• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• The Plant Pathology Journal
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• 제25권2호
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• pp.179-183
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• 2009

To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2002년도 총회 및 추계학술발표회
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• pp.85.1-85
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• 2002

• Journal of Microbiology
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• 제44권4호
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• pp.383-395
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• 2006

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 $transformants/{\mu}g$ DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 $transformants/l0^7$ spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transform ants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

• Journal of Plant Biotechnology
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• 제39권1호
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• pp.1-12
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• 2012

With the increasing advances in Brassicaceae genetics and genomics, considerable progress has been made in the transformation of Brassicaceae. Transformation technologies are now being exploited routinely to determine the gene function and contribute to the development of novel enhanced crops. $Agrobacterium$-mediated transformation remains the most widely used approach for the introduction of transgenes into Brassicaceae. In $Brassica$, the transformation relies mainly on $in$ $vitro$ transformation methods. Nevertheless, despite the significant progress made towards enhancing the transformation efficiencies, some genotypes remain recalcitrant to transformation. Advances in our understanding of the genetics behind various transformations have enabled researchers to identify more readily transformable genotypes for use in routine high-throughput systems. These developments have opened up exciting new avenues to exploit model $Brassica$ genotypes as resources for understanding the gene function in complex genomes. Although many other Brassicaceae have served as model species for improving plant transformation systems, this paper summarizes on the recent technologies employed in the transformation of both $Arabidopsis$ and $Brassica$. The use of transformation technologies for the introduction of desirable traits and a comparative analysis of these as well as their future prospects are also important parts of the current research that is reviewed.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 한국생물과학협회 학술발표대회
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• pp.190.1-190
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• 2001

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• 한국균학회지
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• 제25권3호통권82호
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• pp.233-237
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• 1997

여름느타리버섯의 단포자에 UV를 처리하여 pab 요구성 영양요구주를 작성하였으며 이 균의 균사에서 원형질체를 분리한 후 Coprinus pab 1유전자를 함유하는 plasmid를 이용하여 prototrophy로 형질전환 하였다. 형질전환율은 ${\mu}g$의 DNA 당 5개의 형질전환주를 얻을 수 있었다. 형질전환주는 Southern 분석결과 염색체 DNA 속으로 integration된 것으로 확인되었으며 영양생장과 생식 생장시 모두 안정하게 유지되었다. 형질전환주와 화합성인 다른 영양요구주와 교배후 자실체의 포자를 분리하여 유전분석 결과 pab 생합성 유전자 보다는 그 유전자 주위에 integration이 일어난 것으로 추정되었다.

• Plant Biotechnology Reports
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• 제2권2호
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• pp.93-112
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• 2008

Medicinal and aromatic plants (MAPs) are important sources for plant secondary metabolites, which are important for human healthcare. Improvement of the yield and quality of these natural plant products through conventional breeding is still a challenge. However, recent advances in plant genomics research has generated knowledge leading to a better understanding of the complex genetics and biochemistry involved in biosynthesis of these plant secondary metabolites. This genomics research also concerned identification and isolation of genes involved in different steps of a number of metabolic pathways. Progress has also been made in the development of functional genomics resources (EST databases and micro-arrays) in several medicinal plant species, which offer new opportunities for improvement of genotypes using perfect markers or genetic transformation. This review article presents an overview of the recent developments and future possibilities in genetics and genomics of MAP species including use of transgenic approach for their improvement.

• Molecules and Cells
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• 제41권5호
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• pp.413-422
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• 2018

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.