Yu-Xiang Zeng, Chao-Yue Hu, Yong-Gen Lu, Jin-Quan Li and Xiang-Dong Liu

Transcription

1 Journal of Integrative Plant Biology 2008 Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice Yu-Xiang Zeng, Chao-Yue Hu, Yong-Gen Lu, Jin-Quan Li and Xiang-Dong Liu (Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University, Guangzhou , China) Abstract Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids 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 the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the 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 the eight-nucleate embryo sac stage. The abnormalities that 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 on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting. Key words: indica/japonica hybrid; megagametogenesis; megasporogenesis; Oryza sativa; whole-mount eosin B-staining confocal laser scanning microscopy. Zeng YX, Hu CY, Lu YG, Li JQ, Liu XD (2008). Abnormalities occurring during female gametophyte development result in the diversity of abnormal embryo sacs and leads to abnormal fertilization in indica/japonica hybrids in rice. J. Integr. Plant Biol. doi: /j x Available online at Asian cultivated rice (Oryza sativa L.) has two subspecies, indica and japonica. It is known that strong heterosis expresses in indica/japonica hybrids in both vegetative and reproductive growth, which has attracted the attention of rice breeders for many years (Liu et al. 2004; Song et al. 2005). However, most indica/japonica hybrids are partially sterile, which results in Received 23 Nov Accepted 23 Mar Supported by the National Natural Science Foundation of China ( , ) and the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of MOE, China. Author for correspondence. Tel: ; Fax: ; <xdliu@scau.edu.cn>. C 2008 Institute of Botany, the Chinese Academy of Sciences doi: /j x low seed setting and restricts the direct use of the hybrids in commercial production. Therefore, there has been considerable interest in the rice breeding community to understand the mechanism of partial sterility 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 F 1 sterility (Oka 1974). After the discovery of wide compatibility varieties, which produced fertile F 1 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 series 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.

2 2 Journal of Integrative Plant Biology ; Zhao et al. 2006). The wide-compatibility gene S 5 n 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; Liu et al. 1997b; Zhu et al. 1998; Liu et al. 2004; Song et al. 2005). Embryo sac fertility and pollen fertility were reported to be the 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 contains the egg cell and central cell which, following fertilization, give rise to the embryo and endosperm, respectively. There have been some cytological observations that have attempted to reveal the mechanism of embryo sac abortion in indica/japonica hybrids (Liu et al. 1997b; Zhu et al. 1998; Liu et al. 2004). However, the results obtained in these studies were contrasting. 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). It is still not clear how these abnormal embryo sacs affect the process of fertilization in indica/japonica hybrids. 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). However, the traditional sectioning 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 that can demonstrate the detailed information within the embryo sac is required. 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 the present study, 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 carried out hand pollination, using numerous normal pollens to pollinate 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 the cellular level, and to reveal how the abnormal embryo sacs affect the fertilization in indica/japonica hybrids. 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 (Figure 1A, B), meiotic division stage (Figure 1C, D), functional megaspore stage (Figure 1E), mono-nucleate embryo sac stage (Figure 1F), two-nucleate embryo sac stage (Figure 1G), four-nucleate embryo sac stage (Figure 1H), eight-nucleate embryo sac stage (Figure 1I L), and mature embryo sac stage (Figure 3H). The eight-nucleate embryo sac stage could be subdivided into early (Figure 1I, J), middle (Figure 1K) and late (Figure 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 that occurred during female gametophyte development weresimilarinthetwohybrids. Abnormalities could be found at the early megasporocyte stage (Figure 2A) and the rectangle-like megasporocyte stage (Figure 2B). The abnormal megasporocyte was characterized by the nucleus located in the chalazal-most region (Figure 2A, 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 (Figure 2C, D) and resulted in a lot of degenerated embryo sacs found in the mature stage (Figure 3A). Abnormalities could be found from the mono-nucleate embryo sac stage to the mature embryo sac stage. Most abnormal mono-nucleate, two-nucleate and four-nucleate embryo sacs were characterized by a darkly stained tissue surrounding the female gametophyte (Figure 2E, 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 (Figure 2F). 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

3 Embryo Sac Abortion in indica/japonica Hybrids in Rice 3 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 metaphase II of the meiotic division, arrows indicate the two cells 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 move toward the center of the embryo sac. (K) An embryo sac at the middle stage of eight-nucleate embryo sac development, two nuclei had migrated to the central region. (L) An embryo sac at the late stage of eight-nucleate embryo sac development. Bars, 40 μm.

4 4 Journal of Integrative Plant Biology 2008 Figure 2. Abnormalities that 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 are 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, 40 μm.

5 Embryo Sac Abortion in indica/japonica Hybrids in Rice 5 an abnormal two-nucleate embryo sac (Figure 2H); three nuclei were located at the micropylar end, and only one nucleus was located at the chalazal end in an abnormal four-nucleate embryo sac (Figure 2J). Different kinds of abnormalities were observed from the 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 (Figure 2K). Most of the abnormal embryo sacs found at the middle or late stages of eight-nucleate embryo sac development were characterized by smaller sizes (Figure 2L) compared with the normal ones (Figure 1L). Wrinkled antipodals were found in these embryo sacs (Figure 2L O). Asynchronous nuclear migration (Figure 2M), abnormal nuclear migration (Figure 2N) and abnormal positioning of nuclei (Figure 2P) were observed. Some of the eight-nucleate embryo sacs were found with degenerated egg apparatus (Figure 2N P). Abnormalities that occurred during female gametophyte development resulted in diversity of abnormal embryo sacs in indica/japonica hybrids at anthesis We examined 279 mature embryo sacs in Liaojing 944/Guangluai No. 4 and 350 mature embryo sacs in Taichung 65/Guangluai 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: (i) embryo sac degeneration (ESD, Figure 3A); (ii) abnormal small embryo sac (ASES, Figure 3B) whose size was approximately equal to or less than half of a normal mature embryo sac (Figure 3H), most of the ASES are characterized by abnormal positioning of polar nuclei; (iii) embryo sac without egg apparatus (ESWE; Figure 3C); (iv) embryo sac without female germ unit (ESWF; Figure 3D); (v) embryo sac with abnormal polar nuclei (ESWA; Figure 3E). 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 (Figure 3F). We also found an embryo sac with abnormal egg apparatus, and three nuclei that looked like polar nuclei located in the antipodals (Figure 3G). The ESD and ASES types constituted the majority of abnormal embryo sacs in the two hybrids. For example, the ESD constitutes 33.4% in Liaojing 944/Guangluai No. 4, and the ASES constitutes 37.5%. The ESD constitutes 52.2% in Taichung 65/Guangluai No. 4, and the ASES constitutes 29.5%. The frequencies of various types of abnormal embryo sacs in the twohybridsarelistedintable1. 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 half 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, 75 μm.

6 6 Journal of Integrative Plant Biology 2008 After observation of the megasporogenesis and megagametogenesis in the two hybrids, we found that two types of abnormalities occurred frequently: (i) the degeneration of chalazal-most megaspore; and (ii) the abnormalities occurred at the 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 a result of the degeneration of the chalazal-most megaspore. ASES, ESWE, ESWF and ESWA are a result of the abnormal developmental behavior of the eight-nucleate embryo sac. For example, the degeneration of the egg apparatus at the eight-nucleate embryo sac stage resulted in the ESWE, the degeneration of the female germ unit resulted in the ESWF, and abnormal positioning of the polar nuclei resulted in the ESWA. Since smaller size is characteristic of the majority of the 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 the 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 embryo was observed in a normal fertilized embryo sac in indica/japonica hybrids, and a layer of free endosperm nuclei were suspended in the peripheral cytoplasm of the embryo sac (Figure 4A, 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 (Figure 4C). But very few ASES containing normal female germ units 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 (Figure 4D) compared with the normal ones. Most of the ESWA cannot be fertilized due to the abnormal position in which the polar nuclei locate. However, some of the ESWA were found to form the multi-celled globular embryo, although the polar nuclei did not fuse with the sperm (Figure 4E). ESWE cannot be fertilized due to the absence of an egg cell. However, some of the ESWEs 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 (Figure 4F). In some cases, fertilization was stopped when the pollen tube penetrated into the embryo sac (Figure 4G), demonstrating that even a normal embryo sac cannot ensure a successful fertilization in indica/japonica hybrids. Lagged embryo and endosperm development (Figure 4H) was observed in some embryo sacs. 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, 75 μm. (B) A normal fertilized embryo sac, showing a panorama of the embryo sac. Bar, 150 μm. (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, 75 μm. (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 the normal ones. Bar, 75 μm. (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, 75 μm. (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, 150 μm. (G) Fertilization stopped in a normal embryo sac, the trace of fertilization was observed (arrow). Bar, 75 μm. (H) The primary endosperm (arrow) was not differentiated. Bar, 75 μm.

7 Embryo Sac Abortion in indica/japonica Hybrids in Rice 7 Table 1. Frequencies of various types of abnormal embryo sacs in indica/japonica hybrids grown in March 2005 (all of the ovaries were collected at the mature stage) Total ovaries Cross ESD (%) ASES (%) ESWF (%) ESWE (%) ESWA (%) OAT (%) NES (%) Seed setting (%) examined Liaojing 944/Guangluai No ± 2.6 Taichung 65/Guangluai No ± 2.1 ESD, embryo sac degeneration; ASES, abnormal small embryo sac; ESWF, embryo sac without female germ unit; ESWE, embryo sac without egg apparatus; ESWA, embryo sac with abnormal polar nuclei; OAT, other abnormal types; NES, 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 Total spikelets used Seed setting after Seed setting after Embryo sac fertility Cross in hand pollination hand pollination (%) selfing (%) (total ovaries examined) Taichung 65/Guangluai No ± % (173) 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 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 carried out hand pollination using numerous normal pollens on 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 an effect on the seed setting of Taichung 65/Guangluai No. 4. It also suggested that the effect of abnormal embryo sacs on seed 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 that 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 techniques (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 the mono-nucleate or two-nucleate stages. 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 the nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametophyte from the mono-nucleate embryo sac stage to the four-nucleate embryo sac stage. Most of the abnormalities that happened at the eightnucleate 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 the eight-nucleate embryo sac stage. To gain a comprehensive understanding of the abnormalities that occurred during female gametophyte development in indica/japonica hybrids, our strategy was to examine the mature embryo sacs before we study the megasporogenesis and megagametogenesis. Five major types of abnormalities were found in mature embryo sacs (ESD, ASES, ESWE, ESWF and ESWA), which is consistent with the findings of Zeng et al. (2007). By comparing the abnormalities that occurred during female gametophyte development with the abnormalities found in mature embryo sacs, we found that: (i) the degeneration of the chalazal-most megaspore before the mono-nucleate embryo sac stage resulted in the ESD; and (ii) the majority of abnormalities that happened at the eight-nucleate embryo sac stage mainly resulted in ASES, ESWE, ESWF and ESWA. The degeneration of chalazal-most megaspore and the abnormal eight-nucleate embryo sacs 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 that 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 megasporocytes could undergo meiosis normally and abortion mainly occurred at the stage when the chalazalmost megaspore was beginning to form the mono-nucleate embryo sac, which is also consistent with our findings in the present study. However, it seems that most of the previous

8 8 Journal of Integrative Plant Biology 2008 researchers have neglected the abnormalities that arose at the eight-nucleate embryo sac stage, which occurs frequently in indica/japonica hybrids. We provided detailed pictures of the abnormalities from megasporogenesis to megagametogenesis, and 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 maize. In this study, we observed that 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 still needs to be investigated. The results demonstrate that the embryo sac abnormalities that 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 abnormalities reported in the present study are a consequence of interaction between indica and japonica. Besides embryo sac fertility, 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, and 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 formaldehyde acetic acid (FAA) is a harsh fixative, we also used glutaraldehyde. We found that the images obtained by using FAA were clearer than using glutaraldehyde. Materials and Methods Plant materials Two F 1 sofindica/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, E). Liaojing 944, and Taichung 65 are typical japonica cultivars. Guangluai No. 4isatypicalindica cultivar. The two F 1 s 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, 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 C 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 sulfate 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 (C 20 H 6 N 2 O 9 Br 2 Na 2, FW 624.1, a tissue stain for cell granules and nucleoli) solution (dissolved in 4% sucrose) for h at room temperature. The samples were post-treated in 2% aluminum potassium sulfate 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 (Leica Microsystems, Heidelberg, 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: 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 four to six 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.

9 Embryo Sac Abortion in indica/japonica Hybrids in Rice 9 The numbers of mature embryo sacs examined are listed in Table 1. Hand pollination The F 1 of Taichung 65/Guangluai No. 4 was also planted in July 2005 in the same location as mention above. We carried out hand pollination using numerous normal pollens on 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 of the pollens were mixed and stained with 1% KI- I 2, 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. 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