN115 gene과 표지유전자인 npt II gene을 함유하고 있는 Agrobacterium tumifacience GV 3101 균주를 이용하여 겨울상추품종인 청치마의 잎절편과 공동배양하는 방법으로 형질전환 시켰다. 상추의 잎절편을 Agrobacterium과 공동배양 후 MS 기본배지에 100 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5 mg/L kinetin을 첨가한 선발배지에 치상하였는데, 치상 후 3-4주부터 절편체로부터 multiple shoot들이 생성되기 시작하였다. 선발배지에서 살아남은 선발체들은 1/2 MS배지에 100 mg/L kanamycin, 250 mg/L carbenicillin이 첨가된 발근배지로 옮겨졌다. 한편, 선발된 shoot들은 PCR반응을 이용하여 도입유전자의 삽입여부를 확인하였다. PCR 반응은 표지유전자인 nptII와 저온관련 유전자인 BN115 및 식물에 도입되지 않는 vir G 유전자를 각각 특이적으로 증폭하는 primer를 가지고 실시하였다. PCR 반응 결과 대조구로 쓰인 정상 상추식물체에서는 nptII와 BNl15유전자의 증폭을 볼 수 없는 반면에 형질전환체에서는 두 유전자 모두 PCR 증폭 산물을 확인할 수 있었다. 또한 확인된 식물체의 DNA에서는 vir G유전자가 발견되지 않아 이는 Agrobacterium의 혼입에 의한 결과가 아님을 다시 한번 증명하였다. 또한 선발된 형질전환체를 이용하여 Southern analysis와 RT-PCR을 실시한 결과 내한성 유전자가 상추 식물에 안정적으로 도입되어 발현됨을 확인하였다.

• Journal of Plant Biotechnology
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• 제2권1호
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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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• 제39권2호
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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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• 제19권9호
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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.

• 식물조직배양학회지
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• 제28권6호
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• pp.353-357
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• 2001

인삼의 자엽과 callus에 Biolistic system을 이용한 비선택성 제초제인 bialaphos에 대한 내성을 갖게 하는 PAT 유전자의 형질전환효율 향상 및 형질전환체의 유전분석에 관한 실험을 수행하였다. 자엽의 경우에는 형질전환율이 약했지만 callus의 경우에는 target distance 9 cm, rupture disk-macro-carrier gap distance를 1/3"로 했을 때 가장 양호한 형질전환 결과를 보였다. 형질전환된 인삼식물체에서 PAT 및 NPT 유전자의 존재 여부를 확인하기 위해서 PCR을 수행한 결과 정상 식물체서는 전혀 PCR product가 형성되지 않은 반면 형질전환체 모두에서 PAT (약 300 bp)와 NPT (약 800 bp) 유전자의 band를 확인하여 각각의 유전자가 삽입되어 PAT 및 NPT IIgene이 도입된 형질전환체임을 확인할 수 있었다. 있었다.

• 한국균학회지
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• 제39권3호
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• pp.235-238
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• 2011

팽이균사체의 형질전환을 위하여 Agrobacterium 세포를 사용하였다. 특히, Agrobacterium 세포의 감염단계 전에 약한 NaOH용액을 처리하였으며 이로써 균사체 세포들의 표면 상해 발생을 기대하였다. 그 결과, hygromycin 저항성 ($hyg^r$) 균사체는 NaOH 처리를 거친 경우에서만 출현하였다. 형질전환 균사체의 $hyg^r$ 유전자 도입은 PCR로 확인되었으며 또한 Southern blot hybridization과 western blotting 분석에 의하여 단일 유전자 copy의 삽입과 외래유전자의 발현을 확인할 수 있었다. 본 연구는 팽이균사체에 대한 효율적인 Agrobacterium 이용 형질전환수단을 보여주고 있다.

• 미생물학회지
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• 제33권4호
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• pp.274-276
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• 1997

Coprinus congregatus에서 laccase를 과량생성하는 변이주를 대상으로 phosphinothricin 저항성을 선택표지로 사용하여 형질전환을 수행하였다. 항생물질에 대한 저항성을 부여하는 plasmid DNA(pBARGEM 7-1)를 제한효소로 가수분해하여 원형질체에 형질전환을 수행한 결과 plasmid DNA $1{\mu}g$ 당 약 500여개의 형질전환체를 얻었다. 도입된 plasmid는 형질전환체의 chromosomal DNA에 삽입되어 있음을 Southern blot으로 확인하였다.

• Journal of Plant Biotechnology
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• 제48권2호
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• pp.93-99
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• 2021

This study was conducted to develop an Agrobacterium-mediated genetic transformation protocol for the carnation cv. "Jinju" to counteract its ethylene sensitivity. The new protocol involves the use of an improved shoot regeneration medium, optimized minimal concentrations of the selective agent, a pre-culture period, and co-cultivation periods. Silver nanoparticles (NAg) added at a concentration of 2.0 μM to the Murashige and Skoog (MS) basal shoot regeneration medium supplemented with 0.1 mg/L indole-3-butyric-acid (IBA) and 0.2 mg/L thidiazuron (TDZ) improved the shoot regeneration efficiency, number of shoots per explant, and plant growth compared to the control without the addition of NAg. The phosphinothricin (PPT) concentration of 1.0 mg/L was determined to be the minimal and optimal concentration for the selection of putative transgenic plants. When the explants were infected with Agrobacterium cells harboring the acdS gene, the explants that were pre-cultured for three days induced more putative transgenic plants than those that were co-cultivated for four days. Therefore, we expect that the results of this study will benefit researchers who are developing genetic transformations of carnations.