In VitroCell.Dev.Biol.--Plant32:59.-65.April-June1996 9 1996SocietyforIn VitroBiology 1054-5476/96 105.00+0.00

ACTIN DISTRIBUTION IN SOMATIC EMBRYOS AND EMBRYOGENIC PROTOPLASTS OF WHITE SPRUCE ( P I C E A G L A U C A ) P. BINAROVA,C. CIHALIKOVA,J. DOLEZEL,S. GILMER,ANDL. C. FOWKEt De Montfort University, Norman Borlaug Centrefor Plant Sciences, Institute of Experimental Botan); The Academy of Science of the Czech Republic, Sokolovska 6, 772 O00lomouc, Czech Republic (P. B., C. C., J. D.) and Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada (P. B., S. G., L. C. F.) (Received 24 August 1995; accepted 4 December 1995; editor T. A. Thorpe)

SUMMARY Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and muhinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in Gu and suspensor cells showed signs of DNA degradation. Key words: actin microfilaments; conifer; cytoskeleton; somatic embryogenesis. INTRODUCTION

suspensions yield two types of protoplasts which have different fates in culture: (1) small meristematic protoplasts derived from the embryonal regions which are embryogenic and readily give rise directly to somatic embryos (Attree et al., 1987), and (2) protoplasts from the large, vacuolated suspensor cells, transition cells between the embryonal region and suspensor or free-floating cells which do not regenerate to embryos. Protoplasts from the embryonal region fall within two classes, uninucleate protoplasts from single cells and larger multinucleate protoplasts resulting from spontaneous fusion during enzyme treatment (Fowke et al., 1990). This white spruce protoplast system provides the opportunity to identify cytoskeletal features characteristic of embryogenic cells. The aims of the present study are to (1) examine actin distribution in somatic embryos by improved staining techniques, (2) compare actin distribution in protoplasts capable of regenerating to embryos with those protoplasts which cannot, and (3) examine the nuclear DNA distribution in embryos and embryogenic protoplasts.

Cellular mechanisms controlling the formation of embryos from unorganized somatic cells cultured in vitro are being intensively studied; the same basic cellular mechanisms are likely used in both somatic and zygotic plant embryogenesis (see De Jong et al., 1993). Recently, analysis of Arabidopsis embryo mutants showed that as described for animal systems of embryogenesis, control of cell expansion and asymmetrical cell division are important mechanisms in early plant embryogenesis (Meyer et al., 1993). To elucidate these mechanisms, changes in the cytoskeleton during early embryogenesis have been studied in animals (reviewed by Strome, 1993) but few reports are available concerning cytoskeleton dynamics during plant zygotic and somatic embryogenesis (Gorst et al., 1986; Dijak and Simmonds, 1988; Webb and Gunning, 1991). Conifer somatic embryos provide an excellent experimental system for studying the relationship of the cytoskeleton to early events of embryo formation. Embryogenic suspension cultures of white spruce contain immature embryos at various stages of development as well as separate, large, vacuolated cells (Hakman and Fowke, 1987). The embryos consist of an embryonal region of small meristematic cells subtended by long, highly vacuolated suspensor cells. Embryogenic

MATERIALSAND METHODS Embryogenic suspensioncultures and protoplast cultures. White spruce embryogenic cell suspension cultures were established and maintained, and protoplasts were isolated from these suspension cultures according to the methods of Attree et al. (1989). Protoplastswerecultured in liquid protoplast culture medium (PCM) rather than being embodded in agarose to facilitate

i To whomcorrespondenceshould be addressed. 59

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easier collection and handling of samples for actin staining. Culture of soybean cells and isolation of protoplasts were according to previously published methods (Wang et ol., 1989).

Detection of actin microfilaments without aldehyde fixation (direct staining). The protocol for staining actin filaments was modified from that described by Jung and Wemicke (1991). Briefly, protoplasts were incubated directly in microtubule-stabilizingbuffer (MTSB: 50 mM piperazine-N,N'bis(2-ethanesulfonic acid), 1 mM MgS04, 2 mM ethylene glycol-bis(13-aminoethyl ether)-N, N, N', N'-tetraacetic acid, pH = 6.9) containing 0.1 p.M rhodamine-labeled phalloidin (Rh-ph), 0.1 mM m-maleimidobenzoic acid N-hydroxy-succinimide ester (MBS, Sigma Chemical Co., St. Louis, MO), 5% dimethyl sulfoxide (DMSO), 0.44 M mannitol, 0.01% Triton X-100, 3 mM dithiothreitol, 50 pM leupeptin, 5 mM phenylmethylsulfonyl fluoride, 5% methanol, 0.1% phenylenediamine, and 1 I~g Hoechst 33258/mL (to stain nuclei) in the dark for 30-60 min. Calcoflunr White (0.001%) was used to monitor cell wall formation by cultured protoplasts. Cells and small somatic embryos were treated similarly except the Triton X-100 concentration was increased to 0.05%. Stained cells or protoplasts were embedded in 1% low melting temperature agarose (Sea Plaque, FMC Corporation, Rockland, ME) and mounted on gelatin-covered slides as described in detail in Jung and Wernicke (1991). Localization of actin after fixation with formaldehyde. Cells, protoplasts, and embryos were fixed with 3.7% formalin, 0.02% glutaraldehyde, and 0.2% DMSO prepared in MTSB buffer for 1 h. After being washed in MTSB buffer, cells and protoplasts were attached to poly-L-lysine-coated slides. Mter permeabilization with 0.1% Triton X-100 in phosphate-buffered saline for 30 min, slides were incubated with 0.1 p.M Rh-ph (Amersham, Oakville, On. Canada) for 1 h in the dark. After 5 rain of staining with Hoechst, the specimens were mounted in FluorSave Reagent (Calbiochem-Behring, La Jolla, CA). Cytochalasin B treatment. Embryogenic cell suspension cultures were treated for 2 h with 5 p.M cytochalasin B (CB), the drug was removed by centrifugation, and samples were washed and then collected for actin staining at 3 and 6 h after drug removal. Fluorescence microscopy. Preparations were examined with the aid of a Zeiss Universal epifluorescence microscope equipped with a standard fluorescence filter set. Photographs were taken on Kodak Tmax 400 film. Microspectrophotometry. Somatic embryos from suspension cultures were collected and fixed in 3 : 1 fixative (ethanol : glacial acetic acid) for 1 h at room temperature. Freshly isolated protoplasts were fixed in the same manner except the fixative contained 0.2 M sorbitol. Feulgen staining was according to Dolezel (1989). The quantity of DNA was measured with a Leitz MPV-3 scanning microspectrophotometer interfaced to a computer with a Nucleiscan program (Dolezel, 1989). The DNA content was expressed in arbitrary units (A.U.). RESULTS Microfilaments in Fixed Embryogenic Suspension Cultures The suspension cuhures consisted of immature embryos (Fig. 1) at various stages of development as well as large, free-floating, vacuolated cells, often associated in clusters. The distribution of actin in the fixed embryos confirmed the earlier observations of Hakman et al. (1987). Briefly, the elongated transition cells between suspensor and embryonal region contained bundles of MFs, but very few MFs could be detected in the embryonal region which appeared uniformly bright (Fig. 2). Distinct longitudinally oriented cables of actin MFs were observed in suspeusor cells of somatic embryos (Fig. 3). Our study also showed that the vacuolated cells not associated with embryos contained a MF system consisting of cortical MFs (Fig. 4) and a few MF bundles connecting the nucleus to the cell surface. Microfilaments in Unfixed Embryogenic Suspension Cultures Unfixed suspensor cells (Fig. 5) contained a very similar distribution of stained MF cables to that illustrated for suspensor cells in

fixed material. In the elongated transition cells adjacent to the embryonal region, however, a more extensive actin network was observed in unfixed specimens (Fig. 6). The most dramatic difference between fixed and unfixed specimens was the occurrence of a very fine meshwork of randomly oriented cortical and subcortical biFs in the embryonal regions of unfixed embryos (Fig. 6). Deeper within the cytoplasm, the dense MF meshwork formed cages around the nuclei. The cortical MFs and actin enclosing nuclei remained during mitosis, but little or no actin staining was associated with the spindle (Fig. 7). Actin staining was associated with phragmoplasts; these MFs were resistant to CB treatment (see below). In the vacuolated cells in the culture, thick cortical MF bundles were observed (Fig. 8), and larger bundles were detected throughout the cytoplasm, often linking the nucleus to the cortex. Some cells showed MF cages enclosing nuclei. After CB treatment of embryos, the fine MF meshwork in the embryonal region disappeared and only short fragments and dots were observed, often attached to the cell surface or nuclear envelope (Fig. 9). The elongated transition cells joining the suspensor to the embryonal region contained only short bundles of mainly randomly arranged MFs after CB treatment (Fig. 9). Some actin cables were retained in the suspensor cells after CB treatment (Fig. 10), and the MFs associated with phragmoplasts in dividing cells appeared unaffected (Fig. 11). The treated free-floating vacuolated cells contained short, randomly arranged MF bundles (Fig. 12). During recovery from CB, a network of fine actin MFs was reconstituted in the embryonal regions of many embryos within 1-3 h (Fig. 13) but in some embryos, only dots or diffuse staining remained. During the same time period, the elongated transition cells adjacent to the embryonal region of embryos and the vacuolated cells in the culture had reestablished their normal MF distribution.

Microfilaments in Unfixed Protoplasts Embryogenic protoplasts isolated from the embryonal region of embryos differed in morphology and MF pattern from the nonembryogenic protoplasts derived from vacuolated cells. Muhinucleate embryogenic protoplasts contained a dense meshwork of fine, randomly oriented cortical MFs (Fig. 14). A similar meshwork was observed in the cytoplasm, forming cages around nuclei. Cell wall regeneration was observed by Calcofluor White staining after 24 h of culture, and the protoplasts gradually elongated (Fig. 15). Nuclei underwent division, usually synchronously, and the dense cortical and cytoplasmic meshwork of MFs and nuclear cages were retained (Fig. 15). Uninucleate protoplasts were of two types, small embryogenic protoplasts with small vacuoles derived from the embryonal region of embryos and much larger, highly vacuolated protoplasts derived from the suspensor and other vacuolated ceils in the cultures. The distribution of actin MFs in the uninucleate embryogenic protoplasts was the same as observed in the muhinucleate protoplasts. They contained a fine network of MFs in the cortex as well as deeper in the cytoplasm (Fig. 16). The highly vacuolated uninucleated protoplasts were characterized by pronounced bundles of actin in the cortical and subcortical regions; after cell elongation in culture, the extensive array of MF bundles was retained (Fig. 17). A similar actin pattern was found in protoplasts derived from nonmorphogenic suspension cultures of soybean (Fig. 18). Soybean protoplasts contained actin bundles rather

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FIG. 1. White spruce somatic embryo consisting of meristematic embryonal region (small single arrow), vacuolated transition cells (small double arrow) and elongate, highly vacuolated suspensor cells (large arrow). Bar = 100 ~tm. FIG. 2. Embryonalregion of somatic embryo fixed with formaldehyde and stained with Rh-ph. Most cells are brightly fluorescent and actin MFs are not detectable. Bar = 40 ~m. FIG.3. Suspensor region of somatic embryo fixed with formaldehydeand stained with Rh-ph showingactin MF cables (arrows). Bar = 40 p,m. FIe. 4. Free-floatingvacuolated cell fixed with formaldehydeand stained with Rh-ph showing MF bundles (arrows). Bar = 30 ~m. FIef. 5. Suspensor region of unfixed somatic embryo stained with Rh-ph showing actin MF cables (arrows). Bar = 50 pro. FIG. 6. Embryonal region (E) and transition cells (T) of unfixed somatic embryo. Note the very fine network of MFs in all cells of the embryonal region. Transition cells contain thicker bundles of MFs (arrow) as well as fine MFs. Bar = 40 ttm. FIt. 7(A) Metaphase cell in unfixed somatic embryo stained with Rh-ph to show actin distribution. MFs are located in the conical region of the cell but not in the spindle. Bar = 10 ~tm.(B) Same cell as in Fig. 7 A stained with Hoechst to show chromosomes.Same magnification as in Fig. 7 A. FIG. 8. Unfixedfree-floatingcell clump stained with Rh-ph showingextensive actin MF bundles. Bar = 20 ~tm.

than the very fine actin filameuts seen in embryogenic cells and protoplasts of white spruce. Feulgen microspectrophotometry showed that cells of the embryonal region were distributed in all phases of the cell cycle with DNA content ranging from 2C-4C (Fig. 19), typical of an actively dividing cell population. A similar DNA distribution was found in both uninucleate and multinucleate protoplasts immediately after isolation from the embryonal region (Fig. 20). Transition cells between the embryonal region and the suspensor showed a narrower distribution of DNA content corresponding to G~ and Go phases of the cell cycle (Fig. 19). Cells of the suspensor also showed a narrower distribution of DNA but exhibited higher variability and a decrease in DNA content below 2C.

DISCUSSION Gentle handling combined with the use of the actin cross-linking compound MBS in the Rh-ph staining solution, permitted the detection of a complex network of cortical MFs, subcortical MFs, and MF cages surrounding nuclei in the embryonal region of unfixed embryos. The retention of these MFs is likely due to the inclusion of MBS which is known to stabilize MFs (Sonobe and Shibaoka, 1989) and the absence of a primary aldehyde fixation which has been reported to depolymerize MFs (Lehrer, 1981). Previous investigators using aldehyde-fixed embryos without MBS did not detect this population of fine actin MFs in the embryonal region but revealed only MF cables in the suspensors, adjacent transition cells connecting to the embryonal region, and vacuolated cells free in suspension (Hakman

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Ftc. 9~ Somaticembryotreated with CB and then stained with Rh-ph showing tiny MF fragments and fluorescentdots (small arrows) in the embryonal region (E) and short bundles of randomlyarranged MFs (large arrows) in transition cells (T). Bar = 40 p.m. FIG. 10. Unfixedsomaticembryo treated with CB and then stained with Rh-ph to show few actin MF bundles (arrow) in suspensor cells. Bar = 40 p.m. FIG. 11. Two cells at telophase in unfixed somatic embryo treated with CB and stained with Rh-ph. Strong actin staining is evident in the phragmoplasts(arrows). Bar = 20 ttm. FIG. 12. Unfixedfree-floatingvacuolatedcell treated with CB and stained with Rh-ph to show many short randomly distributed MF bundles. Bar = 20 p.m. I"3G. 13. Unfixedsomatic embryo followingCB treatment and 3 h recovery,stained with Rh-ph and showing reappearance of a fine MF network in embryonalcells (cf. embryonalcells in Fig. 6). Bar = 40 tam.

et al., 1987). These results suggest that MFs in the vacuolated cells are more stable than those in the embryonal region. Stability of MFs in plants has been linked to their association with colocalized microtubules (Kengen and Derksen, 1991), but such a mechanism could not account for the stability of suspensor cell MFs of white spruce somatic emtJryos because the MFs are primarily longitudinal and separated from the plasma membrane, whereas microtubules are adjacent to the plasma membrane and transversely oriented (Fowke et al., 1990). The fine MFs which were sensitive to aldehyde fixation also showed a higher sensitivity to CB treatment. Sensitivity to aldehyde fixation and CB treatment might be a result of more rapid chemical diffusion through the embryonal cell walls which are thinner than those of the suspensor (Hakman et al., 1987). However, these differences in response to aldehydes and CB were also noted in the two different populations of wall-less protoplasts (data not shown), suggesting real differences in sensitivity of MF arrays. Similarly, only particular arrays of F-actin appeared resistant to CB treatment in pea roots (Hush and Overall, 1992) and in leaf segments of rye (Cho and Wick, 1991). The presence of tissue-specific actin isotypes in higher plants (McLean et al., 1990) may explain the classes of MFs differing in their sensitivity to fixation and CB treatment. Microfilaments associated with phragmoplasts in meristematic cells of the cmbryonal region or in embryogenic protoplasts also showed a lower sensitivity to CB treatment than the dense cortical and cytoplasmic MFs. Differences in actin-binding proteins might also contribute to differing sensitivities but very little is known about these proteins in plants (Sun et al., 1995). It would be useful to repeat the CB experiments with actin antibody rather than Rh-ph because in tobacco pollen tubes, CB failed to remove actin MFs which were subsequently detected by actin antibody but not by Rh-ph (Tang et al., 1989).

The current study has clearly distinguished two types of protoplasts in terms of their MF patterns. The embryogenic protoplasts, both uninucleate and multinucleate, have extensive networks of very fine MFs similar to those in the embryonal cells from which they were derived. This pattern of MFs characterizes protoplasts which are capable of direct embryogenesis in culture and which also contain an extensive microtubule network (Attree et al., 1987; Fowke et at., 1990). Microspectrophotometry showed that the distribution of DNA was also similar in the embryonal cells and embryogenic protoplasts and was characteristic of rapidly dividing nuclei. The direct formation of embryos from multinucleate conifer protoplasts is unique (Fowke et al., 1990; yon Aderkas, 1992). The process boars some similarity to the earliest stages of zygotic embryogenesis in conifers (Owens and Blake, 1985) where a free nuclear stage occurs and it is possible that similar mechanisms for nuclear organization and cell formation are operational here. Unfortunately, information is not available regarding microtubule and MF distribution during the free nuclear stage of gymnosperm embryogenesis for comparison. Very extensive networks of microtubules exist in other free-nuclear stages such as in endosperm (Webb and Gunning, 1991) and the coenocytic alga Ernodesmis verticillata (La Claire and Fulginiti, 1991). Nuclei in muhinucleate white spruce protoplasts remain separate and undergo synchronized division to yield somatic embryos (Fowke et al., 1990). The absence of DNA values exceeding 4C it, the muhinucleates in the current study (Fig. 20) confirms the lack of nuclear fusion. The distinct MF cages enclosing individual nuclei likely play a key role in preventing nuclear fusion which frequently occurs in nonembryogenic multinucleate protoplasts of a variety of species (Fowke, 1988). Similarly, in syncytial Drosophila embryos, intact microtubules and actin MFs are necessary to maintain uniform spacing of nuclei (Zalokar and Erk,

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FIe. 14. Freshly isolated unfixed multinucleate spruce protoplast stained with Rh-ph showing dense network of randomly distributed fine actin MFs similar to that observed in the embryonal region of the somatic embryo. Bar = 30 p.m. FIe. 15.(A) Surface view of multinucleate spruce pmtoplast cultured for 3 d and then stained with Rh-ph without fixation to show fine network of cortical MFs. Bar = 30 p.m. (B) Median optical section of same protoplast as in Fig. 15 A showing MF cages (arrows) enclosing individual nuclei. Nuclei were identified by Hoechst staining (not shown). Same magnification as in Fig. 15 A. FIG. 16. Median optical section of freshly isolated uninucleate spruce prutoplast stained with Rh-ph to show network of fine actin MFs. The single nucleus which stained with Hoechst (not shown) appears as a round area lacking actin MFs (arrow). Bar = 30 p.m. FiG. 17. Unfixeduninucleate spruce protoplast isolated from vacuolated free-floating cell, after elongation in culture and staining with Rh-ph to show actin MF bundles. The bright region represents the MFs associated with the nucleus (arrow). Bar = 40 pm. FIG. 18. Unfixedsoybean (SB-1) protoptasts stained with Rh-ph to show extensive network of actin MF bundles. Bar = 20 tim.

1976). Other organelles are known to be enclosed within a network of actin MFs (e.g., protein bodies in corn endosperm; Abe et al.,

1991). In contrast to the embryogenic protoplasts, protoplasts derived from suspensor cells and other vacuolated cells in the suspension culture contained a distinctly different MF pattern. Rather than a network of fine MFs, these protoplasts were characterized by thick MF cables similar to the MF distribution in their cells of origin. Although these protoplasts were capable of limited cell division, they did not give rise to embryos in culture. Actin MF cables, rather than fine MFs, seem to be typical of noumorphogenic cell and protoplast cultures. Soybean (SB-1) protoplasts, for example, contained predominately MF cables (Fig. 18). This rapidly dividing soybean cell line has been in culture for almost 30 years and has never given rise to organs or embryos (see Wang et al., 1989). With staining protocols similar to those used with white spruce, cereal protoplasts which are

not morphogenic nor capable of sustained cell division (Curler et al., 1991) were also characterized primarily by MF cables (Jung et al., 1993). A similar actin pattern is typical of highly vacuolated cells in which actin MFs are known to be involved in premitotic migration and positioning of the nucleus in the phragmosome and division plane alignment (Gunning, 1982; Lloyd, 1991). A different actin distribution is characteristic of meristematic cells which presumably do not require this mechanism for nuclear positioning (Wick, 1991; Liu and Palevitz, 1992). It is interesting that the transition and suspensor cells in somatic embryos characterized by actin bundles rather than fine MFs also exhibited a different distribution of DNA than the morphogenic ceils and protoplasts. Transition and suspensor cells were apparently arrested in G~ and suspensor ceils showed signs of DNA loss. Previous cytological studies of spruce suspensor cells revealed changes in the basipetal region of the suspensor suggestive of senescence (Hakman

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FIG. 20. Feulgen microspectrophotometricanalysis of DNA distribution in protoplasts derived from embryogenic suspension cell cultures. (A) Protoplasts collected from 12% Ficoll-mannitol interface (mixture of small uninucleate and large muhinucleate protoplasts) showing DNA values ranging from 2C-4C. (B) Protoplasts collected from 15%-12% Ficoll interface (small uninucleate protoplasts) showing a DNA distribution similar to that of the protoplasts in A. (A.U. = arbitrary units).

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FEULGEN-DNA CONTENT (A.U.) FIG. 19. Feulgen microspectrophotometricanalysis of nuclear DNA content in immature white spruce somatic embryos. (A) Embryonal region with DNA content ranging from 2C-4C. (B) Transition cells with nuclei characterized by a 2C value of DNA. (C) Suspensor cells with DNA values ranging below the 2C value. (A.U. = arbitrary units).

an extensive network of fine actin MFs which is sensitive to CB. The highly vacuolated suspensor cells do not divide, have nuclei with 2C DNA or less, and contain only actin MF cables, many being resistant to CB. The embryonal cells give rise to uninucleate and multinucleate embryogenic protoplasts which retain the network of fine MFs and are capable of regenerating to somatic embryos in culture. In contrast, suspensor cells yield nonembryogenic vacuolated protoplasts characterized by MF cables. ACKNOWLEDGMENTS

et al., 1987). DNA degradation may account for the inability of protoplasts derived from transition and suspensor cells to divide in culture. In contrast, suspensors in angiosperms often exhibit increased levels of DNA due to endoredupIication (Nag[, 1978).

Special thanks are given to Steve Attree for assistance with the conifer cultures and to Pat Rennie and Dennis Dyck for assistance with the preparation of the manuscript. We gratefullyacknowledgefinancial assistance from the Natural Sciences and Engineering Research Council of Canada in the form of a research grant to L. C. F. and an International Scientific Exchange Award to P. B.

CONCLUSIONS Embryonal cells of white spruce somatic embryos differ markedly from suspensor cells. Embryonal cells divide rapidly, are characterized by nuclei with a DNA content ranging from 2C--4C, and contain

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Kengen, H. M. P.; Derksen, J. Organization of microtubules and mierofilaments in protoplasts from suspension cells of Nicotiana plumbaginifolia: a quantitative analysis. Acta Bot. Neerl. 40:29-40; 1991. La Claire, J. W.; Fulginiti, R. Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kutzing) Bocrgesen. Planta 185:447-457; 1991. Lehrer, S. S. Damage to actin filaments by glutaraldehyde: protection by tropomyosin.J. Cell Biol. 90:459-466; 1981. Liu, B.; Palevitz, B. A. Organization of cortical microfilaments in dividing root ceils. Cell Motil. Cytoskeleton23:252-264; 1992. Lloyd, C. W. Cytoskeletalelements of the phragmosomeestablish the division plane in vacuolated higher plant cells. In: Lloyd, C. W., ed. The cytoskeletal basis of plant growth and form. New York: Academic Press; 1991:245-257. McLean, B. G.; Huang, S.; McKinney, E. C., et al. Plants contain highly divergent actin isovariants. Cell Motil. Cytoskeleton 17:276--290; 1990. Meyer, U.; Buttner, G.; Jurgens, G. Apical-basal pattern formation in the Arabidopsis embryo: studies on the role of the gnom gene. Development 117:149-162; 1993. Nagl, W. Endopolyploidyand polyteny in differentiation and evolution. Elsevier/North-HollandBiomedical Press, Amsterdam; 1978. Owens, J. N.; Blake, M. D. Forest tree seed production. Petawawa Nat. For. Inst. Inf. Rep. PI-X-53; 1985. Sonobe, S.; Shibaoka, H. Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoylN-hydroxysuccinimideester. Protoplasma 148:80-86; 1989. Strome, S. Determinationof cleavage planes. Cell 72:3--6; 1993. Sun, H.-Q.; Kwiatkowska, K.; Yin, H. L. Actin monomer binding proteins. Curt. Opin. Cell Biol. 7:102-110; 1995. Tang, X.; Lancelle, S. A.; Hepler, P. K. Fluorescence microscopiclocalization of actin in pollen tubes: comparison of actin antibody and phalloidin staining. Cell Motil. Cytoskeleton 12:216-224; 1989. Von Aderkas, P. Embryogenesisfrom protoplasts of haploid European larch. Can. J. For. Res. 22:397--402; 1992. Wang, H.; Cutler, A. J.; Fowke, L. C. High frequencies of preprophase bands in soybean protoplast cultures. J. Cell Sci. 92:575-580; 1989. Webb, M. C.; Gunning, B. E. S. The microtubular cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh. Planta 184:187-195; 1991. Wick, S. M. Spatial aspects of cytokinesis in plant cells. Curt. Opin. Cell Biol. 3:253-260; 1991. Zalokar, M.; Erk, I. Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J. Microsc. Biol. Cell 25:97-106; 1976.