Megasporogenesis and Megagametogenesis in Autotetraploid Indica/ Japonica Rice Hybrid

Rice Science, 2010, 17(4): 296 302 Copyright 2010, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(09)60030-5 Megasporogenesis and Megagametogenesis in Autotetraploid Indica/ Japonica Rice Hybrid HU Chao-yue #, ZENG Yu-xiang #, GUO Hai-bin, LU Yong-gen, CHEN Zhi-xiong, Muhammad Qasim SHAHID, LIU Xiang-dong (Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University, Guangzhou 510642, China; # These authors contributed equally to this paper) Abstract: Autotetraploid indica/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid rice hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonica/indica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy (WE-CLSM) technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs (i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac) in autotetraploid japonica/indica hybrid. Embryo sac fertility was lower in diploid japonica/indica hybrid than in autotetraploid japonica/indica hybrid although many abnormal phenomena were observed in autotetraploid hybrid. Key words: Oryza sativa; japonica/indica hybrid; embryo sac; autotetraploid; abnormality; whole-mount eosin B-staining confocal laser scanning microscopy Autotetraploid rice (2n=4x=48) derived from diploid rice (2n=2x=24) through chromosome doubling with colchicine has larger kernel, higher biomass yield, higher protein content and better amino acid content, compared with their original diploid counterparts (Hu et al, 2009; Luan et al, 2007; Song and Zhang, 1992; Tu et al, 2003; Tu et al, 2007). Autotetraploid indica/ japonica hybrid, obtained by crossing 4x indica rice with 4x japonica rice, combines both the advantages of polyploidy and heterosis between indica and japonica (Cai et al, 2001). It has been reported that most of 4x indica/ japonica hybrids have higher seed setting rate than their original 2x indica/japonica hybrids (Hu et al, 2009), which attracts many interests in China, since some researchers believed that the poor seed setting rate of 2x indica/japonica hybrid can be improved at the 4x level (Huang et al, 2004). However, the seed setting rates of most of 4x indica/ Received: 3 March 2010; Accepted: 9 April 2010 Corresponding author: LIU Xiang-dong (xdliu@scau.edu.cn) japonica hybrids were still much lower than the normal level. Embryo sac abortion was reported to be one of the most important factors influencing spikelet fertility of 4x inter-subspecific hybrids (Hu et al, 2009; Xiao et al, 2005). Little information was available concerning the embryo sac abnormality in 4x or 2x indica/japonica hybrid. Since the rice embryo sac is enclosed within the ovary, it is technically challenging to observe the embryo sac using conventional sectioning or whole stain-clearing technique. To facilitate observation of rice embryo sac, a WE-CLSM (whole-mount eosin B-staining confocal laser scanning microscopy) technique was developed in our laboratory (Guo et al, 2006; Xiao et al, 2005; Zhang et al, 2003). WE-CLSM technique is one of the most convenient and effective techniques for embryo sac observation in rice. Diversity of abnormal embryo sacs had been detected in both 4x indica/ japonica hybrid and its corresponding 2x hybrid by using the WE-CLSM technique (Hu et al, 2009; Zeng et al, 2007). The embryo sac formation and development

HU Chao-yue, et al. Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid 297 in 2x indica/ japonica hybrid was studied, and it was reported that the abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs (Zeng et al, 2009). Although different types of abnormal embryo sacs were detected in the mature embryo sacs of 4x indica/ japonica hybrid, the stage when the abnormalities occurred is much less understood, and it is still not clear about the differences between 4x and 2x indica/japonica hybrids during female gametophyte development. In the present study, we investigated the female gametophyte formation and development in a 4x japonica/indica hybrid, in order to clarify the cytological mechanism of embryo sac abortion in 4x indica/ japonica hybrids. MATERIALS AND METHODS Plant materials Two diploid rice varieties, Taichung 65 (japonica) and Guanglu ai 4 (indica) were used. The autotetraploid rice (2n=4x=48) was derived from the diploid variety (2n=2x=24) through chromosome doubling with colchicine. The 4x japonica rice Taichung 65-4x was crossed to the 4x indica rice Guanglu ai 4-4x to produce 4x japonica/indica hybrid. The F 1 of the 4x japonica/indica hybrid (Taichung 65-4x Guanglu ai 4-4x) was used to investigate the female gametophyte formation and development. The diploid variety Guanglu ai 4 was planted at the same time to study megasporogenesis and megagametogenesis in rice. All the plant materials were cultivated in July 2005 at the experimental farm of South China Agricultural University, Guangzhou, China (23 16 N, 113 8 E). Fixation Florets were collected at different developmental stages. When the hybrid was flowering, florets with open glumes, i.e., in which embryo sacs were mature and ready for fertilization, were collected at noon each day. All the samples were fixed in FAA (formaldehyde: acetic acid:50% ethanol=5:6:89) for at least 24 h, then washed with 50% ethanol and stored in 70% ethanol at 4ºC. Eosin B staining and embryo sac scanning The eosin B staining procedure for embryo sac scanning using a laser scanning confocal microscope (WE-CLSM technique) was described by Zeng et al (2007). The measurement of embryo sac size was according to Zeng et al (2007). RESULTS Megasporogenesis and megagametogenesis in autotetraploid japonica/indica hybrid The female gametophyte developmental stages in the 4x japonica/indica hybrid (Taichung 65-4x Guanglu ai 4-4x) were similar to those in its diploid parent Guanglu ai 4. The megasporogenesis and megagametogenesis in the 4x japonica/indica hybrid were described as the following stages: megasporocyte stage (Fig. 1-A, B), meiotic division stage (Fig. 1-C, D), functional megaspore stage (Fig. 1-E), mononucleate stage (Fig. 1-F), two-nucleate stage (Fig. 1-G, I), four-nucleate stage (Fig. 1-J), eight-nucleate stage (Fig. 1-K, N) and mature embryo sac stage (Fig. 3-B). Abnormalities occurred during megasporogenesis in autotetraploid japonica/indica hybrid Abnormalities were observed at the megasporocyte stage, meiotic division stage and functional megaspore stage. In some ovaries, the megasporocyte degenerated, and a new megasporocyte was developed instead (Fig. 1-O). During meiotic division, two degenerating dyad cells were observed (Picture not shown). In some ovaries, four cells of the tetrad were degenerated (Fig. 1-P). The degeneration of the functional megaspores was frequently observed in ovaries at this stage (Fig. 2-A, B). In some cases, two functional megaspores were detected synchronously in one ovary (Fig. 2-C), and one functional megaspore and a diplospory-like megasporocyte were observed together in one ovule (Fig. 2-D). Abnormalities occurred during megagametogenesis in autotetraploid japonica/indica hybrid Abnormalities were also detected from the mononucleate stage to the mature embryo sac stage in the

298 Rice Science, Vol. 17, No. 4, 2010 Fig. 1. Embryo sac development in autotetraploid japonica/indica rice hybrid (Bars=25 μm). A, A megasporocyte (arrow); B, A megasporocyte at pachytene (arrow); C, A dyad (arrows). The bright fluorescence indicates callose around the cells of dyad; D, A tetrad. Arrows indicate callose around the cells of tetrad; E, Functional megaspore (arrow); F, Mono-nucleate embryos sac (arrow); G and H, Two-nucleate embryo sac (arrows); I, The two-nucleate embryo sac at the metaphase of mitotic division. Arrows indicate the two mitotic spindles; J, Four-nucleate embryo sac (arrow); K and L, Eight-nucleate embryo sac development at the early stage. Two nuclei (arrows) had been enlarged and began to move towards the center of the embryo sac; M, An embryo sac at the middle stage of eight-nucleate development. Two nuclei had migrated to the central region (arrows); N, An embryo sac at the late stage of eight-nucleate embryo sac development. The polar nuclei (arrow) had migrated to the egg apparatus; O, A new megasporocyte (arrow head) developed while the original one degenerated (arrow); P, Four cells of the tetrad were degenerated (arrows). 4x japonica/indica hybrid. Some of the degenerated mono-nucleate embryo sacs (Fig. 2-E) or two-nucleate embryo sacs were found. Some abnormal two-nucleate embryo sacs were detected with both nuclei located at the micropylar-most region (Fig. 2-F, G). Moreover, a mono-nucleate embryo sac and a two-nucleate embryo sac were simultaneously detected within an ovary. Different kinds of five-nucleate embryo sacs were found. Some of the five-nucleate embryo sacs contained three nuclei at the micropylar pole and two nuclei at the chalazal pole (Fig. 2-H, I), or three nuclei at the chalazal pole and two nuclei at the micropylar pole (Fig. 2-J). Some of the embryo sacs had five nuclei, which were located at the micropylar pole, and the cells at the chalazal pole were degenerated (Fig. 2-K). The abnormalities that occurred at the eightnucleate stage were diverse. Some embryo sacs contained one more nucleus at the micropylar pole at the late eight-nucleate stage (Fig. 2-L). In a few cases, many nuclei were observed at both the micropylar and the chalazal poles. In some cases, all cells at both the micropylar and the chalazal poles were degenerated (Fig. 2-M, N). At the late eight-nucleate stage, some embryo sacs were detected with the polar nuclei located at abnormal position (Fig. 2-O), and some embryo sacs with abnormal number of polar nuclei were also observed (Fig. 2-P). The degenerated egg apparatus was observed in some ovaries (Fig. 3-A) at the late eight-nucleate stage. At the eight-nucleate

HU Chao-yue, et al. Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid 299 Fig. 2. Abnormalities occurred during embryo sac development in autotetraploid japonica/indica rice hybrid (Bars=25 μm). A and B, The functional megaspore was degenerated (arrow); C, Two functional megaspores (arrows) were found in one ovary; D, One functional megaspore (arrow) and a diplospory-like megasporocyte (arrow head) were found in one ovule; E, The mono-nucleate embryo sac was degenerating (arrow); F, The two nuclei (arrows) were located at the micropylar pole in an abnormal two-nucleate embryo sac; G, The two nuclei (arrows) were still located at the micropylar pole in an abnormal two-nucleate embryo sac although the embryo sac size was increased; H and I, Five-nucleate embryo sac. Three nuclei (arrows) were located at the micropylar pole while two nuclei were located at the chalazal pole; J, Five-nucleate embryo sac. Three nuclei (arrows) were located at the chalazal pole while two nuclei were located at the micropylar pole; K, Five nuclei were found at the micropylar pole, and the cells at the chalazal pole were degenerated (arrow); L, Embryo sac with one more nucleus (arrow) at the micropylar pole; M and N, The cells at both the micropylar and the chalazal poles were degenerated (arrows); O, The polar nuclei (arrow) were located beside the antipodals; P, Three polar nuclei (arrow) existed in the embryo sac. stage, some abnormal embryo sacs with smaller sizes (Fig. 3-I, J) were found. Abnormalities of mature embryo sacs in autotetraploid japonica/indica hybrid A normal mature embryo sac in the 4x japonica/ indica hybrid had one egg apparatus that consisted of one egg cell and two synergids at the micropylar pole, two polar nuclei located above the egg apparatus and a group of antipodal cells at the chalazal pole (Fig. 3-B). The egg apparatus and two polar nuclei formed the female germ unit (Huang and Russell, 1992). We examined 307 mature embryo sacs in the 4x japonica/ indica hybrid, and five major categories of abnormal embryo sacs were frequently observed: (1) Embryo sac degeneration (ESD, Fig. 3-C). (2) Embryo sac without female germ unit (ESWF, Fig. 3-D), but with antipodals. (3) Embryo sac without egg apparatus (ESWE, Fig. 3E, F), but with polar nuclei and antipodals. (4) Embryo sac with abnormal polar nuclei (ESWA). This abnormal type had normal egg apparatus and antipodals, but the polar nuclei either located at an abnormal position (Fig. 3-G), or with abnormal number of polar nuclei (Fig. 3-H). (5) Abnormal small embryo sac (ASES). This type had less than 1/2 of the normal embryo sac size, and often accompanied with abnormal polar nuclei (Fig. 3-K, L). Besides these five types mentioned above, other abnormal types were also found. For example, twin ovules with two abnormal embryo sacs were observed

300 Rice Science, Vol. 17, No. 4, 2010 Fig. 3. Mature embryo sacs in autotetraploid indica/japonica rice hybrid (Bars=25 μm). A, The egg apparatus was degenerated (arrow); B, A normal mature embryo sac; C, Embryo sac degeneration (arrow); D, Embryo sac without female germ unit (arrow); E and F, Embryo sac without egg apparatus (arrows); G, Embryo sac with abnormal polar nuclei. The polar nuclei were located beside the antipodals (arrow); H, Embryo sac with abnormal polar nuclei. Three polar nuclei were observed (arrow); I and L, Abnormal small embryo sac (arrow). at the maturity stage in the 4x japonica/indica hybrid. The frequencies of various abnormal embryo sac types in the 4x japonica/indica hybrid were listed in Table 1. ESD was the most frequent type of abnormality, followed by ESWA (Table 1). DISCUSSION Abnormalities occurred during female gametophyte development resulted in five major types of abnormal embryo sacs in autotetraploid japonica/indica hybrid Five major types of abnormal embryo sacs were detected in the mature embryo sacs of the 4x japonica/ indica hybrid, i.e. ESD, ESWF, ESWA, ESWE and ASES. After the investigation of female gametophyte development, it was obvious to find that ESD was mainly resulted from the degeneration of functional megaspores, because many degenerated functional megaspores were found at the functional megaspore stage. The degeneration of mono-nucleate embryo sac or two-nucleate embryo sac also led to ESD. But abnormalities that occurred at the mono-nucleate embryo Table 1. Frequencies of embryo sac abnormalities in 4x japonica/ indica hybrid. Abnormality of embryo sac Frequency (%) Embryo sac fertility Embryo sac degeneration Abnormal small embryo sac Embryo sac without female germ unit Embryo sac without egg apparatus Embryo sac with abnormal polar nuclei Other abnormal type Number of ovaries observed 61.89 12.70 6.19 6.19 3.26 6.84 2.93 307 All the ovaries were collected at the maturity stage. sac stage or two-nucleate embryo sac stage were not as often as that at the functional megaspore stage. ESWF, ESWA, ESWE and ASES mainly occurred at the eight-nucleate stage, and were supported by cytological proof. For example, we found that some embryo sacs with egg apparatus degenerated at the late eight-nucleate stage. Embryo sacs with abnormal polar nuclei were also observed at the late eight-nucleate stage. Moreover, some of the abnormal embryo sacs were found with smaller sizes compared to normal ones at the eight-nucleate stage, suggesting that ASES occurred at this stage.

HU Chao-yue, et al. Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid 301 Differences between autotetraploid japonica/indica hybrid and its corresponding diploid hybrid during female gametophyte development In the present study, we studied the female gametophyte development in a 4x japonica/indica hybrid (Taichung 65-4x Guanglu ai 4-4x). The developmental process in the 2x japonica/indica hybrid (Taichung 65 Guanglu ai 4) was previously reported by Zeng et al (2009). Thus, it provided an opportunity to discuss the differences between the 4x hybrid and the 2x hybrid. The embryo sac developmental phases were similar in both the 4x and the 2x hybrids. The abnormalities occurred during megasporogenesis and megagametogenesis in the 2x hybrid can also be found in the 4x hybrid, but the abnormalities occurred in the 4x hybrid were more complex compared to those in the 2x hybrid, and some distinct phenomena were detected only in the 4x hybrid. Abnormalities were not observed during the meiosis of the megasporocyte in the 2x hybrid (Zeng et al, 2009), but were found during the meiotic process in the 4x hybrid in the present study. The doubling of chromosomes in the 4x hybrid can result in the doubling of functional megaspores. For example, we found double functional megaspores within an ovary (Fig. 2-C). Since the doubling of megaspores was not detected in ovaries in the 2x hybrid, it suggested that the dosage effect of tetraploid resulted in new abnormalities in the 4x hybrid. Various numbers of nuclei were detected in some abnormal embryo sacs during megagametogenesis in the 4x hybrid. At the four-nucleate stage, we found five-nucleate embryo sac (Fig. 2-H, J). Some fivenucleate embryo sac had one more nucleus at the micropylar pole or at the chalazal pole compared to the normal four-nucleate embryo sac. At the eightnucleate stage, we found nine-nucleate embryo sac that contained one more nucleus at the micropylar pole compared to the normal eight-nucleate embryo sac. However, the abnormal five-nucleate embryo sacs were not observed in the 2x hybrid. Therefore, the doubling of functional megaspores and disordered number of nuclei were the characteristics of 4x japonica/indica hybrid during female gametophyte development. Furthermore, we found diplospory-like megasporocyte (Fig. 2-D) in the 4x hybrid. It seemed that there were connections between 4x hybrid and the apomixis, which needs to be confirmed in future studies. Comparing the mature embryo sacs between 4x and 2x hybrids, we found that the embryo sac fertility was lower in the 2x hybrid (24.3%) (Zeng et al, 2007) than in the 4x hybrid (61.89%). Therefore, although the embryo sac abnormalities were more complex in 4x hybrid, the embryo sac fertility was higher in 4x hybrid than in 2x hybrid. Hu et al (2009) reported that the embryo sac fertility and the seed setting rate were higher in most of 4x indica/japonica hybrids than in 2x hybrids, and they believed that 4x indica/japonica hybrids have potential in rice breeding. In the present study, we studied the female gametophyte development in the 4x japonica/ indica hybrid, and the results broadened our understanding of embryo sac abortion in tetraploid hybrid and provided a theoretical basis for use of 4x indica/japonica hybrid in production. ACKNOWLEDGEMENTS The authors thank Prof. XU Xue-bin, Dr. LI Jin-quan, Ms. YU Shu-hong and HE Jin-hua for help in the laboratory and fields, and thank Assoc. Prof. YANG Bing-yao and SU Wei for technical assistance. This research was supported by the National Science Foundation of China (Grant No. 30771328) and the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of Ministry of Education, China. REFERENCES Cai D T, Yuan L P, Lu X G. 2001. A new strategy of rice breeding in the 21st century: II. Searching a new pathway of rice breeding by utilization of double heterosis of wide cross and polyploidization. Acta Agron Sin, 27: 110 116. (in Chinese with English abstract) Guo H B, Feng J H, Lu Y G, Liu X D. 2006. Abnormal structure of embryo sac in autotetraploid rice. Rice Sci, 13(4): 257 264. Hu C Y, Zeng Y X, Lu Y G, Li J Q, Liu X D. 2009. High embryo sac fertility and diversity of abnormal embryo sacs detected in autotetraploid indica/japonica hybrids in rice by whole-mount eosin B-staining confocal laser scanning microscopy. Plant Breeding, 128: 187 192. Huang B Q, Russell S D. 1992. Female germ unit: Organization,

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