A light and electron microscope study of

Protoplasma (1987) 140:I00-109

PROTOPLASMA 9 by Springer-Verlag1987

A Light and Electron Microscope Study of Picea glauca (White Spruce) Somatic Embryos I. HAKMANI, P. RENNIE, and L. FOWKE* Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan Received December 27, 1986 Accepted June 10, 1987

Summary Somatic embryos in embryogenic callus cultures derived from immature zygotic embryos of Picea glauca (White spruce) were examined by light and electronmicroscopy.Somaticembryosconsist of an embryonicregion of small densely cytoplasmiccells subtended by a suspensor consisting of long highly vaeuolated cells. Mitotic figures are frequent in the embryoniccells but are not observedin the suspensor. Cell divisionsin the embryonicregion apparentlyproduce rows of cells which elongate to form the suspensor. The presence of abundant polysomes, coated membranes and dictyosomes in the cytoplasm of embryonic and upper suspensor cells suggests rapid growth of the embryo. In contrast the basipetal suspensor cells appear to be senescing. Whileonly a few scattered microfilamentsare present in the meristematic celIs, the upper suspensor cells contain numerous bundles of longitudinally oriented microfitaments. These bundles correspond to actin cables observed in light microscope preparations stained with rhodamine labelled phalloidin and are oriented parallel to the direction of active streaming in these cells.

Keywords: Actin; Conifer; Electron microscopy; Picea glauca; Somatic embryo; Tissue culture; Ultrastructure; White spruce.

1. Introduction Only recently has plant regeneration via somatic embryos been attained for conifers. The first species to be successfully cultured and regenerated was Picea abies from which embryogenic cultures were obtained by culturing immature embryos (HAKMAN etal. 1985, HAKMAN and VON ARNOLD 1985, BECWARetal. 1987). i Current address: Institute of Physiological Botany, University of Uppsala, S-751, 21 Uppsala, Sweden. * Correspondenceand Reprints: Department of Biology,University of Saskatchewan, Saskatoon, Saskatchewan STN 0W0, Canada.

Embryogenic cu!tures have since been obtained from cultured mature embryos (VON ARNOLD and HAKMAN 1986, KROGSTRUP 1986) and cotyledons (KRoGSTRUP 1986) of the same species and from immature embryos of Picea glauca and Picea mariana (HAKMAN and Fow~cE 1987a). Embryogenic calli have been used to establish suspension cultures of Picea abies (HAKMAN etal. 1985) and Picea glauca (HAKMAN and FOWKE 1987 b) that continuously produce somatic embryos. In Pinus both Pinus lambertiana and Pinus taeda have yielded somatic embryos (G-UPTA and DURZAN t986, 1987). Embryogenic cultures and plant regeneration have also been achieved with cultured female gametophytes of Larix decidua (NAGMANIand BONGA 1985). In all coniferous species reported so far the whitish translucent embryogenic callus is very characteristic and can easily be recognized and distinguished from non-embryogenic callus which greens in the light (e.g., HAKMAN etal. 1985, BECWARelM. 1987, HAKMANand Fow~:E 1987 a). Somatic embryos were only produced on the whitish translucent callus and never on the green callus. The general morphology of the developing somatic embryos and their growth into plantlets also seem to be quite similar for all these species. Coniferous somatic embryos are characterized by a massive suspensor consisting of elongate highly vacuolated cells. The suspensor subtends the embryonic region of small densely cytoplasmic ceils. Studies of both living isolated embryos and fixed sectioned material demonstrate that the morphology of somatic embryos closely resembles

][. HAKMANet al.: A Light and Electron Microscope Study of Picea glauca (White Spruce) Somatic Embryos t h a t o f z y g o t i c e m b r y o s (HAKMAN a n d YON ARNOLD 1985, KROtSTRUV 1986, BECWAa et al. 1987). T h e function o f the long suspensor cells a n d their role in the d e v e l o p m e n t a l process o f s o m a t i c e m b r y o s is n o t known. This study p r o v i d e s a detailed light a n d electron m i c r o s c o p e d e s c r i p t i o n o f Picea glauca s o m a t i c emb r y o s i s o l a t e d f r o m e m b r y o g e n i c callus. Since active c y t o p l a s m i c s t r e a m i n g was o b s e r v e d in the s u s p e n s o r cells, special a t t e n t i o n was directed t o w a r d actin m i c r o filaments which are believed to p l a y an i m p o r t a n t role in t h a t process (see reviews b y WILLIAMSON 1980, JACKSON 1982). T h e d i s t r i b u t i o n o f m i c r o f i l a m e n t s was e x a m i n e d u l t r a s t r u c t u r a l l y a n d with the light microscope using fluorescently labelled p h a l l o i d i n , a p h a l l o t o x i n with a high affinity for F - a c t i n (WuLF et al. 1979, BARAK etal. 1980).

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50 mM PIPES, 5 mM EGTA, 5 mM MgSO 4, pH 6.9 or in phosphate buffered saline. Triton X- 100 (0.1% w/v) was added to the fixative to reduce shrinkage of the long suspensor cells according to SEAGULL (personal communication). After fixation, somatic embryos were briefly washed in buffer. They were then stained for F-actin with 3.3gM rhodamine conjugated phalloidin (Rh-Ph) (Molecular Probes, Inc., Junction City, Ore.) in the same buffer at room temperature for 20 rain and then washed again with buffer 9After washing, the somatic embryos were spread on glass slides in mounting solution (JoHNsoN and DE C. NOGUEIRAARAUJO 1981), and were separated with fine forceps under a dissection microscope 9 As a control somatic embryos were preincubated in untabelled phalloidin (10-4 M; Sigma) before staining with Rh-Ph. Slides were examined using a Zeiss Universal II microscope equipped with phase contrast and epifluorescence illumination, and filter sets for detecting rhodamine fluorescence. A planapo x 40 lens N.A. 1.0 wasused, andphotographswererecordedonIlfordXP 1 35mmfilm, 400 ASA.

3. Results 2. Material and Methods 2.1. Culture Procedure Somatic embryos of Picea glauca (Moench) Voss. (White spruce) were produced from immature embryos as previously described (HAKMANand FOWKE1987 a). Embryogenic cultures were initiated and maintained on solid LP medium (VONARNOLDand EmKSSON 1981) containing 1% sucrose, 2,4-dichlorophenoxyacetic acid (10-5 M) and N6-benzyladenine (5 x 10--6M). The cultures were incubated at 25 ~ in darkness or in light (16-hour photoperiod) and were subcultured monthly to fresh medium.

2.2. Light and Electron Microscopy Somatic embryos for conventional light microscopy and transmission electron microscopy were prepared according to FOWKE (1984). Briefly, samples ofembryogenic callus were fixed sequentially with 1 and 3% glutaraldehyde (in 0.025M sodium phosphate buffer, pH 6.8) for 1 and 2 hours respectively, at room temperature. After a briefwash with the buffer they were postfixed in 1% OsO4in the same buffer overnight on ice. The samples were slowly dehydrated in ethanol to propylene oxide at 0 ~ infiltrated with resin at room temperature and finally flat embedded in araldite by baking for 48 hours at 60 ~ For light microscope observation the embryos were sectioned at approximately 1 gin, stained with toluidine blue (1% in 1% borax). For electron microscopy, ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 transmission electron microscope (Philips, Eindhoven, The Netherlands).

2.2. Phalloidin Staining of Fixed Somatic Embryos F-actin was localized in cells of whole somatic embryos basically by the method of PARTHASARATHYet al. (1985). The following fixation and staining procedures were carried out in test tubes. Somatic embryos were fixed for 20 minutes using 1.5% (w/v) formaldehyde (prepared freshly from paraformaldehyde) in a buffer containing

3.1. General 9E m b r y o s

Morphology

of

Developing

Somatic

E m b r y o g e n i c callus cultures o f white spruce c o n t a i n s o m a t i c e m b r y o s at different d e v e l o p m e n t a l stages (Figs. 1-4). T h e e m b r y o d e p i c t e d in Fig. 4 is representative o f the m o s t m a t u r e e m b r y o s in such cultures. T h e i n i t i a t i o n o f c o t y l e d o n s a n d further d e v e l o p m e n t to f o r m plantlets requires transfer o f e m b r y o g e n i c callus to a different culture regime (HAKMAN a n d FOWKE 1987a). U n l i k e m o s t a n g i o s p e r m s o m a t i c e m b r y o s , those o f conifers have an extensive s u s p e n s o r consisting o f very l o n g highly v a c u o l a t e d cells e x t e n d i n g f r o m the e m b r y o n i c region which is c o m p o s e d o f small meristematic cells. T h u s two distinct regions can be observed in all d e v e l o p i n g s o m a t i c e m b r y o s . The small cells o f the embryonic region are characterized by a high nucleus-cell v o l u m e ratio. T h e y have relatively dense c y t o p l a s m , thin cell walls a n d generally n u m e r o u s small vacuoles (Figs. 1-5). M i t o t i c activity is a p p a r e n t at all stages o f e m b r y o d e v e l o p m e n t (e.g., Figs. 1, 4, and 5). Cell divisions in the e m b r y o n i c region a p p a r e n t l y give rise to rows o f cells which eventually elongate to f o r m s u s p e n s o r cells (e.g., Fig. 3). The elongate cells o f the s u s p e n s o r are c h a r a c t e r i z e d b y e n o r m o u s vacuoles a n d a thin layer o f p e r i p h e r a l c y t o p l a s m . T h e i n d i v i d u a l cells are usually tightly p a c k e d with only small intercellular spaces (Fig. 6), however, b a s i p e t a l l y the s u s p e n s o r cells seem to be m o r e loosely associated. A l t h o u g h m i t o t i c figures were never o b s e r v e d in suspensors, tiny cells are o c c a s i o n a l l y f o u n d a m o n g the typical elongate s u s p e n s o r cells. These small cells m a y give rise to new s o m a t i c e m b r y o s

Fig. 1. Light micrograph of tiny somatic embryo. Note the mitotic figure (arrow) in the embryonic region. Bar = 40gin Fig. 2. Light micrograph of somatic embryo consisting of small embryonic region and elongate suspensor. Bar = 100 ~na Fig. 3. Light micrograph of somatic embryo. Files of cells (e.g., arrows) can be recognized in the suspensor. Same magnification as Fig. 2 Fig. 4. Light micrograph showing more mature somatic embryo consisting of an enlarged embryonic region and extensive suspensor. Note the mitotic figures (arrows). Same magnification as Fig. 2 Fig. 5. Light micrograph showing mitotic figures in the center of the embryonic region of a somatic embryo. Bar = 40 gm Fig. 6. Light micrograph showing a cross-section through the upper suspensor of a somatic embryo. Note the presence of small intercellular spaces (arrows). Same magnification as Fig. 2 Fig. 7. Light micrograph showing two somatic embryos sharing a common suspensor. Bar = i00 p_m

I. HAKMANet al.: A Light and Electron Microscope Study of Picea glauca (White Spruce) Somatic Embryos by continued cell division. Such a mechanism may explain the frequent occurrence of tiny embryos sharing a common suspensor with more mature embryos (Fig. 7). 3.2. Ultrastructure of Somatic Embryos

Figs. 8 and 9 illustrate the general fine structure of cells in the embryonic region and upper suspensor, respectively. The contrast in cell size, distribution of cytoplasm and vacuole size is striking. The general appearance of the cytoplasm of cells from both the embryonic region and upper suspensor suggests that they are very active metabolically (Fig. 10). The cytoplasm contains numerous ribosomes, many as polysomes. Dictyosomes with associated secretory vesicles are also frequently observed. However, more basipetal suspensor cells do not seem as active and in many cases appear to be senescing. The cell walls of the meristematic cells are generally thinner than those in the suspensor and contain numerous uniformly distributed plasmodesmata (Figs. 8 and 11). In suspensor cells plasmodesmata are generally restricted to transverse cell walls. 3.3. Mitochondria and Plastids

Numerous mitochondria and plastids are observed in both regions of the somatic embryos (Figs. 8, 9, and 12). The plastids in light grown and dark grown embryos are leucoplasts. This is not surprising since embryos do not green under either condition. The leucoplasts contain few internal lamellae and often a single large starch grain. 3.4. Mierobodies and Spherosomes

Cells of the embryonic region contain microbodies typical of other plant species (Fig. 13). They are bounded by a single membrane and exhibit a uniform electron dense matrix. In contrast, microbodies in many suspensor cells seem to contain much less, rather loosely distributed, matrix material (Fig. 17 b). All cells of the somatic embryos examined in this study contain numerous apparently randomly distributed spherosomes (Figs. 17 and 18). 3.5. Coated Membranes and Multivesicular Bodies

Numerous coated membranes and multivesicular bodies are present in both embryonic and upper suspensor cells. The coated membranes are particularly

103

abundant in the meristematic cells. Many coated pits are observed at the cell membrane (Fig. 13) as well as in association with the membrane of developing cell plates at telophase (Fig. 14). Coated vesicles, apparently free in the cytoplasm (Figs. 13 and 16), and partially coated reticulum are also frequently observed (Fig. 15). MuP tivesicular bodies, often bearing distinct plaques, are frequently observed in both regions of the somatic embryos (Fig. I0). 3.6. Cytoskeleton l. Microtubules: Numerous cortical microtubules are observed in embryonic and suspensor cells (Figs. 11, 13, and 16). In suspensor cells the cortical microtubules often appear to be transversely oriented. 2. Actin microfilaments: Suspensor cells exhibit active cytoplasmic streaming with the major flow occurring longitudinally in the thin layer of peripheral cytoplasm lining the elongate cells. Examination of somatic embryos with the electron microscope reveals microfilaments as single filaments or as bundles. In the suspensor cells the microfilaments are frequently present in bundles running roughly parallel to the longitudinal axis of the cell and perpendicular to cortical microtubules seen at the plasmalemma. Such bundles can be seen to run considerable distances within the cell (Fig. 17). Microfilaments are sometimes closely associated with other cell organelles such as plastids, mitochondria (Fig. 18) and often with microtubules. In cross sections of suspensor cells, bundles of microfilaments in section can be detected (Fig. t9). The meristematic cells characteristically contain a few scattered individual filaments, often near the plasma membrane. The abundance and general distribution of Rh-Ph labelled F-actin in cells of somatic embryos is illustrated in Figs. 20-22. It is very difficult to detect specifically stained structures in the embryonic region because of the intense fluorescence which obscures fine details. This staining is partly due to the compact nature of the tissue and possibly also to nonspecific background stain. However, a fine actin network can be detected in some peripheral cells of the embryonic region (Fig. 20) and in slightly elongated cells adjacent to the suspensor. In contrast, the suspensor cells contain brightly stained longitudinally oriented cables which extend the entire length of individual cells (Fig. 21). At the ends of cells the cables curve to follow the contours of the cell walls. Frequently the zone immediately surrounding the nucleus is brightly fluorescent (Fig. 21). Considerable

Fig. 8. Low power electron micrograpi~ of cells in the embryonic region of a somatic embryo. Note the cell size, dense cytoplasm and ~mmeroas plasmodesmata (arrows) in the cell walIs. N nucleus, Bar = 5 gm Fig. 9. Low power electron micrograph showing a small portion of two upper suspensor cells. These cells are characterized by a large central vacuole (lO and thin peripheral layer of cytoplasm. N nucleus, same magnification as Fig. 8 Fig. i0. Electron micrograph showing a grazing section of a cell from the upper portion of the suspensor. Note the dictyosomes (D), polysomes (circled) and multivesicular bodies (arrows) in the cytoplasm. Bar = 1 gm Fig. 11. Electron micrograph showing a grazing section of the cell wall joining a meristematic cell with a suspensor cell. Note the plasmodesmata (arrows) cut in cross section. Bar = 1 lam Fig. 12. Electron micrograph showing mitochondria and leucoplasts typical of the embryonic and upper suspensor ceils. The leucoplasts often contain one or more starch granules (arrows). Bar = 2 gm

I. HAKMANet al.: A Light and Electron Microscope Study of Picea glauca (White Spruce) Somatic Embryos

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Fig. 13. Electron micrograph of embryonic cell showing a microbody (34), coated pits (single arrows), coated vesicle (double arrow) and microtubules along the cell wall. Bar = 0.5 gm Fig. 14. Electron micrograph showing a cell plate in dividing embryonic cell. Note the coated pits (arrows) on the plate. Bar = 0.5 gm Fig. 15. Electron micrograph showing partially coated reticula (arrows) in a meristematic cell. Same magnification as Fig. 13 Fig. 16. Electron micrograph showing a grazing section of a longitudinal suspensor cell wall. Note the numerous microtubules (small single arrows), the bundle of microfilaments (double arrow) and the coated vesicles (large single arrows). C W cell wall. Bar = 0.5 gm b r a n c h i n g a n d a n a s t o m o s i n g o f cables results in a t h r e e - d i m e n s i o n a l n e t w o r k within the cell (Fig. 22). I n a d d i t i o n to the cables, a n e t w o r k o f fine fibrils is evident in the extreme p e r i p h e r y o f the c y t o p l a s m . It is interesting to n o t e t h a t the s u s p e n s o r cells farthest f r o m the e m b r y o n i c r e g i o n d o n o t stain with R h - P h .

In u p p e r s u s p e n s o r cells p r e t r e a t e d with u n l a b e l l e d p h a l l o i d i n as a control, actin cables d o n o t stain w h e n s u b s e q u e n t l y e x p o s e d to R h - P h . H o w e v e r , in meristematic cells t r e a t e d in a similar m a n n e r , a very low b a c k g r o u n d fluorescence remains.

Fig. 17. Low power electron micrograph showing longitudinal section of adjacent cells from upper suspensor. The areas o~tiined in black are shown at higher magnification in Figs. 17a, 17b, 17c. Bar = 0.5gm Fig. 17a. Enlargement from Fig. i7 showing bundles ofmicrofilaments (single arrows) in the cytoplasm. Note the spherosome (double arrow). Bar = 0.5 gm Fig. 17 b. Enlargement from Fig. i7 showing a microbody characteristic of suspensor cells. Same magnification as Fig. 17 a Fig. i7 c. Enlargement from Fig. 17 showing bundle of microfilaments (arrow). Same magnification as Fig. 17 a Fig. 18. Electron micrograph showing a bundle of micro filaments (arrow) attached to a mitochondrion. Bar = 0.5 gm Fig. 19. Electron micrograph showing transverse section of suspensor cell. Note the bundle of microfilaments cut in cross-section (arrow). Bar = 0.25 gm

I. HAKMANet al.: A Light and Electron Microscope Study of Picea glauca (White Spruce) Somatic Embryos

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Fig. 20. Fluorescence micrograph showing the general distribution of actin in the embryonic region of a somatic embryo stained with Rh-Ph. Note the fine actin network (arrows) in the peripheral cells. Bar = 40 lam Fig. 21. Fluorescencemicrograph of upper suspensorcells stained with Rh-Ph. Note the longitudinally oriented actin cables (singlearrows) and the actin associated with the nucleus (double arrow). Same magnification as Fig. 20 Fig. 22. Fluorescencemicrograph showing actin cables that are branching and anastomosing to form a network within the peripheral cytoplasm of upper suspensor cells stained with Rh-Ph. Bar = 40 gm

4. Discussion Embryogenic callus of white spruce is whitish to translucent in colour and resembles embryogenic callus from other conifers (e.g., N o r w a y spruce--HAKMAN et al. 1985, black spruce--HAKMAN and FOWKE 1987 a, sugar pine--GUPTA and DURZAN 1986, l a r c h - NA~MANI and BON~A 1985). The callus contains numerous somatic embryos at various stages of development; even the most mature embryos had not developed cotyledons. The general morphology of the developing embryos is similar to that reported for other somatic embryos and conifer zygotic embryos. Very little is known about the origin and growth of conifer somatic embryos in culture. However, there seem to be at least three different mechanisms which could account for their origin. In suspension cultures of white spruce, embryos apparently arise from single cells or small cell aggregates by an initial asymmetric division which delimits the embryonic and suspensor regions (HAKMAN and Fow~E 1987 b). Sustained division of the smaller cell produces the meristematic region of the embryo, which generates cells that elongate to form the suspensor. A similar mechanism likely operates in embryogenic callus as independent tiny embryos (Fig. 1) are present. Cells derived from the

embryonic region apparently expand to form the suspensor. The presence of distinct files of suspensor cells with plasmodesmata restricted to the end walls of adjacent ceils is consistent with this mode of development. Somatic embryos also seem to develop from a few small meristematic cells within the suspensor. Whether these initials arise by asymmetric divisions of vacuolated suspensor cells or whether they are meristematic cells from the embryonic region which have failed to elongate is not known. In any case, repeated divisions of these small cells result in the formation of new embryos. The tiny embryo sharing a suspensor with the older embryo in Fig. 7 likely formed in this manner. Finally, cultured somatic embryos of various ages apparently can split into multiple embryos by a mechanism similar to that found during early zygotic embryogenesis (cleavage polyembrony) in some conifers (e.g., P i n u s - - B u c H H o L z 1920). The initial separation seems to occur in the embryonic region. Cleavage polyembrony was reported previously for somatic embryos derived from female gametophytes of L a r i x decidua (NAGMANIand BONGA 1985). GUPTA and DURZAN (1986) use the term somatic polyembryogenesis to describe the origin o f adventive embryos from

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I. HAKMANet al.: A Light and Electron MicroscopeStudy of Picea glauca (White Spruce) SomaticEmbryos

zygotic suspensor cells. It is not clear whether the somatic embryos in their established callus cultures were also formed by cleavage. The embryogenic callus cultures grow relatively rapidly and have been maintained for over a year. Furthermore, plantlets can still be regenerated from such callus cultures (HAKMAN and FOWKE unpublished). The high frequency of mitotic figures in the meristematic region of sectioned embryos (Figs. 1, 4, and 5) indicates that individual embryos within the callus are growing rapidly. The appearance of the cytoplasm in the embryonic and upper suspensor cells seems to reflect a high level of metabolic activity (e.g., see Table2 in FOWKE 1986). The frequency of polysomes suggests active protein synthesis which would be necessary for rapid cell growth. Similarly, the frequency and apparent activity of dictyosomes would also be expected with active cell wall formation. Furthermore, these cells contain a striking number of coated membranes particularly in the embryonic region of the embryo. The distribution of coated pits and coated membranes is reminiscent of the high frequency of these organelles in rapidly growing suspension culture cells and protoplasts regenerating new cell walls (FowKE etal. 1983). Coated membranes have recently been implicated in the processes of protein sorting and transport in plants (GRIFFING and FOWKE 1985, GRIFFING etal. 1986) as well as in the process of endocytosis at the cell surface (JOACHIM and ROBINSON 1984, TANCHAK etal. 1984, HUBNERet ai. 1985, H~LLMERet al. 1986, TANCHAKand FOWKE 1987). Cationized ferritin, for example, is taken into plant protoplasts by coated pits and coated vesicles and is delivered to a number of organelles including the partially coated reticulum, dictyosomes, multivesicular bodies and the major lysosomal component of plant cells, the central vacuole (TANCHAKand FOWKE 1984, 1987). In contrast to the cells of the meristematic tip and upper suspensor region, the basipetal suspensor cells seem to be senescing. Active cytoplasmic streaming characteristic of the upper suspensor cells is totally absent. Actin microfilaments are not identified in these cells by RhPh staining (see below). Furthermore, ultrastructural analysis reveals signs of cytoplasmic deterioration (e.g., broken membranes, loosened cytoplasm, swollen organelles). The altered morphology of microbodies in the upper suspensor cells might represent an early stage in the senescence process. Finally, dead or dying cells appear to be sloughed from the base of the suspensor. Fluorescently labelled phallotoxins have recently been

used to demonstrate the presence of actin microfitaments in a variety of plant species and cell types (e.g., CLAYTONand LLOYD1985, PARTHASARATHYet al. 1985, PARTHASARATHY1985) including cells of conifer roots (PESECRETA et al. 1982, PESECRETAand PARTHASARTHY 1984). In the present study, actin microfilaments, often arranged in cables, are easily identified in the upper suspensor cells of somatic embryos by Rh-Ph staining as well as by electron microscopy. The correlation between the direction of active cytoplasmic streaming in these cells and the parallel arrangement of actin cables is not surprising in light of current ideas regarding the function of actin microfilaments in plants. Recent research indicates that actin microfilaments play a central role in the process of cytoplasmic streaming in plant cells (e.g., WILLIAMSON 1980, JACKSON 1 9 8 2 , SHEETZ and SVV,ICH 1983, PARTHASARATHY1985). Furthermore, actin microfilaments are believed to be responsible for movement of some cell organelles (WITZVM and PARTHASARATHY 1985, KACHAR1985). The close association ofmicrofilaments with organelles such as mitochondria (Fig. 18) and plastids, observed in this study, is certainly consistent with this hypothesis. The present study provides a detailed examination of somatic embryos from embryogenic callus cultures of white spruce. Under appropriate conditions these embryos will regenerate plantlets (HAKMAN and FOWKE 1987a). It will be interesting to study the structural changes involved in this transition.

Acknowledgements Financial support from the Program of Researchby Universitiesin Forestry and from the Natural Sciencesand Engineering Research Council of Canada is greatfullyacknowledged.

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