WO2000055339A2 - Method and device for immobilization of polarizable cells of higher plants by the use of a high frequency ac-field - Google Patents

Abstract

Disclosed herein is a method and a device for the immobilization of polarizable cells of higher plants by the use of a high frequency AC-field wherein the surfaces of the cells are polarized by the use of the AC-field and the cells of polarized surface are then contacted with the surface of an electrode under potential. The invention further concerns simple and highly effective microinjection processes performed by the use of the method and device of the invention, which processes are useful for introducing exogenous DNA into different plant cells, e.g. egg cells/zygotes of wheat.

Description

Method and device for immobilization of polari- zable cells of higher plants by the use of a high frequency AC-field

The invention relates to a method and a device for immobilization of polarizable cells of higher plants by the use of a high frequency AC-field.

The invention further concerns simple and highly effective microinjection processes performed by the use of the method and device of the invention, which processes are useful for introducing exogenous DNA into different plant cells, e.g. egg cells/zygotes of wheat.

Using a mechanical dissection method and a novel immobilization approach enabled us to microinject around 15 egg cells of wheat per an hour. Exposing the protoplasts to high frequency AC-field for immobilization, a significantly higher transient expression rate of the injected genes (46% and 52% for egg cells and zygotes, respectively) could be achieved than reported thus far for plant protoplasts by Schnorf et al. (1991) . Whether this high transformation efficiency is due to the high frequency electrical field applied for immobilizing the protoplasts is not known.

The transformation rate appeared to be a factor de- pending upon the time of egg cell isolation. According to the ultrastructural observations this seems to reflect a variation in competence of the egg cells during in situ development.

In order to conduct studies directed towards estab- lishing the optimal time-window for DNA delivery into the fertilized egg cell, the time course of DNA dynamics during zygotic development has been quantified via quantitative microspectrofluorometry. Abbreviations : CCD, charge coupled device; DAE, days after emasculation; FDA, fluorescein diacetate; HAP, hours after pollination; IVF, in vi tro fertilization, LMA low melting-point agarose; LSM, laser scanning microscopy; PCD, programmed cell death, TEM, transmission electron microscopy.

Till the commencement of the ^λ90s progress in the understanding of double fertilization in higher plants was mainly due to the considerable body of information gained from studies using light and electronmicroscopy.

As in vi tro fertilization technique using isolated gametes of maize has been developed (Kranz et al. 1991) and artificially produced zygotes could be regenerated into fertile plants (Kranz and Lorz 1993) the examination of the early developmental processes taking place immediately after fertilization became possible at the molecular, genetic and biochemical level. Deploying the technique of in vi tro gametic fusion approaches aimed at dissecting the genetic factors controlling zygotic embryo- genesis opened up new vistas in developmental biology.

The fusion of gametes of opposite sexes leads to the formation of the zygote which through defined, well- concerted and progressive changes accomplishes a developmental program leading to the stage of a multicellular or- ganism, an entire plant. Gametes as cells predetermined to form zygotes, can therefore be considered to be true progenitor cells being the most useful sources for embryo- logical studies. The fact that artificially produced zygotes grow into mul- ticellular structures with a high frequency of division makes gametes and in vitro produced zygotes very promising target cells for manipulation including electrofusion, electroporation and microinjection. However, there is a considerable time lag in the field of plant embryology as compared to animal embryology, owing mainly to the inaccessibility of the female gametes/zygotes of higher plants. These develop deeply en- closed in surrounding tissue hence making them difficult to manipulate. Thus, questions such as:

1 : how and when is cell polarity established in the zygote? 2: how do processes such as cell division, cell expansion, cell maturation and differentiation unfold bringing the zygote from the unicellular to the multicellular stage at the molecular level? 3 : what genes are involved in triggering the first asym- metrical cleavage of the zygote and how are these genes regulated? remain to be answered.

Some of these questions can only be addressed if systems in which isolated zygotes mimic in planta development (Kumlehn et al. 1997, Ku lehn et al . 1998) are coupled to techniques allowing the efficient insertion of foreign genetic material into gametes/zygotes of higher plants.

When it comes to attempting to transform germ line cells (the number of which is limited in an experiment) the method of choice is introducing genes into zy- gotes/early embryos via microinjection. Since microinjection of DNA has been used successfully in transforming animal cells, attempts have been made to transfer this technology to plants. However, in adapting microinjection to plant cells several problems had to be circumvented, one of the most stubborn of which is the immobilization of plant cells, as plant cells, unlike fibroblasts, are unable to grow attached to glass surfaces. Although several methods of immobilizing plant protoplasts for microinjection have been reported (Steinbish and Stabel 1983, Griesbach 1983, Lawrence and Davis 1985, Morikawa and Yamada 1985) only the holding pipette method developed by Crossway (Crossway and et al . 1986) seems feasible for making it possible to orientate the nucleus relative to the injection pipette.

As the inability of easily targeting cellular compartments (especially the nuclear region) may contribute among other factors to the lack of efficient plant cell transformation based on microinjection, strategies allowing "preferential" immobilization have inherent advantages over conventional methods. This view is supported by the fact that the frequency of transformation in mammalian cells or eggs via nucleus injection has been shown to be higher than that following cytoplasmic injection (Capecchi 1980, Yamaizuni et al. 1983, and Hammer et al. 1985) . Only one paper (Leduc et al. 1997) has demonstrated successful microinjection of maize zygotes but no work on the trans- fection of female gametic cells of higher plants by injection has appeared as yet. Thus, an efficient technique based on micromanipulation is highly desirable in exploring the sexual route for attempting to transform higher plants with agronomic importance. To our knowledge the procedure according to the invention is the first useful method of injecting exogenous DNA into egg cells/zygotes of wheat.

In accordance with the foregoing, the present invention concerns a method for immobilizing cells wherein the surfaces of the said cells are polarized using a high frequency AC-field and the cells of polarized surface are then contacted with the surface of an electrode under potential. The method of the invention is useful for the immobilization of advantageously eukaryotic cells, for example cells of higher plants, advantageously gametes, for example gametes of wheat. The invention also concerns processes for microinject- ing cells immobilized by the method of the invention.

The invention further relates to a device useful for performing the method of the invention, which comprises a microscope observable space compartment suitable for ac- commodating a liquid comprising the cells of interest, the said compartment also comprises electrodes suitable for applying a high frequency AC-field and into which compartment a microinjector can be introduced if desired.

The legends for the figures attached to this patent application are as follows.

Fig.l Egg cells of wheat as targets for microinjection and different steps of karyogamy after fusion in vivo.

(A) Ultrathin section of a cytoplasm region of an egg cell of wheat isolated 6DAE. Bar=l μm; V=vacuole (B) Ultrathin section of a cytoplasm region of an egg cell of wheat isolated 12 DAE. Bar=l μm; V=vacuole, li=lipid body

(C) Immobilization of egg cell of wheat by high frequency AC-field. Bar=30 μm (D) GUS expression in a microinjected, fertilized egg cell after 48h in culture. Bar=30 μm

(E) GFP imaging in a microinjected, premature egg cell after 24h in culture. Bar=15 μm. (F) Sperm-egg cell contact (3 HAP). Bar=20 μm The sperm cell is indicated by arrow

(G) Karyogamy in a fertilized egg cell (6 HAP) . Bar=10 μm

The arrow indicates the sperm cell nucleus Fig.2 Relative nuclear DNA quantities expressed as C values in wheat egg cells/zygotes prior to and at different times after pollination. Frequency histograms show DNA levels of the nuclei analyzed: (A) At time prior to pollination

(B) 3 hours after pollination (3 HAP)

(C) 6 hours after pollination (6 HAP)

(D) 9 hours after pollination (9 HAP)

(E) 12 hours after pollination (12 HAP) (F) 15 hours after pollination (15 HAP)

(G) 18 hours after pollination (18 HAP) (H) 21 hours after pollination (21 HAP) (I) 24 hours after pollination (24 HAP)

Example 1

Materials and methods

Plant material of the winter wheat cultivar Siete Cer- ros were grown in a growth chamber (Conviron, PGV-36) at 20/18°C day/night temperature using a 16-hr light period at a light intensity of 560 μEm-²s^-1 (Tischner et al. 1997) . The florets were emasculated when the spikes were just emerging and still sheathed by the flag leaf. This time the microspores were at mid-uninucleate stage (Timar et al. 1997). Pollination was done by hand at anthesis. Isolation of egg cells/zygotes

The spikes were harvested at consecutive intervals of three days after emasculation (henceforth 3 DAE) and the ovaries were carefully removed from the florets and sterilized for 1 min in 70% ethanol. Following two washes in sterile water the ovaries were placed on slides surface sterilized with UV and washed thoroughly in 70% ethanol before being left to air dry. The pedicel and the two lo- dicules were removed and a cross-section was made at the median position of the ovaries with a sharp sterile blade. About 20 pieces of ovular tissue containing the embryo sac were transferred to sterile 35 mm plastic dishes (Costar) filled up with isolation medium (600 mosmol/kg H₂0) . The female gamete/zygotes where isolated purely mechanically following the procedure of Kovacs et al (1994) . By this method the efficiency of egg cell isolation reached 75%. However, the fertilized egg cells were more difficult to obtain.

Isolated egg cells/zygotes were transferred into an- nitol droplets (600 mosmol/kg) of five microliters each dispensed on the bottom of a plastic dish overlaid with mineral oil (Kranz et al. 1991). The cells were individually injected into the droplets using a microchip controlled microcapillary (nanoliter injector, A203XVZ, WORLD PRECISION INSTRUMENTS, WPI) held by a Zeiss micro anipula- tor arm mounted onto the stage of an inverted microscope (IM35, Carl Zeiss) (Koop and Schweiger 1985) . The individually selected cells were transferred from one droplet into another for washing. Zygotes were isolated the same way at 3, 6, 9, 12, 15, 18, 21 and 24 hours after pollination.

Immobilization of the cells to be injected The immobilization of egg cells was achieved by the following method. The core of the injection set-up was an inverted microscope (IM35, Carl Zeiss, Germany) placed under a vertical airflow hood. A pair of electrodes (platinum wire, diameter 100 μm) was fixed into micromanipulator arms mounted under the condensor and controlled by hydraulic microdrives (Narishige, MO-104) . The immobilization of the egg cells at the electrode was accomplished by dielec- trophoretic force (1 MHz, 50 Vcm^"1) (Kranz and Lδrz 1993) . The surface of the electrode was firepolished so that the area of contact between the cell to be injected and the surface of the electrode was smooth and exceeded the con- tact zone between the surface of the cell and that of the electrode. During injection a 50 Vcm^-1 AC field was applied to the fusion chamber. The dielectrophoretic force was generated by a cell fusion instrument (CF-150, BLS, Hun- gary) . Cell transfer (uptake and release) was performed as described elsewhere (Koop and Schweiger 1985) . Briefly: single cells selected for microinjection were transferred to injection droplets of 5 μl each (0.6 M mannitol) , which were overlayered with mineral oil to avoid evaporation. Following microinjection the cells were picked up by a picking-up pipette and inserted into droplets of 2 μl of fully defined medium (Kao 90) (Kao and Michayluk 1975) . Each microdroplet was placed on the bottom of a plastic dish (diameter: 35 mm, Costar) and individually covered with mineral oil. This individual culture allowed making observations on each injected cell. The dishes were sealed with Parafilm™ and incubated at 23°C in the dark.

Example 2 Injection

Preparation of microneedles

Injection needles were prepared from borosilicate glass capillaries with an inner filament (Clark Electro- medical Instruments, GC 100TF-15, UK) using a horizontal puller (Bachofer, Germany) . Micropipettes could be prepared reproducibly with tip diameter of around 0.5 μm. Before pulling, the capillary tubes were thoroughly cleaned and siliconized with dimethylamino-trimethyl-silane (Sigma-Aldrich) dried at 140°C for 15 min and rinsed with tap water several times. After rinsing, the capillaries were dried at 140°C for 20 min and washed twice with 96% H₂S0₄. After having been left to air dry the micropipettes were overflushed with ultrapure water (Milli-Q50, MILLI- PORE) after rinsing them with detergent (NP 40 10%) . Fil- ter paper was used to infiltrate excess water before baking the capillaries at 130°C. Needles prepared in this way could be stored for a week in special dust free boxes suitable for autoclaving. Prior to use, the microneedles were autoclaved then UV sterilized for 20 min. Injection set-up

Injection pressure was supplied by a nitrogen gas balloon and controlled by a microinjector (Eppendorf 5242) . Microinjections were carried out using a micromotor driven manipulator (Carl Zeiss, Germany) mounted onto the stage of an inverted microscope (IM35, Carl Zeiss, Germany) . The samples to be injected were centrifuged at 10000 x g for 5 min to remove any particles that might clog the orifice of the microneedles (UFC30GV25, MILLIPORE) . In order to re- duce the possibility of the tip being clogged by reenter- ing fluid (either extra or intracellular) during injection the holding pressure was properly adjusted (40 hPa) . To take full advantage of microinjection, namely that injected material reaching the cytoplasm or the nucleus of the cells for which it was intended can be visualized, the microinjection procedure was monitored by semi dark-field fluorescence microscopy under epi-illumination at a magnification of 320x. The microcapillaries were loaded with a mix of DNA, the concentration of which was adjusted to 0.5 mg/ml, in FITC-dextran solution (fluorescein isothiocya- nate dextran MW 4400, FD 105, Sigma Chemical Co., St Louis MO, USA) . FITC-dextran solution was prepared at a concentration of 60 mg/ml in injection buffer as described by Schnorf et al . (1991) . Plasmid DNA was prepared by standard methods (Sambrook et al., 1989). Plasmid constructs, pFF 19G containing the GUS gene (Jefferson et al., 1987) under the control of the 35S promoter and pMNGlOOl plasmid containing the GFP reporter gene driven by the ubiquitin promoter were kindly provided by Professor I. Nagy (Agricultural Biotechnology Center, Hungary) and by Professor L. Tamas (University of Budapest) , conversely. For the transformation experiments the concentration of the plasmids was adjusted to 0.5 μg/μl FITC-dextran solution. Aliquots of 8-12 picoli- tres (estimated by meniscus displacement assuming the volume of a cone for the tip of the pipette) were injected at 180 hPa into each cell.

Example 3

Transient gene expression assays

GUS staining was carried out as described by Mendel et al. (1989). The injected cells embedded in agarose (0.6 M mannitol, 1% LMA) were stained histochemically in micro- titer plates. For GFP imaging LSM was used (Biorad M- 1024) .

DNA measurement

To study the effect of the time of injection upon the efficiency of gene transfer with regard to the cell cycle stage, the dynamics of DNA quantities was determined with particular regard to the inception of DNA synthesis during in vivo fertilization. Relative DNA levels of zygotic nuclei stained with 4, 6-diamidino-2-phenylindole (DAPI) were measured by spectrofluorometry. For measurement, a micro- spectrofluorometer with a digital image analyzing processor coupled to a Zeiss Axioscope microscope equipped with epifluorescence (HBO 50W burner, Carl Zeiss, Germany) and a UN filter set with excitation filter (365 nm) , and a band pass filter (LP397) were used with a Zeiss Plan Νeo- fluar 20 objective.

The relative nuclear DΝA content was measured by summing-up individual fluorescence values gained from serial sections through each nucleus following the standardization of the photometer. Net photometric values were read by the subtractive method: the background fluorescence of cytoplasm and the embedding medium adjacent to the region of interest (nucleus) was subtracted from the initial reading taken for the nucleus . Chemical fixation for cytological studies

The isolated, fertilized egg cells were introduced into 5 μl (1% w/v) ultra-low gelling agarose (in 0.6 M man- nitol, Sigma) droplets. The solidification of the agarose was brought about by cooling the droplets for 5 min at 4°C. This procedure allowed the cells to be placed in the fixative (2.5% [w/v] glutaraldehyde in 0.1 M potassium buffer) solution at precisely defined intervals after pollination.

Staining procedure

DAPI staining was performed as described by Kranz et al. (1991) . The viability of the cells following injection was checked according to Heslop-Harrison (1970) by FDA.

Transmission electron microscopy

To address the question of whether the egg cells were more amenable to integrating exogenous DNA depending on the time they were isolated, the in situ development was followed using TEM (Zeiss EM910) via employing microtech- nical methods described by Pόnya et al (1999) .

Results and discussion

In our hand it proved possible to introduce exogenous DNA into female gametes/zygotes of wheat via microinjection capitalizing on an immobilization technique developed in our laboratory. Hitherto three methods have been developed in immobilizing plant cells:

1) Embedding protoplasts in ultra-low gelling tempera- ture agarose or alternatively in Ca-alginate, (Schnorf et al. 1991) the solidification of which is not temperature- dependent, proved the quickest and mildest method. In this immobilization technique the cell protrudes from the aga- rose, since the movement and control of the glass needle is severely hampered in solidified agarose.

2) Polylysine can also be exploited as an adhesive in immobilizing plant cells. In this case protoplasts have only a small contact zone with the glass slide, so they are less well immobilized than elongated cells that have already started cell wall synthesis.

3) The use of a holding capillary pipette appears thus far to be the most complicated method being the most dif- ficult to perform, partly because of the laborious work needed to pull the holding pipettes, which have specially elaborated orifice. Nonetheless, the holding capillary method has the advantage of allowing the selection of the best position of the nucleus for injecting animal cells (Jaenisch and Mintz 1974, Rusconi and Schaffner 1981, Wagner, et al. 1981, Rubin and Spradling 1982) . The successful transformation of animal cells making use of this method was the impetus to apply this technique to plants in order to genetically modify plant species especially those that are not amenable to Agrobacter um-mediated transformation.

Viability tests (FDA) carried out after the injection procedure showed that the cells had withstood and recovered from microinjection. A total of 175 cells were in- j ected (98 egg cells and 77 zygotes) which yielded 45 and 40 cells giving transient expression of the injected reporter genes, GFP and GUS, respectively (Table 1 and 2) .

The expression of both the GUS and GFP genes was detected with expression rates varying according to the time of isolation (Tables 1 and 2) .

The overall transformation efficiency for zygotes was higher than that reported by Leduc et al. (1996) . Especially in samples microinjected 5 DAE the expression rate obtained differed from that of the cited study by one order of magnitude (46, 52%, respectively) .

As 6 DAE was judged to be the ideal stage for pollina- tion as this corresponded with the anthesis of the control spikes, it is intriguing to note that the highest percentage of transient expression of GFP was significantly higher in egg cells isolated 5 DAE (Table 1) .

The preliminary results obtained from the analysis of marker gene expression studies tempted us to surmise that the markedly higher efficiency of transient gene expression as compared to that of either somatic protoplast or zygote transformation experiments (Leduc et al., 1996) could be due to the impact of AC upon the cells' capabil- ity to integrate foreign DNA.

The exposure of protoplasts isolated from the hypo- cotyl of Helianthus annuus prior to cultivation in a very low density culture seemed to be a perplexingly efficacious factor, which substantially increased the prolifera- tion rate (Keller et al. 1997). Keller's findings are consistent with those of Rech et al. (1988) who also experienced enhanced cell division after exposing cells to electric pulses. It has also been demonstrated that electrical treatment can elicit differentiation such as somatic e - bryogenesis (Dijak et al. 1986) . Electro-stimulation has also been reported to have a discernible effect on DNA synthesis in several species (Dijak et al. 1986, Ochatt et al. 1988, Rech et al. 1987) . Work on nuclear transplantation aimed at the activation of oocytes in mammals (a fun- damental aspect of cloning as the reprogramming of the donor nucleus is the result of activating the recipient oo- cyte) showed that activation among various chemical and physical treatments including alcohols, anaesthetics, protein synthesis inhibitors, hyaluronidase, cold shock and heat shock can be brought about by electrical pulses (Whittingham 1980, Cuthberston and Cobbold 1985) .

At this stage the mechanisms thought to play a role in inducing dramatically enhanced proliferation potential as a result of exposure to high frequency electromagnetic fields are not understood. However, according to studies on mammalian cells the effect is conjectured to be mediated via membrane receptors (Blank 1993) .

To our knowledge no report in the literature has ap- peared yet on the putatively enhancing effect that high frequency AC fields may exercise on the integration of foreign DNA.

The observation that the ability of the egg cells to express the injected reporter genes varied with respect to the time of isolation might reflect some stage-specific features of the 'resistance' of egg cells developing in situ to transcribing exogenous DNA. The nature of the mechanisms presumably involved in the 'acquisition' of a certain physiological state by the egg cell at some points during its maturation which is less recalcitrant to expressing transiently exogenous DNA remains to be elucidated. Findings reported by Collas et al. (1989) indicated that the age of the oocytes is the most important factor involved in activation rates from close to zero using an electrical pulse at the time of ovulation to essentially 100% over a period of 12-14h. This change was not attributable to an abrupt change in calcium in response to the electrical field (an electromagnetic field is thought to cause activation of the oocytes by inducing an elevation of intracellular calcium, Whittingham 1980) the factor inhibiting activation in ovulated oocytes is thought instead to be a cytostatic factor which has recently been identified as the protooncogene product c-mos (Sagata et al. 1989) . As evinced by the cytological characteristics of in si tu egg cell development analyzed at the ultrastructural level using TEM (figures 1A and B) , changes clearly take place (Pόnya et al . 1999) during egg cell maturation: in the cytoplasm of the premature egg cell a large number of mitochondria and numerous ribosomes and vacuoles can be seen, whereas in the mature (i.e. 12 DAE) female gamete the storage materials (starch grains, lipid and protein bodies) are dominant and autophagous vacuoles appear indi- eating cell destruction (Fig. 1A, B) . How these changes may evoke the time-specific activation of cytoplasmic factors, leading to 'less resistant' conditions towards the DNA uptake of the egg cell is not known.

Egg cells developed in situ up to 15 DAE show charac- teristic signs of PCD (programmed cell death) , as they shrink substantially and the plasma membranes become blabbed and ruffled (Pόnya et al. 1999) providing a possible explanation why the frequency of transient expression drops in cells injected 15 DAE. However^ the observation that young (i.e. 5 DAE) egg cells can be transfected more efficiently calls for explanation.

The difference between the transformation efficiency of the two gene constructs (GUS, GFP) is likely to be due to the different level of capability of the two promoters (35S and ubiquitin) to support the expression of the reporter genes used in the female gametes/zygotes.

Nevertheless, since the promoters were different as well as the reporter genes, far-reaching conclusions cannot be drawn from the results. It would be desirable to make use of reporter systems differing only in their promoters, preferably both giving fluorescent signals detectable more accurately and quantitatively by exploiting LSM or CCD-imaging technology. As the transformation frequency in mammalian cells/eggs has been shown to be higher after intranuclear injection compared to cytoplasmic injection (Capecchi 1980, et al., Hammer et al . 1985, Brinster et al. 1985) a procedure was developed in our laboratory which allowed the manipulation of the egg cells/zygotes for the optimal orientation of the nucleus relative to the injection needle, so that intranuclear injection was possible (Fig. 1C) . Although the percentage of cells responding posi- tively in gene expression assays (Fig. 1D,E) following intranuclear injection was higher than when the nucleus was targeted (data not shown) we did not obtain significantly differing data in the two cases.

DNA dynamics in in vivo fertilized egg cells DNA content in egg cells/zygotes isolated mechanically at defined intervals elapsed after hand pollination was estimated using microfluorometry. The data obtained show unequivocally that the nuclei of the egg cells prior to pollination or after 1 hr after pollination were at the—1C DNA level (Fig. 2A) . Although the results of studies on measuring DNA quantities within the egg and zygote nuclei of higher plants are ambiguous and sometimes subject to dispute (Heslop-Harrison 1972), Mogensen and Holm (1995) obtained consistent data pertaining to the DNA amounts in egg and zygote protoplasts of barley. In Gnetum gnemon, a seed plant, related to Angiosperms, expressing a rudimentary pattern of double fertilization, the nuclei of male and female gametes reach the 2C DNA level preceding fertilization (Carmichael and Friedman 1995) thus providing a peculiar example of gametes passing through the synthesis phase of the cell cycle before fertilization ensues. In figure 2 quantitative data descriptive of the DNA quantity of protoplasts isolated at various stages are presented. The data illustrate that karyogamy was in its incipient stage at 2-3 HAP and fusion of the male and female nuclei was completed by 180 min after pollination (Fig. 2B) . This observation is consistent with the findings of Faure et al. (1993) in a study made on the time course of karyogamy in in vi tro cultured zygotes produced by in vi tro fertilization (IVF) . DNA levels began to rise at 12 HAP and the 2C complement was doubled by 16 to 18 HAP (Fig. 2F,G) . In some cases both karyo- and cytokinesis was completed by 24 HAP (Fig. 21) whereas in the bulk of the cells analyzed the first division occurred by 36 HAP (data not shown) . The time course of karyogamy and of the first cell division appeared to indicate variation according to the stage of the egg cells used for fertilization (data not shown) . The reason for this is not known. We were particularly interested in establishing the onset of the synthesis (S) phase of the cell cycle, as information on the timing of DNA synthesis would be indispensable in conducting experiments with the goal of effectuating stable transformation through the microinjection of foreign DNA. According to the data presented in (Fig. 2F,G) progression into the 'S' phase occurred around 16 to 18 HAP. The 4C DNA complement was reached earlier than reported for either cultivar of barley observed by Mogensen and Holm (1995) . The results of the present study corrobo- rate those of Mogensen and Holm (1995) in that during the 15 to 18 HAP period there may have been some unfertilized egg cells (Fig. 2G) in the material containing mainly zygotes still resting in Gl or already completed 'S' phase. We did not implement injection targeted at the zygotic 'S' phase as we judged, based on our observations, that only a limited number of the zygotes would have been ideally synchronized, when microinjected, owing to the inevitable variability in the zygotic cell cycle stages. Additional studies appear to be needed to fathom the relationship between cell cycle stage, DNA levels and karyogamy in flowering plants.

By reconnoitering a novel approach in the immobiliza- tion of plant protoplasts we could expeditiously and re- producibly microinject egg cells/zygotes of wheat. The protoplasts withstood and recovered from the procedure as it was proved by FDA-test. As the zygotes at the time of isolation presented only patches of cell wall material on their surface, AC-field for immobilizing them could also be exploited.

By using reporter systems, the egg cells/zygotes of wheat were demonstrated to be competent target cells for genetic transformation. Upon injection with reporter genes (GUS, GFP) , both the egg cells and the zygotes gave a positive response in transgene expression assays, but the transformation frequency of the GFP gene under the control of the ubiquitin promoter was conspicuously higher. The percentage of transformation carried out preceding t.he commencement of the 'S' phase of the zygotic cycle was one order of magnitude higher than that reported by Leduc et al. (1996) . To establish why the transformation efficiency of either gene construct used was remarkably high further investigations are needed. It also remains to be investi- gated if the integration ratio is higher when linearized DNA is introduced into the female gamete/zygote (Yamaizumi et al. 1983) .

Notwithstanding the fact that Chapecci (1980) has shown that transformation efficiencies seem to be inde- pendent of the number of the DNA molecules injected per cell within the range of 100-400 molecules per nucleus the most expedient (i.e. that resulting in the highest transformation frequency) concentration range of DNA solution to be injected may have to be determined. As high frequency AC field was applied in this study, to the best of our knowledge, for the first time for immobilizing plant protoplasts, it would be intriguing to decipher how exposing cells to an alternating electric field can become a factor presumably responsible for the higher transformation rates achieved. This study raises the question of why egg cells isolated at a certain stage of their development appear to be more 'apt' for micromanipulation? Answering this question would require a detailed scrutiny at the ultramicroscopic level.

The data presented here as to the dynamics of nuclear DNA quantities during the in vivo zygotic development in wheat coupled with IVF (Kranz et al., 1991) may serve as a comparative basis for similar studies on zygotes of wheat produced and cultured in vitro (Kranz and Lδrz 1993) .

Although experiments with more batches of cells should be conducted before broader generalizations can be made, our system may facilitate research aimed at disentangling how the batteries of genes responsible for controlling the first asymmetric division of the zygote are regulated (Sauter et al., 1998) .

The technique described in this paper can offer at the applied level of research a means of delivering genes of interest into higher plants by exploiting the sexual route.

Perhaps the greatest potential of our system may be that it can shed light on how the very early events of the enigmatic program of zygotic embryogenesis, stretching from a single fertilized egg cell to a multicellular, en- tirely new organism, unfold. Table 1 Transient expression of isolated egg cells of wheat after microinjection of GFP reporter gene

Timing of miNo. of microin- No. of egg cells Frequency of croinjection* j ected egg cells showing transient ex- transient expression pression

3 20 11 55,00%

5 23 17 73,91%

6 17 10 58,82%

9 19 4 21,05%

12 10 2 20,00%

15 9 1 11,11% ^rDays after emasculation

Table 2 Transient expression of isolated zygotes of wheat after microinjection of GUS reporter gene

Timing of miNo. of microin- No. of zygotes Frequency of croinjection* j ected zygotes showing transient gene transient gene expres- expression sion (%)

3 24 13 54,17 6 19 11 57,89 9 25 12 48,00 1 9 4 44,44

*Hours after pollination

The immobilization technique of the invention described above is the only method hitherto, to the best of our knowledge, which is experimentally proven to be suit- able for efficient and reproducible immobilization for the female gametes of wheat.

As immobilization of the unfertilized/fertilized egg cells of higher plants is a prerequisite for delivering exogenous DNA into the target cells by microinjection, the technique may be applicable to a wide range of plant species belonging to higher plants.

Since the technique is suitable for injecting other type of materials having biological importance apart from DNA (e.g. proteins, lipids, fluorescent probes, mRNAs, hormones, ions... etc.) it can help in elucidating the regulation of the cell cycle at the molecular level. By employing the technique it also becomes possible to compare somatic and zygotic embryogenesis at the single cell level (egg cell versus somatic protoplast microinjec- tions) .

The method may also be exploitable when attempting to load female gametes fertilized either in vitro/in vivo with dyes used for ratio imaging /e.g. Fura-2 as a Ca⁺⁺ - indicator/ hence circumventing the problem related to poor deesterification of the AM-/acetoxymethil ester/.

Even cells, (e.g. sperm cells) or viroid particles may be considered eligible to be introduced into the cells via capitalizing on the method. As the recently developed technique referred to as "GENE CHIP-technology" is predicted to be revolutionizing molecular biology (sequencing, gene expression studies, hybridization...etc. ) , combining the immobilization method developed in our laboratory with this powerful technique new vistas can be envisaged as to disentangling exciting problems of early embryogenesis of higher plants. Thus, genetic disorders, gene patterns can be searched for by means of resorting to this technology coupled with microinjection made possible with our immobilization technique.

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Claims

CLAIMS :

1. A method for immobilizing cells, characterized in polarizing the surfaces of the said cells using a high frequency AC-field and then contacting the cells of polarized surface with the surface of an electrode under potential .

2. The method as claimed in claim 1, wherein said cells are eukaryotic cells.

3. The method as claimed in claim 2, wherein said cells are higher plant cells.

4. The method as claimed in claim 3, wherein said cells are gametes.

5. The method as claimed in claim 3 or 4, wherein said cells are wheat cells.

6. A process for microinjecting cells, characterized in injecting cells immobilized by the method of anyone of claims 1 to 5.

7. A device for immobilizing and optionally microin- jecting cells, which device comprises a microscope observable space compartment suitable for accommodating a liquid comprising the cells of interest, said compartment also comprises electrodes suitable for applying a high frequency AC-field and into which compartment a microinjector can be introduced if desired.