• Journal of Plant Biotechnology
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• 제43권2호
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• pp.240-247
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• 2016

In this study, routinely used transformation method, which includes transferring explants onto co-cultivation medium after inoculating them with bacterial solution for a while, was compared with 3 different inoculation methods. In every 3 methods, hypocotyl explants excised from 7-day-old sterile flax seedlings having cotyledon leaves and no root system dried under air flow in sterile cabin for 35 min were inoculated with different volumes of bacterial solution at different inoculation periods. GV2260 line of Agrobacterium tumefaciens having 'pBIN 19' plasmid containing npt II (neomycin phosphotransferase II) gene and GUS reporter gene was used in transformation studies. After inoculation, hypocotyl segments of seedlings (0.5 cm in length) - were excised and left to co-cultivation for 2 days. Then, explants were transferred to regeneration medium supplemented with different antibiotics. The presence of npt-II and GUS genes in transformants was confirmed by PCR and GUS analysis. The highest results in all characters examined in all cultivars were obtained from the 2 inoculation method in which hypocotyls excised from seedlings inoculated with $500{\mu}l$ of bacterial solution after drying in sterile cabin for 35 min were used.

• Molecules and Cells
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• 제19권1호
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• pp.16-22
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• 2005

A cDNA encoding ${\gamma}-tocopherol$ methyltransferase (${\gamma}-TMT$) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce ($T_0$) containing the ${\gamma}-TMT$ transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and ${\gamma}-TMT$ activities. The ratio of ${\alpha}-/{\gamma}-tocopherol$ content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the $T_0$ plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 $T_1$ progenies of the $T_0$ transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and ${\gamma}-TMT$ activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in $T_2$ progenies of $T_1$ plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis ${\gamma}-TMT$ transgene in lettuce results in a higher enzyme activity and the conversion of the ${\gamma}-tocopherol$ pool to ${\alpha}-tocopherol$ in transgenic lettuce.

• 한국자원식물학회지
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• 제27권3호
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• pp.242-248
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• 2014

This study was conducted to establish an efficient transformation system by using somatic embryogenesis in an important Korean native conifer, Korean fir (Abies koreana). Embryogenic masses were induced from mature zygotic embryos of the Korean fir on Schenk and Hildebrandt medium, which was supplemented with thidiazuron. For genetic transformation, the embryogenic masses were co-cultivated with a disarmed Agrobacterium tumefaciens strain C58/pMP90 containing the plasmid vector pBIV10 or LBA4404 containing the plasmid vector MP90. Both vectors contain the kanamycin resistance and beta-glucuronidase (GUS) reporter genes. A total of 48 lines of embryogenic masses were selected on mLV medium containing $50{\mu}g/mL$ of kanamycin after 4 weeks of culture, following 3 days of co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 (none of the lines was cultivated with strain LBA4404 carrying MP90). Quantitative real-time PCR was performed, and high levels of GUS transcripts were observed in the 48 putative transgenic lines; however, the control (non-transgenic line) showed negative results. Results of histochemical staining showed that the expression of the GUS reporter gene was observed in somatic embryos that developed from the embryogenic masses of all 48 lines. Stably transformed cultures were successfully produced by co-cultivation with A. tumefaciens strain C58/pMP90 carrying pBIV10 in Korean fir. Here, we have reported an Agrobacterium-mediated gene transfer protocol via somatic embryogenesis that may be helpful in developing breeding and conservation strategies for the Korean fir.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.157-161
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• 2003

Proline is known as an osmoprotectant accumulating in response to salt and dehydration stresses. An increased level of proline is achieved by either an induced synthesis or a reduced degradation of proline. In an attempt to increase salt tolerance in carrot, a P5CS gene from mothbean was introduced via an Agrobacterium-mediated transformation. The resulting carrot cells and the regenerated plants containing the transgene showed increased levels of proline compared to nontransgenics. The transgenic cell line, Pj2 showed about 6 times increased degree of tolerance determined by relative growth after a treatment in 250 mM NaCl. In facts, due to the retarded growth shown in non-saline condition, Pj2 cells grow only about 1.2 times better than nontransgenic control under salt stress condition. Taken together, it appears that a P5CS is a key enzyme in proline biosynthesis and the increased accumulation of proline by overexpression of the enzyme is enough to enhance tolerance to salt stress in carrot.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2001년도 춘계심포지움 및 학술발표회
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• pp.129-129
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• 2001

1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant against nematodes, is a genotoxic carcinogen and also is classified by World Wildlife Fund as endocrine disruptors. DBCP has been extensively studied on genotoxicity, carcinogenicity, and damage in male reproductive-related organs. However, information on precise mechanism of mutagenesis and carcinogenesis of DBCP is yet unknown. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• 제7권3호
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• pp.427-434
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• 1994

The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.

• Journal of Plant Biotechnology
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• 제38권1호
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• pp.15-21
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• 2011

Agrobacterium tumefaciens와 운반체 416을 이용하여 cryIAc1 유전자가 형질전환된 배추 14개체를 선발하였다. Southern blot을 통하여 분석한 결과 1 사본 유전자가 도입된 개체가 6개이었으며 backbone 염기서열이 포함되지 않은 경우는 4개체이었다. LB 인접서열 분석 결과, 416-2과 416-3은 23 bp의 LB 부위가 남아있는 동일한 염기서열을 보였고, 416-9 경우 15 bp가 남았으며, 416-17 경우 LB를 포함하여 안쪽의 염기서열의 최대 36 bp의 결실이 확인되었다. T-DNA의 염기서열을 제외한 배추 gDNA의 499 bp의 LB 인접염기서열은 B. oleracea의 염기서열과 96% 상동성을 가진 유전자로 확인되었다. RB border 인접서열 분석용 primer를 제작하여 PCR을 수행한 결과 모두 834 bp의 염기서열을 확인하였고, vector의 구성 요소 중 cryIAc1의 3' 말단 부위, nos-terminator 부위와 52 bp 및 배추 gDNA RB 인접염기서열로 확인되었다. RB 염기서열은 확인할 수 없었으며 nos-terminator 3' 말단의 21개의 염기를 포함하여 모두 58개의 염기서열이 결실된 것을 확인하였다. 형질전환식물체를 제작할 경우 발생하는 전이유전자나 식물의 염색체에 염기 결실은 매우 다양한 형태로 나타난다. 이번 실험의 경우, 전이유전자가 삽입된 위치에서 약 10 bp의 배추의 gDNA 염기가 결실된 것이나 삽입된 전이유전자의 RB 말단부분이 결실된 것은 예상할 수 있는 결과였지만, 특히 전이유전자의 말단인 nos-terminator 3' 끝에 65 bp 정도의 배추의 다른 유전자가 삽입되어 있다는 것을 확인하였고 이는 기대하지 않았던 결과였다. 삽입된 다른 배추유전자의 절편은 앞으로 더 많은 연구를 통하여 위치와 기능확인이 필요할 것이다.

• 원예과학기술지
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• 제34권1호
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• pp.154-162
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• 2016

유전자 집적에 의해 아황산가스 저항성 증진 페튜니아를 개발하고자 비생물적스트레스 저항성 유전자로 널리 알려진 SOD2와 NDPK2유전자가 각각 도입된 SOD2 형질전환 계통 SOD2-2-1-1-35($T_4$)[S($T_4$)]와 NDPK2 형질전환 계통 NDPK2-7-1($T_2$)[N7-1($T_2$)]간 상호교잡을 실시하여 교잡 제1세대를 획득하였다. 교잡 제1세대 중 SOD2와 NDPK2유전자가 모두 집적된 개체 획득율은 32.1-73.0%이었고, SOD2와 NDPK2유전자가 모두 집적된 개체는 SOD2 또는 NDPK2유전자가 단독 도입된 개체에 비해 아황산가스 30ppm처리 피해율이 2.6-5.1배 낮아 아황산가스 저항성이 증진되었음을 확인하였다. 또한 아황산가스 저항성이 증진된 교잡 제1세대를 자가수분에 의해 세대진전 시켜 획득한 교잡 제2세대의 아황산가스 저항성 검정을 통하여 교잡 제1세대에서의 아황산가스 저항성이 후대에서도 안정적으로 발현함을 확인하였다. 또한, 광합성 효율 증진 조사를 통해 아황산가스 저항성이 증진된 SOD2와 NDPK2유전자 집적 후대가 아황산 가스에 대한 스트레스를 덜 받고 있음을 확인하였고, RT-qPCR분석을 통해 SOD2와 NDPK2유전자 집적 후대의 아황산가스 저항성 증진이 SOD2와 NDPK2 유전자 집적에 의한 효과임을 확인하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제25권5호
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• pp.621-628
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• 2012

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000$^{TM}$. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.

• Weed & Turfgrass Science
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• 제4권1호
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• pp.44-48
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• 2015

형질전환 CPPO06 이벤트 벼를 이용하여 담수직파 재배에서 oxyfluorfen의 적정 처리시기 및 처리량에 대한 연구를 수행하였다. 모본인 동진벼는 10 g a.i. $ha^{-1}$의 농도에서 생육이 억제되었으나, CPPO06 이벤트 벼는 oxyfluorfen 10,000 g a.i. $ha^{-1}$의 농도에서도 생육억제를 보이지 않아 높은 oxyfluorfen 저항성을 보이는 것으로 나타났다. Oxyfluorfen을 써레질 직후와 파종 직후 처리한 결과 가래를 제외한 모든 발생 초종의 방제효과가 뛰어났으며, 파종 5일 후의 경우는 가래를 포함하여 모든 발생 초종이 95% 이상 방제되었다. Oxyfluorfen의 처리시기와 처리량에 따른 생육저해 현상은 없었으며, 안정적인 수확량을 확보할 수 있었다. 담수직파재배에서 oxyfluorfen 처리시기 및 처리량에 따른 CPPO06 이벤트 벼의 안전성 및 잡초방제 효과로 볼 때 담수직파재배면적의 확대가 기대된다.