• 한국초지조사료학회지
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• 제20권2호
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• pp.97-102
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• 2000

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants via Agrobacterium-mediated gene transfer system. Calli induced from scutella were co-cultivated with a A. tumefaciens strain EHAlOl canying a plasmid, pIGhsp21. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of Oshsp2l gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot and immunoblot analyses revealed that the Oshsp21 gene was constitutively expressed and accumulated as mature protein in transgenic plants. Effects of constitutive expression of the OshspZl on thermotolerance were first probed with the chlorophyll fluorescence. Results indicate that inactivation of electron transport reactions in photosystem I1 (PSII), were mitigated by constitutive expression of the Oshsp21. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery during heat stress. (Key words : Thermotolerance, Rice, Transgenic, cDNA)

• 한국초지조사료학회지
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• 제21권3호
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• pp.145-150
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• 2001

This study was conducted to obtain the transformed birdsfoot trefoil (Lotus corniculatus L.) plants with BcHSP17.6 gene using Agrobacterium turnefaciens LBA4404 and we confirmed transformed gene from the regenerated birdsfoot trefoil plants. The expression vector, pBKH4 vector, harboring BcHSP17.6 gene was used for production of transgenic birdsfoot trefoil plants. The callus of birdsfoot trefoil was cocultivated with Agrobacteriurn turnefaciens and transformed calli were selected on kanamycin-containing SH-kc medium to regenerate into plants. The transformed birdsfoot trefoil plants were produced 4 momths after cultivation on BOi2Y medium. The transgenic birdsfoot trefoil plants were analyzed by isolation of genomic DNA and genomic Southern hybridization using a -32P labelled BcHSPl7.6 fragments. (Key words : Birdsfoot trefoil, Transgenic plant. BcHSP17.6 gene, Callus induction, Plant regeneration)

• 한국초지조사료학회지
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• 제33권3호
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• pp.167-170
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• 2013

This study examined the growth performance and field evaluation of the dual herbicide-resistant transgenic creeping bentgrass plants. The effect of glyphosate treatment on the herbicide resistance of the transgenic creeping bentgrass plants was determined, and the non-transgenic control plant withered at the concentration $11{\mu}g/mL$ or higher whereas the transgenic creeping bentgrass plants survived the treatment at the concentration of $3,000{\mu}g/mL$, and the increase of the plant length was repressed as the glyphosate treatment concentration was increased. At field evaluation, glufosinate-ammonium and glyphosate were simultaneously treated to investigate the weed control effect. The results showed that more than 90% of the weeds withered four week after herbicide treatment, while the transgenic creeping bentgrass plants continued to grow normally. Therefore, the dual herbicide-resistant creeping bentgrass plants may be able to greatly contribute to the efficiency of weed control and to the economic feasibility of mowing in places such as golf courses.

• 한국초지조사료학회지
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• 제19권3호
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• pp.281-290
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• 1999

토양 미생물 A. tumefaciens로부터 cytokinin 합성효소 (ipt) 유전자를 분리하여 형질전환 식물을 작성하였다. 도입된 ipt 유전자는 모든 조직에서 발현되었으며, 잎에서의 발현량이 서로 다른 3개의 형질전환 식물체를 선발하였다. 선발한 식물체는 도입유전자의 고정을 위하여 2회 자가수정을 거쳐 homozygous 식물체를 작성한 후 해석하였다. 형질전환 식물체는 노화가 지연되어 녹엽을 유지하였으며, 측아로부터 측지가 분얼하여 그 수가 증가하였을 뿐만아니라, 각 마디에서 복수의 본엽이 생성되었다. 식물체의 노화가 가장 많이 진행된 잎에서의 chlorophyll 함량은 형질전환 식물체에서 1.5~4배 정도로 wild-type에 비하여 월등히 높았다. 이러한 결과는 축적된 cytokinin이 식물의 노화를 지연시킬 뿐만 아니라, 분얼 및 잎수를 증가시켜 식물의 수량을 증가시켰음을 나타낸다.

• Asian-Australasian Journal of Animal Sciences
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• 제25권6호
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• pp.818-823
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• 2012

Tall fescue (Festuca arundinacea Schreb.) is an important cool season forage plant that is not well suited to extreme heat, salts, or heavy metals. To develop transgenic tall fescue plants with enhanced tolerance to abiotic stress, we introduced an alfalfa Hsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by polymerase chain reaction, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. The reduced accumulation of thiobarbituric acid reactive substances indicates that the transgenic plants possessed a more efficient reactive oxygen species-scavenging system. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic tall fescue and may be useful in developing stress tolerance in other crops.

• 한국초지조사료학회지
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• 제21권1호
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• pp.21-26
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• 2001

환경 스트레스에 의해 야기되는 활성 산소종에 의한 피해에 내성을 가지는 목초의 개발을 위하여 오차드그래스의 배반 조직 유래의 캘러스에 배추유래의 cytosolic glutathione reductase 유전자(BcGRl)를 Agrobucterium tumefaciens EHA101을 매개로 형질전환시켰다. Hygromcin으로 선발된 캘러스로부터 재분화된 식물체는 야생형과 비교하여 형태적으로 차이를 나타내지 않았다. PCR 및 Southern blot 분석을 통하여 형질전환 식물체의 염색체 내에 BcGRl 유전자가 integration 되었음을 확인하였다. 오차드그래스의 잎으로부터 total RNA를 분리하여 Northern blot 분석을 실시한 결과, 도입된 유전자가 형짙전환 식물체 내에서 지속적으로 발현된다는 것을 확인하였다.

• 한국초지조사료학회지
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• 제34권3호
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• pp.187-192
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• 2014

Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world. It has been demanded to establish the efficient transformation system in commercial varieties of alfalfa for forage molecular breeding and production of varieties possessing new characteristics. To approach this, genetic transformation techniques have been developed and modified. This work was performed to establish conditions for effective transformation of commercial alfalfa cultivars, Xinjiang Daye, ABT405, Vernal, Wintergreen and Alfagraze. GUS gene was used as a transgene and cotyledon and hypocotyl as a source of explants. Transformation efficiencies differed from 0 to 7.9% among alfalfa cultivars. Highest transformation efficiencies were observed in the cultivar Xinjiang Daye. The integration and expression of the transgenes in the transformed alfalfa plants was confirmed by polymerase chain reaction (PCR) and histochemical GUS assay. These data demonstrate highly efficient Agrobacterium transformation of diverse alfalfa cultivars Xinjiang Daye, which enables routine production of transgenic alfalfa plants.

• 한국초지조사료학회지
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• 제26권1호
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• pp.1-8
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• 2006

조사료로서 소화율을 향상시킨 신품종 형질 전환 오차드그래스를 개발할 목적으로 lignin 합성경로에 있어서 중요한 효소 유전자 중의 하나인 COMT 유전자를 cloning하여 그 특성을 해명하였다. 오차드그래스의 COMT 유전자는 식물체의 전 조직에서 발현되고 있었으며, 특히 줄기와 뿌리조직에서 높은 발현량을 나타냄으로서 목질화에 크게 관여하는 lignin 생합성 유전자일 것으로 판단되었다. Dgcomt 유전자의 발현을 억제시킨 형질전환 오차드그래스를 개발하기 위하여 Dgcomt 유전자를 RNAi 발현벡터에 도입한 후, Agrobacterium 형질전환시스템을 이용하여 오차드그래스에 도입하였다. PCR, Southern 및 Northern 분석 결과 RNAi 발현벡터가 genome에 도입되었으며, Dgcomt 유전자의 발현이 상당한 수준으로 저하되었음을 확인하였다. Dgcomt 유전자의 발현억제는 식물체의 목질화와 더불어 증가되는 lignin의 축적량을 감소시킬 것으로 기대되며 향후 소화율이 증가된 고품질 신품종 목초의 개발에 유용하게 활용될 것으로 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.33-40
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• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.425-441
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• 2010

Forages are the backbone of sustainable agriculture. They includes a wide variety of plant species ranging from grasses, such as tall fescue and bermudagrass, to herbaceous legumes, such as alfalfa and white clover. Abiotic stresses, especially salinity, drought, temperature extremes, high photon irradiance, and levels of inorganic solutes, are the limiting factors in the growth and productivity of major cultivated forage crops. Given the great complexity of forage species and the associated difficulties encountered in traditional breeding methods, the potential from molecular breeding in improving forage crops has been recognized. Plant engineering strategies for abiotic stress tolerance largely rely on the gene expression for enzymes involved in pathways leading to the synthesis of functional and structural metabolites, proteins that confer stress tolerance, or proteins in signaling and regulatory pathways. Genetic engineering allows researchers to control timing, tissue-specificity, and expression level for optimal function of the introduced genes. Thus, the use of either a constitutive or stress-inducible promoter may be useful in certain cases. In this review, we summarize the recent progress made towards the development of transgenic forage plants with improved tolerance to abiotic stresses.