Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants
Identifieur interne : 002D23 ( Main/Merge ); précédent : 002D22; suivant : 002D24Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants
Auteurs : Randall P. Niedz [États-Unis] ; W. L. Mckendree [États-Unis] ; R. G. Jr Shatters [États-Unis]Source :
- In vitro cellular & developmental biology. Plant [ 1054-5476 ] ; 2003.
Descripteurs français
- Pascal (Inist)
- Wicri :
- topic : Plante transgénique.
English descriptors
- KwdEn :
Abstract
Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin]. Electric field strength (375-450 V cm-1), vector DNA concentration (100 μg ml-1), carrier DNA concentration (100 μg ml-1), electroporation buffer (pH 8), and pre-electroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24 h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined, GUS activity was 7714pmol 4-methylumbelliferone (MU) mg-1 (protein) min-1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S-35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.
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Osbeck) and regeneration of transformed plants</title><author><name sortKey="Niedz, Randall P" sort="Niedz, Randall P" uniqKey="Niedz R" first="Randall P." last="Niedz">Randall P. Niedz</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft.</s1><s2>Pierce, FL 34945-3030</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="Mckendree, W L" sort="Mckendree, W L" uniqKey="Mckendree W" first="W. L." last="Mckendree">W. L. Mckendree</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft.</s1><s2>Pierce, FL 34945-3030</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="Shatters, R G Jr" sort="Shatters, R G Jr" uniqKey="Shatters R" first="R. G. Jr" last="Shatters">R. G. Jr Shatters</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft.</s1><s2>Pierce, FL 34945-3030</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">In vitro cellular & developmental biology. Plant</title><title level="j" type="abbreviated">In vitro cell. dev. biol., Plant</title><idno type="ISSN">1054-5476</idno><imprint><date when="2003">2003</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">In vitro cellular & developmental biology. Plant</title><title level="j" type="abbreviated">In vitro cell. dev. biol., Plant</title><idno type="ISSN">1054-5476</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Biological transport</term><term>Citrus sinensis</term><term>Efficiency</term><term>Electroporation</term><term>Endoplasmic reticulum</term><term>Gene expression</term><term>Genetic transformation</term><term>Green fluorescent protein</term><term>Localization</term><term>Optimization</term><term>Protoplast</term><term>Regeneration</term><term>Transfection</term><term>Transgenic plant</term><term>Transient response</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Electroporation</term><term>Protoplaste</term><term>Régénération</term><term>Plante transgénique</term><term>Transformation génétique</term><term>Transfection</term><term>Optimisation</term><term>Expression génique</term><term>Réponse transitoire</term><term>Citrus sinensis</term><term>Efficacité</term><term>Transport biologique</term><term>Localisation</term><term>Réticulum endoplasmique</term><term>Protéine fluorescente verte</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Plante transgénique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin]. Electric field strength (375-450 V cm<sup>-1</sup>), vector DNA concentration (100 μg ml<sup>-1</sup>), carrier DNA concentration (100 μg ml<sup>-1</sup>), electroporation buffer (pH 8), and pre-electroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24 h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined, GUS activity was 7714pmol 4-methylumbelliferone (MU) mg<sup>-1</sup> (protein) min<sup>-1</sup>. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S-35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.</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="Niedz, Randall P" sort="Niedz, Randall P" uniqKey="Niedz R" first="Randall P." last="Niedz">Randall P. Niedz</name></region><name sortKey="Mckendree, W L" sort="Mckendree, W L" uniqKey="Mckendree W" first="W. L." last="Mckendree">W. L. Mckendree</name><name sortKey="Shatters, R G Jr" sort="Shatters, R G Jr" uniqKey="Shatters R" first="R. G. Jr" last="Shatters">R. G. Jr Shatters</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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