Embryological Evidence of Apomixis in Eulaliopsis binata

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1 Acta Botanica Sinica 2004, 46 (1): Embryological Evidence of Apomixis in Eulaliopsis binata YAO Jia-Ling *, YANG Ping-Fang, HU Chun-Gen, ZHANG You-De, LUO Bin-Shan (College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan , China) Abstract: Embryological investigation was carried out on apomixis in Eulaliopsis binata (Rotz) C. E. Hubb by using paraffin section method. The results indicated that the development pattern of the embryo sac was apospory. In the early stage of the ovule development, a few of the nucellar cells developed into aposporous initial cells, which differentiated later into two forms of mature embryo sac: (1) one form of the mature embryo sac contained one egg cell, one synergid and two polar nuclei; (2) another form possessed one egg cell, two synergids and one polar nucleus. The former occupied about 67.6% and the later only 32.4%. The development pattern of the aposporous embryo sac resembled the Panicum type. Multiple initial cells of apospory might undergo development simultaneously to form two- or multiple-embryo sacs. The ratio of multiple mature embryo sacs in one ovule was 17.7%. No sexual embryo sac was found in the observed ovules. The genesis of the embryo could be classified into two types according to their initial time: (1) the pre-genesis embryo (74%), which originated from unreduced egg cell before the division of the polar nucleus, was observed at one to two days earlier than anthesis. (2) the late-genesis embryo (26%) which was observed at one or two days after anthesis and formation of free endosperm nuclei. The endosperm was derived from the polar nucleus or secondary nucleus without fertilization. The process of the embryonic development followed the sequence of the sexual embryo. The frequency of polyembryony observed was 13%. Key words: apomixis; embryology; apospory; Eulaliopsis binata Asexual seed production (agamospermy) in flowering plants via gametophytic apomixis is accomplished by apomeiosis followed by parthenogenesis. Two major types of gametophytic apomixis have been described, namely aposporous and diplosporous apomixis, depending on the origin of the unreduced megagametophytes. In apospory, unreduced female gametophytes develop from a cell of the nucellus, and in diplospory megasporocytes arise directly from mitotic or mitotic-like divisions of the megasporocyte. Parthenogenetic development of the unreduced egg cell gives rise to embryos that are genetically identical to the maternal parent (Asker and Jerling, 1992), a feature of great value for plant breeding and seed production. If apomictic nature could be introduced into sexual crops, it would greatly simplify breeding schemes and allow the fixation of any genotype, including that of F1 hybrids (Grossniklaus et al., 1998; Spillane et al., 2001a). There are more than 400 plant species which reproduce asexual seed by apomixis, but typically, apomixis is a facultative phenomenon in which an apomictic plant usually produces a few meiotic embryos beside a majority of ameiotic embryos (Sherwood et al., 1980; Spillane et al., 2001b), which variation in the expressivity of apomixis may create an additional complication in genetic studies and breeding field. For nearly two decades, the genetic control of apomixes has been elucidated in very few species. Recently, however, inheritance studies for several natural apomicts have been published that shed new light on the genetic control of this important developmental process (Noyes and Riesseberg, 2000; Grossniklaus and Nogler, 2001). So, it has become an intensively studied field seeking the genetic resources for obligate apomixis. Eulaliopsis binata is a perennial grass, which belongs to the subtribe Apocopidinae in Gramineae, widely distributed in the south part of Qinling moutain in China (Liu, 1988), and has been used in the conservation of water and soil for its thrive roots. It is also an excellent material in the paper making industry for its long vegetable fiber (Zhou,1990; Zhang et al., 1996). Recently, apomixis phenomenon was observed in E. binata (Zhang et al., 1996). However, the information about its reproduction is still limited. Whether or not the sexual reproduction and apomixis coexists in E. binata, and whether the stimulation of pollination is necessary to the development of the embryo and endosperm is really not known. In order to reveal the pattern and degree of apomixis (facultative or obligate) in E. binata, careful Received 27 Mar Accepted 7 Aug Supported by the National Natural Science Foundation of China ( ). * Author for correspondence. Tel: +86 (0) ; Fax: +86 (0) ; <yaojlmy@mail.hzau.edu.cn>.

2 YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata embryological investigation is needed. This article presents the results of our embryological investigation. 1 Materials and Methods All materials were obtained from a population of slender-leaf and red haulm ecotype Eulaliopsis binata (Rotz) C. E. Hubb which were grown in the experimental field of Huazhong Agricultural University. At the flowering stage, samples were collected every 1 3 h. Each time five panicles (about 350 to 400 flowers) were harvested and fixed in Carnoy s Fluid (95% alcohol : acetic acid, 3:1) then stored in 70% alcohol solution. Before anthesis and after blossom, samples were collected and fixed once every day. Ovaries were peeled and stained with Ehrlich s haematoxylin. Sequential paraffin sections 8 10 µm thick were made. Observation and photography were conducted under an Olympus Vanox AH3 microscope. 2 Results 2.1 The origin and development process of embryo sac The inflorescence is a digitiform panicle, consisting of many pedunculate spikelets. There are two kinds of florets in one ear of E. binata, one is hermaphroditic and the other one is single male. The flower has three stamens and possesses a unilocular ovary with an anatropous ovule, which is bitegmic and tenuinucellatae. In the early stage of ovule development, one or more nucellar cells could be distinguished by its slightly enlarged size and obvious nuclei and nucleoli (Figs.1, 2). These specialized nucellar cells were found usually in the 4th to 6th cell layer of the nucellar tissue. They were round in shape at first and then, gradually changed into a long rectangular shape with an increase in cell volume. With the cytoplasm tenuating gradually, the nuclear and nucleoli seemed to be more obvious and distinctive (Figs.1, 2). Later, the specialized cells developed into aposporous embryo sac. Though the sexual archesporial cell and megasporocyte was observed under the epidermis of nucellus in very few (about 1%) ovules, they degenerated soon without any further development (Fig.3). The primary aposporous cells continued to enlarge with the nucleus in the center until many small vacuoles emerged in cytoplasm and later combined into a large vacuole. Thus the aposporous uninucleate embryo sac was formed (Figs. 4, 5). Later on, the nucleus moved toward the micropylar end and underwent the first division to form a two-nucleate embryo sac. The mitosis usually occurred in a perpendicular direction to the vertical section of the embryo sac and the two nuclei were located side by side near the micropyle (Fig.6). After another mitosis division, 4-nucleate embryo sac was formed (Fig.7). Then, two types of mature embryo sacs were produced as follows: one type of mature embryo sac contained one egg cell, one synergid and one central cell of two polar nuclei (Figs.10,11); while another type possessed one egg cell, two synergids and one central cell of single nucleus (Fig.12). The former occupied about 67.6% and the latter only 32.4%. Antipodal cells were completely lacking in the both types. The mature embryo sac had a polarity with micropyle or integument end located egg apparatus and the centralized polar nucleus (Figs ). Along with the development of aposporous embryo sac, the adjacent nucellar cells continued to disintegrate and resulted in an enlargement of the cavity in the embryo sac. Development of the sexual embryo sac was not observed in the observed ovules. Several initial cells of apospory embryo sacs could be observed coexisting in one ovule and co-developed synchronously or asynchronously (Figs.1, 2, 4 9). According to the statistics, two or more mature multiple embryo sacs were observed simultaneously in 17.7% of the ovules. The membranes of the aposporous embryo sacs are not so easily identified. In this case, there should be one mature embryo sac near the micropylar end, while others distributed randomly, they might be observed either at the chalazal end or in the center or in elsewhere in the ovules (Figs.13 15). 2.2 The genesis and development of embryo and endosperm The egg cell became intensively vacuolized, and enlarged with strict polarity. The nucleus was near the chalazal end while the large vacuole was located in the micropylar end (Figs. 12,16). According to the initial time, the embryogenesis could be divided into two types. One was the pre-generated embryo (74%), which happened 1 2 d earlier than anthesis. Another type was the late-generated embryo (26%), which could be found 1 2 d later than anthesis. The pre-generated embryo was derived from the unreduced egg cell which produced pro-embryo (Fig.16) before division of the polar nucleus. When the globular embryo formed the polar nucleus had not divided yet (Fig. 17). As to the latter, when the egg was about to divide, the polar nucleus had already divided into several free endosperm nuclei, and meanwhile, the synergids had disappeared as a result of degeneration (Fig.18). No matter when they were produced, both types of the embryos derived from egg cells would undergo development following the process similar to sexual embryo differentiation (Figs. 19, 20). At the last, the matured embryo (Fig.21) possessed

3 Figs Aposporous initial cells (arrows), Enlarged aposporous initial cells (arrows), A degenerated archesporial cell, An aposporous initial cell (arrow) and 1-nucleate aposporous embryo sac with a vacuole (V), Two 1-nucleate aposporous embryo sac (arrow), A 2-nucleate aposporous embryo sac (T) and two 1-nucleate embryo sacs (arrows), A 4-nucleate embryo sac (F) with a vacuole and two degenerated cavities (D), Showing three nuclei (arrows) in one 4-nucleate embryo sac, while three cells in another embryo sac, Adjacent optical sections 9A and 9B. 9A shows a polar nucleus (P), synergids (S), one degenerated 2-nucleate embryo sac (T), and a degenerated cavity (D); 9B shows an egg in the mature embryo sac, A mature embryo sac with two polar nuclei (P) and one synergid in degeneration (DS), one egg cell (E) is division, A mature embryo sac with one polar nucleus (P), one egg cell (E) and one synergid S, Sections 12A and 12B are adjacent optical sections. 12A shows one polar (P) and the nuclei of two synergids (S and arrows), 12B shows the egg cell, 540.

4 YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata Figs Multiple mature embryo sacs (ES1), (ES2) and a disintegrated embryo sac (ES3), A showing multiple mature embryo sacs ES1, ES2, a 2-nucleate embryo sac T and a degenerated embryo sac (D); 14B showing the egg cell (E), synergid (S) and polar nucleus (P) in ES2; 14C showing the other mature embryo sac (ES3) located in the micropylar end, Showing three embryo sacs, two of them at mature stage (ES1 and ES2) located in micropylar end and chalaza end respectively, and in another embryo sac (ES3), two polar nuclei (P) were seen, Showing synergid (S), the 2-celled pro-embryo EM, A globular embryo EM and polar nucleus (P), Specialized egg cell (E) and several free endosperm nuclei (arrows), Two embryos, one of which was pro-embryo (EM1), another was in differentiation (EM2), Two embryos EM1 and EM2 in differentiation, A mature embryo (EM), 100. shoot apex, coleoptile, radicle, coleorhiza, scutellum, etc. The endosperm derived from polar nucleus or secondary polar nucleus. While the embryo was in the pearshaped stage, the free endosperm nuclei started to form

5 endosperm cell from the micropylar end firstly then to other positions. In about 7.6% ovules, no endosperm generated, which caused the arrest of the embryo development, leading to shrunken seeds. Because in observed more than two thousand ovules, no symptoms of fertilization, such as entrance of pollen tube into embryo sac, were observed, and moreover, the synergids remained integrating while the egg cell initiated mitotic division. These results indicated that the embryo derived by parthenogenesis and the endosperm formed by division of polar nucleus autonomously in E. binata. The seed formation was fertilization independent. Poly-embryo phenomena were easily found in apospory plants. In E. binata, poly-embryo was observed at a frequency of 13%. They originated from the egg cells in various embryo sacs. They might be in different development stage (Figs.19, 20). 3 Discussion 3.1 Characteristics of the genesis and development of apospory embryo sac in Eulaliopsis binata As documented in other aposporous plants, the initial cell of apospory embryo sac usually originated from nucellar cell (Nogler, 1984; Dujardin and Hanna, 1984; Li et al., 1996; Yao et al., 1997; Wen et al., 1998). Cytological studies on guinea grass (Panicum maximum) indicate all megaspores degenerate; one or more adjacent somatic cells of the nucellus will grow and divide into aposporous embryo sacs after a normal division (Warmks, 1954). However, while initial cells of the aposporous embryo sac formed, archesporial cell and megasporocyte of sexual embryo sac were seldom observed in Eulaliopsis binata. The possible reason might be that they degenerated at very early stage. Therefore, the genesis modes of apospory embryo sac might be variable in different species. According to Brown and Emery (1958), apospory embryo sac in Gramineae could be divided into two types: Panicum type and Hieracium type. Based on our investigation, the generation of aposporous embryo sac in E. binata belonged to the former. One or more specialized nucellar cells vacuolized at first, then the nuclei divided twice to produce 4-nucleus embryo sac. With further differentiation, two types of mature embryo sac were constructed as mentioned above, which is in accordance with the report in Panicum maximum (Warmks, 1954). However, in Pennisetum squamulatum and P. ciliare (Hanna, 1992; Wen et al., 1998), another species of the Panicum type, only the second type of mature embryo sac was formed. Why are there two kinds of apospory embryo sacs in E. binata simultaneously and what kinds of mechanism are there in E. binata? Koltunow (1995) described the enormous variability existing in apomoctic processes. In Hieracium, apomictically derived embryo sacs usually reproduce through apospory. However, several loci modify the timing of apomictic initiation, the frequency at which apomictic embryo sacs are formed, and the mode of progression of apomictic development (Koltunow, 2000). These findings indicate that the major locus associated with apomixis might create a competence for a variety of reproductive developmental processes in the ovule. Our results add support to this opinion. The developmental mechanisms employed by different apomoctic species, however, are remarkably varied, possibly reflecting the apparent polyphyletic origin of this trait among flowering plants. Also, we noted in E. binata that there were several matured embryo sacs presented in the same one ovule with the frequency of 17.7%. Among the apospory species, one or more initial cells in one nucellus undertake development at the same time. Usually, only mature embryo sac is located in one nucellus, but the descriptions about polyembryo sacs were reported in Pennisetum and Poa (Nogler, 1984). Producing more female gametophytes may be a reproductive strategy to apomictic plants. Dujardin and Hanna (1984) and Wen et al. (1998)considered Pennisetum ciliare and P. squamulatum as an obligate apospory plant that only the aposporous embryo sac would develop further, whereas the sexual embryo sac would degenerate in certain stage. In more than two thousand ovules, the development of sexual embryo sac was not observed at all, only the aposporous embryo sac was generated in E. binata. Therefore, based on the development of embryo sac, it might be suggested that E. binata should be an obligate apospory species. 3.2 Development of embryo and endosperm in Eulaliopsis binata In aposporous plants, the embryo could develop without stimulation of fertilization. The unreduced egg cell initiated embryogenesis in the absence of fertilization (parthenogenesis) in E. binata as well as in other aposporous species. Interestingly, the initial time of parthenogenesis of egg cell was different, which was consistent with the reports in Pennisetum aquamulatum (Wen et al., 1998). For agricultural applications, it is essential that endosperm development in engineered apomicts is normal. Most apomictic seed formation requires fertilization of the central cell (pseudogamy) (Nogler, 1984; Asker and Jerling, 1992; Spillane et al., 2001b). We had paid much attention to

6 YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata making sure if fertilization be involved in endosperm formation in E. binata. However in two years investigation, we had not find evidence to support this usual opinion. During flowering time, we fixed samples every 1 3 h but we thoroughly failed to observe any symptom of fertilization. We did not find trace of pollen tube or sperm nucleus in embryo sac. We also applied fluorescence method to check if pollen tube entered the embryo sac. We found that only few of pollen could germinate on stigma but none of them could elongate further and stretch into ovule (data not shown). This result also suggested that no stimulation of male gamete and no pseudo-fertilization were involved in egg cell division and endosperm formation. Similar results were reported in Hieracium, Alnus rugosa, Allium nutans, Malus hupehensis and Chondrilla spp. (Nogler, 1984; Grossniklaus et al., 2001), but no records from Gramineae. Our work provided an exception in apomictic monocots. Koltunow reported that a dominant locus is required for apospory and autonomous embryo/endosperm formation in apomictic Hieracium, and the dissection of this locus is a major objective in their laboratory (Koltunow et al., 2000). Why do the autonomous apomictics not need a sexual endosperm, and how do they manage without one? Is the genetic regulation in E. binata identical with dicots or not? Further work is necessary in order to understand its reproductive characteristic. In our study, polyembryony was observed with the frequency of 13%, which came from different embryo sacs in the same ovule. Moreover, the embryo and endosperm may come respectively from different embryo sacs. The results interrelate with the poly-mature embryo sac. After seed germination, twin- or multiple-seedlings were observed in a frequency of 8.4%, which suggested that most of the polyembryos could develop into mature stage. Although the conclusion that Eulaliopsis binata should be an obligate apomixis plant needs to be corroborated through genetic and cytological investigation, it will be another new valuable genetic resource in Gramineae in addition to species, such as Pennisetum ciliare, Tripsacum dactyloides, which has shown great potential in crop breeding. Acknowledgements: We would like to thank Prof. Anna KOLTUNOW of CSIRO Plant Industry Horticulture Unit (Waite Campus, Australia) for her critical comments on this investigation, and Dr. June HAMMOND of Sydney University for her kind amending of this manuscript in English. References: Asker S E, Jerling L Apomixis in Plant. Boca Raton, Finland: CRC Press. Brown W V, Emery W H P Apomixis in the Gramineae: Panicoideae. Am J Bot, 45: Dujardin A K, Hanna W Microsporogenesis, reproductive behavior and fertility in five Pennisetum species. Theor Appl Genet, 67: Grossniklaus U, Koltunow A, Lookeren Campagne M A bright future for apomixis. Trends Plant Sci, 3: Grossniklaus U, Nogler G A How to avoid sex: the genetic control of gametophytic apomixes. Plant Cell, 7: Grossniklaus U, Spillane C, Page D R, Kohler C Genomic imprinting and seed development: endosperm formation with and without sex. Curr Opin Plant Sci, 4: Hanna W W Transfer of apomixes in Pennisetum. James H J R, Miksche J P. Proceedings of Apomixis Workshop USDA- ARS ARS. Vol Koltunow A, Bicknell R, Chaudhury A Apomixis: molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol, 108: Koltunow A, Johnson S, Bicknell R Apomixis is not developmentally conserved related, genetically characterized Hieracium plants of varying ploidy. Sex Plant Reprod, 12: Li H-P, Sun M-X, Wang Z-A Cai D-T Embryology studies on Kentucky bluegrass.. Multiple embryo sacs and polyembryony. J Wuhan Bot Res, 14: ( in Chinese with English abstract) Liu L Study on classification and evolution of Gramineae subfamily Panicoideae. Acta Phytotax Sin, 26: (in Chinese) Nogler G A Gametophytic apomixes. Johri B M. Embryology of Angiosperms. Berlin: Springer-Verlag Noyes R, Riesseberg L Two independent loci control agamospermy (apomixes) in the triploid flowering plant Erigeron annuus. Genetics, 155: Sherwood B F, Young B A, Bashow E C Facultative apomixis in Buffelglass. Crop Sci, 20: Spillane C, Steimer A, Grossniklaus U. 2001a. Apomixis in agriculture: the quest for clonal seeds. Sex Plant Reprod, 14: Spillane C, Vielle-Calzada J P, Grossniklaus U. 2001b. APO2001: a sexy apomixer in como. Plant Cell, 13: Warmks A Apomixis in Panicum maximum. Am J Bot, 41: Wen Q-S, Ye X-L, Li Y-Q, Chen Z-L Xu S-X Embryological studies on apomixis in Pennisetum squamulatum. Acta Bot Sin, 40: (in Chinese with English abstract) Yao J-L, Cai D-T, Ma P-F Zhu H Embryological studies on apospory in rice HDAR. Chin J Rice Sci, 11: (in

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