Hetero-fertilization together with failed egg-sperm cell fusion supports single fertilization involved in in vivo haploid induction in maize

Abstract

In vivo doubled-haploid technology is widely applied in commercial maize breeding programs because of its time-saving and cost-reducing features. The production of maize haploids primarily depends on the use of Stock6-derived haploid inducer lines. Although the gene underlying haploid induction, MTL/ZmPLA1/NLD, was cloned recently, the mechanism of haploid induction is still unknown. Hetero-fertilization can occur via a single fertilization, which provides a means to investigate single-fertilization events by studying the hetero-fertilization phenomenon. In this study, we found that the hetero-fertilization rate increased significantly when female maize lines were first individually crossed with pollen from the inducer CAU5 in dual-pollination experiments 4 h before a second pollination with common lines. We also examined embryogenesis during haploid induction by confocal laser-scanning microscopy and observed single-fertilized ovules, indicating that single fertilization occurred during haploid induction. We therefore postulate that both single fertilization and chromosome elimination contribute to haploid induction in maize. We also propose a scheme for the formation of hetero-fertilized and haploid kernels. Our results provide an efficient approach to identify hetero-fertilized kernels for research on interactions between embryo and endosperm.

Fig. 1.

Diversity of kernel types formed during haploid induction. (A) Kernels on an ear singly pollinated with the inducer CAU5, showing substantial R1-nj pigmentation. (B) Kernels on an ear pollinated by the high-oil content line GY923 4 h after initial pollination with the inducer CAU5. (C) A kernel from a type-1 hetero-fertilization (HF) showing a colored scutellum and a colorless aleurone. (D) A putative haploid kernel, showing a colored aleurone and a colorless scutellum, which can be associated with haploid or type-2 HF kernels. (E) A diploid kernel from a Bo × CAU5 cross showing a colored scutellum and a colored aleurone. (F) An embryo-aborted kernel. (G) An endosperm-aborted kernel. The B73os1 line was used as the female parent throughout.

Fig. 2.

Comparison of the type-1 hetero-fertilization (HF) rate and putative haploid-production rate (PHPR) for kernels from each dual-pollination experiment. The type-1 HF rate (A) and PHPR (B) were greater when the inducer CAU5 was the first to pollinate the line B73os1 in the dual-pollination. By undergoing type-1 HF and type-2 HF, some endosperm-aborted kernels and embryo-aborted kernels were rescued at a very early stage, which led to a decrease in the endosperm-abortion rate (EnAR) and induced crossed-diploid (ICD) rate (C), and in the embryo-abortion rate (EmAR) (D) in the dual pollinations in comparison with the single pollinations. Data are means (±SD). Significant differences from GY923+CAU5 were determined using Student’s t-test; *P<0.05.

Fig. 3.

Identification and verification of type-2 HF kernels. (A) Variation in the oil content (OC) in kernels formed in the single- and dual-pollination experiments. (B) Field performance of haploid plants (red arrows) and type-2 HF plants (black arrows).

Fig. 4.

Comparison of pollen viability and pollen-tube growth rates associated with inducer and non-inducer pollen grains. No significant differences were found for pollen viability (A, B) and the growth rates (C, D) associated with inducer and non-inducer pollen grains used for cultivation in the 2016 (A, C) and HN (B, D) field tests conducted at Shangzhuang Experimental Station, Beijing, and Sanya Experimental Station, Hainan Island, respectively. Different letters in (A) and (B) indicate significant differences according to the results of multiple comparisons (P<0.05). Data are means (±SD) in (A) and (B). The box-plots in (C) and (D) show the mean, the first and third quartiles, and the maximum and minimum values.

Fig. 5.

Single fertilizations occur during haploid induction. (A–L) Embryogenesis during haploid induction from 1 to 6 d after pollination (DAP). (A) Unfertilized ovule. (B) At 1 DAP, cell fusion has begun. (C) At 2 DAP, cell divisions occur in doubly fertilized ovules. (D) At 3 DAP, the young embryo has formed in doubly fertilized ovules. (E) At 2 DAP, some ovules contained an unfertilized egg cell and a fertilized central cell. (F) At 3 DAP, the egg cell has not fused with sperm cells in single fertilized ovules. (G–I) Development of doubly fertilized ovules between 4 and 6 DAP. (J–L) Aberrant development of singly fertilized ovules between 4 and 6 DAP. EC, egg cell; SC, sperm cell; EMB, embryo; END, endosperm; UEC, unfertilized egg cell; CAV, cavity. A cavity forms in the embryo region when the embryo has not developed properly. Scale bars are 20 μm.

Fig. 6.

Proposed scheme for haploid and hetero-fertilization (HF) formation during dual pollination. Most ovules doubly fertilized by inducer sperm cells will develop into colored diploid kernels, although chromosome elimination may occur in the zygotes of other ovules leading to formation of haploid kernels. Singly fertilized ovules often may not fully develop, or produce defective kernels. In some cases, the central cells of ovules will only be fertilized by an inducer sperm cell, and the ovules will develop into haploid kernels by parthenogenesis. In these singly fertilized ovules, to ensure that reproduction can occur, the second synergid cell may attract a second pollen tube, which delivers sperm cells (in our experiments those of GY923 or CAU5) to complete the double fertilization and form type-2 HF or type-3 HF kernels, respectively. Haploid and type-2 HF kernels have the same pigmentation phenotype, meaning that a different phenotype must be used to differentiate between them. We therefore used their oil content (oc) to differentiate between them. In other cases, an ovule with only a fertilized egg cell may have its central cell fertilized during the second fertilization, forming a type-1 HF or type-4 HF kernel. CC, central cell; EC, egg cell; SY, synergid; SYN, synergid nucleus; VCN, vegetable cell nucleus; SC, sperm cell; FCC, fertilized central cell; FEC, fertilized egg cell; DSY, degenerated synergid; PT1, pollen tube 1; PT2, pollen tube 2.