A GFP-containing binary vector for Agrobacterium tumefaciens-mediated plant transformation
Identifieur interne : 002500 ( Main/Exploration ); précédent : 002499; suivant : 002501A GFP-containing binary vector for Agrobacterium tumefaciens-mediated plant transformation
Auteurs : V. Orbovic [États-Unis] ; G. Pasquali [États-Unis] ; J. W. Grosser [États-Unis]Source :
- Acta horticulturae [ 0567-7572 ] ; 2007.
Descripteurs français
- Pascal (Inist)
- Wicri :
- topic : Agrume, Plante transgénique.
English descriptors
- KwdEn :
Abstract
A new binary vector (pT-LAB21) that contains a modification of the Aequorea victoria green fluorescent protein-GFP (erGFP7INT) as a reporter gene was constructed for the use in citrus transformation experiments. The gfp gene was excised from pLMNC95 plasmid and cloned into the pPZP212 binary vector containing an nptll gene for selection of putatively transformed shoots and the aadA gene for bacterial selection. pT-LAB21 was mobilized into EHA101, a hypervirulent disarmed strain of A. tumefaciens, by the freeze-thaw method. In order to test the plasmid, we performed transformation experiments on Carrizo citrange, 'Marsh' grapefruit, and 'Valencia' sweet orange by stem segment infection. Transformed plants of all three citrus cultivars were easily obtained, despite varying transformation efficiencies that were observed in these experiments. Highest efficiency of transformation was recorded for Carrizo citrange and the lowest for 'Valencia' orange. The possibility of detecting GFP fluorescence in seedlings by observing them under the microscope at an early stage and without doing any destructive assay has facilitated early screening of transgenic plants while maintaining their maximum vigor. To confirm the presence of foreign DNA in genomes of transformed citrus seedlings, we performed PCR and Southern analysis. This new binary vector is available for the transformation of citrus and other plants.
Affiliations:
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Pasquali</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center University of Florida 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850-2299</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author><author><name sortKey="Grosser, J W" sort="Grosser, J W" uniqKey="Grosser J" first="J. W." last="Grosser">J. W. Grosser</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center University of Florida 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850-2299</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">INIST</idno><idno type="inist">07-0528060</idno><date when="2007">2007</date><idno type="stanalyst">PASCAL 07-0528060 INIST</idno><idno type="RBID">Pascal:07-0528060</idno><idno type="wicri:Area/PascalFrancis/Corpus">000438</idno><idno type="wicri:Area/PascalFrancis/Curation">000566</idno><idno type="wicri:Area/PascalFrancis/Checkpoint">000504</idno><idno type="wicri:doubleKey">0567-7572:2007:Orbovic V:a:gfp:containing</idno><idno type="wicri:Area/Main/Merge">002594</idno><idno type="wicri:Area/Main/Curation">002500</idno><idno type="wicri:Area/Main/Exploration">002500</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">A GFP-containing binary vector for Agrobacterium tumefaciens-mediated plant transformation</title><author><name sortKey="Orbovic, V" sort="Orbovic, V" uniqKey="Orbovic V" first="V." last="Orbovic">V. Orbovic</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center University of Florida 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850-2299</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author><author><name sortKey="Pasquali, G" sort="Pasquali, G" uniqKey="Pasquali G" first="G." last="Pasquali">G. Pasquali</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center University of Florida 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850-2299</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author><author><name sortKey="Grosser, J W" sort="Grosser, J W" uniqKey="Grosser J" first="J. W." last="Grosser">J. W. Grosser</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center University of Florida 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850-2299</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Acta horticulturae</title><idno type="ISSN">0567-7572</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Acta horticulturae</title><idno type="ISSN">0567-7572</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agrobacterium tumefaciens</term><term>Binary system</term><term>Citrus fruit</term><term>Citrus paradisi</term><term>Citrus sinensis</term><term>Genetic transfer</term><term>Genetic transformation</term><term>Green fluorescent protein</term><term>Plasmid</term><term>Reporter gene</term><term>Structural gene</term><term>Transfection</term><term>Transgenic plant</term><term>Vector</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Agrume</term><term>Plante transgénique</term><term>Transfection</term><term>Transfert génétique</term><term>Transformation génétique</term><term>Vecteur</term><term>Système binaire</term><term>Gène indicateur</term><term>Gène structure</term><term>Agrobacterium tumefaciens</term><term>Citrus paradisi</term><term>Citrus sinensis</term><term>Plasmide</term><term>Protéine fluorescente verte</term><term>Gène gfp</term><term>Citroncirus webberi</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Agrume</term><term>Plante transgénique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A new binary vector (pT-LAB21) that contains a modification of the Aequorea victoria green fluorescent protein-GFP (erGFP7<sup>INT</sup>) as a reporter gene was constructed for the use in citrus transformation experiments. The gfp gene was excised from pLMNC95 plasmid and cloned into the pPZP212 binary vector containing an nptll gene for selection of putatively transformed shoots and the aadA gene for bacterial selection. pT-LAB21 was mobilized into EHA101, a hypervirulent disarmed strain of A. tumefaciens, by the freeze-thaw method. In order to test the plasmid, we performed transformation experiments on Carrizo citrange, 'Marsh' grapefruit, and 'Valencia' sweet orange by stem segment infection. Transformed plants of all three citrus cultivars were easily obtained, despite varying transformation efficiencies that were observed in these experiments. Highest efficiency of transformation was recorded for Carrizo citrange and the lowest for 'Valencia' orange. The possibility of detecting GFP fluorescence in seedlings by observing them under the microscope at an early stage and without doing any destructive assay has facilitated early screening of transgenic plants while maintaining their maximum vigor. To confirm the presence of foreign DNA in genomes of transformed citrus seedlings, we performed PCR and Southern analysis. This new binary vector is available for the transformation of citrus and other plants.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Floride</li></region></list><tree><country name="États-Unis"><region name="Floride"><name sortKey="Orbovic, V" sort="Orbovic, V" uniqKey="Orbovic V" first="V." last="Orbovic">V. Orbovic</name></region><name sortKey="Grosser, J W" sort="Grosser, J W" uniqKey="Grosser J" first="J. W." last="Grosser">J. W. Grosser</name><name sortKey="Pasquali, G" sort="Pasquali, G" uniqKey="Pasquali G" first="G." last="Pasquali">G. Pasquali</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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