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

## TL;DR

This study shows how to efficiently create hybrid maize seeds using CRISPR/Cas9 to edit a fertility gene in a non-transgenic way.

## Contribution

A novel in vivo CRISPR/Cas9 approach to generate non-transgenic GMS and MGM maize lines with minimal linkage drag.

## Key findings

- Precise editing of the Ms26 gene in Z372 achieved 98.74% recovery for GMS and 96.32% for MGM in the BC2F2 generation.
- The Z372-GMS line is non-transgenic and production-ready, avoiding linkage drag.
- The method enables rapid generation of hybrid seed lines in recalcitrant maize inbreds.

## 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.