Introduction

In angiosperms, the sporophytic generation is initiated by double fertilization, resulting in the formation of seeds (reviewed in Raghavan 2003). Upon double fertilization, a sperm cell from a pollen grain fuses with an egg cell, and the resultant zygote develops into an embryo. The central cell fuses with a second sperm cell and develops into the endosperm (reviewed in Russell 1992). In contrast to animals and lower plants, which use naturally free-living gametes, in angiosperms, fertilization and post-fertilization events such as gamete fusion, embryogenesis and endosperm development occur in the embryo sac deeply embedded in ovular tissue. The difficulties associated with research directly addressing the biology of the female gamete, zygote and early embryo have impeded investigations into the molecular mechanisms of fertilization and embryogenesis; thus, these investigations have been predominantly conducted through mutant analyses using Arabidopsis. However, for a decade, in vitro fertilization (IVF) has been utilized as a tool to directly observe and analyze fertilization and post-fertilization processes (reviewed in Wang et al. 2006). The IVF system used in angiosperms includes a combination of three basic micro-techniques: (1) the isolation and selection of male and female gametes; (2) the fusion of pairs of gametes and (3) single cell culture (Kranz 1999). Procedures for the isolation of viable gametes have been reported for a wide range of plant species including maize, wheat, tobacco, rape, rice, barley, Plumbago zeylanica and Alstroemeria (Dupuis et al. 1987; Kranz et al. 1991; Holm et al. 1994; Kovács et al. 1994; Tian and Russell 1997a; Katoh et al. 1997; Cao and Russell 1997; Uchiumi et al. 2006; Hoshino et al. 2006). These isolated gametes can be fused electrically (Kranz et al. 1991; Kranz and Lörz 1993) or chemically using calcium (Faure et al. 1994; Kranz and Lörz 1994; Khalequzzaman and Haq 2005) or polyethyleneglycol (Sun et al. 1995; Tian and Russell 1997b), since gametes are generally protoplasts. By calcium-based fusion of maize gametes, Antoine et al. (2001) demonstrated that an influx of calcium is triggered by gamete fusion, and that calcium influx induces cell wall formation, an event known as egg activation. In addition, fusion behavior and gamete interaction have been traced by video-enhanced microscopy using PEG-mediated gamete fusion (Sun et al. 2002). However, tobacco zygotes produced by calcium- or PEG-fusion arrested (Tian and Russell 1997b; Sun et al. 2002) and maize zygotes produced by calcium-fusion did not fully develop (Kranz and Lörz 1994), suggesting that the procedure of chemical fusion has to be optimized to obtain sufficient zygotes for studies of embryogenesis. A complete IVF system was developed by Kranz and Lörz (1993) using maize gametes and electrical fusion procedures. A maize zygote produced in vitro by the electrical fusion of an egg cell with a sperm cell developed into an asymmetrical two-celled embryo, proembryo and transition phase embryo via zygotic embryogenesis in a similar manner to that in planta (Kranz et al. 1995). Moreover, the in vitro-produced embryo continued to develop and grew into the fertile plant (Kranz and Lörz 1993). This maize IVF system has been successfully used to observe and analyze post-fertilization events including karyogamy in zygotes (Faure et al. 1993), zygote development (Kranz et al. 1995), decondensation of paternal chromatin in zygotes (Scholten et al. 2002), changes in the microtubular architecture in zygotes (Hoshino et al. 2004) and identification of fertilization-induced/suppressed genes (Okamoto et al. 2005).

Rice (Oryza sativa L. cv. Nipponbare) is an excellent model plant among the monocot crop species based on the fact that it has a relatively small genome of about 440 Mb. The whole genome sequence (Ito et al. 2005), Tos17 retrotransposon insertion plants (20,000 independent loci; Miyao et al. 2003) and over 28,000 full-length cDNA clones (Kikuchi et al. 2003) are available, and these databases and resources have been released for academic usage. To utilize these resources in the investigation of the mechanisms involved in fertilization and early embryogenesis, we planned to establish a rice IVF system and recently reported procedures for the isolation of viable egg and sperm cells from rice flowers (Uchiumi et al. 2006). In the present study, we report the establishment of a rice IVF system consisting of zygote production by electrofusion, zygote development into a globular-like embryo and the regeneration of fertile plants from the embryo.

Materials and methods

Plant materials and isolation of gametes

All rice plants (O. sativa L. cv Nipponbare, kindly supplied by Dr S. Komatsu, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan) used in this study were grown in environmental chambers (K30-7248, Koito Industries Ltd, Minato-ku, Tokyo, Japan) under conditions of 25°C in a light (13 h)/dark (11 h) cycle with a photosynthetic photon flux density of 150 μmol photons/m2/s.

Rice egg and sperm cells were isolated as previously reported (Uchiumi et al. 2006) except for the use of mannitol solution with osmolality of 370 mosmol/kg H2O.

Electrofusion of an egg cell with a sperm cell and culture of in vitro-produced zygotes

An isolated egg and a sperm cell were transferred into a 0.5–1.0 μl fusion droplet of mannitol solution (370 mosmol/kg H2O) overlaid with mineral oil on a coverslip, and electrofusion was conducted according to Kranz and Lörz (1993) with some modifications. During alignment of an egg cell and a sperm cell on one of the electrodes under an AC field (1 MHz, 0.4 kV/cm), 0.5–1.0 μl of the mannitol solution (520 mosmol/kg H2O) was gently added to the fusion droplet using a thin glass capillary connected to a NanoSpuit (Ikeda Rika, Chiyoda-ku, Tokyo, Japan). Subsequently, fusion was induced by a single negative DC pulse (50 μs, 14–15 kV/cm) using an electrofusion apparatus (PA-4000, Cyto Pulse Science, Columbia, MD, USA).

The fusion products, zygotes, were washed by transferring them into fresh mannitol droplets (450 mosmol/kg H2O) twice, and then transferred onto the membranes of 12-mm-diameter Millicell-CM dishes (Millipore, Billerica, MA, USA). These dishes were placed in 35-mm-diameter plastic dishes filled with 3 ml of N6Z medium prepared as described previously (Kumlehn et al. 1998), except for the use of a commercially supplied N6 basal salt mixture (Sigma, St Louis, MI, USA) and adjustment of the osmolality to 450 mosmol/kg H2O with glucose. To these dishes, 50 μl of a rice suspension cell culture (Line Oc, provided by Riken Bio-Resource Center, Tsukuba, Japan) were added as feeder cells. After overnight culture of zygotes at 26°C in darkness without shaking, cultures were continued with gentle shaking (40 rpm). After 110–124 h of fusion, feeder cells were removed by transferring the Millicell dishes, in which embryos were developing, into clean 35-mm-diameter dishes filled with 3 ml of fresh N6Z medium. Subsequent cultures were maintained at 26°C in darkness with shaking (40 rpm).

Regeneration of in vitro-produced embryos

The cell colonies that developed from in vitro-produced zygotes after 18–19 days in culture were regenerated to produce fertile rice plants according to Hiei et al. (1994), with some modifications. Briefly, cell colonies were subcultured on a regeneration medium consisting of MS salt, MS vitamin, 100 mg/l myo-inositol, 2 g/l casamino acid, 30 g/l sucrose, 30 g/l sorbitol, 0.2 mg/l NAA, 1 mg/l kinetine and 0.3% gelrite, under light (photosynthetic photon flux density of 80 μmol photons/m2/s), at 30°C for 12–30 days. The differentiated shoots were transferred into a hormone-free medium consisting of MS salt, MS vitamin, 100 mg/l myo-inositol, 30 g/l sucrose and 0.3% gelrite, cultured under a light (13 h)/dark (11 h) cycle (photosynthetic photon flux density of 55 μmol photons/m2/s) at 28°C for 11–13 days, and the resulting plantlets were transferred to soil pods and grown in environmental chambers as described above. Seeds were harvested from the regenerated plants and germinated under conditions of 28°C in a light (13 h)/dark (11 h) cycle with a photosynthetic photon flux density of 95 μmol photons/m2/s.

Fluorescent staining

For cell wall staining, egg cells or IVF-produced zygotes cultured for 5 h were transferred into mannitol drops (450 mosmol/kg H2O) containing 50 μg/ml Fluorescent Brightener 28 (Sigma, St Louis, MI, USA) and 0.1 M Tris–HCl, pH 8.8, and incubated for 15 min at room temperature. After washing by transferring the cells into fresh mannitol droplets twice, cells were observed using an inverted fluorescence microscope with 360–370 nm excitation and 420–460 nm emission wavelengths (Olympus BX-71, U-MNUA2 mirror unit).

For nuclear staining, in vitro-produced embryos were fixed in mannitol droplets (450 mosmol/kg H2O) containing 1% paraformaldehyde, 1 mM cacodylate, 1 mM collidine and 1 × PBS for 5 min. After washing by transferring the embryos into droplets of mannitol solution containing 1 ×  PBS twice, they were transferred into droplets of mannitol solution containing 2 μg/ml DAPI and 1 ×  PBS, and incubated for 10 min. After washing by transferring the cells into mannitol droplets containing 1 ×  PBS twice, the embryos were observed as described above.

Observation of egg cells, zygotes and early embryos in ovules

For observation of rice egg cells, ovules were isolated from flowers before flowering using forceps and a syringe as described (Uchiumi et al. 2006), and fixed in 2.37% glutaraldehyde (mixture of 2.0% monomer and 0.37% polymer forms as calculated from the absorbance ratio at 235/280 nm), 1.9% paraformaldehyde and 0.25% hydrogen peroxide in a mixture of three kinds of buffer (3.75 mM cacodylate buffer, pH 7.4; 3.75 mM collidine buffer, pH 7.4; and 3 ×  PBS; at a ratio of 1:1:1, by vol.) for 1 h at room temperature. The recipe of mixed buffers was chosen because this mixture provided better quality images than fixation in a single buffer. After washing the ovules three times with PBS, ovules were cleared according to the method of Berleth and Jürgens (1993) with a modification. Briefly, fixed ovules were cleared with a graded series of chloral hydrate:glycerol:water solution (8:1:6, w:v:v), and observed using an inverted microscope.

For observation of zygotes and early embryos, ovaries were harvested from flowers at 6, 24 or 30 h after flowering, and fixed as described above. After fixation for 2 h at room temperature, the ovaries were washed three times with PBS and cut transversely with a razor blade through the middle position. Ovules were isolated from the basal portion of cut ovaries using forceps and a syringe, and dehydrated in a graded ethanol series. Subsequently, ovules were cleared in benzyl-benzoate-four-and-a-half fluid (Herr Jr 1982) and observed under a microscope (Zeiss Axioplan) using Nomarski optics.

Results

Cytological polarities of egg cells in planta and isolated egg cells

The egg cells of angiosperms are generally known to have polarity, and it has been suggested that egg cell polarity is important or prerequisite for the development of the zygote (Mansfield et al. 1991; Raghavan 1997). However, to the best of our knowledge, polarity in rice egg cells has not yet been reported. Therefore, we first observed egg cells in ovules that were picked from rice ovaries. As a clear-cut image of an egg cell in the ovule could not be obtained when several kinds of commonly used fixative were employed, we used a unique fixative as described in Sect. “Materials and methods,” resulting in the detection of cytological polarity in rice egg cells. The rice egg cell was oblong-shaped with a relatively sharp angle at the micropylar end (Fig. 1 a). The cytoplasm, nucleus and possible starch granules were located at the micropylar end, whereas vacuoles were localized at the chalazal side, suggesting that rice egg cells also possess polarity. Interestingly, the organelle distribution in the rice cell showed an opposite pattern to that in the Arabidopsis egg cell, in which the nucleus and vacuoles are localized at the chalazal and micropylar ends, respectively (Mansfield et al. 1991).

Fig. 1
figure 1

Egg cell in planta (a), isolated egg and sperm cells (b, c), in vitro fusion of rice gametes (d–f) and early development of a zygote produced by IVF (h–m). a A rice egg cell in an embryo sac. The cell outline is shown by a dashed line. Arrowhead indicates nucleolus. V vacuole. b Two isolated egg cells. c A bursting pollen grain and two released sperm cells (arrowheads). d Alignment of an egg cell with a sperm cell (arrowhead) on one of the electrodes under an AC field in a fusion droplet. e Aligned egg and sperm cells after the addition of mannitol solution with a higher osmolality to the fusion drop. The shape of the sperm cell became oblong (arrowhead). f Fusion of gametes following a negative DC pulse. Arrowhead indicates fusion point. g A zygote 10 s after fusion. h and i Cell wall staining of an egg cell visualized by brightfield and fluorescence microscopy, respectively. j and k Cell wall staining of an IVF-produced zygote visualized by bright field and fluorescence microscopy, respectively. l An isolated egg cell. Arrowhead indicates a nucleolus. m A zygote 4 h after fusion. Two nucleoli are indicated by arrowheads. Bars, 50 μm in a, b, h, j, l and m, 10 μm in c

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When egg cells were isolated from ovaries they become spherical due to the lack of cell wall. However, asymmetric distribution of cytoplasm, nucleus and vacuoles could be clearly observed in the isolated egg cells (Fig. 1b). Although it remains open whether vacuolar and cytoplasm rich regions consistently correspond to chalazal and micropylar ends, respectively, at least this result shows that the egg cell maintains cytological polarity even after isolation of the cell from an embryo sac. In addition, the existence of cytological polarity in the isolated egg cell, which is probably important for zygote development, provides the possibility that an isolated egg cell fused with a sperm cell will enter into the developmental process of zygotic embryogenesis.

Gamete fusion and early cytological events in the zygote

Isolated egg and sperm cells (Fig. 1b, c) in a fusion droplet (370 mosmol/kg H2O mannitol) were aligned on one of the electrodes under a dielectrophoretic AC field (Fig. 1d), and then mannitol solution (520 mosmol/kg H2O) was added to the fusion droplet during cell alignment. The addition of mannitol solution with a higher osmolality changed the shape of the sperm cell to oblong (Fig. 1e), and made the attachment of the egg cell to the electrode more stable. Without this treatment, egg cells were often released from the electrode upon fusion induced by a DC pulse, and fusion efficiency was greatly reduced. After addition of the mannitol solution, fusion was induced by a single negative DC pulse (Fig. 1f). The fusion process progressed within 1 s, and the shape of the zygote on the electrode recovered to a spherical shape around 10 s after fusion (Fig. 1g). The efficiency of successful electrofusion was 86.6% (n = 1,043) under optimal conditions. Twenty to 50 egg cells could be isolated from 100 processed ovaries, and 20–30 egg cells could be fused with sperm cells by one experimenter in a day.

Cell wall formation and the emergence of an additional nucleolus are known fertilization-induced early events in zygotes (Kranz et al. 1995; Higashiyama et al. 1997). The rice zygotes produced by IVF started to form cell walls (Fig. 1j, k), whereas formation of cell walls was not induced in egg cells even if they were stimulated by a single DC pulse and cultured under the same conditions as zygotes (Fig. 1h, i). Two nucleoli were observed in a zygote at least 4 h after fusion (Fig. 1l, m). These indicate that the in vitro-fertilized egg was activated, and that nuclear fusion was completed in the zygote within 4 h of fusion.

Development of in vitro-produced zygotes into globular-like embryos

Using the zygote culture system described in Sect. “Materials and methods,” the development of 97 zygotes produced by IVF was monitored (21 independent experiments, 2–8 zygotes/experiment). At around 12 h after fusion, well-developed granular organelles, probably starch granules, were visible in the zygotes (Fig. 2 a). Eighty-nine zygotes (91.8%) started to divide, and the first cell division of the zygotes was observed at 15–24 h after fusion (Fig. 2b and Table 1). The first cell division frequently represented the establishment of a cleavage furrow (arrowhead in Fig. 2b) as observed in the two-celled embryo of wheat (Kumlehn et al. 1999). After the first division, the two-celled embryos continued to develop into early embryos consisting of 4–8 cytoplasmic dense cells by 30 h after fusion (Fig. 2c, e, f). At 40–50 h after fusion, 75 of the embryos had developed into globular-like embryos. Approximately 70% of these embryos showed a typical globular-shape and putative traces of the first cell division were often visible (Fig. 2d). The inset image in Fig. 2d represents a globular-like embryo in which a putative trace of the first cell division can be observed very clearly; this embryo is representative of approximately 30% of the globular-like embryos. Among the remaining 14 early embryos, 6 developed into globular-like embryos by 62–72 h after fusion and 8 ceased their development. In total, 83.5% of the IVF-produced zygotes developed into globular-like embryos (Table 1). Next, to estimate the number of cells in early and globular-like embryos, the number of nuclei stained with DAPI was counted (Fig. 2e, f, g, h). The results indicated that embryos cultured for 30 and 48 h after fusion were composed of about 5 and 15–16 cells, respectively (Table 2).

Fig. 2
figure 2

Early embryonic development of zygotes produced by the IVF system (a–h) or in planta (i–k). a A zygote 12 h after fusion. b A two-celled embryo 19 h after fusion. Arrowhead indicates the cleavage furrow. c An early embryo 30 h after fusion. Arrowhead indicates a possible trace from the cleavage furrow. d Two globular-like embryos 49 h after fusion with a typical globular shape. A possible trace of the cleavage furrow is indicated by arrowhead. The inset shows a globular-like embryo 47 h after fusion, in which a possible trace of the cleavage furrow is clearly observed (arrowhead). e and f Nuclear staining of an embryo 30 h after fusion, visualized by brightfield and fluorescence microscopy, respectively. g and h Nuclear staining of an embryo 48 h after fusion, visualized by brightfield and fluorescence microscopy, respectively. i A zygote and a synergid in an ovule 6 h after flowering. The arrow and arrowhead indicate the zygote and a synergid, respectively. j An early embryo in an ovule 24 h after flowering. Arrowhead indicates a nucleolus. k A globular embryo in an ovule 30h after flowering. Bars, 50 μm

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Table 1 Growth of zygotes produced by IVF
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Table 2 Estimated number of cells in embryos produced by the IVF system or in planta
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Early embryonic development in planta

To compare the manner of development of IVF-produced zygotes into globular-like embryos with that of zygotes in embryo sacs, zygotes and early stage embryos in ovules were observed. Figure 2i represents a zygote and a synergid in an ovule 6 h after flowering. Vacuoles in the zygote were less developed than those in the egg cell, and cytoplasmic density appeared to increase in the zygote after fertilization. Fertilization-induced cell elongation/enlargement was not observed in rice zygotes, although zygote elongation/enlargement has been reported in some species; for example, Arabidopsis (Mansfield and Briarty 1991), Capsella bursa-pastoris (Schulz and Jensen 1968) and Cypripedium insigne (Poddubnaya-Arnoldi 1967). At 24 h after flowering, an early embryo consisting of several cells was observed (Fig. 2j). At 30 h after flowering, embryos were globular-shaped, but their size appeared to be unchanged or only slightly enlarged (Fig. 2k). Synergids disappeared until this stage.

Since a nucleolus could be detected in each cell of the early embryos (arrowhead in Fig. 2j; George et al. 1979), we counted the number of nucleoli in these embryos to estimate the number of cells in each embryo. The results indicated that the average cell number in embryos at 24 and 30 h after flowering is about 6 and 14, respectively (Table 2), consistent with the report that rice embryos are composed of 5–6 cells at 24 h after fertilization (Jones and Rost 1989).

Plant regeneration and fertility

The globular-like embryo produced by the IVF system (Fig. 2d) developed into a cell mass with an irregular shape around 5 days after fusion (Fig. 3a). The cell mass grew rapidly into a white cell colony with a diameter >1 mm during the 13 days of subsequent culture (18 days after fusion; Fig. 3b). After 4 days of subculture of the cell colony on a solidified-regeneration medium (22 days after fusion), green spots became visible (Fig. 3c), and the emergence of multiple shoots was observed after 8 days of subculturing (26 days after fusion; Fig. 3d). After 12–30 days of subculturing (30–48 days after fusion), the regenerated shoots were transferred into a hormone-free medium and cultured for 11–13 days (41–61 days after fusion), resulting in the formation of plantlets (Fig. 3e). These plantlets were capable of growth in soil (Fig. 3f), and flowered about 60 days after transferring the plantlets to soil pods (about 100–120 days after fusion; Fig. 3g). The seeds harvested from the regenerated plants were germinated and grew normally (Fig. 3h). Throughout this study, 149 plantlets were prepared, of which 30 were grown in soil pods. Finally, 27 fertile plants were obtained.

Fig. 3
figure 3

Development and regeneration of globular-like embryos produced by IVF. a A cell mass 5 days after fusion, which developed from the globular-like embryo. b A white cell colony 18 days after fusion. c A developed cell colony 4 days after transferring the white cell colony (panel b) into regeneration medium (22 days after fusion). Green spots are visible in/on the cell colony. d Regenerated shoots. Generation of shoots was observed after 8 days of subculturing the white cell colony (26 days after fusion). e A plantlet after 12 days of subculturing a regenerated shoot on a hormone-free medium (43 days after fusion). f A regenerated plant 42 days after planting the plantlet in a soil pod (85 days after fusion). g Flowers of a regenerated plant (100 days after fusion). h 13-day-old seedlings. Seeds harvested from regenerated plants were germinated and grown as described in the materials and methods. Bars, 50 μm in a, 1 mm in b, c and d, 1 cm in e

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Discussion

In planta, rice egg cells are highly polarized, with chalazally located vacuoles and a micropylar nucleus and cytoplasm. The polar distribution of vacuoles disappeared in zygotes, and zygotes developed into early embryos, consisting of about 6 cells, by 24 h after flowering, and then globular embryos, composed of about 14 cells, by 30 h after flowering. When the size of egg cells, zygotes and these embryos were compared, their sizes were almost equal, with only a slight enlargement being observed in the 30 h embryo. This indicates that zygotes and the cells of early embryos divide without cell elongation or enlargement during the early embryogenesis of rice.

Isolated egg cells maintaining their polarity were electrically fused with sperm cells, resulting in zygotes. During the culture of zygotes, these cells developed into early embryos, consisting of about 5 cells, by 30 h after fusion, and then into globular-like embryos, composed of about 15–16 cells, by 48 h after fusion. Notably, the cell numbers in embryos at 30 and 48 h after fusion were comparable to those of embryos in ovules at 24 and 30 h after flowering, respectively. In addition, the diameters of isolated egg cells, IVF-produced zygotes and early/globular-like embryos were unchanged, or showed a slight enlargement in globular-like embryos. These suggest that cell elongation and enlargement do not occur during the culturing of zygotes into globular-like embryos, consistent with observations of early zygotic embryogenesis in planta. It was suggested that in vitro-produced zygotes develop into globular-like embryos via an embryogenic process similar to that in zygotes developing in the embryo sac, although the development of in vitro-produced zygotes is relatively slower than the development of zygotes in planta. By in vitro culture of rice zygotes isolated from pollinated ovaries, Zhang et al. (1999) successfully regenerated fertile plants. However, the cultured zygotes developed into multicellular structures with irregular shapes after one to three rounds of cell division. This may be due to the composition of the medium used for zygote culture and/or the culture conditions.

Rice embryogenesis can be separated into 10 stages, Em1-10 (Itoh et al. 2005). Em1 represents the zygote stage, and the globular embryo stage is divided into three stages: Em2, the early globular stage; Em3, the middle globular stage; and Em4, the late globular stage. Globular embryos at Em2 are composed of 2 to ca. 25 cells, and rapid cell division has been reported in these embryos (Itoh et al. 2005). Based on the number of cells in IVF-produced embryos 30 and 48 h after fusion, they could be categorized as being in the Em2 stage. Although the in vitro-produced globular-like embryos did not develop into a middle or late globular stage, but into irregularly shaped embryos, our IVF system can at least be considered to reproduce the zygotic embryogenesis of rice during the Em1 and Em2 stages.

In angiosperms, the formation of a globular embryo is a general event during early embryogenesis. Morphogenetic events for organ differentiation occur after the globular embryo stage, although fixed and variant patterns of cell division during the development of zygotes into globular embryos have been reported depending on the species (Pollock and Jensen 1964; Schulz and Jensen 1968; Tykarska 1979; Schel et al. 1984; Mansfield and Briarty 1991). Recently, it was proposed that a globular embryo can be divided into domains, demarcated by gene expression patterns, with distinct developmental fates (Bowman and Eshed 2000; Laux et al. 2004). It has been known that the homeobox gene WUSCHEL, which regulates stem cell fate in the Arabidopsis shoot meristem, is first expressed in the apical subepidermal cells at the 16-cell stage of embryogenesis (Mayer et al. 1998), although the tunica-corpus structure, a characteristic of shoot apical meristems, becomes evident in the late heart or torpedo stage embryo. In addition, OSH1, a KNOX-family homeobox gene, is expressed in the ventral region of rice globular embryos, where the shoot apex will later differentiate (Sato et al. 1996). Therefore, investigations into early embryogenesis, from the zygote to the globular embryo stage, will be of great importance to understand how the subdomains of globular embryos are specified and/or zonated. The rice IVF system reported in this study will provide an opportunity to directly analyze such early embryogenic events. Because molecular and biochemical analyses using limited materials have recently become possible, as reported for egg cells, zygotes and early embryos of maize and wheat (Okamoto et al. 2004, 2005; Le et al. 2005; Sprunck et al. 2005), rice zygotes and embryos produced by IVF are also available for investigations at the molecular and biochemical levels. Moreover, the abundant resources for research of rice plants can be utilized when this rice IVF system is employed. We are currently conducting microarray analysis, using 100 egg cells and 100 zygotes, as the first effort to monitor gene expression profiles after fertilization.

Because the IVF-produced globular-like embryos developed into irregular shapes, we adjusted the composition of phytohormones and/or nutrients in the embryo culture medium in an effort to develop the globular-like embryos into embryos at later globular or organ-differentiating stages as in planta. However, these experiments resulted in developmental arrest or the formation of irregularly shaped embryos. Kumlehn et al. (1998) reported that isolated wheat zygotes develop into differentiated embryos only when sporophytically induced barley microspores are co-cultured with the zygotes. Utilizing microspores as feeder cells in the rice IVF system might induce the embryonic development of globular-like embryos.

The in vitro-produced globular-like embryos regenerated into fertile plants with complete seed sets through possible callus-derived shoot regeneration. The rice IVF system described here might become an important technique for generating new cultivars with desirable characters.

Isolated zygotes from barley, rice and wheat have been successfully used to monitor the zygote development and make regenerated plants, as the procedure for isolating zygotes from pollinated ovaries is much simpler than making zygotes by IVF of an egg cell with a sperm cell (Holm et al. 1994; Kumlehn et al. 1998; Zhang et al. 1999). The IVF system described here will have advantages for the determination of exact fusion time, for observing very early-stage events in zygote development, such as cell wall formation, and for producing zygotes derived from the gametes of different cultivars.

A complete IVF system, from zygotes to fertile plants, was first established by Kranz and Lörz (1993) using maize gametes. Therefore, the rice IVF system reported here is the second case for establishing a complete IVF system. The present work describes a method that can be used for studies of IVF and subsequent early embryogenesis and plant formation in rice, which is a crop of considerable scientific and economical importance.