An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus
Identifieur interne : 001C99 ( Main/Exploration ); précédent : 001C98; suivant : 001D00An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus
Auteurs : M. Dutt [États-Unis] ; J. W. Grosser [États-Unis]Source :
- Plant cell reports : (Print) [ 0721-7714 ] ; 2010.
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- Pascal (Inist)
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Abstract
A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en" level="a">An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus</title><author><name sortKey="Dutt, M" sort="Dutt, M" uniqKey="Dutt M" first="M." last="Dutt">M. Dutt</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 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/IFAS, 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 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">10-0504849</idno><date when="2010">2010</date><idno type="stanalyst">PASCAL 10-0504849 INIST</idno><idno type="RBID">Pascal:10-0504849</idno><idno type="wicri:Area/PascalFrancis/Corpus">000209</idno><idno type="wicri:Area/PascalFrancis/Curation">000791</idno><idno type="wicri:Area/PascalFrancis/Checkpoint">000259</idno><idno type="wicri:doubleKey">0721-7714:2010:Dutt M:an:embryogenic:suspension</idno><idno type="wicri:Area/Main/Merge">001D43</idno><idno type="wicri:Area/Main/Curation">001C99</idno><idno type="wicri:Area/Main/Exploration">001C99</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus</title><author><name sortKey="Dutt, M" sort="Dutt, M" uniqKey="Dutt M" first="M." last="Dutt">M. Dutt</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 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/IFAS, 700 Experiment Station Road</s1><s2>Lake Alfred, FL 33850</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Floride</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Plant cell reports : (Print)</title><title level="j" type="abbreviated">Plant cell rep. : (Print)</title><idno type="ISSN">0721-7714</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Plant cell reports : (Print)</title><title level="j" type="abbreviated">Plant cell rep. : (Print)</title><idno type="ISSN">0721-7714</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agrobacterium tumefaciens</term><term>Callus</term><term>Cell culture</term><term>Citrus sinensis</term><term>Genetic transformation</term><term>Mandarin</term><term>Somatic embryo</term><term>Suspension culture</term><term>Tissue culture</term><term>Transfection</term><term>Vegetals</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Embryon somatique</term><term>Culture tissu</term><term>Culture en suspension</term><term>Culture cellulaire</term><term>Transformation génétique</term><term>Agrobacterium tumefaciens</term><term>Transfection</term><term>Citrus sinensis</term><term>Cal</term><term>Mandarine</term><term>Végétal</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.</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="Dutt, M" sort="Dutt, M" uniqKey="Dutt M" first="M." last="Dutt">M. Dutt</name></region><name sortKey="Grosser, J W" sort="Grosser, J W" uniqKey="Grosser J" first="J. W." last="Grosser">J. W. Grosser</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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