Central cell fertilization and endosperm development in vitro

Erhard Kranz1, Petra von Wiegen, Hartmut Quader, and Horst Lörz

Oral presentation at the XVI International Botanical Congress, Saint Louis, USA, August 1-7, 19991

Center for Applied Plant Molecular Biology, AMP II, Institute for General Botany, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany

E-mail: ekranz@botanik.uni-hamburg.de

Central cell isolation
Central cell - sperm fusion
Endosperm development
Further Prospects

One hundred years ago it was discovered that two fertilization events occur in angiosperm species (Nawaschin, 1898; Guignard, 1899). One sperm of a pollen grain or tube fuses with the egg and the resulting zygote develops into an embryo. The other fuses with the central cell forming a primary endosperm cell which subsequently develops into endosperm. With the possibility of central cell fertilization using isolated gametes, now these two fertilization processes can be performed at the single cell level in vitro. In vitro fertilization with single gametes (IVF) has been performed mainly with maize, (for recent reviews see Dumas et al., 1998; Kranz et al., 1999; Kranz and Kumlehn, 1999). The application of single cell culture techniques allows single zygotes to develop into embryos and fertile plants, as well as single in vitro fertilized central cells to develop into endosperm in culture. Although double fertilization in vitro is clearly different from the situation in planta, it has been demonstrated that both the embryo and the endosperm are able to self-organize without mother tissue in a manner similar to that in vivo. Moreover, in culture the zygote without an endosperm and the primary endosperm cell without an embryo develop to what is observed in vivo. An experimental system for investigations of zygotic embryogenesis and endosperm development is now available to dissect more precisely the early developmental processes of higher plants.

IVF techniques with single higher plant sperm, egg and central cells presuppose their isolation, because the embryo sac is generally deeply embedded in the ovule, and the sperm cells are enclosed in pollen grains or tubes. The isolated plant gametes are protoplasts and therefore can be fused by an externally applied electrical pulse, by polyethylene glycol (PEG)- or by calcium. The fusion of pairs of gametic cells can be performed in small droplets of fusion medium and followed under microscopic observation. These techniques allow a precise timing after gamete fusion and thus experimental access to detailed investigations of early events of zygote, embryo and endosperm formation.

Central cell isolation

Central cells are isolated manually by fine tipped needles from pre-dissected ovule tissue. Reasonable quantities of central cells were isolated from maize (Kranz et al., 1998 ) and tobacco (Fu et al., 1997). In maize, a softening of the nucellar tissue by a brief treatment with cell wall degrading enzymes (cellulase, hemicellulase, pectinase, pectolyase) prior to the microdissection of central cells is useful. Three to eight maize central cells can be isolated from about 160 nucellar tissue pieces within 2 to 3 hours. Plasmolysis of the maize central cell prior to its manual isolation is useful. Compared to egg cells, a longer treatment of cell wall degrading enzymes is suitable for the isolation of these cells. The hard maize embryo sac wall is not digested after treatment with commonly used cell wall degrading enzymes. Thus, the manual isolation procedure determines the final yield of central cells.

Compared to maize egg cells having a diameter of about 65 - 77 µm, isolated maize central cells are very large cells, having a diameter of about 200 µm (Fig. 1).

Figure 1. Isolated, adherent cells of the embryo sac of maize: egg cell (E), central cell (CC) and two synergids (SY) are shown.

Therefore they are not easy to handle. As the isolated egg cells, central cells are protoplasts and therefore are spherical. In maize, the nuclei of these highly polar cells are surrounded by the main cytoplasm, including the other cell organelles. This cluster is mainly located at the cell periphery, thus a pronounced polarity is maintained in the isolated central cells.

Isolated sperm, egg and central cells of several species are available to explore sperm movement (Russell, 1996), adhesion and putative gamete specific recognition events of higher plant gametes. A recognition assay might be developed for cell adhesion and to test the function of putative gamete specific surface molecules, including an efficient procedure for the alignment of gametic cells. It is not known whether gamete-specific receptors are involved in gametic interactions. Also, the gene expression status of single gametes can be investigated by the use of reverse transcriptase polymerase chain reaction (RT-PCR) methods (Richert et al., 1996; Sauter et al., 1998).

Central cell - sperm fusion and karyogamy

In vitro fusion of these cells presupposes an alignment of the two protoplasts. This is accomplished manually, by the use of a microneedle or, with high efficiency by the use of an AC-field. Electrically mediated fusion of pairs of sperm and central cell protoplasts were performed in maize (Kranz et al., 1991 ; 1998) using the conditions elaborated for somatic protoplast fusion (for review see Zimmermann, 1996) and the device and conditions for fusion of two selected somatic protoplasts (Koop and Schweiger, 1985 ). Fusion is induced by one or a few short DC-pulses while the gametes are aligned at one of the two tiny electrodes (Fig. 2)


Figure 2 (left). Maize sperm (S) and central cell (CC) aligned on one electrode before cell fusion. (from Kranz et al., 1998)

Figure 3 (right). The maize sperm nucleus (SN) is located inside the in vitro fertilized central cell. The in vitro fertilized central cell was stained with DAPI one hour after cell fusion and followed by epifluorescence microscopic analysis. Note the large vacuole (V). (from Kranz et al., 1998)

Electrofusion of gametic protoplasts is highly efficient. For example, in maize sperm-egg fusions the mean fusion frequency is 85%. Due to more difficult handling, central cell-sperm fusion frequency is lower (44%) (Fig. 3).

Also in maize, calcium mediated fusions of sperm and central cells were performed (Kranz et al., 1998).

The application of media with suitable osmolalities during the different steps of isolation and of fusion procedures is an important criterion for the efficiency of these IVF methods (Kranz, 1999). As with egg cells, isolated sperm cells fuse fast, generally in less than one second with central cells. This effect may be attributed to the large differences between the size of these cells. Cell fusion was more effective and faster with spindle shaped sperm cells than with round ones. Before cell fusion, spindle-shaped sperm cells were aligned during dielectrophoresis by one of the tail-like ends to the egg or central cell membrane (Fig. 4).

Figure 4. Alignment of a tail-like extension of a maize sperm cell with an egg cell membrane during dielectrophoresis. (from Kranz et al., 1995)

This was observed in egg-sperm and central cell- sperm fusion. The spindle-like shape of sperm cells is maintained by the cytoskeleton, thus the latter might contribute to the efficiency of gamete fusion in vitro. It provides a tiny end of one tail which aligns in a point-to-point attachment to the egg cell membrane. Interestingly, somatic protoplasts are efficiently electro-fused only when the protoplasts of both partners have a high turgor. This results in a stable spherical shape of the protoplasts and provides a point-to-point attachment and efficient cell fusion. These observations should be included in considerations and investigations to elucidate processes of gamete fusion occurring in vivo. The occurrence of spindle shaped sperm cells during fertilization was already observed by Nawaschin (1898).

The point-to-point attachment of the membranes after alignment of a spindle-shaped sperm cell with an egg or central cell is one reason for efficient cell fusion. However, proteins in the membrane may well be present to bind the egg or central cell membrane with the sperm membrane to promote fusion of the phospholipid vesicles of these cells, which are naturally predestined for fusion. Although it might be difficult to discriminate between adhesion or binding and fusion, antibodies can be used to prevent the fusion process, once putative fusion proteins are isolated. Using calcium as a fusion mediating agent, further conditions might be investigated which promote membrane fusion of gametic cells.

Figure 5. Isolated secondary nucleus of maize with integrated sperm nucleus (SN). Karyogamy at 150 min after sperm-central cell in vitro fusion. Note one large nucleolus (N). Light/epifluorescence microscopy after DAPI staining. (from Kranz et al., 1998)

Following IVF, karyogamy was observed after 1 h in central cells (Fig. 5).

It was observed in egg cells 35 min (Tirlapur et al., 1995) to 45 min (Faure et al., 1993) after in vitro gamete fusion. It is completed both in the egg and in the central cell within 2 h after fertilization in vitro (HAF). As in planta in maize (Mól et al., 1994), two types of karyogamy were observed in in vitro fertilized central cells. The sperm nucleus fuses either with one of the two polar nuclei or with the secondary nucleus which can be formed prior to pollination and fertilization. The time course of karyogamy can rapidly be determined by using isolated, DAPI-stained nuclei of fertilized egg and central cells (Fig. 5). The application of this method is useful when the nuclei of these cells are clustered together with other organelles and therefore not easy to observe.

Endosperm development

The availability of an efficient culture system which allows sustained growth of a few in vitro fertilized central cells is necessary. The culture system should allow rapid growth of endosperm. This has been exclusively achieved by co-cultivation of in vitro fertilized central cells and feeder cells. Compared to somatic cereal protoplasts and zygotes, in vitro development of in vitro fertilized central cells turned out to be genotype­independent.

IVF of maize central cells results in endosperm formation and development in culture (Kranz et al., 1998 ). Single fertilized central cells develop into a characteristic, heterogeneous tissue without embryo, embryo sac cells and mother tissue, comparable to the in vivo situation. In planta, the primary maize endosperm cell develops into a syncytium after rapid nuclear divisions without cell wall formation until the third day after pollination (DAP), followed by the formation of uninucleate cells via a cellularization process within about 5 DAP (Clore et al., 1996 ; Becker et al., 1999).

The in vitro fertilized central cell develops into a bipolar, highly polarized oblong cell with a characteristic narrowing while still in the syncytium stage (Fig. 6).


Figure 6 (left). In vitro-produced maize primary endosperm cell after 1 day in culture. (from Kranz et al., 1998)

Figure 7 (right). Incomplete cellularization in the globular part of the in vitro-produced maize endosperm after 4 days in culture. (from Kranz et al., 1998)

As in vivo, the transition from the syncytium to the stage of cellularization of in vitro maize endosperm occurs within 3 - 5 DAF. Division of the in vitro produced zygote occurs as early as 29 HAF (E. Kranz, unpublished data), but generally 42-46 HAF (Kranz and Lörz, 1993; Kranz et al., 1995). In planta, maize zygotes are dividing about 16 h after karyogamy (Mól et al., 1994). Cellularization extended centripetally from the periphery of the primary endosperm cell (Fig. 7).

Figure 8. Feulgen-stained in vitro-produced maize endosperm after 5 days in culture. (from Kranz et al., 1998)

In the cellularized endosperm tissue in planta, rapid mitotic divisions occur within about 12 DAP in the centrally located cells and within 20 to 25 DAP in the peripheral tissue. The oblong primary endosperm cell further develops into endosperm which consists of two parts: one globular part containing small cells with dense cytoplasm, and one oblong part characterized by a gradient of fewer nuclei towards the pole opposite to the globular part which ended in large cells (Fig. 8).

The globular part is developing more rapidly than the oblong part is. In vivo, maize endosperm develops initially more rapidly in the micropylar than in the antipodal area of the fertilized embryo sac (Randolph, 1936). The heterogeneity of endosperm tissue is characterized in maize by densely cytoplasmic cells predominately located at the base of the suspensor and larger vacuolated cells in other regions near the embryo (Schel et al., 1984 ). The globular part of the bipolar in vitro endosperm might derive from the micropylar pole of the central cell, but this remains to be determined.

Figure 9. Three dimensional survey of a section of 4-day-old in vitro-produced maize endosperm. Note the highly synchronized cell divisions. Early prophases. (from Kranz et al., 1998)

After cellularization, cell division occurred at a high frequency and highly synchronized (Fig. 9).

The cells of the 4- to 6-day-old in vitro endosperms were predominantly in prophase and only occasionally in early stages of metaphase and anaphase. The examined chromosome numbers of 25 to 30 indicate the triploid nature of the in vitro-produced endosperm.

In planta, endosperm development is terminated. The stability of the in vitro endosperm tissue in long term cultures as well as the ability of organ and plant regeneration have still to be investigated. In some species, shoot buds were formed in cultured endosperm and plants regenerated from callus derived from excised and cultured endosperm (for review see Bhojwani 1984 ). Although maize endosperm explants were cultured (Straus, 1954 ) and callus and suspension cultures were established from excised, immature endosperm (Tabata and Motoyoshi 1965; Shannon and Lui 1977 ), plant regeneration has not been reported.

The fertilization of single central cells allows studies comparative with investigations of all those processes occurring at and after sperm-egg in vitro fusion; for example, the involvement of signaling molecules in endosperm development. Processes involved in the transition from coenocytic to cellularized endosperm are conserved in barley, wheat, rice and maize (Brown et al., 1994 , 1996a, 1996b; Olsen et al., 1995). Such early events, as the suppression of phragmoplast formation between nuclei, the mitotic hiatus, the synchronized re-initiation of mitosis, the periclinal phragmoplast formation, the initiation of cellulariation via formation of nucleocytoplasmic domains (NCD) of a radial microtubular array, alveolation, if they occur in vitro, might well be studied by access to manipulation under defined conditions (Olsen, 1998). The mechanisms behind the programming of nuclear location and division planes during cell wall formation in the syncytium in endosperm seem to be important for later kernel development, however they have not been elucidated (McClintock, 1987; Walbot, 1994). Single fertilized central cells are promising target cells for gene expression studies by using genes, especially endosperm specific ones and for further functional analysis of gene products during early endosperm development (Olsen 1992; Hueros, 1995; Doan et al., 1996 ; Opsahl-Ferstad et al., 1997; Becker et al., 1999).

Both a single egg and a single central cell can now be fertilized in vitro. These fusion products, the zygote and the primary endosperm cell can be cultured in the same medium. Development occurs in a simple medium, widely used in tissue culture. No special media are needed for both the developing embryo and the endosperm. The development of an embryo and endosperm, respectively, occurs in culture independently from each other and without female tissue. Biochemical exchanges between co-cultured embryos and endosperms can be investigated to study the interactions during their development. These interactions are complex and not well understood. As they are able to self-organize under these conditions, it might be of interest to compare developmental processes in both products of fertilization and to dissect similarities and divergences of such processes during early stages of individual growth. The processes are unknown, which cause the zygote to develop into an embryo and finally into a plant, while the primary endosperm cell develops into a unique storage tissue (Lopes and Larkins 1993 ). Although there are these distinctly different developmental fates, there is a pronounced morphological similarity between the in vitro endosperm and the embryo at early developmental stages under the same culture conditions. Both the embryo and the endosperm are composed of a globular part containing small cells rich in cytoplasm and another part consisting of large cells, which represent the suspensor and a haustorium-like tissue, respectively. This similarity in morphological polarization might indicate underlying similar developmental processes. One might consider the central cell as a modified egg cell and early endosperm development as a special kind of embryogenesis (Favre-Duchartre, 1984; Dumas et al., 1998 ). The similarities in embryo and endosperm development might have a common origin. One hypothesis has suggested, that endosperm tissue evolved from a second embryo (Sargant, 1900; Friedman, 1995).

Further Prospects

These in vitro studies clearly demonstrate that in vitro and in vivo zygotes, and the in vitro-produced primary endosperm cell as well, are able to self-organize in culture in a typical manner similar to that in vivo. Development of both the embryo and the endosperm occurs independently from maternal tissues. Furthermore, the embryo develops without endosperm, and the endosperm develops without the embryo in culture. Thus, previously developed techniques are valuable experimental tools for the elucidation of various processes of double fertilization and early development of embryo and endosperm in angiosperms under defined conditions. Certainly, important progress towards a better understanding of these processes will continue to come from analyses of mutants. However, experimental access to single higher plant gametes and zygotes will facilitate studies on double fertilization and early developmental processes which are difficult to investigate in plants. Manipulations with single egg cells and zygotes under defined conditions together with gene cloning, protein isolation and its three-dimensional characterization will also allow a comparison with fertilization-induced processes occurring in lower plants and animals (Epel, 1990; Vacquier, 1998).

Analyses of the initiation of activation of egg and central cells, fertilization-induced signal transduction events, changes in the cytoskeleton and nuclear movement are now possible under more controlled conditions, e.g. an exact time after gamete fusion. Comparable to in somatic cell culture (Dudits et al., 1995 ), a short treatment of high amounts of 2,4-D can trigger cell division in cultured isolated egg and central cells leading to a multicellular structure formation (Kranz et al., 1995 ; Fu et al., 1997). The egg activation and fertilization-induced signaling events have been widely studied in animal and lower plant systems. For example, little is known about calcium signaling during angiosperm fertilization (Zhang and Cass, 1997). Membrane calcium and the calcium receptor protein calmodulin are mainly localized in the vicinity of nuclei of egg cells and in vitro zygotes (Tirlapur et al.,1995). A transient elevation of free cytosolic calcium was observed in maize in vitro zygotes (Digonnet et al., 1997 ). Investigations like these are now also feasible by using single central and primary endosperm cells.

Although the amount of central cells and primary endosperm cells is small, molecular analyses will be possible. cDNA libraries from few egg cells and in vitro zygotes were already constructed by using RT/PCR techniques to isolate and to study the function of the cloned egg- and fertilization induced genes (Dresselhaus et al., 1994; 1996). PCR protocols were also developed for expression studies of known genes by use of single cells (Richert et al., 1996), for example, to study gene expression in a time course during development of the fertilized central cell, as performed with in vitro zygotes (Sauter et al., 1998 ). Further methods (e.g. embedding, fixation and staining) were developed for electron-(Faure et al., 1992; 1993), and for laser scanning microscopic (Tirlapur et al., 1995; Kranz et al., 1998 ) analyses of single egg cells, zygotes and young endosperm. Other existing protocols such as whole mount in situ hybridization and immunocytochemical techniques for RNA and protein detection have to be adapted to single cells and to small cell aggregates. The established experimental tools that allow embryogenesis from zygotes in culture, surely will provide a valuable contribution to the elucidation of common features and differences in zygotic and somatic embryonic processes.

What is generally the role of the sperm cell in the onset of egg and central cell division? To follow the re-entering of the gametes into the cell cycle between IVF and the first cell division, the expression of some cell cycle genes was investigated in single gametes and zygotes of maize (Sauter et al., 1998). Sperm cells express the cell division cycle-specific genes cdc2ZmA/B and Zeama;CycA1;1, whereas the other mitotic cyclins Zeama; CycB1;2 and Zeama;CycB2;1 are not expressed in these cells. The same picture of expression of these genes was found after analyses of isolated egg and central cells. These cyclin genes are differentially expressed during the first cell division cycle of the zygote. There are no gene expression data from the developing primary endosperm cell. The function of cell cycle regulatory genes in the first zygotic cell cycle and during endosperm development also remains to be elucidated.

The analyses of events like these can be performed both in the zygote and in the primary endosperm cell allowing comparative studies. Further biochemical and molecular studies will give a more precise picture of co-ordinated processes during early developmental stages of the embryo and the endosperm.


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Peter v. Sengbusch - b-online@botanik.uni-hamburg.de