• Title/Summary/Keyword: NPT II

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DNA-mediated gene transfer in plant protoplasts (식물 원형질체에서의 marker gene 삽입)

  • U, Zang-Kual;Riu, Key-Zung;So, In-Sup;Hong, Kyung-Ae
    • Applied Biological Chemistry
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    • v.36 no.6
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    • pp.557-561
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    • 1993
  • The neomycin phosphotransferase II gene (nptII) was introduced into geranium (Pelargonium zonale hybrids) protoplast by using PEG or electroporation method. The presence of the introduced DNA in the protoplast and the expressions of the gene in the transformed cells were examined. The presence of the nptII DNA in the protoplasts were detected by polymerase chain reaction. The expressions of nptII gene in the transformed cells were confirmed by the nptII assay.

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Organ Specific Expression of the nos-NPT II Gene in Transgenic Hybrid Poplar (형질 전환된 포플러에 대한 nos-NPT II 유전자의 기관별 발현 특성)

  • Chun, Young Woo;Klopfenstein, Ned B.
    • Journal of Korean Society of Forest Science
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    • v.84 no.1
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    • pp.77-86
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    • 1995
  • To effectively modify tree function with genetic engineering, transgenes must be expressed at the proper level in the appropriate tissues at suitable developmental stages. Toward understanding the spatial and temporal expression of transgenes in woody plants, transgene expression was evaluated in three greenhouse-grown, transgenic lines of Populus alba ${\times}$ P. grandidentata hybrid clone 'Hansen'. All transgenic poplar lines possess constructs containing the bacterial nopaline synthase(nos) promoter linked to a neomycin phosphotransferase II(NPT II) selectable marker gene. In addition, each transgenic poplar line contains one of the following gene constructs : 1) a wound-inducible potato proteinase inhibitor II (pin2) promoter linked to a chloramphenicol acetyltransferase(CAT) reporter gene. 2) a nos promoter linked to a PIN2 structural gene : or 3) a Cauliflower Mosaic Virus 35s promoter linked to a PIN2 structural gene. Polymerase chain reaction(PCR) was used to verify the presence of foreign genes in the poplar genome. Enzyme-linked immunosorbent assays(ELISAs) were used to evaluate organ specific expression of the nos-NPT II construct. NPT II expression was detected in leaves, petioles, stems, and roots of transgenic poplar, thereby indicating that the nos promoter is potentially effective for general constitutive expression of transgenes. NPT expression varied among transgenic poplar lines and among organs for one transgenic line, Tr15. With Tr15, NPT II levels were highest in older leaves and petioles. These results indicate that screening of several transgenic lines may be required to identify lines with optimal transgene expression.

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Differential Expression of a Chimeric nos-npt II Gene in 9 Years Old Hybrid Poplars (Populus koreana x P. nigra)

  • Noh, Eun Woon;Lee, Jae Soon;Choi, Young Im;Lee, Hyo Shin;Bae, Eun Kyung;Lee, Ji Hee
    • Journal of Plant Biotechnology
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    • v.6 no.1
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    • pp.15-19
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    • 2004
  • The expression of a chimeric transgene (nos-npt II) has been examined in 9 years old transgenic poplars (Populus koreana x P. nigra) growing in a nursery. The expression of the gene in twenty six independentely transformed plants were examined by 1) enzyme (NPT II) assay, 2) RT-PCR, and 3) resistance to kanamycin. High NPT II activities in young leaves of all the transformed plants were found even without a selection pressure for antibiotics for 9 years. However, the activity varied with the positions of leaves in the stem in that young leaves showed higher activity than did mature tissues. When leaf segments were cultured in the presence of 150 mg/l kanamycin, only those from young leaves produced vigorously growing callus. However, as in the case of NPTII assay, the leaf segments from mature leaves did not form callus well on the media. RT-PCR with nptII specific primers also showed that amplification products were observed only when RNAs from young tissues were used. The total RNA gel showed that while RNA in young leaves are relatively stable and in a large quantity, those in old leaves were mostly degraded. All the above results suggest that the gene is transcriptionally active only in young tissue even though it is attached to a constituitive promoter. Therefore, the expression of foreign gene in poplar plants seemed to be affected by the metabolic state of the cells and thus vary greatly with the developmental stages and the age of tissue.

Production of Transgenic Petunia hybrida cv. Rosanpion Using Agrobacterium-mediated Transformation

  • Ko, Jeong-Ae;Kim, Young-Sook;Kim, Myung-Jun;Kim, Hyun-Soon
    • Plant Resources
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    • v.4 no.1
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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.

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Direct Regeneration of Transgenic Buckwheat from Hypocotyl Segment by Agrobacterium-mediated Transformation

  • Kim, Hyun-Soon;Kang, Hyeon-Jung;Lee, Young-Tae;Lee, Seung-Yeob;Ko, Jeong-Ae;Rha, Eui-Shik
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.46 no.5
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    • pp.375-379
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    • 2001
  • Transgenic plants from hypocotyl segments of buckwheat were produced with the Agrobacterium strain LBA4404 harboring the binary vector pBI121 containing chimeric genes of neomycin phosphotransferase II (npt II) and $\beta$-glucuronidase (gus). Two weeks after co-cultivation with Agrobacterium, most of the hypocotyl segments gradually became brown and died on the selection medium containing 100mg/$\ell$ of kanamycin. Plants regenerated from the hypocotyl explants grown on selection medium were GUS-positive in the leaf, stem and vascular tissues by histochemical assay, and varied in gus activity (440-2568 pmol, 4-MU/mg protein) by fluorimetry. The plants showing GUS activity were confirmed of containing GUS and NPT-II genes by polymerase chain reaction (PCR). Within 3 months, transgenic buckwheat plants were able to obtained from the hypocotyl segments.

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Optimization of Cymbidium transformation system by the particle gun techniques (DNA 입자총에 의한 Cymbidium속 난의 형질전환 조건 검토)

  • Hong, Kyung-Ae;So, In-Sup;Lee, Ok-Young;Cheong, Choong-Duk;Riu, Key-Zung;U., Zang-Kual
    • Applied Biological Chemistry
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    • v.39 no.4
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    • pp.260-264
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    • 1996
  • Process of particle bombardment for efficient transformation of Cymbidium virescence rhizome microcross sections was investigated using Biolistic particle delivery system with pBI121 harboring the ${\beta}-glucuronidase$(GUS) and the neomycin phosphotransferaseII(nptII). The best result was obtained from the combination of $1.11{\;}{\mu}m$ tungsten particles coated with pBl121, $77.33kg/cm^2$ helium pressure, 6.35 mm gap distance, and 7.0 cm target distance. Transient expression of the reporter gene, GUS, bombarded into the rhizome microsections was observed by the histochemical assay. The marker gene, nptII, delivered by bombarding the tungsten particles coated with the plasmid DNA was identified in the transformed rhizome by polymerase chain reaction.

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Agrobacterium Mediated Transformation from Callus Pretreated with Particle Bombardment in Lilium lancifolium Thunb. (Particle Bombardment에 의해 전처리 된 참나리(Lilium lancifolium Thunb.) 캘러스의 Agrobacterium tumefaciens을 통한 형질전환)

  • Nam, Sang-Wook;Kim, Hei-Young
    • Journal of Plant Biotechnology
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    • v.31 no.1
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    • pp.13-17
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    • 2004
  • To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

Transformation of Fuji Apple Plant Harboring the Coat Protein Gene of Cucumber mosaic virus

  • Lee, C.H.;Hyung, N.I.;Lee, G.P.;Choi, J.Y.;Kim, C.S.;Choi, S.H.;Jang, I.O.;Han, D.H.;Ryu, K.H.
    • The Plant Pathology Journal
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    • v.19 no.3
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    • pp.162-165
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    • 2003
  • Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

The use of cotyledonary-node explants in Agrobacterium tumefaciensmediated transformation of cucumber (Cucumis sativus L.) (Agrobacterium에 의한 오이 형질전환에서 자엽절 절편의 이용)

  • Jang, Hyun-A;Kim, Hyun-A;Kwon, Suk-Yoon;Choi, Dong-Woog;Choi, Pil-Son
    • Journal of Plant Biotechnology
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    • v.38 no.3
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    • pp.198-202
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    • 2011
  • Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

Effects of Wounding and Inoculation Time on Agrobacterium -mediated Transformation in Capsicum annuum L. (상처처리와 접종시간이 Agrobacterium에 의한 고추 형질전환에 미치는 영향)

  • Jeon, Young-Ju;Park, Young-Doo;Choi, Geun-Won
    • Horticultural Science & Technology
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    • v.18 no.6
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    • pp.797-801
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    • 2000
  • The present study was conducted to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in hot pepper. Both regeneration ratio and transformation frequency after the cocultivation with A. tumefaciens were affected by inoculation time and artificial wounding. Transformation frequency was increased over 50% by combining artificial wounding with 120 s of inoculation treatment. Confirmation for the transformation of regenerated shoots was carried out by histochemical ${\beta}$-glucuronidase assay and polymerase chain reaction analysis using npt II primer.

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