Please use this identifier to cite or link to this item: http://hdl.handle.net/2067/49452
Title: In-depth characterization of greenflesh tomato mutants obtained by CRISPR/Cas9 editing: a case study with implications for breeding and regulation
Authors: Mazzucato A 
Comino C
Moglia A
Acquadro A
García-Carpintero V
Diretto G
Sevi F
Rambla JL
Dono G,
Valentino D
Moreno-Giménez E,
Fullana-Pericàs M
Conesa MÀ
Galmès J
Lanteri S
Gianoglio C
Orzáez D
Granell A
Journal: FRONTIERS IN PLANT SCIENCE 
Issue Date: 2022
Abstract: 
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.
URI: http://hdl.handle.net/2067/49452
ISSN: 1664-462X
DOI: https://doi.org/10.3389/fpls.2022.936089
Rights: Attribution 4.0 International
Appears in Collections:A1. Articolo in rivista

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