• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.120.2-120
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• 2003

Agrobacterium vitis is a causal agent of crown-gall disease on grapevine. In Korea, grapevine variety (GeoBong) have severely been infected by the bacteria since stems of the variety were buried in soil for overwintering. Infection ratio over 70-80％ was observed on 7 years old GeoBong grapevine in Ansung and Cheonan. PCR specific primers for A. vitis strains were designed using nucleotide sequences of vir A gene in Ti-Plasmid, pheA gene in chromosomal DNA and a URP-PCR polymorphic band. Three hundred bacterial strains were isolated from the different 80 galls formed on GeoBong grapevine in Cheonan and Ansung of Korea and were screened to identify A. vitis using the three specific PCR primers for Agrobacterium vitis. Twenty-four bacterial strains that are detected by the primers were further confirmed by pathogenicity and biochemical methods. To investigate the genomic diversity of the bacterial strains, twenty primers of 20 mer referred to universal rice primers (URP) were applied for PCR fingerprinting, Of them, URP2R and URP2F primers could effectively be used to detect polymorphism within the bacterial strains.

• 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.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.45-49
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• 1999

Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

• Plant Resources
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• 제6권3호
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• pp.205-210
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• 2003

Ginseng(Panax ginseng C.A. Meyer) is a perennial herbaceous plant which grows very slowly. It takes about 3 to 4 years from seeding to collecting the ripe seeds and the ginseng propagation is very difficult. and so, it is very difficult to breed ginseng plant. Ginseng tissue culture was started from at 1960, and ginseng commercial product by in vitro callus culture was saled, however upto now, regenerants were not planted to soil normally. Recently, plant genetic engineering to produce transgenic plants by introducing useful genes has been advanced greatly. In a present paper, transformation of ginseng plants was achieved by co-cultivation with Agrobacterium harboring the binary vector coding Proteinase-II gene, which confer resistant or tolerant to insect pests, The binary vector for transformation was constructed with disarmed Ti-plasmid and with double 35S promoter. The NPT II gene and introduced genes of the transgenic ginseng plants were successfully identified by the PCR. Especially the transgenic ginseng plants were regenerated using new techniques such as repetitive single somatic embryogenesis.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.335-341
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• 2007

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1995년도 식물학심포지움 식물로부터 유용 2차대사산물의 생산 PRODUCTION OF USEFUL SECONDARY METABOLITES FROM PLANTS
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• pp.67-78
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• 1995

Monoclonal antibodies (MoAbs) are a highly diversified class of proteins with major research and commercial applications such as diagnostics and therapeutics. Currently, the dominant method for producing MoAbs is through the hybridoma technique. However, this technique is slow, tedious, labor intensive, and expensive. The production of MoAbs in cultured transgenic plant cells can offer some advantages over that in the over that in the mammalian systems. The media to cultivate plant cells are well defined and inexpensive. Contamination by bacteria or fungi is easily monitored in plant tissue cultures. Furthermore, these contaminants are usually not potent pathogens to human beings. In our interdisciplinary research efforts, heavy chain monoclonal antibody (HC MAb) was inserted into Ti plasmid vector and transferred into A. tumefaciens for the transformation in tobacco cells. It was found that 76% of the transformants produced HC MAb. The presence of HC MAb in the cell membrane fraction indicated that the signal peptide was functional and efficient. The change of the HC MAb concentration during a batch culture followed a similar trend as dry cell concentration, indicating that the production of HC MAb was growth related. The long-term repeated subcultures of 11 cell lines showed that there was no obvious trend of neither the decrease nor the increase of the productivity with the repeated subcultures.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 1994년도 Proceedings of International Symposium on BIOLOGICAL CONTROL OF PLANT DISEASES Korean Society of Plant Pathology
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• pp.11-26
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• 1994

Crown gall of stonefruit and nut trees is one of the very few plant diseases subject to efficient biological control. The disease is caused by the soil-inhabiting bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes and the original control organism was a non-pathogenic isolate of A. rhizogenes strain K84. Control is achieved by dipping planting material in a cell suspension of strain K84 which specifically inhibits pathogenic strains containing a nopaline Ti plasmid. Because the agrocin 84-encoding plasmid (pAgK84) is conjugative, it can be transmitted from the control strain to pathogenic strains which, as a result, become immune to agrocin 84 and cannot be controlled. To prevent this happening, the transfer genes on pAgK84 were located and then largely eliminated by recombinant DNA technology. The resulting construct, strain K1026, is transfer deficient but controls crown gall just as effectively as does strain K84. Field data from Spain confirm that pAgK84 can transfer to pathogenic recipients from strain K84 but not from strain K1026. The latter has been registered in Australia as a pesticide and is the first genetically engineered organism in the world to be released fro commercial use. It is recommended as a replacement for strain K84 to prevent a breakdown in the effectiveness of biological control of crown gall. Several reports indicate that both strains K84 and K1026 sometimes control crown gall pathogens that are resistant to agrocin 84. A possible reason for this is that both strains produce a second antibiotic called 434 which inhibits growth of nearly all isolates of A. rhizogenes, both pathogens and non-pathogens. Crown gall of grapevine is caused by another species, Agrobacterium vitis. It is resistant to agrocin 84 and cannot be controlled by strains K84 or K1026. It is different from other crown gall pathogens in several characteristics, including the fact that, although a rhizosphere coloniser, its also lives systemically in the vascular tissue of grapevine. Pathogen free propagating material can be obtained from tissue culture or, less surely, by heat therapy of dormant cuttings. A number of laboratories are searching for a biocontrol strain that will prevent, or at least delay, reinfection. A non-pathogenic A. vitis strain F/25 from South Africa looks very promising in this regard.

• Journal of Ginseng Research
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• 제10권1호
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• pp.45-54
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• 1986

인삼에 이용할 수 있는 vector system의 개발연구의 일환으로 우선 Agrobacterium spp.를 인삼의 잎, 줄기 및 뿌리에 접종하여 crown gall tumor의 형성 및 탈분화 그리고 Agrobacterium spp.의 opine화합물의 이용정도등을 조사하였던 바 그 결과를 요약하면 다음과 같다. 1. Agrobacterium tumefaciens C58은 인삼의 모든 부위에서 crown gall tumor를 형성하였으나 secondary tumor나 teratoma는 형성하지 못했다. 2. Wild type Agrobacterium tumefaciens Y101, Y104, Y109는 crown gall tumor를 형성하였으며, tumor의 형태, 크기 그리고 생장 정도는 strain별로 차이가 있었다. 3. Agrobacterium tumefaciens Y194는 특히 amorphic tumor를 형성하였다. 4. 줄기에서 형성된 tumor조직에서 callus를 유기하고자 phytohormone free배지 및 2,4-D 첨가 배지에 접종한 결과 전혀 callus가 형성되지 않았다. 5. 뿌리에서 형성된 callus가 형성되긴 하였으나 출현빈도가 극히 낮았으며, 정상 조직과는 달리 2,4-D의 효과가 미미하였다. 6. Agrobacterium spp.에 의한 opone화합물의 이용능력을 조사한 결과, Agrobacterium tumefacciens Y104, Y110 과 C58은 nopaline type이었고 Y109는 octopine type이었으며, Y101은 nopaline과 octopine 어느것도 이용하지 못하였다.

• Journal of Plant Biotechnology
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• 제42권4호
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• pp.409-413
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• 2015

본 연구는 토양에 오염된 중금속을 제거하기 위한 식물정화공정에 사용할 식물체를 개발하기 위해 $TgMTP_1$ 유전자를 CaMV35S 상시 발현 할 수 있도록 Ti-plasmid 벡터를 구축하여 형질전환식물을 육성하였다. 유전자가 도입된 형질전환 애기장대에서 TgMTP유전자를 과발현하는 호모 TG-116 (T3 generation) 계통을 육성하여 그 특성을 조사하였다. 호모계통으로 육성한 TG-116 계통은 callus 및 식물체에서 중금속에 대한 저항성을 보였다. 특히 RT-PCR 및 Western 분석에서 유전자의 발현은 잎에서 높게 나타났으며, Ni 흡수 및 축적이 많이 일어났다. 따라서 MTP1 유전자가 발현되어 액포에 중금속을 축적하는 실험결과를 활용한다면 식물정화공정에 사용할 수 있는 다양한 유전자원으로 기대할 수 있다.

• Journal of Applied Biological Chemistry
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• 제49권1호
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• pp.1-7
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• 2006

Differential display reverse transcription PCR (DDRT-PCR) analysis was performed on lily 'Marcopolo' bulb scale for isolation of expressed genes during bulblet formation. Cu/Zn lily-superoxide dismutase (LSOD) of 872 bp gene, with ability to scavenge reactive oxygen in stress environment, was isolated. Northern blot analysis showed expression levels of LSOD maximized 12 days after bulblet formation. Ti plasmid vectors were constructed with sense and antisense expressions of LSOD gene and transformed into potato. Southern blot analysis of transgenic potatoes revealed different copies of T-DNA were incorporated into potato genome. In transgenic potatoes, lily SOD gene was overexpressed in sense lines and not in antisense lines. In native polyacrylamide gel electrophoresis analysis, additional engineered LSOD was detected in sense overexpressed transgenic line only. Transgenic potatoes were subjected to oxidative stress, such as herbicide methyl viologen (MV). Transgenic potato lines with sense orientation exhibited increased tolerance to MV, whereas in antisense lines exhibited decreased tolerance. In vitro tuberization of transgenic potato with sense orientation was promoted, but was inhibited in transgenic potato with antisense orientation.