Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.
Identifieur interne : 000334 ( Main/Exploration ); précédent : 000333; suivant : 000335Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.
Auteurs : Ganeshan Sivanandhan [Corée du Sud] ; Gnajothi Kapil Dev [Inde] ; Jeevaraj Theboral [Inde] ; Natesan Selvaraj [Inde] ; Andy Ganapathi [Inde] ; Markandan Manickavasagam [Inde]Source :
- PloS one [ 1932-6203 ] ; 2015.
Descripteurs français
- KwdFr :
- MESH :
- effets des médicaments et des substances chimiques : Transformation génétique.
- génétique : Agrobacterium, Transformation génétique, Withania.
- microbiologie : Withania.
- pharmacologie : Réactifs sulfhydryle.
- Sonication.
English descriptors
- KwdEn :
- MESH :
- chemical , pharmacology : Sulfhydryl Reagents.
- drug effects : Transformation, Genetic.
- genetics : Agrobacterium, Transformation, Genetic, Withania.
- microbiology : Withania.
- Sonication.
Abstract
In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.
DOI: 10.1371/journal.pone.0124693
PubMed: 25927703
PubMed Central: PMC4416026
Affiliations:
Links toward previous steps (curation, corpus...)
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wicri:level="1"><nlm:affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu</wicri:regionArea><wicri:noRegion>Tamil Nadu</wicri:noRegion></affiliation></author><author><name sortKey="Selvaraj, Natesan" sort="Selvaraj, Natesan" uniqKey="Selvaraj N" first="Natesan" last="Selvaraj">Natesan Selvaraj</name><affiliation wicri:level="1"><nlm:affiliation>Plant Tissue Culture Laboratory, Department of Botany, Periyar E.V.R College (Autonomous), Tiruchirappalli 620 023, Tamil Nadu, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Plant Tissue Culture Laboratory, Department of Botany, 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last="Manickavasagam">Markandan Manickavasagam</name><affiliation wicri:level="1"><nlm:affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu</wicri:regionArea><wicri:noRegion>Tamil Nadu</wicri:noRegion></affiliation></author></analytic><series><title level="j">PloS one</title><idno type="eISSN">1932-6203</idno><imprint><date when="2015" type="published">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agrobacterium (genetics)</term><term>Sonication (MeSH)</term><term>Sulfhydryl Reagents (pharmacology)</term><term>Transformation, Genetic (drug effects)</term><term>Transformation, Genetic (genetics)</term><term>Withania (genetics)</term><term>Withania (microbiology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Agrobacterium (génétique)</term><term>Réactifs sulfhydryle (pharmacologie)</term><term>Sonication (MeSH)</term><term>Transformation génétique (effets des médicaments et des substances chimiques)</term><term>Transformation génétique (génétique)</term><term>Withania (génétique)</term><term>Withania (microbiologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Sulfhydryl Reagents</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Transformation, Genetic</term></keywords><keywords scheme="MESH" qualifier="effets des médicaments et des substances chimiques" xml:lang="fr"><term>Transformation génétique</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Agrobacterium</term><term>Transformation, Genetic</term><term>Withania</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Agrobacterium</term><term>Transformation génétique</term><term>Withania</term></keywords><keywords scheme="MESH" qualifier="microbiologie" xml:lang="fr"><term>Withania</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Withania</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Réactifs sulfhydryle</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Sonication</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Sonication</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. </div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">25927703</PMID><DateCompleted><Year>2015</Year><Month>12</Month><Day>09</Day></DateCompleted><DateRevised><Year>2019</Year><Month>02</Month><Day>22</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Electronic">1932-6203</ISSN><JournalIssue CitedMedium="Internet"><Volume>10</Volume><Issue>4</Issue><PubDate><Year>2015</Year></PubDate></JournalIssue><Title>PloS one</Title><ISOAbbreviation>PLoS One</ISOAbbreviation></Journal><ArticleTitle>Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.</ArticleTitle><Pagination><MedlinePgn>e0124693</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1371/journal.pone.0124693</ELocationID><Abstract><AbstractText>In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. </AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Sivanandhan</LastName><ForeName>Ganeshan</ForeName><Initials>G</Initials><AffiliationInfo><Affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India; Molecular Genetics and Genomics Laboratory, Department of Horticulture, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305 764, South Korea.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kapil Dev</LastName><ForeName>Gnajothi</ForeName><Initials>G</Initials><AffiliationInfo><Affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Theboral</LastName><ForeName>Jeevaraj</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Selvaraj</LastName><ForeName>Natesan</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Plant Tissue Culture Laboratory, Department of Botany, Periyar E.V.R College (Autonomous), Tiruchirappalli 620 023, Tamil Nadu, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ganapathi</LastName><ForeName>Andy</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Manickavasagam</LastName><ForeName>Markandan</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2015</Year><Month>04</Month><Day>30</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>PLoS One</MedlineTA><NlmUniqueID>101285081</NlmUniqueID><ISSNLinking>1932-6203</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D013439">Sulfhydryl Reagents</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D060054" MajorTopicYN="N">Agrobacterium</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013010" MajorTopicYN="Y">Sonication</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013439" MajorTopicYN="N">Sulfhydryl Reagents</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014170" MajorTopicYN="N">Transformation, Genetic</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="N">drug effects</QualifierName><QualifierName UI="Q000235" 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first="Gnajothi" last="Kapil Dev">Gnajothi Kapil Dev</name></noRegion><name sortKey="Ganapathi, Andy" sort="Ganapathi, Andy" uniqKey="Ganapathi A" first="Andy" last="Ganapathi">Andy Ganapathi</name><name sortKey="Manickavasagam, Markandan" sort="Manickavasagam, Markandan" uniqKey="Manickavasagam M" first="Markandan" last="Manickavasagam">Markandan Manickavasagam</name><name sortKey="Selvaraj, Natesan" sort="Selvaraj, Natesan" uniqKey="Selvaraj N" first="Natesan" last="Selvaraj">Natesan Selvaraj</name><name sortKey="Theboral, Jeevaraj" sort="Theboral, Jeevaraj" uniqKey="Theboral J" first="Jeevaraj" last="Theboral">Jeevaraj Theboral</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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