National Grassland Research Institute

Detection of an unfertilized polar nucleus with a fertilized egg cell

--Akio Kato

It has been known for about forty years that maize produces a few maternal haploids in its progenies (the incidence is about 0.1%) (Chase, SS, Genetics 34:328-332, 1949) and that in the case of double fertilization, the maize embryo sac, egg cell and polar nucleus are fertilized by different sperms originating from different pollen grains. This phenomenon is called "heterofertilization" (Sarkar et al., J. Hered. 62:118-120, 1971; Robertson, DS, J. Hered. 75:457-461, 1984). The most likely explanation for this phenomenon is that "A part of the maize pollen tubes has only one sperm cell or releases one sperm cell when the tubes enter the embryo sac." However this hypothesis had not been verified. In this paper I report data which strongly support the hypothesis. I detected an unfertilized polar nucleus with a fertilized egg cell using purple embryo marker and dual pollination.

Two normal maize lines, i.e. X18G (supplied by the Maize Genetics Cooperation Stock Center) and a sweet corn variety, Silver Honey Bantam, were used (Table 1). The X18G line carries a dominant pigment-conditioning gene R-scm2. The R-scm2 gene, in conjunction with the dominant pigment-conditioning genes A1, A2, C1, C2, induces a deep pigmentation of the aleurone and scutellum. This color is visible in the dormant kernel. The seeds of Silver Honey Bantam are colorless due to the absence of one or more of the major dominant color genes.

Table 1. Description and genotypes of the stocks used.
 

Line	Genotype	Phenotype
X18G	A1 A1 A2 A2 C1 C1 C2 C2 R-scm2 R-scm2 Y Y Sh2 Sh2	colored scutellum, colored aleurone, yellow normal endosperm
Silver Honey Bantam	y y sh2 sh2	colorless scutellum, colorless aleurone, white shrunken endosperm

I put a small amount of X18G pollen on the silks of detasseled Silver Honey Bantam very carefully. Twenty four hours later, I deposited an appropriate amount of Silver Honey Bantam pollen on the same silks (dual pollination). Fourteen ears were pollinated and about 3500 seeds were obtained. Then I examined the characters of the aleurone, endosperm and scutellum (Table 2).

Table 2. Segregation of seed color after dual pollination.
 

	colored aleurone with normal endosperm		colorless aleurone with shrunken white endosperm
Cross	colored scutellum	colorless scutellum	purple scutellum	colorless scutellum
Silver Honey Bantam X X18G X Silver Honey Bantam (24h later)	1602	4	5	1871

The seeds with colored aleurone and colored scutellum were those that had been fertilized by the X18G pollen while the seeds with colorless aleurone and colorless scutellum were fertilized by the Silver Honey Bantam pollen (one or more seeds should be haploid because of the above-mentioned spontaneous haploid production). Of the four seeds with a colored aleurone and colorless scutellum, three were haploid (n=10) and one was diploid (2n=20) based on root tip chromosome counting. I planted the diploid plant and selfed it. The genotype was Y/y, Sh2/sh2 and all the selfed seeds showed a colorless scutellum and aleurone, presumably due to contamination associated with heterofertilization between X18G and the pollen. The three haploid plants which were sterile and exhibited marked haploid features (narrow leaves, white stripes, dwarfism) were considered to be maternal haploids based on the color of the scutellum.

The presence of five seeds with colorless aleurone and colored scutellum is interesting. These findings suggest that the eggs were fertilized by the X18G sperm, while 24 hours later the polar nuclei were fertilized by the Silver Honey Bantam sperm and double fertilization occurred. Since the fertilization of maize occurs within 24 hours after pollination (Styles, ED et al., Maydica 32:139-150, 1987), it is suggested that the pollen tube of Silver Honey Bantam which was pollinated 24 hours later could not compete with the X18G pollen tube which was pollinated before. It is unlikely that the pollen of X18G germinated slowly on the silks or the pollen tubes extended slowly to the silks. The possibility that the pollen tube of X18G reached the ovule at the same time as the pollen tube of Silver Honey Bantam and that they heterofertilized the polar nucleus and the egg cell is also remote. It is obvious that if two pollen tubes (that of X18G and Silver Honey Bantam) enter the ovule and release sperm cells at the same time, double heterofertilization may occur: namely the egg cell is fertilized by the X18G sperm cell and the polar nucleus is fertilized by the Silver Honey Bantam sperm cell. Conversely the egg cell may be fertilized by the Silver Honey Bantam sperm cell and the polar nucleus may be fertilized by the X18G sperm cell. Since the latter case was not observed in the current experiment, it is suggested that the presence of five seeds with colorless aleurone and colored scutellum indicates the occurrence of single fertilization, namely unfertilized polar nuclei with fertilized egg cells. It is reasonable to assume that unfertilized egg cells with fertilized polar nuclei may also occur, and that the eggs develop apomictically and produce haploid embryos. If this hypothesis is valid, it is suggested that the embryonic development of the unfertilized egg cell starts within 24 hours after the fertilization of the polar nucleus, as indicated by the presence of the three haploid plants in this experiment.

Heterofertilization may be involved in this phenomenon too. Some investigators state that simultaneous insertion of the pollen tube results in heterofertilization. However, simultaneous insertion is not always necessary for heterofertilization to occur. In this experiment heterofertilization occurred in spite of the wide interval of pollination.

The presence of this aberrant form of fertilization was first reported in the maize line Stock 6 (Kato, A MNL 64:109-110, 1990), which has been known to induce a high frequency of haploid stocks (the incidence of haploids is 3%) (Coe, EH Am. Nat. 93:381-382, 1959; Sarkar, KR et al., Genetics 54:453-464, 1966). However, since the normal line used in this experiment exhibited the same pattern of aberrant fertilization, it is suggested that single fertilization may be widespread in maize.

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