A green fluorescent protein-engineered haploid inducer line facilitates haploid mutant screens and doubled haploid breeding in maize

Abstract

Doubled haploid (DH) technology is a useful tool for maize breeding and gene discovery. The production of a large number of DH lines has been possible recently with the development of various in vivo haploid inducer lines based on the discovery of the initial stock 6. Most of the haploid induction systems use the R1-navajo (R1-nj) gene whose expression in the kernel produces diploid kernels with colored aleurone crowns and scutella and haploid kernels with colored aleurone crowns but colorless scutella. However, the R1-nj gene expression depends on various genetic and environmental factors, which can mask the typical R1-nj phenotype. To solve this problem, we introduced a dominant green fluorescent protein (GFP) marker gene into a maize haploid inducer, RWS, to generate a RWS-GFP inducer. This line allows the identification of haploids in the early germination stage by visualizing the GFP expression of germinated kernels. Germinated diploid seeds will produce GFP fluorescence in emerged radicles and coleoptiles, but haploids will be GFP negative because of the lack of paternal GFP gene during hybridization with the haploid inducer. We demonstrated that this system can be used to screen haploid mutants and to produce haploids from various commercial sweet corn hybrids, which have a genetic background that prevents haploid identification by other systems. Haploid plants were treated with colchicine to produce DH plants. The GFP-engineered haploid inducer line will be a powerful tool in maize genetic studies and DH breeding.