Wheat (Triticum aestivum L.) is one of the most important staple crops globally. Doubled haploid technology enables rapid development of pure lines and has been extended from maize to several other crop species. A key step in DH breeding is the identification of haploids from diploids, which requires accurate and convenient phenotypic markers. In this study, we generated two wheat haploid inducers carrying different markers by a one-step strategy. One harbored a dual fluorescent marker system consisting of eGFP and TagRFP, the other carried a RUBY reporter. Both markers enabled near 100% accuracy of haploid identification at the immature embryo, mature embryo, and germinating seedling stages. Moreover, both lines consistently exhibited a high and stable haploid induction rate (~20%). This study not only provides efficient wheat haploid inducers but also establishes a convenient pipeline for developing haploid induction systems in other crop species.

The identification of haploid induction genes has promoted the advancement of several breeding technologies. Haploid induction genes in wheat, coupled with visual marker, have led to the establishment of a novel in vivo doubled-haploid (DH) technology. When combined with dominant male sterile genes, this innovative DH method presents a promising avenue for high-throughput production of DH lines. Furthermore, the application of haploid induction genes has facilitated the establishment of other innovative breeding technologies, such as HI-Edit and cyto-swapping in creating cytoplasmic male sterility lines, as well as synthetic apomixis. This review summarizes the progress of DH technology in wheat and presents examples of application of haploid induction genes in accelerating breeding practices, aiming to promote the development of these innovative technologies in wheat and enhancing wheat breeding efficiency.

Doubled haploid (DH) technology is an important tool in crop breeding because it can significantly accelerate the breeding process. ZmPLA1/MATL/NLD and ZmDMP are two key genes controlling haploid induction (HI) in maize, exhibiting a synergistic effect. However, it is unknown whether knock out of ZmDMP orthologs can stimulate HI in rice. In this study, a ZmPLA1 ortholog (OsPLA1) and two ZmDMP orthologs (OsDMP3 and OsDMP6) were identified in rice. All three genes encode plasma membrane-localized proteins and were highly expressed in mature anthers. Knockout of OsPLA1 in both Minghui 63 and Nipponbare resulted in reduced seed setting rate (SSR) and caused HI. The osdmp3, osdmp6 and the double mutant failed to trigger HI independently, nor increased the haploid induction rate (HIR) when combined with ospla1. Repeated pollinations operations of QX654A with the ospla1 mutant significantly improve SSR, while reducing HIR. RNA-seq profiling of mature ospla1 mutant anthers indicated that a large number of differentially expressed genes (DEGs) were enriched in redox homeostasis and lipid metabolic GO terms, plant hormone signal transduction, and MAPK signaling pathways. These findings provide important insights towards construction of an efficient DH breeding technology and study of the molecular mechanism of HI in rice.