• Title/Summary/Keyword: LBA

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Characteristics of Resistance to Potato Virus Y in Transgenic Tobacco Plants Mediated with Complimentary DNA (cDNA) of PVY Replicase Mutant Genes

  • Chae, Soon-Yong;Park, Eun-Kyung;Kim, Young-Ho;Kim, Sang-Seock;Paek, Kyung-Hee
    • Journal of the Korean Society of Tobacco Science
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    • v.20 no.1
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    • pp.57-65
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    • 1998
  • This study was conducted to develop a resistant tobarro against Potato virus Y (PVY) by transformation of the plants with genetically engineered viral genes. The complimentary DNAs (cDNAS) of potato virus Y-necrosis strain (PVY-Vn) replicase mutant genes (3'-deleted, 5'-deleted and ADD-mutant Nlbs) were synthesized through RT-PCR by using purified PVY-VN RNA and synthesized primers, and cloned in the sense orientation into a plant expression vector (pMBPI), The cDNAS of the genes were transferred into Agrobacterium tumefaciens LBA 4404, and then transformed into tobacco (Nicotiana tabacum cv. Burley 21) plants. Regenerated plants were tested for PVY resistance by inoculation test; 13 transgenic plants including 7 for 3'-deleted Nlb, 3 for 5'-deleted Nlb, and 3 for ADD-mutant Nlb appeared to be resistant at 4 weeks after inoculation with PVY-VN. Among the 13 transgenic tobacco plants, 8 plants had no symptom up to 14 weeks after inoculation. The progenies ($T_1$) from self-fertilization of the transgenic lines varied 0.0% to 81.2% in their resistance (% of resistant plants). The analysis of Nlb-31deleted, -5'deleted and -ADD mutant in the $T_1$ plants by polymerase chain reaction (PCR) showed that Nlb-3'deleted, -5'deleted and -ADD mutants were detected in all of the resistant plants. These results suggest that the PVY resistance was inherited in the $T_1$ generation.

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Mycological Characteristics of Phytophthora nicotianae var. nicotianae Causing Phytophthora Rot of Strawberry and Resistance of Strawberry Cultivars to the Pathogen (딸기 역병균 Phytophthora nicotianae var. nicotianae의 균학적 특성 및 딸기 품종간 저항성)

  • 송주희;노성환;박현철;문병주
    • Korean Journal Plant Pathology
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    • v.14 no.6
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    • pp.646-650
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    • 1998
  • Mycological characteristics of Phytophthora nicotianae var. nicotianae SPC10 (A1 type) causing Phytophthora rot of strawberry and the resistances of 11 strawberry cultivars against the pathogen were examined. Optimum temperature for the mycelial growth of the pathogen was obtained in the range of 30~35$^{\circ}C$, and the growth was completely stopped under 13$^{\circ}C$ or over 42$^{\circ}C$. Aerial mycelia were abundant on oatmeal agar (OMA), V-8 juice agar (V8A) and lima bean agar (LBA) medium, although there were slight differences, however, on cornmeal agar (CMA) medium, it was a shape of stellate without aerial mycelia. The colony shape on potato dextrose agar (PDA) medium was rough and irregular whereas the mycelial growth was slow, and some aerial mycelia were only produced in the middle of PDA medium. Optimum temperature for sporangial formation was 3$0^{\circ}C$, and zoospores were mostly released at $25^{\circ}C$ from the sporangia. Sporangia were more produced in C/Z solution with pH 5. 0~6.$0^{\circ}C$ than sterilized distilled water (DSW) and distilled water (DW), and zoospores were also released much more than other solutions. Eleven strawberry cultivars such as Reiko, Hokowase, Eyeberry, Akaneko, Sistakara, Toyonoka, Nyoho, Sulhong, Suhong, Myhong and Wonkyo #3104 revealed the disease incidence up to 88.9~100% by the leaf inoculation with mycelial disk. However, Nyoho and Suhong showed higher level of resistance against the pathogen by root inoculation.

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Photosynthetic Characterization of Transgenic Tobacco Plant, by Transformation of Chlorophyll a/b Binding Protein Gene of Korean Ginseng (인삼의 Chlorophyll a/b Binding Protein유전자를 도입한 연초의 광합성 특성)

  • 이기원;채순용;김갑식;박성원;황혜연;이영복
    • Journal of the Korean Society of Tobacco Science
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    • v.23 no.2
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    • pp.109-114
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    • 2001
  • A CAB cDNA vector(pKGCAB), encoding the light harvesting chlorophyll a/b binding protein in Korean ginseng (Panax ginseng C. A. Meyer), was constructed with the CaMV35S promoter of plant expression vector. The chimeric vector was transformed into tobacco(Nicotiana tabacum cv. NC 82) using Agrobacterium tumefaciens LBA 4404 strain, and the transgenic tobacco plant CAB-TP2 was selected. Photosynthetic rates of the CAB-TP2 plant at before-flowering stage were increased about 20% under low irradiance conditions of quantum 100 and 500 $\mu$mol.m$^{-2}$ s$^{-1}$ , however, the rates were similar to those of NC 82 under quantum 1000 and 2000 $\mu$mol.m$^{-2}$ s$^{-1}$ conditions. The plants were germinating under low- or normal irradiance condition and the quantum yield of photosystem III were measured. The differences of the Fv/Em values between conditions were 0.07 and 0.01 in NC 82 and CAB-TP2, respectively. The mature leaves in the position 8-10 of the CAB-TP2 at before-flowering stage revealed l0% higher Fv/Fm values in range of 0.759 to 0.781 and 40% more chlorophyll contents of 70-93mg/$m\ell$ than those of normal NC 82. These data suggest the possibility that the increase in photosynthetic activity of leaves under low light intensity in the canopy of CAB-TP2 transgenic tobacco might lead to increase the quality of lower tobacco leaves.

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Genetic Transformation and Plant Regeneration of Codonopsis lanceolata Using Agrobacterium (Agrobacterium에 의한 더덕의 형질전환과 식물체 재분화)

  • 최필선;김윤성;유장렬;소웅영
    • Korean Journal of Plant Tissue Culture
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    • v.21 no.5
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    • pp.315-318
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    • 1994
  • To obtain transformed plants, we cocultured cotyledonary explants of Codonopsis lanceolata with Agrobacterium tumefaciens LBA4404, a disamed strain harboring a binary vector pBI121 carrying the CaMV35S promoter-$\beta$-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker in MS liquid medium with 1mg/L BA. After 48 h of culture, explants were transferred onto MS solid medium with Img/L BA, 250mg/L carbenicillin, and 100mg/L kanamycin sulfate and cultured in the dark. Numerous adventitious buds formed on the cut edges of the explants after 2 weeks of culture. When subjected to GUS histochemical assay buds showed a positive response at a frequency of 15%. Explants formed adventitious shoot at a frequency of 56.7%, after 6 weeks of culture. Upon transfer onto the basal medium, most of the shoots were rooted and subsequently the regenerants were transplanted to potting soil. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants.

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Development of transgenic disease-resistance root stock for growth of watermelon.(oral)

  • S.M. Cho;Kim, J.Y.;J.E. Jung;S.J. Mun;S.J. Jung;Kim, K.S.;Kim, Y.C.;B.H. Cho
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.65.2-65
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    • 2003
  • To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

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Ballistic Resistance Performance of Kevlar Fabric Impregnated with Shear Thickening Fluid (전단농화유체가 함침된 Kevlar 재료의 방탄특성)

  • Song, Heung-Sub;Yoon, Byung-Il;Kim, Chang-Yun;Park, Jong-Lyul;Kang, Tae-Tin
    • Composites Research
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    • v.20 no.3
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    • pp.1-7
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    • 2007
  • Manufacturing process of the shear thickening fluid(STF) and evaluation of the ballistic penetration resistance performance of the Kevlar-STF composites were studied. STF was made from silica and ethylene glycol, and the Kevlar-STF composite was made by impregnating the STF into the Kevlar fabric. Specimens including neat Kevlar woven fabrics and Kevlar-STF composites with two types of silica were prepared and carried out the ballistic tests. From the results STFs represented shear thickening behavior irrespective of the silica type, and Kevlar-STF composite with spherical silica showed best ballistic penetration resistance performance among them. Especially the specimens of Kevlar-STF composites with spherical silica showed radial fiber deformation by the projectile during the tests, that was somewhat different deformation behavior from those of the neat Kevlar fabrics shown fiber pull-out phenomena or fracture.

Plant regeneration and transformation of grape (Vitis labrusca L.) via direct regeneration method (포도 (Vitis labrusca L.)의 직접 재분화 방법을 이용한 식물체 재분화와 형질전환)

  • Kim, Se Hee;Shin, Il Sheob;Cho, Kang Hee;Kim, Dae Hyun;Kim, Hyun Ran;Kim, Jeong Hee;Lim, Sun-Hyung;Kim, Ki Ok;Lee, Hyang Bun;Do, Kyung Ran;Hwang, Hae Seong
    • Journal of Plant Biotechnology
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    • v.40 no.4
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    • pp.210-216
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    • 2013
  • Efficient regeneration methods and transformation system are a priority for successful application of genetic engineering to vegetative propagated plants such as grape (Vitis labrusca L.). This research is to establish shoot regeneration system from plant explants for 'Campbell Early', 'Tamnara', 'Heukgoosul', 'Heukbosek' using two types of plant growth regulators supplemented to medium. The highest adventitious shoot regeneration rate of 5% was achieved on a medium containing of Murashige and Skoog (MS) inorganic salts and Linsmaier and Skoog (LS) vitamins, 2 mg/L of TDZ and 0.1 mg/L of IBA. Leaf tissue of 'Campbell Early', was co-cultivated with Agrobacterium strains, LBA4404 containing the vector pBI121 carrying with CaMV 35S promoter, gus gene as reporter gene and resistance to kanamycin as selective agent, the other Agrobacterium strains, GV3101 containing the vector pB7 WG2D carrying with mPAP1-D gene. mPAP1-D is a regulatory genes of the anthocyanin biosynthetic pathway. 'Campbell Early' harboring mPAP1-D gene was readily able to be selected by red color due to anthocyanin accumulation in the transformed shoot. These results might be helpful for further studies to enhance the transformation efficiency in grape.

Transformation of Populus nigra × P. maximowiczii Using Agrobacterium tumefaciens vectors (Agrobacterium tumefaciens vector를 이용(利用)한 양황철의 형질전환(形質轉換))

  • Son, Suk Gyu;Hyu, Jung Oh
    • Journal of Korean Society of Forest Science
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    • v.87 no.2
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    • pp.164-172
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    • 1998
  • This study was conducted to find the optimum transformation condition using Agrobacterium harboring promoterless GUS gene. The optimal medium for shoot induction from leaves of Populus nigra${\times}$P. maximowiczii was MS medium supplemented with $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP(94% regeneration frequency and 11.5 average number of shoot) According to the test using pBI121, the concentration of antibiotics for selection marker gene was $100mg/{\ell}$ kanamycin or $60mg/{\ell}$ geneticin in the SIM(shoot inducing medium) 3. Two weeks later, callus was induced in the SIM 3 and this callus grew up to 0.5-1cm shoots after 6 weeks in the new SIM 3. And the treatment with methylation inhibitor(5-azacytidine) led to a dramatic increase in foreign gene expression rate from 5.7% to 26.7%. The vector systems showed. different transformation efficiencies based on the fluorometric and histochemical GUS assay. In this study the vector systems used for transformation seemed to affect transformation frequency, in which pEHA101 yielded more transformants(35.9%) than LBA4404/pBI121 did(5.7%). This result indicated that pEHA101 was effective to insert the promoterless foreign gene into a poplar genome.

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Distinct Spatio-temporal Expression Patterns of Patatin Promoter-GUS Gene Fusion in Transgenic Potato Microtubers (형질전환 감자 소괴경의 발달단계에 따른 Patatin Promoter-GUS 유전자의 발현 분석)

  • Youm, Jung-Won;Kim, Mi-Sun;Lee, Byoung-Chan;Kang, Won-Jin;Jeon, Jae-Heung;Joung, Hyouk;Kim, Hyun-Soon
    • Journal of Plant Biotechnology
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    • v.30 no.1
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    • pp.13-18
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    • 2003
  • This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.

Molecular Breeding of Tobacco Plants Resistant to TMV and PVY (분자생물학적 TMV 및 PVY 저항성 연초 육종)

  • E.K. Pank;Kim, Y.H.;Kim, S.S.;Park, S.W.;Lee, C.H.;K.H.Paik
    • Proceedings of the Korean Society of Tobacco Science Conference
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    • 1997.10a
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    • pp.134-152
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    • 1997
  • Plant viruses of tobacco including tobacco mosaic virus (TMV) and potato virus Y (PVY) cause severe economic losses in leaf-tobacco production. Cultural practices do not provide sufficient control against the viruses. Use of valuable resistant cultivars is most recommendable for the control of the viruses. However, conventional breeding programs are not always proper for the development of virus-resistant plants mostly owing to the frequent lack of genetic sources and introduction of their unwanted properties. Therefore, we tried to develop virus-resistant tobacco plants by transforming commercial tobacco cultivars, NC 82 and Burley 21, with coat protein (CP) or replicase (Nlb) genes of TMV and PVY necrosis strain (PVY-VN) with or without untranslated region (UTR) and with or without mutation. Each cDNA was cloned and inserted in plant expression vectors with 1 or 2 CaMV 35S promotors, and introduced into tobacco leaf tissues by Agrobacterium tumefaciens LBA 4404. Plants were regenerated in kanamycin-containing MS media. Regenerated plants were tested for resistance to TMV and PVY In these studies, we could obtain a TMV-resistant transgenic line transformed with TMV CP and 6 genetic lines with PVY-VN cDNAs out of 8 CP and replicase genes. In this presentation, resistance rates, verification of gene introduction in resistant plants, stability of resistance through generations, characteristics of viral multiplication and translocation in resistant plants, and resistance responses relative to inoculum potential and to various PVY strains will be shown. Yield and quality of leaf tobacco of a promising resistant tobacco line will be presented.

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