In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phen...

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Autores: Gianoglio, Silvia, Comino, Cinzia, Moglia, Andrea, Acquadro, Alberto, García-Carpintero, Víctor, Diretto, Gianfranco, Sevi, Filippo, Rambla, José Luis, Dono, Gabriella, Valentino, Danila, Moreno Giménez, Elena, Fullana Pericàs, Mateu, Conesa, Miguel Ángel, Galmés, Jeroni, Lanteri, Sergio, Mazzucato, Andrea, Orzáez, Diego, Granell, Antonio
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/280671
Acceso en línea:http://hdl.handle.net/10261/280671
https://api.elsevier.com/content/abstract/scopus_id/85134672851
Access Level:acceso abierto
Palabra clave:CRISPR-Cas9
Breeding
Greenflesh
Nutritional quality
Pathogen resistance
Staygreen
Tomato
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spelling In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and RegulationGianoglio, SilviaComino, CinziaMoglia, AndreaAcquadro, AlbertoGarcía-Carpintero, VíctorDiretto, GianfrancoSevi, FilippoRambla, José LuisDono, GabriellaValentino, DanilaMoreno Giménez, ElenaFullana Pericàs, MateuConesa, Miguel ÁngelGalmés, JeroniLanteri, SergioMazzucato, AndreaOrzáez, DiegoGranell, AntonioCRISPR-Cas9BreedingGreenfleshNutritional qualityPathogen resistanceStaygreenTomatoGene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.This work was supported in part by the PID2019-108203RB-I00 project of the Spanish Plan Nacional I+D, Spanish Ministry of Science and Innovation and by HARNESSTOM Innovation Action EU-H2020-SFS-2020-1 (Contract number: 101000716). Finally, this work was supported by COST Actions ROXy CA18210 and EUROcarotene CA15136 for networking.Peer reviewedFrontiers MediaMinisterio de Ciencia e Innovación (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202220222022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/280671https://api.elsevier.com/content/abstract/scopus_id/85134672851reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-108203RB-I00info:eu-repo/grantAgreement/EC/H2020/101000716Frontiers in plant sciencehttps://doi.org/10.3389/fpls.2022.936089Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2806712026-05-22T06:33:51Z
dc.title.none.fl_str_mv In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
title In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
spellingShingle In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
Gianoglio, Silvia
CRISPR-Cas9
Breeding
Greenflesh
Nutritional quality
Pathogen resistance
Staygreen
Tomato
title_short In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
title_full In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
title_fullStr In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
title_full_unstemmed In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
title_sort In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation
dc.creator.none.fl_str_mv Gianoglio, Silvia
Comino, Cinzia
Moglia, Andrea
Acquadro, Alberto
García-Carpintero, Víctor
Diretto, Gianfranco
Sevi, Filippo
Rambla, José Luis
Dono, Gabriella
Valentino, Danila
Moreno Giménez, Elena
Fullana Pericàs, Mateu
Conesa, Miguel Ángel
Galmés, Jeroni
Lanteri, Sergio
Mazzucato, Andrea
Orzáez, Diego
Granell, Antonio
author Gianoglio, Silvia
author_facet Gianoglio, Silvia
Comino, Cinzia
Moglia, Andrea
Acquadro, Alberto
García-Carpintero, Víctor
Diretto, Gianfranco
Sevi, Filippo
Rambla, José Luis
Dono, Gabriella
Valentino, Danila
Moreno Giménez, Elena
Fullana Pericàs, Mateu
Conesa, Miguel Ángel
Galmés, Jeroni
Lanteri, Sergio
Mazzucato, Andrea
Orzáez, Diego
Granell, Antonio
author_role author
author2 Comino, Cinzia
Moglia, Andrea
Acquadro, Alberto
García-Carpintero, Víctor
Diretto, Gianfranco
Sevi, Filippo
Rambla, José Luis
Dono, Gabriella
Valentino, Danila
Moreno Giménez, Elena
Fullana Pericàs, Mateu
Conesa, Miguel Ángel
Galmés, Jeroni
Lanteri, Sergio
Mazzucato, Andrea
Orzáez, Diego
Granell, Antonio
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv CRISPR-Cas9
Breeding
Greenflesh
Nutritional quality
Pathogen resistance
Staygreen
Tomato
topic CRISPR-Cas9
Breeding
Greenflesh
Nutritional quality
Pathogen resistance
Staygreen
Tomato
description Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.
publishDate 2022
dc.date.none.fl_str_mv 2022
2022
2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/280671
https://api.elsevier.com/content/abstract/scopus_id/85134672851
url http://hdl.handle.net/10261/280671
https://api.elsevier.com/content/abstract/scopus_id/85134672851
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-108203RB-I00
info:eu-repo/grantAgreement/EC/H2020/101000716
Frontiers in plant science
https://doi.org/10.3389/fpls.2022.936089
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
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
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