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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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-07-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2022.936089/full |
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| author | Silvia Gianoglio Cinzia Comino Andrea Moglia Alberto Acquadro Víctor García-Carpintero Gianfranco Diretto Filippo Sevi Filippo Sevi José Luis Rambla José Luis Rambla Gabriella Dono Danila Valentino Elena Moreno-Giménez Elena Moreno-Giménez Mateu Fullana-Pericàs Miguel A. Conesa Jeroni Galmés Sergio Lanteri Andrea Mazzucato Diego Orzáez Antonio Granell |
| author_facet | Silvia Gianoglio Cinzia Comino Andrea Moglia Alberto Acquadro Víctor García-Carpintero Gianfranco Diretto Filippo Sevi Filippo Sevi José Luis Rambla José Luis Rambla Gabriella Dono Danila Valentino Elena Moreno-Giménez Elena Moreno-Giménez Mateu Fullana-Pericàs Miguel A. Conesa Jeroni Galmés Sergio Lanteri Andrea Mazzucato Diego Orzáez Antonio Granell |
| author_sort | Silvia Gianoglio |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-10T16:53:46Z |
| format | Article |
| id | doaj.art-bcb6a8b7cfe441b08fccd0ba5db5f700 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-10T16:53:46Z |
| publishDate | 2022-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-bcb6a8b7cfe441b08fccd0ba5db5f7002022-12-22T01:40:48ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2022-07-011310.3389/fpls.2022.936089936089In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and RegulationSilvia Gianoglio0Cinzia Comino1Andrea Moglia2Alberto Acquadro3Víctor García-Carpintero4Gianfranco Diretto5Filippo Sevi6Filippo Sevi7José Luis Rambla8José Luis Rambla9Gabriella Dono10Danila Valentino11Elena Moreno-Giménez12Elena Moreno-Giménez13Mateu Fullana-Pericàs14Miguel A. Conesa15Jeroni Galmés16Sergio Lanteri17Andrea Mazzucato18Diego Orzáez19Antonio Granell20Departamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainDipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, ItalyDipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, ItalyDipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, ItalyDepartamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainItalian Agency for New Technologies, Energy and Sustainable Development (ENEA), Rome, ItalyItalian Agency for New Technologies, Energy and Sustainable Development (ENEA), Rome, ItalyDepartment of Agricultural Sciences, University of Naples Federico II, Naples, ItalyDepartamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainDepartment of Biology, Biochemistry and Natural Sciences, Universitat Jaume I, Castellón de la Plana, SpainDepartment of Agriculture and Forest Sciences (DAFNE), Università degli Studi della Tuscia, Viterbo, ItalyDipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, ItalyDepartamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainDepartamento de Biotecnología, Instituto de Agroquímica y Tecnología de los Alimentos (IATA-CSIC), Paterna, SpainInstituto de Investigaciones Agroambientales y de Economía del Agua (INAGEA), Research Group on Plant Biology Under Mediterranean Conditions, Universitat de les Illes Balears, Palma, SpainInstituto de Investigaciones Agroambientales y de Economía del Agua (INAGEA), Research Group on Plant Biology Under Mediterranean Conditions, Universitat de les Illes Balears, Palma, SpainInstituto de Investigaciones Agroambientales y de Economía del Agua (INAGEA), Research Group on Plant Biology Under Mediterranean Conditions, Universitat de les Illes Balears, Palma, SpainDipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, ItalyDepartment of Agriculture and Forest Sciences (DAFNE), Università degli Studi della Tuscia, Viterbo, ItalyDepartamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainDepartamento de Biotecnología de Cultivos, Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universitat Politécnica de Valéncia (UPV), Valencia, SpainGene 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.https://www.frontiersin.org/articles/10.3389/fpls.2022.936089/fullCRISPR-Cas9tomatostaygreengreenfleshnutritional qualitypathogen resistance |
| spellingShingle | Silvia Gianoglio Cinzia Comino Andrea Moglia Alberto Acquadro Víctor García-Carpintero Gianfranco Diretto Filippo Sevi Filippo Sevi José Luis Rambla José Luis Rambla Gabriella Dono Danila Valentino Elena Moreno-Giménez Elena Moreno-Giménez Mateu Fullana-Pericàs Miguel A. Conesa Jeroni Galmés Sergio Lanteri Andrea Mazzucato Diego Orzáez Antonio Granell In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation Frontiers in Plant Science CRISPR-Cas9 tomato staygreen greenflesh nutritional quality pathogen resistance |
| title | 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_short | 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 |
| topic | CRISPR-Cas9 tomato staygreen greenflesh nutritional quality pathogen resistance |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2022.936089/full |
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