Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. T...

ver descrição completa

Detalhes bibliográficos
Autores: Corredoira, Elena, San José, M. Carmen, Viéitez Martín, Ana María, Allona, Isabel, Aragoncillo, Cipriano, Ballester, Antonio
Formato: artículo
Fecha de publicación:2016
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/178267
Acesso em linha:http://hdl.handle.net/10261/178267
Access Level:acceso abierto
Palavra-chave:Agrobacterium tumefaciens
blight disease
Cisgenics
Chitinases
Pathogenesis related proteins
silver thiosulphate
id ES_3d652a32a7d757bcb6d9ed7bc3b7afc3
oai_identifier_str oai:digital.csic.es:10261/178267
network_acronym_str ES
network_name_str España
repository_id_str
spelling Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase geneCorredoira, ElenaSan José, M. CarmenViéitez Martín, Ana MaríaAllona, IsabelAragoncillo, CiprianoBallester, AntonioAgrobacterium tumefaciensblight diseaseCisgenicsChitinasesPathogenesis related proteinssilver thiosulphateChestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20%). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.Peer reviewedSpringer NatureConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192016info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/178267reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1782672026-05-22T06:33:51Z
dc.title.none.fl_str_mv Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
title Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
spellingShingle Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
Corredoira, Elena
Agrobacterium tumefaciens
blight disease
Cisgenics
Chitinases
Pathogenesis related proteins
silver thiosulphate
title_short Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
title_full Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
title_fullStr Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
title_full_unstemmed Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
title_sort Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene
dc.creator.none.fl_str_mv Corredoira, Elena
San José, M. Carmen
Viéitez Martín, Ana María
Allona, Isabel
Aragoncillo, Cipriano
Ballester, Antonio
author Corredoira, Elena
author_facet Corredoira, Elena
San José, M. Carmen
Viéitez Martín, Ana María
Allona, Isabel
Aragoncillo, Cipriano
Ballester, Antonio
author_role author
author2 San José, M. Carmen
Viéitez Martín, Ana María
Allona, Isabel
Aragoncillo, Cipriano
Ballester, Antonio
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Agrobacterium tumefaciens
blight disease
Cisgenics
Chitinases
Pathogenesis related proteins
silver thiosulphate
topic Agrobacterium tumefaciens
blight disease
Cisgenics
Chitinases
Pathogenesis related proteins
silver thiosulphate
description Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20%). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.
publishDate 2016
dc.date.none.fl_str_mv 2016
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/178267
url http://hdl.handle.net/10261/178267
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869406437118050304
score 15.812429

Registos relacionados