Expediting Next-Generation Hybrid Technology in Recalcitrant Maize Inbreds through In Vivo Targeted Activity of CRISPR/Cas9

Abstract

The Manipulated Genic Male Sterile Maintainer (MGM) system, a next-generation hybrid seed technology, enables efficient production of sortable seeds from genic male sterile (GMS) lines. However, implementing robust MGM systems in commercial maize inbred lines requires stable transformation, a genotype-specific and laborious process. This study aimed to integrate MGM technology into the commercial maize inbred line Z372, developing both GMS and MGM lines. We utilized the MGM line ZC01-3A-7, which contains the MS26ΔE5 editor T-DNA and MGM T-DNA, previously established in the highly transformable ZC01 recipient plants. Through a combination of crossing and backcrossing with Z372, we targeted the fertility gene Ms26 within the Z372 genome for mutation using the in vivo CRISPR/Cas9 activity within the MS26ΔE5 editor T-DNA construct. This approach facilitated precise editing of the Ms26 locus, minimizing linkage drag associated with the Ms26 mutation. Whole-genome SNP analysis achieved a 98.74% recovery rate for GMS and 96.32% for MGM in the BC2F2 generation. Importantly, the Z372-GMS line with the ms26ΔE5 mutation is non-transgenic, avoiding linkage drag and demonstrating production readiness. This study represents a significant advancement in maize breeding, enabling the rapid generation of GMS and MGM lines for efficient hybrid seed production.

Keywords: CRISPR/Cas9; Zea mays L.; genic male sterile; molecular breeding; next-generation hybrid technology.

Figure 1

Schematic overview of the efficient creation of Z372 GMS and MGM lines with genetic background restoration. (A) The panel depicts the ZC01-3A-7 donor line, which carries both the MS26ΔE5 editor T-DNA and MGM T-DNA constructs. (B) The rational design and experimental strategy underlying the efficient generation of Z372 GMS and MGM lines, with a focus on genetic background restoration.

Figure 2

Assessment of MGM T-DNA copy number in Z372-MGM candidate material using ddPCR technology. (A) Represents the 1D droplet digital PCR (ddPCR) plot displaying the count of MGMT-DNA-positive droplets (blue droplets and orange droplets) and ZmADH1 gene droplets (green droplets and orange droplets) for ten candidate Z372-MGM plants. (B) Presents the detailed 2D ddPCR data for a single plant, Z372-MGM2. (C) Depicts the calculated MGM T-DNA copy numbers for plants Z372-MGM1 to Z372-MGM10.

Figure 3

Phenotypic analysis of fertility traits in Z372, Z372-MGM, and Z372-GMS lines. The upper panel represents the tassels of the plants, the middle panel displays the anthers, and the lower panel showcases the pollen. The panels on the left depict the inbred line Z372, while those on the right illustrate the Z372-GMS8. The panels situated between Z372 and Z372-GMS8 correspond to the Z372-MGM2 line.

Figure 4

Ear phenotypes of ZC01-3A-7, Z372 inbred line, and Z372-MGM2 under bright and 550 nm excitation light. The left panel displays the ear phenotype of ZC01-3A-7 under bright and 550 nm excitation light, the central panel shows the ear phenotype of the Z372 inbred line under the same lighting conditions, and the right panel illustrates the ear phenotype of Z372-MGM2 under bright and 550 nm excitation light.

Figure 5

Plant phenotype and agronomic traits of Z372-MGM and GMS lines. (A) Phenotypes of ZC01-3A-7, Z372, Z372-MGM, and Z372-GMS plants, bar = 20 cm. (B) Statistical analysis of plant height, kernel row number (KRN), kernel number per row (KNR), and blade number in ZC01-3A-7, Z372, Z372-MGM, and Z372-GMS materials.

Figure 6

Genome background recoveries of Z372-MGM2 and Z372-GMS6.