• Journal of Plant Biotechnology
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• 제34권4호
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• pp.293-298
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• 2007

Robinia pseudoacacia var. umbraculifera, commonly called umbrella black locust were regenerated after co-cultivation of internode segments with Agrobacterium tumefaciens which included yeast cadmium factor 1 (YCF 1) gene. The tolerance to cadmium and lead for plants can be increased by the YCF1 gene expression. Moreover, the recent studies have shown that YCF1 gene transgenic plants increase the accumulation of cadmium and lead into plant vacuoles. The effect of plant growth regulator such as 2,4-dichlorophenoxyacetic acid (2,4-D), ${\alpha}$-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron (TDZ) were studied to evaluate the propagation of plants through internode explants. The efficient induction of multiple adventitious shoots and callus were observed on a medium supplemented with 0.1 mg/L TDZ + 0.2 mg/L BA. To induce shoot elongation and rooting, regenerated shoots were transferred into basal MS medium without any plant growth regulator. Successful Agrobacterium tumefaciens mediated transformation was obtained by 20 min vacuum-infiltration with $50{\mu}M$ acetosyringone on the optimal multiple shoot induction medium with 30 mg/L hygromycin and 300 mg/L cefotaxime. To confirm the integration and expression of transgene, Polymerase Chain Reaction (PCR) and Reverse Transcriptase PCR (RT-PCR) were performed with specific primers. The frequency of transformation was approximately 18.94%. This study can be used to genetic engineering of phytoremediator.

• Journal of Plant Biotechnology
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• 제31권2호
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• pp.109-113
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• 2004

산화스트레스에 내성을 지닌 형질전환 감자 식물체를 개발하기 위하여 산화스트레스에 의해 발현이 강하게 유도되는 SWPA2 프로모터에 CuZnSOD와 APX 유전자가 엽록체에서 동시에 발현되도록 연결한 형질전환 벡터 (pSSA-K)를 제작한 후 Agrobacterium 매개로 형질전환 하였다. 기관 발생 경로에 의해 kanamycin 저항성 식물체를 재분화 시킨후 Southern 분석으로 외래 유전자가 안정적으로 감자 게놈내로 삽입되었음을 확인하였다. 형질전환 감자 식물체의 잎 조직에 10$\mu$M methyl viologen을 처리하여 산화스트레스 내성 검정을 조사한 결과 형질전환체는 MV에 대해 강한 내성을 지님을 확인하였다. 내성을 보인 개체 중에서 환경스트레스에 대한 내성 조사를 위하여 품종별로 2 개체씩 선발하였다. 선발된 식물체는 건조, 고온 등의 여러 가지 환경스트레스 내성검정에 이용될 것이며 향후 복합재해 내성 감자 품종이 개발될 수 있을 것으로 기대한다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.234-241
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• 2008

Agrobacterum tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with co-cultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.

• 한국산림과학회지
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• 제79권3호
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• pp.278-284
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• 1990

우리나라에서 육성(育成)한 교잡종(交雜種)인 양황철나무에 대해 A. tumefaciens strain 6044(pGA472)에 대한 감염력(感染力)을 조사(調査)하였다. 침으로 자극(刺戟)한 양황철나무 잎에서 callus유발은 MS-2, 4-D 0.5 mg/l-BA 0.1 mg/l 배지에서 높게 나타났으며, 줄기는 MS-2, 4-D 0.1 mg/l-BA 0.2 mg/l 배지에서 많이 발생하였다. 형질전환(形質轉換)에 사용(使用)한 A. tumefaciens strain 6044는 AB 액체배지에서 OD 0.5 일때 접종하였는데 이때 박테리아의 농도(濃度)는 1ml당 $4{\times}10^8$개 였다 기내배양(器內培養)한 양황철나무옆에 대한 kanamycin 감수성(感受性)을 조사(調査)한 결과(結果) 10 mg/l에서 심한 생장(生長) 장애(障碍) 현상(現象)을 나타내어 식물체(植物體) 재분화(再分化)가 일어나지 않았다. 침으로 자극한 양황철나무 잎은 A. tumefaciens 6044와 공배양(共培養)한 후 carbenicillin 300 mg/l와 cefotaxime 200 mg/l에서 엽표면에 붙어 있는 박테리아를 제거하였다. 공배양(共培養)한 잎은 kanamycin 10 mg/l가 첨가된 배지(培地)에서 재분화(再分化)되었으며, 줄기 재분화율(再分化率)은 약 10%에 달했다.

• 식물조직배양학회지
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• 제22권2호
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• pp.95-99
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• 1995

본 연구는 이미 확립되어 있는 고려인삼의 체세포배발생을 통한 식물체 재분화와 Agrobacterium을 매개로 한 형질전환 시스템을 이용하여 항곰팡이성 인삼을 개발하고자 염기성인 강낭콩 키틴가수분해효소 유전자를 인삼으로 도입하였다. CaMV 35S promoter-강낭콩 키틴가수분해효소 유전자와 선발표지로서의 neomycin phosphotransferase II (NPT II) 유전자를 가진 pChi/748 binary 벡터를 pGA748로부터 제조하여 이를 도입한 A. tumefacience LBA4404와 인삼 접합배의 자엽절편을 1 mg/L 24-D, 0.1 mg/L kinetin이 첨가된 MS 액체배지에서 48시간 동안 공동배양한 후 동일배지에 100 mg/L kanamycine 500 mg/L carbenicillin을 첨가한 고체 배지에 옮겨 배양하였다. 배양 한달 후부터 절편의 절단면 부근으로부터 캘러스가 유도되기 시작하였으며 이어서 수많은 체세포배가 형성되었다. 이들 체세포배를 BA와 GA3가 각각 1 mg/L 첨가된 배지로 옮겨서 5주 경과되었을 때 식물체로 전환되었다. 재분화된 개체 중 선발된 8개의 식물체로부터 PCR과 이 산물의 Southern분석 결과 6개의 재분화 개체에서 강낭콩 키틴가수분해효소 유전자가 도입되었음을 확인하였다.

• 한국초지조사료학회지
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• 제25권4호
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• pp.267-274
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• 2005

고온 내성 오차드그라스를 개발하기 위하여, 재조합 DgP23 유전자에 CaMV 35S 프로모터를 붙여 발현벡터를 제작, Agrobacterium 형질전환 방법으로 오차드그라스 형질전환체를 생산하였다. Genomic DNA를 분리하여 PCR 및 Southern blot 분석을 실시한 결과, PCR 분석에서 DgP23 유전자의 DNA band가 관찰되었고, Southern blot 분석에서도 X-ray film 상에 hybridization signal이 관찰되어, 오차드그라스 genome에 DgP23 유전자의 도입이 확인되었으며, wild type 및 empty vector control에서는 DNA band 및 hybridization signal이 관찰되지 않았다. 또한 RT-PCR 및 이들 산물의 Southern blot 분석 결과, DgP23 유전자의 정상적인 발현이 확인되었다. 형질전환 오차드그라스를 온실 및 포장에서 재배하며 생육특성을 조사한 결과, 비형질전환체와 비교하여 형태적 차이는 나타내지 않았다. 실험실 조건에서 고온내성을 조사한 결과, 고온내성이 확인되지 않았기 때문에 형질전환 종자를 생산하여 포장조건에서 고온내성을 검정할 계획이며, 재배시험에서는 내성이 강한 개체를 선발할 수 있을 것으로 예상된다.

• Journal of Plant Biotechnology
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• 제42권2호
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• pp.83-87
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• 2015

Genetic transformation was affected by material of explant, age of callus, and medium of regeneration. Two rice seed cultivars (Ilpum and Baekjinju) and mediums were investigated in this study for enhancing regeneration of transgenic rice expressed AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor. Regeneration rate of Ilpum rice transformant in gelrite of 5 and 8 g were 27.4% and 18.0%, respectively. In Baekjinju, regeneration rate of transformant was 5.4% and 4.3% in 5 and 8 g gelrite, respectively. The highest number of transformant plant in this study was regenerated from Ilpum cultivar on MS medium (30.4%) and was applied for the subsequent experiment. The callus regeneration rate of transformant were 40.7% in callus infection of up-side, it was higher regeneration then in the down-side (3.9%). The regeneration rate of callus of 25 days and 35 days were 14.7% and 38.6%, respectively. The most important application of this work is in genetic transformation of rice, particularly for improvement transgenic plant tissue culture protocol with high frequency of plant regeneration.

• Journal of Plant Biotechnology
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• 제43권2호
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• pp.255-260
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• 2016

This research was conducted to improve the cold tolerance of the gerbera cv. Gold Eye by introduction of the Arabidopsis $Ca^{2+}/H^+$ antiporter gene (CAX1) via Agrobacterium-mediated transformation. Prior to genetic transformation, we optimized a combination of plant growth regulators; $1.0mgl^{-1}$ 6-Benzyladenine (BA) and $0.1mgl^{-1}$3-indole-acetic acid (IAA) were found to lead to proper in vitro shoot regeneration from petiole explants. In addition, $50mgl^{-1}$ kanamycin was determined to be the minimal concentration useful for selection of putative transgenic plants. In this study, transgenic gerbera expressing the Arabidopsis $Ca^{2+}/H^+$ antiporter gene (CAX1) were obtained using the optimized concentrations. We expect that introduction of the gene to the cultivar will improve cold tolerance, which will be important in the winter months.

• Mycobiology
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• 제51권3호
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• pp.169-177
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• 2023

To further explore the molecular mechanism of triterpenoid biosynthesis and acquire high-value strain of Sanghuangporus baumii, the Agrobacterium tumefaciens-mediated transformation (ATMT) system was studied. The key triterpenoid biosynthesis-associated gene isopentenyl diphosphate isomerase (IDI) was transformed into S. baumii by ATMT system. Then, the qRT-PCR technique was used to analyze gene transcript level, and the widely targeted metabolomics was used to investigate individual triterpenoid content. Total triterpenoid content and anti-oxidant activity were determined by spectrophotometer. In this study, we for the first time established an efficient ATMT system and transferred the IDI gene into S. baumii. Relative to the wild-type (WT) strain, the IDI-transformant (IT) strain showed significantly higher transcript levels of IDI and total triterpenoid content. We then investigated individual triterpenoids in S. baumii, which led to the identification of 10 distinct triterpenoids. The contents of individual triterpenoids produced by the IT2 strain were 1.76-10.03 times higher than those produced by the WT strain. The triterpenoid production showed a significant positive correlation with the IDI gene expression. Besides, IT2 strain showed better anti-oxidant activity. The findings provide valuable information about the biosynthetic pathway of triterpenoids and provide a strategy for cultivating high-value S. baumii strains.

• Mycobiology
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• 제43권1호
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• pp.37-42
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• 2015

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.