• Molecules and Cells
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• 제39권9호
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• pp.705-713
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• 2016

The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

• 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.

• Journal of Plant Biotechnology
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• 제2권2호
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• pp.89-96
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• 2000

For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.

• Journal of Ginseng Research
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• 제27권1호
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• pp.37-42
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• 2003

인삼의 형질전환 시스템의 개발과 내병성관련 유전자의 도입에 관한 연구의 일환으로 soybean에서 cloning한 chitinase 유전자와 내병성 관련유전자(DR gene)가 식물에서 발현 및 형질전환될 수 있도록 운반체를 재조합하여 Agrobacterium을 이용해서 인삼에 형질전환시키고자 수행하였다. 식물세포에서 발현될 수 있는 promoter(35S-35S-AMV)에 내병성유전자인 DR-49 gene을 부착한 후 다시 식물형질전환용 binary vector에 도입하여 인삼에 도입되어 발현될 수 있도록 재조합하였으며, Disarmed Ti-plasmid 함유 Agrobacterium에 내병성유전자인 chitinase(pCH18)와 disease resistant gene(pDR)가 도입되었다. 인삼 callus 배양 과정을 거치지 않고 바로 multi-shoots를 생산하여 형질전환체 획득에 사용하고자 인삼 embryo와 petiole를 이용하여 direct shoots 생산을 유도한 결과 2,4-D 1 mg/ι와 kinetin 0.5 mg/ι 복합처리구에서 multi-shoot가 형성되었다. 또한 인삼 자엽을 이용한 형질전환에서도 MS 기본배지에 2,4-D 1 mg/ι와 kinetin 0.5 mg/ι를 첨가한 복합처리구에서 가장 효과적이었으며, dieases resistant와 chitinase 유전자에 의한 형질전환율은 각각 14%,그리고 18%를 나타내었다. 자엽으로부터 유기된 형질전환된 조직은 pre-embryoid 상태이기 때문에 성숙배의 과정을 거쳐 정상적인 shoot의 형성이 필요하였다.

• KSBB Journal
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• 제8권2호
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• pp.104-109
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• 1993

한 개의 pladsmid에 Gus gene과 Hygromycin resistant gene(Hpt)을 합친 vector를 개발하였다. 이 재조합된 DNA를 벼의 건조한 종자에 imbibition시켜 형질전환했을 때, hygromycin 배지에서 선별한 결과 그 발현율은 single Hpt vector와 차이 가 없었으며, 주로 뿌리의 생장점이나 자엽초에서 Gus expression을 볼 수 있였다. 또한 hygromycin 배지에서 선별되어 성체가 된 개체에서 그 genomic DNA를 뽑아 PCR을 한 결과 1Kb Hpt gene을 확인하였다. 그리고 생체에서 추출한 총 RNA에서 cDNA를 만든 후 reverse transcription PCR을 통하여, 외부 유전자의 발현을 증명하였다.

• Journal of Plant Biology
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• 제32권1호
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• pp.23-32
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• 1989

We have developed a plasmid vector, pKCH1, for the purpose of higher plant transformation. It contains the promoter region of cauliflower mosaic virus 35S transcript (P35s) and the terminator region of nopaline synthase gene (Tnos) with unique cloning sites, Bam HI and Xba I, between them. After inserting a foreing gene at the cloning sites, P35s-foreign gene-Tnos cassette can be recovered by using a restriction enzyme Hind III.

• 농업생명과학연구
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• 제45권6호
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• pp.175-181
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• 2011

Trichoderma harzianum 균사체를 $Al_2O_3$ 입자와 마찰시킴으로써 Agrobacterium을 이용한 형질전환에 효과적으로 이용할 수 있었다. Hygromycin 저항성 균사체 출현을 비교한 결과 형질전환 효율이 20% 정도로 나타났으며 대조군의 경우 형질전환균사체의 출현은 없었다. 2차례의 연속적인 항생제배지에서 선발을 거친 형질전환균사체들은 PCR에 의하여 안정적 DNA도입이 확인되었으며 RT-PCR에 의하여 target gene의 mRNA발현을 확인할 수 있었다. 현재까지 Agrobacterium을 이용한 T. harzianum 형질 전환은 보고된 바 없다.

• Mycobiology
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• 제31권1호
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• pp.42-45
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• 2003

For transformation of Pleurotus ostreatus, two novel vectors, pPhKM1 and pPhKM2, were constructed, using the regulatory sequences of the P. sajor-caju $\beta$-tubulin gene(TUB1) and the ble gene encoding phleomycin binding protein. pPhKM1 contains ble fused to the TUB1 promoter and the Schizophyllum commune GPD terminator. pPhKM2 contains ble fused to the promoter and terminator regions of P. sajor-caju TUB1. To confirm phleomycin-resistance activity, each vector was cotrans-formed with pTRura3-2 into the P. ostreatus homokaryotic $ura^-$ strain. The transforming DNA was stably integrated into the genomic DNA. Subsequently, phleomycin resistance was conferred on wild-type dikaryotic P. ostreatus by transformation with pPhKM1 or pPhKM2. This transformation system generated stable phleomycin-resistant transformants.

• 식물조직배양학회지
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• 제24권4호
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• pp.225-231
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• 1997

For genetic engineering to be commercially viable, an efficient transformation system is needed to produce transgenic plane from diverse genotypes ("generalized protocol"). Development of such a system requires optimization of a number of components such as gene transfer agent, plant tissues competent for both regeneration and transformation, and control of transgene expression. Although several novel gene transfer methods have been developed for plane, a majority of stably transformed plane express the introduced genes at low levels. Moreover, silencing of selectable marker genes shortly after their incorporation into plant chromosomes may result in low recovery of transgenic tissues from selection. Matrix attachment regions (MARs) are DNA sequences that bind to the cell's proteinaceous nuclear matrix to form DNA loop domains. MARs have been shown to increase transgene expression in tobacco cells, and reduce position in mature transgenic plants. Flanking an antibiotic resistance transgene with MARs should therefore lead to improved rates of transformation in a diversity of species, and may permit recalcitrant species and genotypes to be successfully transformed. Literature review and recent data from my laboratory suggest that MARs can serve as a transformation booster in recalcitrant plant species.

• 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.