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 A, Galmés Jeroni, Lanteri Sergio, Mazzucato Andrea, Orzáez Diego, Granell Antonio
Departamento 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, Spain.
Dipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Plant Genetics and Breeding, University of Turin, Turin, Italy.
Front Plant Sci. 2022 Jul 11;13:936089. doi: 10.3389/fpls.2022.936089. eCollection 2022.
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 () 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 locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T 'MoneyMaker' 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 leaves and fruits with 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 locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.
基因编辑已被证明是作物育种中产生突变体的宝贵工具，但其对农业的最终影响将取决于基因编辑技术产生的作物如何受到监管，以及我们对突变对植物表型影响的表征能力。评估基于基因编辑的植物育种方法的一个初步操作策略可能包括以作物和位点特异性方式评估诱导突变的效果：这涉及分析作物不同品种中的编辑效率、评估潜在的脱靶突变，以及对携带不同突变等位基因的编辑品系进行表型评估。在这里，我们针对两个番茄品种（“MoneyMaker”和“San Marzano”）中的（）位点，并评估了与该基因的CRISPR/Cas9活性相关的效率、特异性和突变模式。该位点编码一种负责启动叶绿素降解的镁螯合酶；在突变体中，成熟果实同时积累类胡萝卜素和叶绿素。对两个具有不同突变等位基因（1个核苷酸的小插入和123 bp的较大缺失）的无转基因T“MoneyMaker”品系进行了表型评估。与野生型（WT）相比，这两个品系除了叶绿素降解减少外，在果实成熟过程中类胡萝卜素和生育酚水平也显著增加。用感染“MoneyMaker”叶片和果实后，与野生型相比，感染面积和病原体增殖显著减少。我们的数据表明，CRISPR/Cas9介导的番茄位点突变是高效、特异且可重复的，并且产生的表型稳定，与先前用不同育种技术获得的突变体一致，同时还揭示了诸如维生素E过度积累和病原体抗性等新性状。这使得该位点成为培育具有改良栽培特性、消费者认可度和健康特性的番茄品种的有吸引力的目标。
