• Journal of Plant Biotechnology
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• 제7권4호
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• pp.219-223
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• 2005

We investigated chemical-wounding effect on Agrobacterium-mediated Chinese cabbage transformation via vacuum infiltration. Pre-germinated or germinating Chinese cabbage seeds were infiltrated with Agrobacterium tumefaciens LBA4404 cells carrying either GUS gene (pBI121) or hepatitis B virus surface antigen DNA (pBIHBsAg). Prior to agroinfiltration process, the seeds were soaked in sodium hydrosulfite (SHS) solution or just in sterile water as a control. Comparative transformation efficiency was determined by both of histochemistry and ELISA. We could demonstrate that SHS solution treatment especially to 1-day or 2-days old germinating seeds efficiently improved transformation process, and therefore, transient expression level. This strongly indicated that Agrobacterium infection could be facilitated indeed by SHS-causing wounds on Chinese cabbage seeds.

• Mycobiology
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• 제34권2호
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• pp.104-107
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• 2006

Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.683-688
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• 2005

A transformation system mediated by Agrobacterium tumefaciens is routinely used for the genetic engineering of plants. Here, we report an efficient and stable method for transformation of heterogeneous genes into an industrial Cephalosporium acremonium by using a similar transformation system established in plants. Both the phleomycin-resistant gene and vgb gene were used as screening markers to confirm the success of transformation by either Southern hybridization or PCR amplification. It was found that acetosyringone (AS) was necessary only for protoplast transformation and the heterogeneous genes transferred were integrated into the genome of C. acremonium. The transformation efficiency obtained with this system was much higher than the conventional techniques used for transformation of C. acremonium.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.235-243
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• 2011

The non-edible plant Jatropha curcas L. is one of the most promising feedstock for sustainable biodiesel production as it is not a source of edible vegetable oils, produces high amounts of oil (approx. 30-60% in dry seeds) and does not require high-cost maintenance. However, as with other undomesticated crops, the cultivation of J. curcas presents several drawbacks, such as low productivity and susceptibility to pests. Hence, varietal improvement by genetic engineering is essential if J. curcas is to become a viable alternative source of biodiesel. There is to date no well-established and efficient transformation system for J. curcas. In this study, we tested various physical wounding treatments, such as sonication and sand-vortexing, with the aim of developing an efficient Agrobacterium-mediated transformation for J. curcas. The highest stable transformation rate (53%) was achieved when explants were subjected to 1 min of sonication followed by 9 min of shaking in Agrobacterium suspension. The transformation frequency achieved using this protocol is the highest yet reported for J. curcas.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.185-190
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• 2004

Agrobacterium을 이용한 GUS 유전자를 효과적으로 발현시키기 위하여 수행되어진 실험 결과를 요약하면 다음과 같다. 박테이라 농도별 실험을 수행한 결과 균의 전처리 배양 농도 O $D_{600nm}$ 0.3일때 원심분리한 후 얻어진 균을 희석한 후의 최종 농도는 O $D_{600nm}$ 0.8로 맞춘 실험 처리구에서 GUS 유전자 발현율이55%로 가장 높게 나타났다. 병원성 유도 배지 내에 Acetosyringone (AS)이 첨가되지 않은 경우 GUS 유전자가 발현된 고추 잎을 얻을 수 없었으나, 200$\mu$M을 첨가했을 때 90%의 가장 높은 GUS 유전자 발현율을 나타내어 많은 수의 GUS spots을 관찰할 수 있었다. Agrobacterium에 의한 고추 잎의 감염 정도를 조사한 바 Agrobacterium으로 감염시킨지 3일째부터는 박테리아 에한 감염 정도가 심해져서 GUS 유전자 발현 정도가 약해지므로 Agrobacterium으로 감염시킨지 2일째 되었을 때 GUS 유전자 발현이 가장 강하게 나타난 것을 확인하였다. 이 같은 결과는 박테리아에 의한 감염이 일어난지 3일째 부터는 식물체의 감염부위 고사를 일으키는 것과 관련된 것으로 보인다.

• 한국육종학회지
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• 제40권2호
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• pp.128-135
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• 2008

Herbicide resistance is the most common trait being tested and thus herbicide?resistant genetically modified plants are now the most widely cultivated worldwide. Here we developed herbicide?resistant transgenic Agrostis mongolica Roshev. by employing an efficient Agrobacterium?mediated transformation procedure with 25.2% of transformation efficiency. The identification and employment of regenerable and reproducible type of callus was one of the most critical factors to ensure success in this study. PCR analysis confirmed that the bar transgene was integrated into the genome of transgenic plants. The expression of 35S?bar gene was confirmed by Northern blot analysis. The transgenic plants showed complete resistance to herbicide, indicating that the bar gene is functional in transgenic plants.

• 농업생명과학연구
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• 제44권1호
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• pp.9-15
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• 2010

단자엽 식물인 보리는 Agrobacterium을 이용한 형질전환이 비교적 까다로운 편이다. 본 연구에서는 큰알1호, 내쌀보리, 올보리, 새찰쌀보리, 서둔찰보리, 풍산찰쌀보리의 유묘에 알칼리, 산화제, 환원제 등을 처리하여 화학적 상처를 유발하였으며 이들에 감압진공을 이용한 Agrobacterium 형질전환을 실시한 후 GUS 유전자발현을 분석하였다. 그 결과, 보리묘 생육을 일부 저하시킬 수 있는 농도의 화학물질 처리는 각기 다른 보리 품종의 형질전환율을 전반적으로 증대시킬 수 있는 것으로 판단되었다. 화학물 중에서는 특히 hydrogen peroxide 처리가 비교적 우수한 것으로 나타났다.

• 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.

• Journal of Plant Biotechnology
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• 제35권2호
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• pp.109-114
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• 2008

Agrobacterium 공동배양을 적용하여 소포자에 Agrobacterium-mediated genetic transformation이 가능한지를 조사하기 위하여 소포자 형질전환에 대한 조건을 구축하고자 하였다. 선발 배지에서 Kanamycin에 대한 소포자의 배발생율을 조사하였는데 Kanamycin 농도 10 mg/L, 50 mg/L, 100 mg/L를 처리하였을 때 배발생율이 각각 4배, 8배, 10배 이하로 감소하였고 Kanamycin이 형질전환 선발마커로 사용할 경우 형질 전환율이 매우 낮아질 것으로 사려 된다. GFP 유전자 발현을 이용하여 visual reporter로서 활용하고자 Agrobacterium과 공동배양 후 배발생 유도 배지에 치상하고 소포자가 배로 발생하는 과정을 현미경으로 조사하면서 GFP 발현을 관찰하였다. 소포자는 배양 12일째서부터 소포자 분열로 cluster를 형성하였으며 24일째에는 반복되는 분열을 통하여 큰 mass의 배로 발달된 모습을 각각 GFP 발현을 통해 볼 수 있었다. 배양48일째도 GFP발현이 계속 보이며 총 8 개의 배에서 GFP 발현 재현성을 보임으로서 형질전환은 된 것으로 보이지만 더 이상 성숙된 배로 자라지 않아 소포자를 이용한 Agrobacterium 형질전환 조건을 더 개선할 필요가 있다.

• 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.