TY - JOUR

T1 - Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol

AU - Hernandes-Lopes, José

AU - Pinto, Maísa Siqueira

AU - Vieira, Letícia Rios

AU - Monteiro, Patrícia Brant

AU - Gerasimova, Sophia V

AU - Nonato, Juliana Vieira Almeida

AU - Bruno, Maria Helena Faustinoni

AU - Vikhorev, Alexander

AU - Rausch-Fernandes, Fernanda

AU - Gerhardt, Isabel R

AU - Pauwels, Laurens

AU - Arruda, Paulo

AU - Dante, Ricardo A

AU - Yassitepe, Juliana Erika de Carvalho Teixeira

N1 - This work was funded by grant 2016/23218-0 “Genomics for Climate Change Research Center (GCCRC)” from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). JH-L received a postdoctoral fellowship from EMBRAPA and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (381669/2019-0). MP and LV received postdoctoral fellowships from FAPESP (2022/04929-3 and 2020/10677-1, respectively). JN and MB received technical training fellowships from FAPESP (2022/04930-1 and 2022/04441-0, respectively). PM received a postdoctoral fellowship from CNPq (167867/2022-9). SG is supported by a visiting researcher grant by FAPESP (2022/09418-7). IG is recipient of CNPq productivity fellowship (308896/2020-3). Copyright © 2023 Hernandes-Lopes, Pinto, Vieira, Monteiro, Gerasimova, Nonato, Bruno, Vikhorev, Rausch-Fernandes, Gerhardt, Pauwels, Arruda, Dante and Yassitepe.

PY - 2023

Y1 - 2023

N2 - The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

AB - The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85180523537&origin=inward&txGid=349f4b7b43ea030b0018a5e1d431e0fe

U2 - 10.3389/fgeed.2023.1241035

DO - 10.3389/fgeed.2023.1241035

M3 - Article

C2 - 38144709

VL - 5

JO - Frontiers in genome editing

JF - Frontiers in genome editing

SN - 2673-3439

M1 - 1241035

ER -