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Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants

Identifieur interne : 000291 ( PascalFrancis/Curation ); précédent : 000290; suivant : 000292

Electroporation 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 :

RBID : Pascal:04-0060572

Descripteurs français

English descriptors

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.
pA  
A01 01  1    @0 1054-5476
A03   1    @0 In vitro cell. dev. biol., Plant
A05       @2 39
A06       @2 6
A08 01  1  ENG  @1 Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants
A11 01  1    @1 NIEDZ (Randall P.)
A11 02  1    @1 MCKENDREE (W. L.)
A11 03  1    @1 SHATTERS (R. G. JR)
A14 01      @1 Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft. @2 Pierce, FL 34945-3030 @3 USA @Z 1 aut. @Z 2 aut. @Z 3 aut.
A20       @1 586-594
A21       @1 2003
A23 01      @0 ENG
A43 01      @1 INIST @2 15474A @5 354000118917290060
A44       @0 0000 @1 © 2004 INIST-CNRS. All rights reserved.
A45       @0 1 p.1/2
A47 01  1    @0 04-0060572
A60       @1 P
A61       @0 A
A64 01  1    @0 In vitro cellular & developmental biology. Plant
A66 01      @0 USA
C01 01    ENG  @0 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.
C02 01  X    @0 002A32D02B
C02 02  X    @0 002A31C02A5B
C02 03  X    @0 215
C03 01  X  FRE  @0 Electroporation @5 01
C03 01  X  ENG  @0 Electroporation @5 01
C03 01  X  SPA  @0 Electroporación @5 01
C03 02  X  FRE  @0 Protoplaste @5 02
C03 02  X  ENG  @0 Protoplast @5 02
C03 02  X  SPA  @0 Protoplasto @5 02
C03 03  X  FRE  @0 Régénération @5 03
C03 03  X  ENG  @0 Regeneration @5 03
C03 03  X  SPA  @0 Regeneración @5 03
C03 04  X  FRE  @0 Plante transgénique @5 04
C03 04  X  ENG  @0 Transgenic plant @5 04
C03 04  X  SPA  @0 Planta transgénica @5 04
C03 05  X  FRE  @0 Transformation génétique @5 05
C03 05  X  ENG  @0 Genetic transformation @5 05
C03 05  X  SPA  @0 Transformación genética @5 05
C03 06  X  FRE  @0 Transfection @5 06
C03 06  X  ENG  @0 Transfection @5 06
C03 06  X  SPA  @0 Transfección @5 06
C03 07  X  FRE  @0 Optimisation @5 07
C03 07  X  ENG  @0 Optimization @5 07
C03 07  X  SPA  @0 Optimización @5 07
C03 08  X  FRE  @0 Expression génique @5 08
C03 08  X  ENG  @0 Gene expression @5 08
C03 08  X  SPA  @0 Expresión genética @5 08
C03 09  X  FRE  @0 Réponse transitoire @5 09
C03 09  X  ENG  @0 Transient response @5 09
C03 09  X  SPA  @0 Respuesta transitoria @5 09
C03 10  X  FRE  @0 Citrus sinensis @2 NS @5 10
C03 10  X  ENG  @0 Citrus sinensis @2 NS @5 10
C03 10  X  SPA  @0 Citrus sinensis @2 NS @5 10
C03 11  X  FRE  @0 Efficacité @5 33
C03 11  X  ENG  @0 Efficiency @5 33
C03 11  X  SPA  @0 Eficacia @5 33
C03 12  X  FRE  @0 Transport biologique @5 34
C03 12  X  ENG  @0 Biological transport @5 34
C03 12  X  SPA  @0 Transporte biológico @5 34
C03 13  X  FRE  @0 Localisation @5 35
C03 13  X  ENG  @0 Localization @5 35
C03 13  X  SPA  @0 Localización @5 35
C03 14  X  FRE  @0 Réticulum endoplasmique @5 36
C03 14  X  ENG  @0 Endoplasmic reticulum @5 36
C03 14  X  SPA  @0 Retículo endoplásmico @5 36
C03 15  X  FRE  @0 Protéine fluorescente verte @5 37
C03 15  X  ENG  @0 Green fluorescent protein @5 37
C03 15  X  SPA  @0 Proteína fluorescente verde @5 37
C07 01  X  FRE  @0 Rutaceae @2 NS
C07 01  X  ENG  @0 Rutaceae @2 NS
C07 01  X  SPA  @0 Rutaceae @2 NS
C07 02  X  FRE  @0 Dicotyledones @2 NS
C07 02  X  ENG  @0 Dicotyledones @2 NS
C07 02  X  SPA  @0 Dicotyledones @2 NS
C07 03  X  FRE  @0 Angiospermae @2 NS
C07 03  X  ENG  @0 Angiospermae @2 NS
C07 03  X  SPA  @0 Angiospermae @2 NS
C07 04  X  FRE  @0 Spermatophyta @2 NS
C07 04  X  ENG  @0 Spermatophyta @2 NS
C07 04  X  SPA  @0 Spermatophyta @2 NS
C07 05  X  FRE  @0 Agrume @5 39
C07 05  X  ENG  @0 Citrus fruit @5 39
C07 05  X  SPA  @0 Agrios @5 39
N21       @1 040
N82       @1 PSI

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Pascal:04-0060572

Le document en format XML

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<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>
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<term>Electroporation</term>
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<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>
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<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>
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<s0>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.</s0>
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<s5>01</s5>
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<s5>02</s5>
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<s5>02</s5>
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<s5>02</s5>
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<s5>03</s5>
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<s5>03</s5>
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<s5>05</s5>
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<s5>05</s5>
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<s5>06</s5>
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<s0>Transfection</s0>
<s5>06</s5>
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<s0>Transfección</s0>
<s5>06</s5>
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<s0>Optimisation</s0>
<s5>07</s5>
</fC03>
<fC03 i1="07" i2="X" l="ENG">
<s0>Optimization</s0>
<s5>07</s5>
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<s5>08</s5>
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<s0>Gene expression</s0>
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<s5>08</s5>
</fC03>
<fC03 i1="09" i2="X" l="FRE">
<s0>Réponse transitoire</s0>
<s5>09</s5>
</fC03>
<fC03 i1="09" i2="X" l="ENG">
<s0>Transient response</s0>
<s5>09</s5>
</fC03>
<fC03 i1="09" i2="X" l="SPA">
<s0>Respuesta transitoria</s0>
<s5>09</s5>
</fC03>
<fC03 i1="10" i2="X" l="FRE">
<s0>Citrus sinensis</s0>
<s2>NS</s2>
<s5>10</s5>
</fC03>
<fC03 i1="10" i2="X" l="ENG">
<s0>Citrus sinensis</s0>
<s2>NS</s2>
<s5>10</s5>
</fC03>
<fC03 i1="10" i2="X" l="SPA">
<s0>Citrus sinensis</s0>
<s2>NS</s2>
<s5>10</s5>
</fC03>
<fC03 i1="11" i2="X" l="FRE">
<s0>Efficacité</s0>
<s5>33</s5>
</fC03>
<fC03 i1="11" i2="X" l="ENG">
<s0>Efficiency</s0>
<s5>33</s5>
</fC03>
<fC03 i1="11" i2="X" l="SPA">
<s0>Eficacia</s0>
<s5>33</s5>
</fC03>
<fC03 i1="12" i2="X" l="FRE">
<s0>Transport biologique</s0>
<s5>34</s5>
</fC03>
<fC03 i1="12" i2="X" l="ENG">
<s0>Biological transport</s0>
<s5>34</s5>
</fC03>
<fC03 i1="12" i2="X" l="SPA">
<s0>Transporte biológico</s0>
<s5>34</s5>
</fC03>
<fC03 i1="13" i2="X" l="FRE">
<s0>Localisation</s0>
<s5>35</s5>
</fC03>
<fC03 i1="13" i2="X" l="ENG">
<s0>Localization</s0>
<s5>35</s5>
</fC03>
<fC03 i1="13" i2="X" l="SPA">
<s0>Localización</s0>
<s5>35</s5>
</fC03>
<fC03 i1="14" i2="X" l="FRE">
<s0>Réticulum endoplasmique</s0>
<s5>36</s5>
</fC03>
<fC03 i1="14" i2="X" l="ENG">
<s0>Endoplasmic reticulum</s0>
<s5>36</s5>
</fC03>
<fC03 i1="14" i2="X" l="SPA">
<s0>Retículo endoplásmico</s0>
<s5>36</s5>
</fC03>
<fC03 i1="15" i2="X" l="FRE">
<s0>Protéine fluorescente verte</s0>
<s5>37</s5>
</fC03>
<fC03 i1="15" i2="X" l="ENG">
<s0>Green fluorescent protein</s0>
<s5>37</s5>
</fC03>
<fC03 i1="15" i2="X" l="SPA">
<s0>Proteína fluorescente verde</s0>
<s5>37</s5>
</fC03>
<fC07 i1="01" i2="X" l="FRE">
<s0>Rutaceae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="01" i2="X" l="ENG">
<s0>Rutaceae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="01" i2="X" l="SPA">
<s0>Rutaceae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="02" i2="X" l="FRE">
<s0>Dicotyledones</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="02" i2="X" l="ENG">
<s0>Dicotyledones</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="02" i2="X" l="SPA">
<s0>Dicotyledones</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="03" i2="X" l="FRE">
<s0>Angiospermae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="03" i2="X" l="ENG">
<s0>Angiospermae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="03" i2="X" l="SPA">
<s0>Angiospermae</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="04" i2="X" l="FRE">
<s0>Spermatophyta</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="04" i2="X" l="ENG">
<s0>Spermatophyta</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="04" i2="X" l="SPA">
<s0>Spermatophyta</s0>
<s2>NS</s2>
</fC07>
<fC07 i1="05" i2="X" l="FRE">
<s0>Agrume</s0>
<s5>39</s5>
</fC07>
<fC07 i1="05" i2="X" l="ENG">
<s0>Citrus fruit</s0>
<s5>39</s5>
</fC07>
<fC07 i1="05" i2="X" l="SPA">
<s0>Agrios</s0>
<s5>39</s5>
</fC07>
<fN21>
<s1>040</s1>
</fN21>
<fN82>
<s1>PSI</s1>
</fN82>
</pA>
</standard>
</inist>
</record>

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   |texte=   Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants
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