Download Abnormalities occurred during female gametophyte development

Title: Abnormalities occurred during female gametophyte development
result in the diversity of abnormal embryo sacs and lead to the abnormal
fertilization in indica/japonica hybrids in rice
Running title: Embryo sac abortion in indica/japonica hybrids in rice
Yu-xiang Zeng1, Chao-yue Hu1, Yong-gen Lu1, Jin-quan Li1 and Xiang-dong Liu1*
(1Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South
China Agricultural University, Guangzhou 510642, China)
*
Author for correspondence.
（0） 85280205 ;
Fax: +86 （0） 85285705 ;
Tel: +86
E-mail: <[email protected]>.
1
Abstract
Embryo sac abortion is one of the major reasons causing sterility in indica/japonica
hybrid in rice. To clarify the causal mechanism of embryo sac abortion, we studied the
female gametophyte development in two indica/japonica hybrids via an eosin B staining
procedure for embryo sac scanning using confocal laser scanning microscope. Different
types of abnormalities occurred during megasporogenesis and megagametogenesis were
demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the
chalazal-most megaspores were degenerated before mono-nucleate embryo sac stage.
Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of
the abnormal female gametes from mono-nucleate to four-nucleate embryo sac stage. The
abnormalities occurred from early stage of eight-nucleate embryo sac development to
mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals.
Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of
egg apparatus were also found at eight-nucleate embryo sac stage. The abnormalities
occurred during female gametophyte development resulted in five major types of
abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand
pollination using normal pollens upon the spikelets during anthesis showed that normal
pollens could not exclude the effect of abnormal embryo sac on seed setting.
Keywords: Indica/japonica hybrid; Megasporogenesis; Megagametogenesis; Oryza
sativa; whole-mount eosin B-staining confocal laser scanning microscopy(WE-CLSM).
.
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Indica and japonica are two subspecies in Asian cultivated rice (Oryza sativa L.). It is
known that strong heterosis expresses in indica/japonica hybrids in both vegetative and
reproductive growth, which has attracted the attentions of rice breeders for many years
(Liu et al. 2004; Song et al. 2005). However, the majority of indica/japonica hybrids are
partial sterile, which results in low seed setting and restricts the direct utilization of the
hybrids in commercial production. Therefore, there have been considerable interests in
rice breeding community to understand the mechanism of partial sterile in
indica/japonica hybrids.
Genetic analysis has been extensively carried out to reveal the mechanism of
inter-subspecific hybrid sterility. Oka (1953, 1957) proposed a “duplicate gametic lethal
model” to explain the genetic basis of sterility in distantly related hybrids, and
demonstrated that several sets of duplicate genes were responsible for gametophytic F1
sterility (Oka 1974). After the discovery of “wide compatibility varieties”, which
produced fertile F1 hybrids when crossed to either indica or japonica, Ikehashi and Araki
(1986) proposed a “one-locus sporo-gametophytic interaction” model to explain
indica/japonica hybrid sterility. Besides the S5 locus (Ikehashi and Araki 1986), a serious
of other loci causing hybrid sterility, such as S7, S8, S9, S15, S16, S17, S29, S30 and S31
were identified and mapped by using molecular markers (Wan et al.1993,1996; Wan and
Ikehashi 1995; Zhu et al. 2005; Zhao et al. 2006). The wide-compatibility gene S5n for
overcoming partial abortion of female gametes was mapped within a 50 kb region (Ji et
al. 2005). To date, more than 30 loci conferring pollen fertility, embryo sac fertility or
spikelet fertility in indica/japonica hybrids have been identified.
Cytological observations have shown that defects in the developmental process of
pollen and embryo sac are causes for sterility in indica/japonica hybrids (Yokoo 1984;
Zhu et al. 1998; Liu et al.1997b; Liu et al. 2004; Song et al. 2005). Embryo sac fertility
and pollen fertility were reported to be two most important factors influencing spikelet
fertility (Liu et al. 2004; Song et al. 2005). The embryo sac plays a pivotal role in sexual
reproduction in rice. It is the structure that containing egg cell and central cell which,
following fertilization, give rise to the embryo and endosperm, respectively. There have
been some cytological observations that trying to reveal the mechanism of embryo sac
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abortion in indica/japonica hybrid (Liu et al.1997b; Zhu et al. 1998; Liu et al. 2004).
However, the results obtained in these studies were different. Although some abnormal
phenomena during female gametophyte development have been reported (Liu et al.1997b;
Zhu et al. 1998; Liu et al. 2004), it is still not clear whether the different types of
abnormal embryo sacs occurred in a specific stage or several continuous stages (Zhu et al.
1998; Liu et al. 2004). And it is still not clear how these abnormal embryo sacs affect the
process of fertilization in indica/japonica hybrid.
Although some techniques for embryo sac examination in rice have been
introduced, such as the whole-stain clearing technique (Yang 1986) or methyl salicylate
clearing procedure for embryo sac scanning using confocal laser scanning microscope
(Ren et al.1998). Traditional sectioning technique, which is an arduous job especially in
genetic analysis when a lot of embryo sacs have to be examined, is still the major
technique used in most of the previous studies (Liu et al. 2001). To investigate the
mechanism of embryo sac abortion in rice, a rapid and convenient method which can
demonstrate the detailed information within the embryo sac is demanded. To meet this
need, we developed a simple eosin B staining procedure for embryo sac scanning using
confocal laser scanning microscope based on the previous studies (Shotton 1989; Shaw et
al.1992; Ren et al.1998; Zhang et al. 2003). This technique was named “WE-CLSM”
(whole-mount eosin B-staining confocal laser scanning microscopy) (Zeng et al. 2007). It
facilitates the observation of abnormal cells and nuclei in different stages of female
gametophyte development in rice without requiring continuous sections of the sample.
In this paper, we investigated the female gametophyte formation and development
in two typical indica/japonica hybrids (Liaojing 944/Guangluai No.4, Taichung
65/Guangluai No.4) via WE-CLSM. The embryo sacs at 24 h after pollination were
observed to examine the embryogenesis and endosperm development. We also performed
hand pollination, using numerous normal pollens to pollinate upon the spikelets of an
indica/japonica hybrid, to examine whether the effect of abnormal embryo sacs on
fertilization can be excluded. The objective was to clarify the causal mechanism of
embryo sac abortion at cellular level, and to reveal how the abnormal embryo sacs affect
the fertilization in indica/japonica hybrids.
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Results
Abnormalities occurred during megasporogenesis and megagametogenesis in
indica/japonica hybrids
The megasporogenesis and megagametogenesis in parental varieties (Guangluai
No.4 and Liaojing 944) were observed. We found that the embryo sac formation and
development in the two parental varieties were similar to those reported by Tai and Tseng
(1964) and Liu et al. (1997a). The female gametophyte formation and development were
described as the following stages: megasporocyte stage (Fig. 1 a and b), meiotic division
stage (Fig. 1c and d), functional megaspore stage (Fig. 1e), mono-nucleate embryo sac
stage (Fig. 1f), two-nucleate embryo sac stage(Fig. 1g), four-nucleate embryo sac stage
(Fig. 1h), eight-nucleate embryo sac stage (Fig. 1i-l) , and mature embryo sac stage (Fig.
3h). The eight-nucleate embryo sac stage could be subdivided into early (Fig. 1i and j),
middle (Fig. 1k) and late (Fig. 1l) stage.
Abnormalities were observed during megasporogenesis and megagametogenesis in
the two indica/japonica hybrids (Liaojing 944/Guangluai No.4, Taichung 65/Guangluai
No.4). The abnormalities occurred during female gametophyte development were
similar in the two hybrids.
Abnormalities could be found at early megasporocyte stage (Fig. 2a) and the
rectangle-like megasporocyte stage (Fig. 2b). The abnormal megasporocyte was
characterized by the nucleus located in the chalazal-most region (Fig.2 a and b). However,
abnormalities were not observed during the meiosis of the megasporocyte. Most of the
abnormalities occurred after the meiosis. At the stage when the chalazal-most megaspore
was developing into the mono-nucleate embryo sac, some abnormal megaspores could
not elongate along the micropylar-chalazal axis and form the normal mono-nucleate
embryo sac, instead they degenerated (Fig. 2 c and d) and resulted in a lot of degenerated
embryo sacs found in the mature stage (Fig. 3a).
Abnormalities could be found from mono-nucleate embryo sac stage to mature
embryo sac stage. The majority abnormal mono-nucleate, two-nucleate and four-nucleate
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embryo sacs were characterized by a darkly stained tissue surrounding the female
gametophyte (Fig.2 e, g , i, j), indicating that the nucellus tissue was abnormal.
In very few cases, abnormality was also found in the metaphase of the first mitotic
division (Fig.2 f). During the mitotic division stage, disordered positioning of the nucleus
was another characteristic of the abnormal female gametophyte. For example, both nuclei
were located at the micropylar-most end in an abnormal two-nucleate embryo sac (Fig.2
h); three nuclei were located at the micropylar end, only one nucleus was located at the
chalazal end in an abnormal four-nucleate embryo sac (Fig.2 j).
Different kinds of abnormalities were observed from early stage of eight-nucleate
embryo sac development to the mature embryo sac stage. In very few cases, the abnormal
eight-nucleate embryo sac was without the differentiation of the normal embryo sac
cavity, but the eight nucleoli could be found clearly (Fig.2 k). Most of abnormal embryo
sacs found at middle or late stage of eight-nucleate embryo sac development were
characterized by smaller sizes (Fig. 2 l) compared with normal ones (Fig. 1l). Wrinkled
antipodals were found in these embryo sacs (Fig. 2 l-o). Asynchronous nuclear migration
(Fig.2 m), abnormal nuclear migration (Fig.2 n) and abnormal positioning of nuclei
(Fig.2 p) were observed. Some of the eight-nucleate embryo sacs were found with
degenerated egg apparatus (Fig.2 n-p).
Abnormalities occurred during female gametophyte development resulted in the
diversity of abnormal embryo sacs in indica/japonica hybrids at anthesis
We examined 279 mature embryo sacs in “Liaojing 944/Guanglu’ai No.4” and 350
mature embryo sacs in “Taichung 65/Guanglu’ai No.4”, and found that 67.7% and 47.1%
of the embryo sacs were abnormal in the two hybrids, respectively (Table 1). The
abnormal embryo sacs could be categorized into five major types: (1) embryo sac
degeneration (ESD, Fig.3 a); (2) abnormal small embryo sac(ASES, Fig.3 b) whose size
approximately equal to or less than 1/2 of a normal mature embryo sac (Fig. 3h), most of
the ASES are characterized by abnormal positioning of polar nuclei; (3) embryo sac
without egg apparatus (ESWE; Fig.3 c); (4) embryo sac without female germ unit
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(ESWF; Fig.3 d); (5) embryo sac with abnormal polar nuclei (ESWA; Fig.3 e).
Besides the five categories mentioned above, some other abnormal embryo sacs were
also found, but since their frequencies were very low, we defined them as “other
abnormal type”(OAT). For example, the embryo sac with its egg cell and polar nuclei
located in an abnormal position (Fig. 3 f). We also found an embryo sac with abnormal
egg apparatus, and three nuclei which looked like polar nuclei located in the antipodals
(Fig. 3g).
ESD and ASES constituted the majority of abnormal embryo sacs in the two hybrids.
For example, the ESD constitutes 33.4% in “Liaojing 944/Guanglu’ai No.4”, and the
ASES constitutes 38.1%. The ESD constitutes 52.2% in “Taichung 65/Guanglu’ai No.4”,
and the ASES constitutes 29.7%. The frequencies of various types of abnormal embryo
sacs in the two hybrids are listed in Table 1.
After observation of the megasporogenesis and megagametogenesis in the two
hybrids, we found that two types of abnormalities occurred frequently: (1) the
degeneration of chalazal-most megaspore (2) the abnormalities occurred at eight-nucleate
embryo sac stage. It coincides with the statistic data of the mature embryo sacs, that ESD
and ASES are two major abnormal types. It is obvious that ESD are resulted from the
degeneration of chalazal-most megaspore. And ASES, ESWE, ESWF and ESWA are
resulted from the abnormal developmental behavior of the eight-nucleate embryo sac.
For example, the degeneration of the egg apparatus at eight-nucleate embryo sac stage
resulted in the ESWE, the degeneration of the female germ unit resulted in the ESWF,
abnormal positioning of the polar nuclei resulted in the ESWA. Since smaller size is
characteristic of the majority of eight-nucleate embryo sacs, ASES constituted a large
proportion of the abnormal eight-nucleate embryo sacs in the two hybrids.
Defects observed in embryo sacs at 24 h after pollination in indica/japonica hybrids
After the pollen tube penetrates the embryo sac through the synergid, one sperm
fuses with egg cell and develops into the embryo, another sperm fuses with polar nuclei
and develops into the endosperm. At 24 h after pollination, a multi-celled globular
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embryo was observed in normal fertilized embryo sac in indica/japonica hybrids, and a
layer of free endosperm nuclei suspended in the peripheral cytoplasm of the embryo sac
(Fig. 4 a and b).
Abnormalities were observed in the embryo sacs of the two hybrids at 24 h after
pollination. Most of the ASES cannot be fertilized due to the abnormal positioning of
polar nuclei (Fig.4 c).But very few ASES containing normal female germ unit were
found to form the multi-celled globular embryo, indicating a successful fusion of egg cell
and sperm, but the size of the fertilized embryo sac was still smaller (Fig.4 d) compared
to normal ones. Most of the ESWA cannot be fertilized due to the abnormal position in
which the polar nuclei locate. But some of the ESWA were found to form the multi-celled
globular embryo, although the polar nuclei did not fuse with the sperm (Fig.4 e). ESWE
cannot be fertilized due to the missing of egg cell. But some of the ESWE were found
with the free endosperm nuclei at 24 h after pollination, indicating a successful fusion of
the polar nuclei and the sperm, although the egg apparatus was degenerated (Fig.4 f).
In some cases, fertilization was stopped when the pollen tube penetrated into the
embryo sac (Fig.4 g), demonstrating that even a normal embryo sac cannot ensure a
successful fertilization in indica/japonica hybrids. Lagged embryo and endosperm
development (Fig.4 h) was observed in some embryo sacs.
Seed setting of Taichung 65/Guangluai No.4 after hand pollination
The pollen fertility of Taichung 65/Guangluai No.4 was 60.5% when it was grown
in March 2005. When grown in July 2005, its pollen fertility was 29.9%. To exclude the
effect of pollen fertility, and to analyze the real effect of embryo sac fertility on seed
setting, we performed hand pollination using numerous normal pollens upon Taichung
65/Guangluai No.4. The seed setting of Taichung 65/Guangluai No.4 after selfing was
9.6% when it was grown in July 2005 (Table 2). After hand pollination, the seed setting
increased to 21.2%, which was very close to the embryo sac fertility (Table 2). These
results indicated that the pollen fertility had effect on the seed setting of Taichung
65/Guangluai No.4. It also suggested that the effect of abnormal embryo sacs on seed
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setting could not be excluded by using normal pollens, because the abnormal embryo
sacs were abortive in Taichung 65/Guangluai No.4.
Discussion
Some abnormalities occurred during megagametogenesis in indica/japonica hybrid
were reported in previous studies. The abnormality of the functional megaspore which
results in the degeneration of the entire embryo sac has been observed by using
sectioning technique (Liu et al.1997b; Zhu et al.1998; Liu et al. 2004). Liu et al. (2001)
reported that the degeneration of female gametophyte occurred from mono-nucleate or
two-nucleate stage. Liu et al. (2004) observed abnormal antipodals in one
indica/japonica hybrid. In the present study, we found that the abnormalities could
happen before the meiosis, i.e. abnormal positioning of the nucleus in the megasporocyte,
which is not reported in previous studies. During megagametogenesis, we found that
disordered positioning of nucleus, abnormal nucellus tissue were characteristics of the
abnormal female gametophyte from mono-nucleate embryo sac stage to four-nucleate
embryo sac stage. Most of the abnormalities happened at eight-nucleate embryo sac stage
were characterized by smaller sizes and wrinkled antipodals. Asynchronous (or abnormal)
nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus
were also found at eight-nucleate embryo sac stage.
To make a comprehensive understanding of the abnormalities occurred during
female gametophyte development in indica/japonica hybrid, our strategy is to examine
the mature embryo sacs before we study the megasporogenesis and megagametogenesis.
Five major types of abnormalities were found in mature embryo sacs i.e. ESD, ASES,
ESWE, ESWF and ESWA, which is consistent with the findings of Zeng et al. (2007). By
comparing the abnormalities occurred during female gametophyte development with the
abnormalities found in mature embryo sacs, we found that (1) The degeneration of the
chalazal-most megaspore before the mono-nucleate embryo sac stage resulted in the ESD.
(2) The majority of abnormalities happened at eight-nucleate embryo sac stage mainly
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resulted in ASES, ESWE, ESWF and ESWA. The degeneration of chalazal-most
megaspore and the abnormal eight-nucleate embryo sacs were frequently occurred during
embryo sac development, which coincided with the statistic data of the mature embryo
sacs.
The results reported in this study are consistent with the finding of Oka and Doida
(1962), who speculated the abortion occurred before the first mitotic division of the
megaspore, and of Ouyang and Li (1995), who suggested that the abortion occurred at
the functional megaspore stage. Liu et al. (1997b) found that all megasporocyte could
undergo meiosis normally and abortion mainly occurred at the stage when the
chalazal-most megaspore beginning to form the mono-nucleate embryo sac, which is also
consistent with our findings in the present study. But it seems that most of the previous
researchers have neglected the abnormalities arose at eight-nucleate embryo sac stage,
which occurs frequently in indica/japonica hybrids. Besides, we provided detailed
pictures of the abnormalities from megasporogenesis to megagametogenesis, the results
obtained in this study would be more comprehensive.
The polarity of nuclei is mainly manifested by the regular pattern of positioning of
nuclei during embryo sac development (Huang and Sheridan 1994). Huang and Sheridan
(1994) observed that the DNA-containing organelles were predominantly localized at the
chalazal end of the megaspore mother cell before meiosis and established the premeiotic
megasporocyte polarity in maze. In this study, we observed abnormal positioning of the
nucleus occurred from before the meiosis to the mature embryo sac stage. The abnormal
positioning of the nuclei may distort the polarity of the cell and cause the abnormalities
in indica/japonica hybrids. Furthermore, it seems that the abnormal nucellus tissue has
some relationship with the abnormal female gametophyte, which is still need to be
investigated.
The results demonstrate that the embryo sac abnormalities occurred in
indica/japonica hybrids are very complicated. Significant genetic differentiation between
indica and japonica varieties was detected by using various molecular markers in
previous studies (Zhang et al. 1997). The various abnormalites reported in this paper are
consequence of interaction between indica and japonica. Besides embryo sac fertility,
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pollen fertility was also considered a major factor affecting seed setting. Liu et al. (2004)
reported that the seed setting was highly dependent on adherence of pollen on stigma. In
the present study, the seed setting of the two hybrids were lower than their embryo sac
fertility (Table 1), indicating that there are other factors affecting the seed setting besides
embryo sac fertility.
In this study, we used WE-CLSM to investigate the embryo sac development and
fertilization status in indica/japonica hybrids and demonstrated a lot of abnormal
phenomena within the female gamete. WE-CLSM highlights the nucleolus structure,
therefore the position of the nucleus can be shown (Eosin B is a tissue stain for cell
granules and nucleoli) (Zeng et al. 2007). Since FAA is a harsh fixative, we also used
glutaraldehyde. And we found the images obtained by using FAA were clearer than using
glutaraldehyde.
Materials and methods
Plant materials
Two F1s of indica/japonica hybrids (Liaojing 944/Guangluai No.4, Taichung
65/Guangluai No.4) were cultivated in March 2005 at the experimental farm of South
China Agricultural University, Guangzhou, China (23°16’N, 113°8’E). Liaojing 944, and
Taichung 65 are typical japonica cultivars. Guangluai No.4 is a typical indica cultivar.
The two F1s were used to investigate the female gametophyte formation and development
in indica/japonica hybrid. The parental varieties (Guangluai No.4 and Liaojing 944) were
also planted to study the megasporogenesis and megagametogenesis in rice.
Fixation
Florets were collected at different developmental stages. Florets with open glumes,
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i.e., in which embryo sacs were mature and ready for fertilization, were collected at noon
each day. At anthesis, the florets were labeled when pollination began and collected at 24
h after pollination. All collected materials were fixed in FAA (formaldehyde: acetic acid:
50% ethanol = 5:6:89) immediately for at least 24 h, then washed with 50% ethanol and
stored in 70% ethanol at 4.
Eosin B staining
The ovaries were dissected in 70% ethanol under a binocular dissecting microscope,
and hydrated sequentially in 50% ethanol, 30% ethanol and distilled water. After that, the
ovaries were pretreated in 2% aluminum potassium sulphate for 20 min to allow the dye
to enter the embryo sac more readily. Then the ovaries were stained with 10 mg/L eosin
B (C20H6N2O9Br2Na2, FW 624.1, a tissue stain for cell granules and nucleoli) solution
(dissolved in 4% sucrose) for 10-12 h at room temperature. The samples were
post-treated in 2% aluminum potassium sulphate for 20 min in order to remove some dye
from the ovary walls. The samples were rinsed with distilled water three times, and
dehydrated with a series of ethanol solutions (30%, 50%, 70%, 90% and 100%).
Subsequently, the dehydrated samples were transferred into a mixture of absolute ethanol
and methyl salicylate (1:1) for 1 h, and then cleared in pure methyl salicylate solution for
at least 1 h (Zeng et al. 2007).
Embryo sac scanning
The samples were scanned under a Leica SP2 laser scanning confocal microscope
(Germany). Excitation wavelength was 543 nm, and emitted light was detected between
550 and 630 nm. All embryo sacs were placed in a specific orientation on the slide, i.e.
the plane constituted by the two stigmas was perpendicular to the plane of the glass slide.
The images of different focal planes of a sample were recorded. A composite of 4-6
images from different focal planes of a sample was shown (Zeng et al. 2007). The ovaries
for embryo sac scanning were randomly collected from five plants of each hybrid. The
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numbers of mature embryo sacs examined were listed in Table 1.
Hand pollination
The F1 of Taichung 65/Guangluai No.4 was also planted in July 2005 in the same
location as mention above. We performed hand pollination using numerous normal
pollens upon the spikelets of Taichung 65/Guangluai No.4. Hand pollination was
conducted every day at noon when the glumes were open, until the anthesis was over.
The normal pollens used in hand pollination were from some elite varieties in China.
Examination of pollen fertility
Five mature florets were collected from each panicle of a plant. All the pollens were
mixed and stained with 1% KI-I2, and observed under a microscope. The darkly stained
and round pollen grains were regarded as fertile pollen grains, and all others were
classified as sterile. About 500 pollens per plant were analyzed, the pollen fertility was
the mean of three plants.
Acknowledgements
The authors thank Prof. Xue-Bin Xu, Hai-Bin Guo, Ms. Shu-Hong Yu and Jin-Hua He
for help in the laboratory and field, and thank Assoc. Prof. Bing-Yao Yang and Wei Su for
technical assistance. This research was supported by the National Science Foundation of
China (30270814, 30771328) and the Teaching and Research Award Program for
Outstanding Young Teachers in Higher Education Institutions of MOE, P.R.C.
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Zhu XH, Cao XZ, Zhu QS (1998). Investigation on gametophytic sterility and it’s
contribution to spikelet sterility of F1 plants of Indica/Japonica in rice. Acta Agron
Sin 24, 421-430.
Figure 1. The megasporogenesis and megagametogenesis in rice (In each frame, the
orientation of the sample is with the micropylar pole towards the lower end of the
picture). (a) An early stage megasporocyte with the nucleus (arrow) located toward the
micropylar region. (b) A rectangle-like megasporocyte with the nucleus (arrow) located a
little toward the micropylar region. (c) The metaphaseof the meiotic division, arrows
indicate the two cell of the dyad. (d) A tetrad, arrows indicate the four nuclei. (e) Three
megaspores at the micropylar end were degenerated, only the chalazal-most megaspore
(arrow) survived. (f) A mono-nucleate embryo sac. (g) A two-nucleate embryo sac. (h) A
four-nucleate embryo sac. (i) An early stage of eight-nucleate embryo sac development,
the eight nucleoli were observed. (j) Two nuclei (arrow heads) had enlarged and began to
16
move toward the center of the embryo sac. (k) An embryo sac at middle stage of
eight-nucleate embryo sac development, two nuclei had migrated to the central region. (l)
An embryo sac at late stage of eight-nucleate embryo sac development. Bars = 40µm.
Figure 2. Abnormalities occurred during embryo sac development in indica/japonica
hybrids (In each frame, the orientation of the sample is with the micropylar pole towards
the lower end of the picture). (a) Abnormal early stage megasporocyte (Taichung
65/Guangluai No.4) with the nucleus located in the chalazal-most region (arrow). (b)
Abnormal rectangle-like megasporocyte (Liaojing 944/Guangluai No.4) with the nucleus
located in the chalazal-most region (arrow). (c) The chalazal-most megaspore was
degenerating (arrow). (d) The chalazal-most megaspore was degenerated (arrow). (e)
Abnormal mono-nucleate embryo sac, arrow indicates the darkly stained abnormal
nucellus tissue. (f) Abnormal embryo sac at metaphase of the first mitotic division. (g)
Abnormal two-nucleate embryo sac, arrow indicates the abnormal nucellus tissue. (h)
The two nuclei (arrow) were located at the micropylar pole in an abnormal two-nucleate
embryo sac. (i) Abnormal four-nucleate embryo sac, arrow indicates the abnormal
nucellus tissue. (j) Abnormal four-nucleate embryo sac, with three nuclei (arrows)
located at the micropylar end, while only one nucleus (arrow head) located at the chalazal
end. (k) Abnormal eight-nucleate embryo sac without the differentiation of the normal
embryo sac cavity, eight nucleoli can be observed. (l) Abnormal small embryo sac at
eight-nucleate embryo sac stage, the antipodal cells were wrinkled. (m) Asynchronous
nuclear migration at eight-nucleate embryo sac stage, one nucleus (arrow) was almost
migrated to the center of the embryo sac while another nucleus (arrow) still positioned
near the embryo sac wall. (n) Abnormal nuclear migration, the polar nuclei were located
at the embryo sac wall, the egg apparatus was degenerated. (o) Abnormal eight-nucleate
embryo sac with degenerated egg apparatus. (p) Abnormal eight-nucleate embryo sac
with degenerated egg apparatus, the polar nuclei were located near the antipodals. Bars
= 40µm.
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Figure 3. Five major types of abnormalities found in the mature embryo sacs in
indica/japonica hybrids. (a) Embryo sac degeneration, without the differentiation of
embryo sac cavity. (b) Abnormal small embryo sac, the size of the embryo sac was less
than 1/2 of a normal mature embryo sac. (c) Embryo sac without egg apparatus (arrow),
the polar nuclei still existed. (d) Embryo sac without female germ unit (arrow). (e)
Embryo sac with abnormal polar nuclei (arrow) located beside the antipodals. (f)
Abnormal embryo sac whose female germ unit (arrow) located in an abnormal position.
(g) An embryo sac with abnormal egg apparatus and three nuclei (arrow head) located in
the antipodals. (h) A normal mature embryo sac. Bars = 75µm.
Figure 4. Embryo sacs in indica/japonica hybrids at 24 h after pollination. (a) Part of a
normal fertilized embryo sac, the multi-celled globular embryo (arrow head) was
observed, a layer of free endosperm nuclei (arrows) suspended in the peripheral
cytoplasm of the embryo sac. Bar=75µm.
(b) A normal fertilized embryo sac, showing
a panorama of the embryo sac. Bar= 150µm. (c) Abnormal small embryo sac with polar
nuclei located in an abnormal position which was too far to fuse with the sperm, the trace
of fertilization was observed (arrow). Bar=75µm. (d) Abnormal small embryo sac with
multi-celled globular embryo and free endosperm nuclei, indicating that the female germ
unit should be normal, but the size of the embryo sac was smaller than normal ones.
Bar=75µm. (e) An abnormal embryo sac with the polar nuclei located in a position which
was too far to fuse with the sperm, but the multi-celled globular embryo (arrow) was
formed, indicating a successful fusion of egg cell and sperm. Bar=75µm. (f) An abnormal
embryo sac without egg apparatus (arrow), but the free endosperm nuclei were observed,
indicating a successful fusion of polar nuclei and sperm. Bar= 150µm. (g) Fertilization
stopped in a normal embryo sac, the trace of fertilization was observed (arrow).
Bar=75µm. (h) The primary endosperm (arrow) was not differentiated. Bar=75µm.
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Table 1. Frequencies of various types of abnormal embryo sacs in indica/japonica hybrids grown in
March 2005 (All the ovaries were collected at mature stage.)
Cross
ESD
a
(%)
b
c
ASES
ESWF
ESWE
(%)
(%)
(%)
d
e
ESWA
OAT
(%)
(%)
f
g
NES
Tatal
Seed
(%)
ovaries
setting
(%)
examined
Liaojing
22.6
25.4
3.6
3.6
8.6
3.9
32.3
279
27.4±2.6
24.6
13.9
0.6
1.1
6.0
0.9
52.9
350
36.0±2.1
944/Guangluai No.4
Taichung
65/Guangluai No.4
a
Embryo sac degeneration
b
Abnormal small embryo sac
c
Embryo sac without female germ unit
d
Embryo sac without egg apparatus
e
Embryo sac with abnormal polar nuclei
f
Other abnormal types
g
Normal embryo sac
Table 2. The comparison of seed setting after selfing, seed setting after hand pollination and embryo
sac fertility when the hybrid was grown in July 2005
Cross
Total spikelets
Seed setting
Seed setting
Embryo sac
used in hand
after hand
after selfing
Fertility
pollination
pollination
(%)
(Tatal ovaries
(%)
Taichung
504
examined)
9.6±1.9
21.2
24.3%
(173)
65/Guangluai
No.4
19
Figure 1
20
Figure 2
21
Figure 3
22
Figure 4
23
24