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Haploid frailty (HF), the reduced vigour of haploids compared to their diploid counterparts, limits the efficiency of doubled haploid (DH) breeding in maize. This study evaluated 192 BS39 perfect isogenic haploid–diploid pairs across two replications to quantify HF in eight agronomic traits and integrate analyses of HF with haploid fertility, genomic prediction, and genome-wide association studies (GWAS). Haploids were on average 30–40% shorter in plant and ear height, while traits such as tassel branch number showed minimal or even negative frailty, indicating the presence of naturally vigorous haploids. Heritability was consistently high for diploid (0.8–0.9) and haploid performance (0.7–0.9), but lower for HF (0.1–0.7), reflecting its complexity as a derived trait. Correlation analyses revealed strong diploid–haploid relationships (r = 0.5–0.9) and moderate negative associations between haploid performance and HF (r = –0.4 to –0.6). Importantly, haploid female fertility correlated positively with vigour traits, suggesting that dual improvement of vigour and fertility is possible. Genomic prediction using diploid data showed moderate accuracy for haploid traits (0.3–0.5) but weak performance for HF, showing the need for direct haploid evaluations. GWAS detected significant SNPs across traits, including 14 for plant height, 30 for ear height, 20 for flag leaf length, 19 for flag leaf width, 23 for tassel length, 2 for spike length, and 6 for tassel branches, with one SNP shared between haploid and diploid tassel branches. Candidate genes included regulators of cell cycle control (Myb3R-1), auxin signalling (SRS1, SAUR-like proteins), cytoskeletal organization (PRONE-domain proteins), and oxidative stress protection (HGGT1).

Efficient production and use of doubled haploid lines can greatly accelerate genetic gains in maize breeding programs. One of the critical steps in standard doubled haploid line production is doubling the haploid genome using toxic and costly mitosis-inhibiting chemicals to achieve fertility in haploids. Alternatively, fertility may be spontaneously restored by natural chromosomal doubling, although generally at a rate too low for practical applications in most germplasm. This is the first large-scale genome-wise association study to analyze spontaneous chromosome doubling in haploids derived from tropical maize inbred lines. Induction crosses between tropicalized haploid inducers and 400 inbred lines were made, and the resulting haploid plants were assessed for haploid male fertility which refers to pollen production and haploid fertility which refers to seed production upon self-fertilization. A small number of genotypes were highly fertile and these fertility traits were highly heritable. Agronomic traits like plant height, ear height and tassel branch number were positively correlated with fertility traits. In contrast, haploid induction rate of the source germplasm and plant aspect were not correlated to fertility traits. Several genomic regions and candidate genes were identified that may control spontaneous fertility restoration. Overall, the study revealed the presence of large variation for both haploid male fertility and haploid fertility which can be potentially exploited for improving the efficiency of doubled haploid derivation in tropical maize germplasm.

Groundbreaking advances in gene editing technologies are transforming modern plant breeding by enabling precise genetic modifications that dramatically accelerate crop improvement. Haploid and diploid induction systems have emerged as particularly powerful tools in this landscape, offering both efficient gene editing capabilities and rapid production of homozygous lines while seamlessly integrating with the advanced genome-editing platforms such as CRISPR-Cas systems. This review synthesizes the current state of knowledge regarding the mechanisms, applications, and recent progress in haploid and diploid induction systems for gene editing. We examine their transformative potential for enhancing genetic gains and compressing breeding timelines, with significant implications for global food security. Additionally, we provide a critical analysis of emerging challenges of genome editing in crops and outline promising future directions for research and development.

Summary In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1‐nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background‐independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.

Spontaneous haploid genome doubling (SHGD) is a valuable trait in maize breeding, enabling the development of doubled haploid (DH) lines without chemical chromosome doubling. However, SHGD is a rare phenotype, expressed in only a small fraction of maize germplasm, making its identification resource-intensive. This study evaluated the efficiency of a two-stage field screening approach designed to identify maize genotypes with high haploid male fertility (HMF), a key indicator of SHGD potential. Simulation analyses showed that evaluating 50 haploid plants per genotype, combined with a 25% HMF threshold, provides an optimal balance between detection accuracy and resource efficiency. Across three growing seasons, HMF exhibited a highly skewed distribution, with most genotypes showing low HMF and a small subset exceeding 30% HMF. Field evaluations conducted in 2022, 2023, and 2024 consistently identified high-performing genotypes, including A427, N525, N516, and NK778, which maintained stable HMF expression across years. A genome-wide association analysis identified genomic regions associated with HMF. Our two-stage screening approach identified both SHGD donor lines and genomic loci and candidate genes for HMF.

This research investigated the variation in haploid frailty (%HF), which is the difference in trait performance between isogenic haploid and diploid maize lines, and the heritability of haploid frailty for different agronomic traits. A total of 48 isogenic pairs was evaluated in three environments, and 192 isogenic line pairs were evaluated in two environments for plant height (PH), ear height (EH), flag leaf length (FLL) and width (FLW), tassel length (TL), spike length (SL), stem diameter (SD) and tassel branch (TB) number. We found that the qshgd1 locus, associated with spontaneous haploid genome doubling (SHGD), plays a crucial role in improving haploid performance by reducing %HF and promoting diploid-like vigor. The BS39+SHGD genotypes exhibited significantly lower HF% rates compared to the BS39 group, with consistent reductions across multiple traits. Environmental factors also contributed to %HF variation, but genetic influences such as the presence of SHGD proved to have a greater impact on haploid frailty. Leveraging SHGD to enhance both vigor and fertility of haploid plants, is likely to benefit breeding programs in maize and perhaps other crops by more economic and efficient production of DH lines.

Doubled haploid (DH) technology becomes more routinely applied in maize hybrid breeding. However, some issues in haploid induction and identification persist, requiring resolution to optimize DH production. Our objective was to implement simultaneous marker-assisted selection (MAS) for qhir1 ( MTL/ZmPLA1/NLD ) and qhir8 ( ZmDMP ) using TaqMan assay in F 2 generation of four BHI306-derived tropical × temperate inducer families. We also aimed to assess their haploid induction rate (HIR) in the F 3 generation as a phenotypic response to MAS. We highlighted remarkable increases in HIR of each inducer family. Genotypes carrying qhir1 and qhir8 exhibited 1 – 3-fold higher haploid frequency than those carrying only qhir1 . Additionally, the qhir1 marker was employed for verifying putative haploid seedlings at 7 days after planting. Flow cytometric analysis served as the gold standard test to assess the accuracy of the R1-nj and the qhir1 marker. The qhir1 marker showed high accuracy and may be integrated in multiple haploid identifications at early seedling stage succeeding pre-haploid sorting via R1-nj marker.

• Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. • We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). • Chemical assays detected a secondwave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. • Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.

Maize doubled haploid (DH) lines are usually created in vivo, through crosses with maternal haploid inducers. These inducers have the inherent ability of generating seeds with haploid embryos when used to pollinate other genotypes. The resulting haploid plants are treated with a doubling agent and self-pollinated, producing completely homozygous seeds. This rapid method of inbred line production reduces the length of breeding cycles and, consequently, increases genetic gain. Such advantages explain the wide adoption of this technique by large, well-established maize breeding programs. However, a slower rate of adoption was observed in medium to small-scale breeding programs. The high price and/or lack of environmental adaptation of inducers available for licensing, or the poor performance of those free of cost, might explain why smaller operations did not take full advantage of this technique. The lack of adapted inducers is especially felt in tropical countries, where inducer breeding efforts are more recent. Therefore, defining optimal breeding approaches for inducer development could benefit many breeding programs which are in the process of adopting the DH technique. In this manuscript, we review traits important to maize maternal haploid inducers, explain their genetic basis, listing known genes and quantitative trait loci (QTL), and discuss different breeding approaches for inducer development. The performance of haploid inducers has an important impact on the cost of DH line production.

Doubled haploid (DH) line production through in vivo maternal haploid induction is widely adopted in maize breeding programs. The established protocol for DH production includes four steps namely in vivo maternal haploid induction, haploid identification, genome doubling of haploid, and self-fertilization of doubled haploids. Since modern haploid inducers still produce relatively small portion of haploids among undesirable hybrid kernels, haploid identification is typically laborious, costly, and time-consuming, making this step the second foremost in the DH technique. This manuscript reviews numerous methods for haploid identification from different approaches including the innate differences in haploids and diploids, biomarkers integrated in haploid inducers, and automated seed sorting. The phenotypic differentiation, genetic basis, advantages, and limitations of each biomarker system are highlighted. Several approaches of automated seed sorting from different research groups are also discussed regarding the platform or instrument used, sorting time, accuracy, advantages, limitations, and challenges before they go through commercialization. The past haploid selection was focusing on finding the distinguishable marker systems with the key to effectiveness. The current haploid selection is adopting multiple reliable biomarker systems with the key to efficiency while seeking the possibility for automation. Fully automated high-throughput haploid sorting would be promising in near future with the key to robustness with retaining the feasible level of accuracy. The system that can meet between three major constraints (time, workforce, and budget) and the sorting scale would be the best option.