• 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.

• Journal of Yeungnam Medical Science
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• 제2권1호
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• pp.175-181
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• 1985

Polyethylene glycol(PEG) 처리에 의한 Agrobacterium tumefaciens spheroplast와 연초 엽육 protoplast의 상호작용을 연구하기 위하여, 효소적 방법으로 분리한 연초엽육 protoplast와 carbenicillin 및 lysozyme의 처리에 의해 제조된 Agrobacterium tumefaciens ATCC 15955 spheroplast를 섞어서 polyethylene glycol (PEG) 및 high pH-high $Ca^{2+}$ buffer를 처리한 후 시료를 취하여 전자현미경으로 관찰한 결과, spheroplast는 초기 단계에서 protoplast membrane에 부착하고, 시간이 경과함에 따라 endocytosis에 의해 protoplast의 세포질 내부로 도입된 다음, 점차로 그 형체(cell integrity)가 파괴되어지는 것을 관찰할 수 있었다. 이러한 관찰 결과로부터 spheroplast는 polyethylene glycol(PEG)에 의해 protoplast내부로 endocytosis되어짐을 알 수 있었다.

• The Plant Pathology Journal
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• 제24권2호
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• pp.207-210
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• 2008

In March of 2003, tumors (galls) were observed on ginseng seedling roots in ginseng seedbeds at Yeoju, Gyeonggi province, Korea. Symptoms were spherical or galls with about 0.5-1.0cm in diameter formed on the upper through middle parts of the primary roots. Bacterial isolates obtained from the root galls were Gram-negative, rod-shaped with peritrichous flagella, aerobic, not forming yellow or orange colonies on nutrient glucose agar, yeast extract-dextrose $CaCO_3$ agar and nutrient-broth yeast extract agar, non-fluorescent on King's B agar, and non-spore forming, which were identical to characteristics of the genus Agrobacterium. They were identified as Agrobacterium tumefaciens with 0.732-0.993 similarities in 100% probability by the Biolog analyses. The 16S rRNA gene partial sequences of the six isolates tested (Genbank Accession EF486308-EF486313) were 100% homologous to those of other A. tumefaciens strains (GenBank accession AF501343, AY701900, AY701898, AY701899). The above results confirmed that this bacterium is A. tumefaciens. Pathogenicity of the bacteria was proved by the inoculation test on carrot root discs and tomato seedlings. This is the first description of A. tumefaciens causing root gall in ginseng seedling. The disease occurred locally and sparsely, but considering its appearances in seedbeds suggests that the ginseng root gall may become a threat to ginseng in Korea.

• Journal of Plant Biotechnology
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• 제6권2호
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Journal of Plant Biology
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• 제38권4호
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• pp.365-371
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• 1995

Binary vectors, pBI-ActR1, pBI-ActF1 and pBSH-ActR1, were constructed using pGA642, the replication origin of pTi12 and the rice actin promoter. The sizes of pBI-ActR1, pBI-ActF1 and pBSH-ActR1 were 12.9 kb, 13.2 kb and 11.95 kb, respectively. These vectors containing a rice actin promoter followed by a GUS structural gene could induce stronly the expression of GUS gene in transformed rice cells. Rice explants from 3-4 day old seedlings after germinatin were cocultured with A. tumefaceins harboring pBI-ActR1, pBI-ActF1 or pBSH-ActR1, and then GUS expression in the explants was assayed. Transformation of rice explants by these binary vectors was tissue-specific, such that the meristematic regions of shoot apex, root and hypocotyl were transformed by these binary vectors.

• Mycobiology
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• 제45권2호
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• pp.84-89
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• 2017

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

• Mycobiology
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• 제29권3호
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. 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 $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.

• 미생물학회지
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• 제4권2호
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• pp.1-4
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• 1966

As a part of studies of plant tumor induction, this experiment was prepared for the purpose of studying the ability of tumor induction and the tendency of tumor initiation in some Korean plants using the various Agrobacterium tumefaciens strains. Results obtained from this experiment are as follows. The virulences of five strains used in this experiment were gradually decreased in order of strain A6Kl, B6, 11BV7, T37 and 11 BNV6. Especially strain T37 which is known to the host limited strain showed virulent effect to the most of plants given for the materials as well as strain A6Kl, B 6 and 11BV7. Concerning the grade of tumor development, in plants which has tough stem, for example, Glycine max Meer, tumor induction was not well developed after the inoculation of all strains. Particullary in Ricinus communes Linne all strains showed virulent effect but tumor tissues were declined in relation to the development of lignification. It was also confirmed that the induction of tumor tissues on plants is to delay according to the increase of the age of host plants.

• Plant Resources
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• 제4권1호
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• pp.36-40
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• 2001

Transgenic Petunia hybrida cv. Rosanpion was produced by Agrobactepium tumefaciens LBA4404 harboring a binary vector pBI 121 containing $\beta$-glucuronidase (gus) and neomycin phosphotransferase (nptII). For genetic transformation, leaf discs were precultured on MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BA (MNB) for 2 days and cocultured for 15 mins with A. tumefaciens. For selection of transformant, leaf discs were transferred to fresh MNB containing 50 mg/L kanamycin and 500 mg/L cefotaxime. Eighteen plants were regenerated and four were confirmed by PCR for detection of gus and nptII gene integrated into the nuclear genome of petunia ‘Rosanpion’. Using this transformation system, we expect that transgenic petunia ‘Rosanpion’ incorporating a useful gene can be produced.

• 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.