Download Single Fertilization in Maize

Single Fertilization in Maize
A. Kato
Single fertilization events were detected in seven maize lines (Zea mays L.) of different genetic background. Detection of single fertilization was achieved by a dual
pollination method, that is, pollen parent 1 (y1/y1, white endosperm) was pollinated
onto the silks of Oh43 (Y1/Y1, yellow endosperm) and 24 h later the same silks
were pollinated with pollen of X18G (R1-scm2/R1-scm2, purple aleurone and purple
embryo). Seventy-nine kernels with purple aleurone and white scutellum (Pw) were
observed in 29,999 examined kernels. Determination of chromosome numbers and
progeny tests revealed that 31 plants were maternal haploid, 4 were monosomics
between Oh43 and X18G, 36 originated from single fertilization events by pollen
parent 1 (0.4%), and 5 triploids are thought to have originated from single fertilization by a diploid sperm cell of pollen parent 1. At least one-fifth of heterofertilization
events in maize can be the result of single fertilization.
From National Grassland Research Institute, Ministry
of Agriculture, Forestry, and Fisheries, Nishinasuno, Tochigi, Japan. A. Kato is currently at Tucker Hall, University of Missouri, Columbia, MO 65211-7400, or email: [email protected]. I thank E. H. Coe
for providing stock 6 and reviewing the manuscript.
q 1999 The American Genetic Association 90:276–280
276
Maize plants produce maternal haploids
in progenies at a frequency of 0.1%
(Chase 1949, 1952; Greenblatt and Bock
1967; Nanda and Chase 1966), and attempts to utilize haploids for breeding
purposes were proposed by Chase
(1969). Several haploid-inducing lines
were developed in maize (Chalyk 1994;
Lashermes and Beckert 1988; Sarkar et al.
1972). Stock 6 is one of the haploid-inducing lines discovered by Coe (1959).
The incidence of haploids in self-pollinated progenies of stock 6 is 3%, and 0.5–
1.0% when crossed to other stocks. Both
maternal and paternal effects were detected, and the haploid-inducing character is a heritable trait (Aman and Sarkar
1978; Coe and Sarkar 1964; Lashermes
and Beckert 1988; Sarkar and Coe 1966;
Sarkar et al. 1972). The exact mechanism
of haploid induction is not known.
Heterofertilization, another abnormality of fertilization exhibited by maize, was
first reported by Sprague (1929, 1932)
and was detected as kernels with genetically different embryo and endosperm. In
heterofertilization the egg cell and central cell in an ovule are fertilized by
sperm cells produced by different pollen
grains. The incidence of heterofertilization is about 1% in diverse lines of maize
(Robertson 1984), detected as yellow kernels with purple embryo (if concordant
cases are accounted, the incidence becomes 2%). Origins of heterofertilized
kernels were considered by Sarkar and
Coe (1971a), and they did not detect any
cases involving diploid sperm cells.
Recently the author ( Kato 1997b) reported single fertilization events in maize
in which fertilization of egg cells occurred
even though central cells were not fertilized. In the experiment, artificially induced bicellular pollen was used. The bicellular pollen grains contained one diploid mitotically arrested generative cell
and one vegetative nucleus, thus a high
frequency of single fertilization events occurred. The author also reported spontaneous single fertilization events in preliminary experiments ( Kato 1990, 1992).
Parthenogenesis and heterofertilization
in maize might be related to spontaneous
single fertilization events, which have not
been detected in maize conclusively. In
this model a haploid embryo is developed
from an ovule whose central cell is singly
fertilized by a haploid sperm cell from a
pollen grain and the egg cell is not fertilized, but the unfertilized egg cell develops
into a haploid embryo apomictically. If an
ovule has an egg cell that is singly fertilized, and the central cell is fertilized by a
sperm cell of a different pollen grain, the
case will be conceived as a heterofertilization.
The purpose of this study is to detect
single fertilization events in diverse lines
of maize.
Table 1. List of lines used in the experiment
Line
Seed parent
Oh43
Pollen parent 1
907E
A188
GA203
Kohattyo
Na5
Silver honey bantam
Stock 6
Pollen parent 2
X18G
Genotype
Phenotype
Origin
Y1/Y1,wx1/wx1,v1/v1,
c1/c1,r1/r1,Bz2/Bz2
y1/y1,C1-I/C1-I,wx1/wx1
y1/y1
y1/y1
y1/y1
y1/y1
y1/y1,sh2/sh2
y1/y1,C1-I/C1-I
Yellow waxy endosperm, colorless
aleurone and embryo, virescent seedling
White endosperm, colorless aleurone and
embryo, normal seedling
Y1/Y1,Wx1/Wx1
R1-scm2/R1-scm2,bz2/bz2
Yellow endosperm, pale purple aleurone
and embryo, normal seedling
MGCSC, 916A
MGCSC
A dent inbred line of USA
A dent inbred line of USA
A hybrid variety of China
A Japanese flint inbred line
A sweet corn variety
Highly haploid inducing line
(Coe and Sarkar 1964), MGCSC
MGCSC
MGCSC 5 Maize Genetics Cooperation Stock Center, Urbana, Illinois.
Materials and Methods
A maize inbred line, Oh43, homozygous
for Y1 (yellow endosperm), wx1 (waxy endosperm), and v1 (virescent seedling, pale
green or yellowish white seedling later
turns green), received from Maize Genetics Cooperation Stock Center ( Urbana, Illinois) was used as seed parent. The dualpollination method employed in this experiment is basically the same as that described by Kato (1990, 1997b). The ears of
Oh43 were covered with paper bags prior
to silk emergence and all tassels of seed
parents were removed to minimize contamination. Silks were cut back to 1–2 cm
length and a small amount of pollen of pollen parent 1 (y1/y1, white endosperm, normal seedling; Table 1) was pollinated on
the silks. Then 24 h later the same silks
were pollinated with an ample amount of
pollen produced by pollen parent 2, X18G
(R1-scm2/R1-scm2, bz2/bz2, Y1/Y1, pale
purple aleurone and embryo, yellow endosperm, and normal seedling). The R1scm2 gene generally induces deep purple
pigmentation in the aleurone layer and
scutellum, and the combination with recessive bz2 turns deep purple color to
pale purple ( bronze phenotype). Kernels
of Oh43 crossed with X18G have deep purple aleurone and scutellum because the
recessive bz2 gene is hidden by dominant
Bz2 in Oh43. Thirty to 60 ears were dual
pollinated in each line and 13–26 well-pollinated ears were selected based on distribution of yellow kernels and purple kernels on the ears. The characters of kernels
concerning aleurone color and scutellum
color were examined. In some cases, observation of embryo color was difficult, so
the edge of the pericarp on the scutellum
was cut and scutellum color was determined in those cases. Oh43 and the pollen
parents 1 lack one or more color-conditioning genes or carry the C1-I gene (907E
and stock 6) and have colorless aleurone
and embryo. One pollen parent 1, 907E, is
homozygous for wx1 and has waxy endosperm, and Silver honey bantam is homozygous for sh2 (shrunken endosperm).
Purple kernels with white scutellum
(Pw) and yellow kernels with purple scutellum ( Yp) were separated. They were
germinated in moist vermiculite and roottip chromosome numbers were determined according to the method described
by Kato (1997a). Seedlings after chromosome determination were planted in the
greenhouse and genotypes were determined by self-pollination in diploid plants
or by cross-pollination with appropriate
marker stocks in monosomics and triploids.
Results
Among 118 ears examined, 9239 yellow
kernels with white scutellum ( Yw), 20,673
purple kernels with purple scutellum (Pp),
79 purple kernels with white scutellum
(Pw), and eight yellow kernels with purple
scutellum ( Yp) were obtained ( Figure 1,
Table 2). All eight seedlings germinated
from Yp kernels were diploid (2n 5 20)
and plants strictly resembled the hybrid
between Oh43 and X18G morphologically.
On the selfed ears of the eight Yp plants,
segregation of deep purple, bronze, and
yellow kernels (colorless aleurone) was
observed. Iodine staining showed that the
endosperm of two Yp kernels obtained
from dual pollination with 907E (wx1/wx1)
was nonwaxy phenotype. This indicates
that the endosperm of the two Yp kernels
is not the product of fertilization between
Oh43 (wx1/wx1) and 907E (wx1/wx1).
The 79 Pw kernels were germinated and
chromosome numbers were determined,
except that one kernel failed to germinate
( Table 3). There were 31 haploids (n 5
10), four monosomics (2n 5 19), 38 diploids (2n 5 20), and five triploids (3n 5
30) among the seedlings. In the control, all
five Pw cases exhibited virescent phenotype and were maternal haploids of Oh43.
Among the 31 haploid seedlings from
Pw kernels from dual pollination, 21 exhibited virescent seedling phenotype, 6 were
apparently normal seedlings, and 4 died
just after germination. The surviving 27
haploids exhibited striking haploid features ( low plant height, narrow leaves,
and high sterility) and all they were morphologically identical to Oh43 haploids.
Four monosomics (2n 5 19) exhibited
lower plant height (1.5 m) and semisterility, and their morphology was similar.
They were pollinated with bronze stocks
(R1-scm2/R1-scm2, bz2/bz2) and both
bronze kernels and deep purple kernels
were produced on the ears of the four
monosomics. Pollen collected from these
four monosomics was pollinated onto y1/
y1 stocks (Silver honey bantam) and all
the resultant kernels on Silver honey bantam were yellow and had colorless aleurone.
The 38 diploid plants germinated from
Pw kernels exhibited hybrid vigor and normal fertility. Selfed ears of 36 plants produced yellow and white kernels in a 3:1
ratio and the genotypes were Y1/y1, of
which three were from dual pollination
with Silver honey bantam (y1/y1, sh2/sh2)
segregated normal and shrunken kernels
in a 3:1 ratio also. Differences in the incidence of Y1/y1 cases against Yw kernels in
each line (0.12–0.69%) were not statistically significant. Ears of two plants (stock 617 and GA203-12) in the 38 Pw cases produced only yellow kernels with colorless
aleurone and the fertility was normal. The
morphology of the two plants resembled
the hybrid between Oh43 and X18G. Each
of 10 progenies of stock 6-17 and GA20312 were grown in the nursery and were
pollinated with X18G (R1-scm2/R1-scm2,
bz2/bz2), and 5 and 7 progenies of each
Kato • Single Fertilization in Maize 277
Figure 1. Four kinds of kernels are observed in dual-pollinated ears of Oh43, y1/y1 stocks as the first pollinator
and X18G (R1-scm2/R1-scm2) as the second pollinator. Yellow kernels with white scutellum ( Yw, top left) result
from fertilization by pollen parent 1. Purple kernels with purple scutellum (Pp, top right) result from fertilization
by X18G (R1-scm2/R1-scm2). Yellow kernels with purple scutellum ( Yp, bottom left) are thought to result from
fertilization of the egg by X18G (R1-scm2/R1-scm2), accompanied by a mutation or deletion in the other sperm of
X18G. Kernels with purple aleurone and white scutellum (Pw, bottom right) contain haploids, monosomics, diploids
(Y1/y1 and Y1/Y1), and triploids. The diploid Y1/y1 cases and triploids are thought to be the result of single
fertilization by pollen parent 1.
Table 2. Segregation of kernel phenotype exhibited after dual pollination of seven lines
Pollen parent 1
Ears examined
Yellow kernel
with white
scutellum ( Yw)
907E
A188
GA203
Kohattyo
Na5
Silver honey bantam
Stock 6
Total
Control, Oh43 3 X18G
16
16
19
15
13
13
26
118
38
868
1580
1004
1678
938
1168
2003
9239
4
Purple kernel
with purple
scutellum (Pp)
Purple kernel Yellow kernel
with white
with purple
scutellum (Pw) scutellum ( Yp)
1677
2956
2968
2849
2797
2343
5083
20,673
11,642
8
13
14
13
6
8
17
79
5
2
2
1
1
1
0
1
8
3
Ears of Oh43 (Y1/Y1) were pollinated with pollen parent 1 (y1/y1) first, 24 h later the same ears were pollinated
with pollen parent 2 ( X18G, R1-scm2/R1-scm2, purple aleurone and scutellum).
278 The Journal of Heredity 1999:90(2)
produced bronze kernels on the ears, and
the genotypes of stock 6-17 and GA203-12
were considered to be Y1/Y1, Bz2/bz2.
Among the five triploids obtained from
the Pw cases, three (907E-3, 907E-4, and
907E-8) were from dual pollination with
907E (C1-I/C1-I) and two (GA203-8, GA20314) were from GA203 ( Table 3). Silks of the
three triploids obtained from dual pollination with 907E were pollinated by R1scm2 stocks (R1-scm2 /R1-scm2, purple aleurone purple embryo). The ears produced various sizes of kernels, as is a feature of triploids. Plump kernels were
selected and the numbers of colorless or
colored aleurone kernels were determined: (colorless : colored) 217:44 (907E3), 39:13 (907E-4), and 45:13 (907E-8). Chisquare tests revealed the segregation ratio
was not consistent with a 1:1 ratio (x 2
5114.7, x 2 5 13.0, and x2 5 17.7, all cases
P , .001), and agreed with a 5:1 ratio (x2
5 0.0 ns, x2 5 2.6 ns, and x2 5 1.4 ns),
which is expected from C1-I /C1-I /c1 genetic constitution. The two triploids, GA2038 and GA203-14, were crossed with y1/y1
stock. The ears also produced various size
of kernels and considerably sterile, plump
kernels were selected and the segregations of endosperm type yellow : white
were 10:9 (GA203-8) and 8:20 (GA203-14).
The ratios agreed with 1:1 segregation (x 2
5 0.05 ns and x2 5 5.1, P , .05), which is
expected from Y1/y1/y1 genetic constitution and did not agree with a 5:1 ratio (x 2
5 12.9 and x2 5 60.5, both P , .001),
which is expected from Y1/Y1/y1 genetic
constitution.
Discussion
The Yw kernels are considered to be produced by the first pollination (Oh43 3 pollen parents 1) and the Pp kernels by the
second pollination (Oh43 3 X18G) in this
experiment. Pw kernels obtained in this
experiment proved to have various origins
( Table 3). All the haploids of the Pw cases
are considered to be spontaneous maternal Oh43 haploids induced by the second
pollination. Six haploids with Oh43 haploid morphology which did not exhibit virescent phenotype would be the result of
marginal expression of the v1 constitution.
The ratio of Pw haploids relative to Pp kernels is 0.15% (31/20,673) and is consistent
with the spontaneous occurrence of haploids described in previous reports (Chase
1949, 1952).
The four monosomics (2n 5 19) carrying the bz2 gene are considered to originate from fertilization between an Oh43
Table 3. Ploidy and genotypes exhibited by seedlings of Pw cases obtained from dual-pollinated ears
a
Haploid
n 5 10
Monosomics
2n 5 19
Diploid 2n 5 20
Pollen parent 1
Seedlings
examined
907E
A188
GA203
Kohattyo
Na5
Silver honey bantam
Stock 6
Total
Control, Oh43 3 X18G
8
13
14
13
6
8
17
79
5
4
5
3
5
4
5
5
31
5
0
1
0
2
1
0
0
4
0
1 (0.12)
7 (0.44)
7 (0.69)
6 (0.36)
1 (0.11)
3 (0.26)
11 (0.55)
36
0
Y1/y1
a
Y1/Y1
Triploid
3n 5 30
Ungerminated
0
0
1
0
0
0
1
2
0
3
0
2
0
0
0
0
5
0
0
0
1
0
0
0
0
1
0
Percentage of Y1/y1 diploids against the correspondent number of Yw cases ( Table 2).
egg cell (n 5 10) and a sperm cell of X18G
with nine chromosomes that lost one
chromosome carrying either of the colorconditioning genes (C1 or R1-scm2) while
the central cell was fertilized by a sperm
cell carrying both C1 and R1-scm2 genes.
This is considered a case of abnormal
X18G pollen grains resulting from nondisjunction in the second pollen mitosis.
The diploids of the 38 Pw cases contained plants of two different origins: 36
Y1/y1 and the 2 Y1/Y1 cases. The 36 plants
with genotype Y1/y1 are considered to be
hybrids between Oh43 and pollen parent
1 because of the presence of the y1 gene
of pollen parent 1 (or the sh2 gene in dual
pollination with Silver honey bantam).
These originated from single fertilization
events, namely, egg cells were first fertilized by the sperm cells released from the
pollen tubes of pollen parents 1 (y1/y1),
then 24 h later central cells were fertilized
by the sperm cells released from pollen
tubes of pollen parent 2, X18G, and double
fertilization was completed. The fate of
the other sperm cell, which must be contained in the pollen grains at first pollination, is elusive. Loss of a sperm cell during
migration in pollen tubes or loss of a
sperm cell at the entrance of the micropyle might cause single fertilization events
by pollen parent 1. Existence of single fertilization events in maize was first speculated by Sarkar and Coe (1971c), and Kato
(1997b) proved it using artificially induced
bicellular pollen of maize.
The average single fertilization rate exhibited by untreated maize pollen in this
experiment is 0.4%. In usual pollination, an
excess amount of pollen is pollinated on
silks and singly fertilized ovules must be
fertilized by sperm cells from other pollen
grains and conceived as heterofertilized
kernels. Heterofertilization is usually detected by crosses between stocks homozygous recessive and heterozygous dominant, for example, Yp cases in r1/r1 3 R1-
scm2/r1 cross, and the incidence is about
1%, though the actual heterofertilization
rate is 2% if concordant cases are included
(Robertson 1984). The single fertilization
rate in this experiment (0.4%) implies at
least one-fifth of heterofertilized kernels
can result from spontaneous single fertilization events in maize.
Single fertilization was observed in all
seven genetically different lines (0.11–
0.69%), and the rates were not statistically
different. The rates must also be affected
by the pollen distribution on the silks at
first pollination. Stock 6, which induces
haploids 5–30 times higher than usual
lines, did not exhibit an exceptionally high
single fertilization rate. The relationship
between haploid-inducing ability and single fertilization is elusive, though spontaneous single fertilization must have some
role in the occurrence of spontaneous
haploids in maize.
The two diploid plants, which were homozygous for the Y1 gene and heterozygous for the bz2 gene in Pw cases, are considered to be hybrids between Oh43 and
X18G. Mutation of a color-conditioning
gene after the second pollen mitosis may
be responsible for absence of scutellum
color in the two cases.
The five triploids of Pw cases are intriguing. Considering the segregation rate,
the three triploids (907E-3, 907E-4, and
907E-8) obtained from the dual pollination
of 907E have two doses of the C1-I gene
and their genetic constitution is C1-I/C1-I/
c1. This means there were two genome
contributions from pollen parent 907E. In
the two triploids GA203-8 and GA203-14,
the chi-square test indicates their genetic
constitutions are Y1/y1/y1 and that they
also received two genome contributions
from the pollen parent 1. Thus the five
triploids are considered to have originated by the same mechanism. Spontaneously produced bicellular pollen (containing
one diploid sperm cell and one vegetative
cell) could be responsible for the occurrence of these five triploids. Bicellular pollen in maize can be produced by chemical
treatment by inhibiting the second pollen
mitosis ( Kato 1997b), and bicellular pollen
grains were observed in normal maize pollen grains at a frequency of 0.25% ( Kato
1998). Occasional plump kernels in tetraploid 3 diploid crosses (Sarkar and Coe
1971b) and occasional shriveled kernels
(1–3 per ear) in diploid 3 diploid crosses
on maize ears may also be caused by
these pollen grains. These triploid cases
suggest that a small part of heterofertilized kernels must have a triploid embryo,
though Sarkar and Coe (1971a) did not detect any triploid cases in their 117 heterofertilized progenies.
The eight Yp cases might have resulted
from a second pollination (Oh43 3 X18G)
where color loss in the endosperm occurred by chromosome loss or deletion,
or by mutation of color-conditioning
genes occurring in a sperm cell of X18G
after the second pollen mitosis, after
which the mutated sperm cell fertilized
the central cell (the reverse phenomenon
of the two Y1/Y1 plants in the Pw cases).
Yp kernels also might be produced by a
single fertilization event in which the central cell was first fertilized by a sperm cell
of pollen parent 1 and the egg cell was fertilized by a sperm cell of X18G 24 h later.
The latter possibility would be low, because Yp kernels are usually observed
(0.03%) in the r1/r1 3 R1-scm2/R1-scm2
cross (Robertson 1984), and the incidence
of Yp kernels (0.039%, 8/20,673) is almost
the same as that of the control (0.026%, 3/
11,642). Furthermore, the nonwaxy endosperm of the two Yp kernels of dual-pollination 907E (wx1/wx1) indicates that the
endosperm did not result from the first
pollination in these two cases.
In this study egg side single fertilization
events have successfully been detected.
The detection of central cell side single
fertilization, which would develop into
kernels with a haploid embryo or heterofertilized kernels resembling Yp cases as
observed in this experiment, requires another large-scale experiment with appropriate embryo and endosperm markers.
References
Aman MA and Sarkar KR, 1978. Selection for haploidy
inducing potential in maize. Indian J Genet Plant Breed
38:452–457.
Chalyk ST, 1994. Properties of maternal haploid maize
plants and potential application to maize breeding. Euphytica 79:13–18.
Chase SS, 1949. Monoploid frequencies in a commercial
Kato • Single Fertilization in Maize 279
double cross hybrid maize, and in its component single
cross hybrids and inbred lines. Genetics 34:328–332.
Kato A, 1997a. An improved method for chromosome
counting in maize. Biotech Histochem 72:249–252.
Sarkar KR and Coe EH, 1971a. Analysis of events leading to heterofertilization in maize. J Hered 62:118–120.
Chase SS, 1952. Production of homozygous diploids of
maize from monoploids. Agron J 44:263–267.
Kato A, 1997b. Induced single fertilization in maize. Sex
Plant Reprod 10:96–100.
Sarkar KR and Coe EH, 1971b. Anomalous fertilization
in diploid-tetraploid crosses in maize. Crop Sci 11:539–
542.
Chase SS, 1969. Monoploids and monoploid-derivatives
of maize (Zea mays L.). Bot Rev 35:117–167.
Coe EH, 1959. A line of maize with high haploid frequency. Am Nat 93:381–382.
Coe EH and Sarkar KR, 1964. The detection of haploids
in maize. J Hered 55:231–233.
Greenblatt IM and Bock M, 1967. A commercially desirable procedure for detection of monoploids in maize. J
Hered 58:9–13.
Kato A, 1998. Hematoxylin procedure for staining mature pollen grains in maize with dimethylsulfoxide as a
clearing agent. Biotech Histochem 73:1–5.
Lashermes P and Beckert M, 1988. Genetic control of
maternal haploidy in maize (Zea mays L.) and selection
of haploid inducing lines. Theor Appl Genet 76:405–410.
Sarkar KR and Coe EH, 1971c. Origin of parthenogenetic diploids in maize and its implications for the production of homozygous lines. Crop Sci 11:543–544.
Sarkar KR, Panke S, and Sachan JKS, 1972. Development
of maternal-haploidy-inducer lines in maize (Zea mays
L.). Indian J Agric Sci 42:781–786.
Nanda DK and Chase SS, 1966. An embryo marker for
detecting monoploids of maize (Zea mays L.). Crop Sci
6:213–215.
Sprague GF, 1929. Hetero-fertilization in maize. Science
69:526–527.
Kato A, 1990. Heterofertilization exhibited by using
highly haploid inducing line ‘‘Stock 6’’ and supplementary cross. Maize Genet Coop Newslett 64:109–110.
Robertson DS, 1984. A study of heterofertilization in
diverse lines of maize. J Hered 75:457–462.
Sprague GF, 1932. The nature and extent of hetero-fertilization in maize. Genetics 17:358–368.
Kato A, 1992. Detection of an unfertilized polar nucleus
with a fertilized egg cell. Maize Genet Coop Newslett
66:105–106.
Sarkar KR and Coe EH, 1966. A genetic analysis of the
origin of maternal haploids in maize. Genetics 54:453–
464.
280 The Journal of Heredity 1999:90(2)
Received March 19, 1998
Accepted September 30, 1998
Corresponding Editor: Gary Hart