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. 2023 Dec 7:5:1241035.
doi: 10.3389/fgeed.2023.1241035. eCollection 2023.

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

José Hernandes-Lopes  1   2 , Maísa Siqueira Pinto  1   2 , Letícia Rios Vieira  1   2 , Patrícia Brant Monteiro  1   2 , Sophia V Gerasimova  1   2   3 , Juliana Vieira Almeida Nonato  1   2 , Maria Helena Faustinoni Bruno  1   2 , Alexander Vikhorev  4 , Fernanda Rausch-Fernandes  1   2   5 , Isabel R Gerhardt  1   2   5 , Laurens Pauwels  6   7 , Paulo Arruda  1   2   8 , Ricardo A Dante  1   2   5 , Juliana Erika de Carvalho Teixeira Yassitepe  1   2   5
Affiliations

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

José Hernandes-Lopes et al. Front Genome Ed. .

Abstract

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.

Keywords: Agrobacterium; B104; BABY BOOM; CRISPR/Cas9; VIRESCENT YELLOW-LIKE; WUSCHEL; off-target; protoplast.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) VYL (Chr.9_ClpP5) and VYL-MODIFIER (Chr.1_ClpP5) target and off-target sequences, respectively, in different maize lines. Red, underlined letters: mismatches compared to the sgRNA spacer sequence; gray boxes: spacer “seed” region; blue “AGG”: PAM site. (B) Protoplast transfection efficiency counted as the proportion of GFP positive cells. (C) Assessment of indel frequencies in leaf-derived protoplasts transfected with pLAPAU17-VYL. (D) Mutation frequency counted as the proportion of mutated to WT reads normalized by the transformation efficiency of corresponding samples.
FIGURE 2
FIGURE 2
Explants (immature zygotic embryos–IZEs) and regenerant plants of maize B104 inbred line. (A) Transient expression of mRuby, 3 days post infection. (B) Initiation of somatic embryos observed 8 days post infection. (C) Somatic embryos developing at the surface of the immature embryo 17 days post infection. (D–F) Regenerant plants showing the pale-yellow vyl loss-of-function phenotype. (G) Control embryo 3 days post infection. (H) WT plant. White scale bar = 1 mm; Black scale bar = 1 cm.
FIGURE 3
FIGURE 3
Explants and regenerant plants of tropical maize lines. (A–C) IZEs of CML360. (A) Initiation of somatic embryos observed 9 days post infection. (B) Callus structure forming on the scutellum of an IZE, 15 days post infection. (C) Callus showing initiation of organogenesis. (D–G) Regenerant plants of CML444 showing the pale-yellow vyl loss-of-function phenotype. White scale bar = 1 mm; Black scale bar = 2 cm.
FIGURE 4
FIGURE 4
Genotyping of T0 events. (A) Frequency of excision of the morphogenic genes expression cassette. (B) Frequency of T0 events harboring diverse indel types. (C) Zygosity analysis of the VYL locus in T0 events.
FIGURE 5
FIGURE 5
VYL-MODIFIER off-target editing in tropical maize lines. (A) T0 events showing leaves with mosaic albino stripes. The pot diameter is 30 cm. (B) Zygosity analysis of the edited VYL-MODIFIER locus in T0 events. (C) Frequency of tropical T0 events harboring diverse indel types.
FIGURE 6
FIGURE 6
Analysis of T1 plants of two edited independent events of different maize lines. (A) Phenotypic gradient observed in seedlings (left) and leaves (right). (B) Genotyping of T1 plants, each row corresponding to an individual B104 (62GC1_VI), CML360 (3CML_III) or PCL1 (1PCL_II) T0 event. Boxes represent different individuals (1–16) in the T1 progeny. Plus and minus signs denote transgenic and non-transgenic individuals, respectively. Missing boxes represent seeds that failed to germinate. Scale bar = 2 cm.
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