Planta
Planta 135, 257-266 (1977)
9 by Springer-Verlag 1977
Fine Structure of Fusion Products from Soybean Cell Culture and Pea Leaf Protoplasts L.C. Fowke* Department of Biology, University of Saskatchewan, Sakatoon, Saskatchewan S7NOW0, Canada
F. Constabel and O.L. Gamborg Prairie Regional Laboratory, National Research Council, Saskatoon, Sakatehewan S7NOW9, Canada
Abstract. Protoplasts from pea (Pisum sativum L.)
leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contact was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide. Key words: Cell culture plasts fusion.
Glycine
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Pisum
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Proto-
Introduction
Protoplasts of various plant genera have been fused by using polyethylene glycol (PEG) and the resulting fusion products regenerated cell walls and divided (Kao etal., 1974; Kartha etal., 1974; Constabel et al., 1975, 1976). A prerequisite for tight agglutination and subsequent fusion is the complete absence of cell wall microfibrils (Weber et al., 1976; Williamson et al., 1977). When intergeneric fusion products are cultured there is evidence of nuclear fusion. Fusion of inter* Supported by National Research Council of Canada, Grant A6304 Abbreviations." PEG polyethylene glycol ; SEM scanning electron microscopy; TEM=transmission electron microscopy
phase nuclei has been illustrated by light microscopy using a differential staining procedure (Constabel etal., 1975; Dudits etal., 1976; Kao and Wetter, 1976). Division has occurred in fusion products of protoplasts from widely different plant genera and there was no direct evidence of incomparability in the cell progeny from the fusion products (Constabel et al., 1976; Kao and Wetter, 1976). Some aspects of protoplast fusion have been studied by electron microscopy. These include intraspecific (Withers and Cocking, 1972; Burgess and Flemming, 1974a; Wallin et al., 1974) and also intergeneric (Fowke et al., 1975b) protoplast fusions. In a recent report, we described some of the ultrastructural changes occurring in cultured sweet clover-soybean fusion products (Fowke et al., 1976). In the present study the process of plasma membrane adhesion is examined by both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ultrastructural studies also include observations on cultured fusion products and daughter cells from fused pea and soybean protoplasts. The observations provide a possible structural basis for nuclear fusion and illustrate the hybrid nature of nuclei and cells arising from the fusion products.
Materials and Methods 1. Plant Material
Pea plants (Pisum sativum L. cv. Century) were grown under greenhouse conditions with 18 h light of a minimum of 2000 lx at the level of the plants provided by daylight and additional fluorescent lamps. Before pea leaves were used for protoplast isolation, shoot cuttings were kept in the dark at room temperature for 30 h. Soybean (Glycine max (L.) Merr. cv. Mandarin) cells were grown in 50 ml l-B5 medium (Gamborg et al., 1968) and cultured in Delong flasks on gyratory shakers (150rpm) under continuous light of 800 lx at about 28 ~ C. Protoplasts were isolated from 2 and 3 day old subcultures.
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2. Protoplast Isolation Pea leaflets were surface sterilized by immersion in 70% ethanol for 90-120 s. They were then given two washes in 0.55 M sorbitol (5 min each) at pH 5.8. The lower epidermis was removed and about 0.5 g tissue incubated with 10 ml of enzyme solution in 100 x 15 mm petri dishes. The enzyme solution contained 0.5% pectinase, 1.0% Driselase (Kyowa Hakko Kogyo Co., Tokyo, Japan) and 0.55 M sorbitol, pH 6.8. The petri dishes were sealed with Parafilm | wrapped in aluminum foil and placed on a gyratory shaker (30 rpm) at room temperature for 4-6 h. The protoplasts were filtered through an 85 pm stainless steel sieve to remove debris and then centrifuged at 100 g for 3 min. The supernatant was discarded and the sediment was washed in 10 ml 0.55 M sorbitol solution, pH 5.8, centrifuged and resuspended in 1 ml of sorbitol solution. The leaf protoplasts were then mixed with those from cultured soybean cells at a ratio of 1:2. The isolation of protoplasts from soybean cell cultures has been described previously (Kao et al., 1970; Constabel, 1975).
3. Protoplast Aggregation and Fusion Droplets of 0.2 ml of the protoplast suspension were transferred to 60 x 15 mm plastic petri dishes or glass coverslips (for SEM). After the protoplasts had settled, three 0.1-0.2 ml drops of PEG solution were gently added. To prepare the PEG solution, 5g of PEG 1540 (Baker pure) were dissolved in 10 ml of distilled water containing 10 mM CaClz2H20, 0.7 m M KH2PO 4 and 0.1 M glucose (pH 5.8). After 10 to 15 rain the PEG was slowly diluted by dropwise addition o f rinse solution at intervals of several minutes. The rinse solution contained 0.3 M glucose, 0.2 M sorbitol, pH 9.0. About 15 rain after the dilution had begun, the petri dishes were flooded with a thin layer of the rinse solution. After a further 20 min the supernatant was discarded and the protoplasts adhering to the petri dishes were covered with a thin layer of culture medium. The medium contained mineral salts and vitamins after Gamborg et al. (1968), 20 mg/ml sucrose, 36 mg/ml sorbitol, 36 mg/ml mannitol, 0.25mg/ml CaH4(PO4)2-H20 , 0.3mg/ml L-glutamine, 1 mg/ml casein hydrolysate (N-Z-amine, Sheffield Chemicals, Norwich, N.Y.), 1 mg/ml ribose, 1 rag/1 2,4-D, 2 rag/1 kinetin, pH 5.8. The protoplasts were then cultured for 7 days at 26 ~ C with continuous low intensity light (200-300 lx). Samples for electron microscopy were fixed 1-2 rain after dilution of the PEG with rinse solution and after one week of culture.
4. Microscopy Samples for TEM and SEM were fixed and dehydrated together. The methods have been reported in detail (Fowke, 1975) and are only briefly outlined below. Samples were fixed in 1% glutaraldehyde for 1 h followed by 3% glutaraldehyde for 2-3 h at room temperature. The glutaraldehyde was prepared in culture medium or 0.55 M sorbitol (pH 5.8). The protoplasts or cells adhering to the culture dishes were removed by a stream of fixative from a fine bore hypodermic needle and fixed along with those floating in the culture medium. They were washed in 0.05 M sodium phosphate buffer (0 ~ C) for 2 h (with 3 changes) and then postfixed overnight in 1% osmium tetroxide in the same buffer (0 ~ C). After a brief wash they were slowly dehydrated in ethanol (0 ~ C).
a) Scanning Electron Microscopy (SEM) : The dehydrated proto plasts were brought to room temperature and transferred to n-amyl acetate by 25% increments. They were critical point dried, coated with gold and examined in a Cambridge scanning electron microscope.
L.C. Fowke et al. : Fine Structure of Fusion Products
b) Transmission Electron Microscopy (TEM) : The dehydrated samples were transferred gradually to propylene oxide (0 ~ (2) and infiltrated with Araldite at room temperature. They were embedded in a thin layer of Araldite in glass petri dishes previously coated with a release agent (MS 122, Miller-Stephenson Chemical Co. Ltd., 156 Front St., Toronto, Canada). Silver sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, mounted on coated grids and examined in a Philips 300 electron microscope.
c) Light Microscopy: Thick sections (0.5-1.0 gm) were cut on glass knives, mounted on slides, stained with toluidine blue and examined with a Zeiss light microscope.
Results
1. Agglutination Protoplasts fixed 1-2 rain after commencing dilution of the P E G were agglutinated over large areas of their surface (Figs. 1-3). Groups of protoplasts, usually containing both pea and soybean protoplasts, and single pea-soybean pairs (Fig. 2) were observed. The pea protoplast in Figure 2 is recognizable by the outlines of the chloroplasts against the inner surface of the plasma membrane. Some protoplasts lost their outer membranes or were completely disrupted when prepared for SEM. The plasma membrane has been lost from one protoplast in Figure 1 exposing the peripheral chloroplasts. Intact pea and soybean protoplasts appear to be completely free of cellulose microfibrils. TEM revealed that tight adhesion of the plasma membranes was discontinuous along the agglutinated surfaces (Fig. 3). Considerable variation was noted in the thickness of cytoplasm adjacent to adhering membranes.
2. Cultured Soybean and Pea Protoplasts During the culture period soybean protoplasts regenerated cell walls, divided and formed cell clusters. Thus the nucleus and cytoplasmic organelles, particularly the leucoplasts, in co-cultured soybean cells and fusion products could be directly compared. The soybean cells were characterized by numerous leucoplasts containing small spherica! starch grains and very few lamellae (Fig. 4). The nuclei were relatively isodiametric and contained very little heterochromatin. It was not possible to compare the structure of co-cultured pea cells because pea protoplasts deteriorated under the culture conditions. Organelles in the fusion products derived from the pea protoplasts (nucleus, chloroplasts) were recognized by comparison with those in freshly prepared pea protoplasts. The nuclei of pea protoplasts were smaller than those in
L.C. Fowke et al. : Fine Structure of Fusion Products
259
Fig. 1". Scanning electron micrograph showing agglutinated soybean (5) protoplasts. The pea (P) protoplast has lost its plasma m e m b r a n e revealing n u m e r o u s chloroplasts (arrows), x 1800 Fig. 2. Scanning electron micrograph showing agglutinated pea (P) and soybean (5) protoplasts. Note the outlines of the chloroplasts under the plasma m e m b r a n e of the pea protoplast, x 1800 Fig. 3. Electron micrograph showing agglutination between pea (P) and soybean (5) protoplasts. Areas of tight adhesion (arrows) are separated by gaps. • 7200
Abbreviations used on Figures."
C = chloroplast ; L = leucoplast ; M = mitochondrion ; N ~ nucleus ; N u = nucleolus ; P = pea; S = soybean ; V = vacuole. The figures in series (e.g. 5 and 5a) represent micrographs of the same fusion products
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L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 4. Electron micrograph of regenerated soybean cells containing numerous leucoplasts (arrows). Note the small spherical starch granules in the plastids, x 1800 Fig. 5. Light micrograph of fusion product containing a soybean nucleus (single arrow) and a pea nucleus (double arrow), x700. a Electron micrograph of the same fusion product as in Figure 5. The heterochromatic pea (P) nucleus is linked to the soybean (S) nucleus by a narrow nuclear envelope bridge (arrow). x 8300
s o y b e a n a n d were densely h e t e r o c h r o m a t i c . T h e clor o p l a s t s c o n t a i n e d n u m e r o u s g r a n a a n d l a c k e d starch grains (Fig. 3).
3. Cultured Fusion Products C u l t u r e d fusion p r o d u c t s were r e c o g n i z e d b y the coo c c u r r e n c e of green c h l o r o p l a s t s a n d s o y b e a n leuco-
plasts. F u s i o n p r o d u c t s r e g e n e r a t e d cell walls a n d div i d e d to f o r m aggregates of cells. D i v i s i o n figures were o b s e r v e d w i t h the light m i c r o s c o p e . S o m e single cell m u l t i n u c l e a t e fusion p r o d u c t s c o n t a i n i n g b o t h p e a a n d s o y b e a n nuclei survived the 7 d a y s in culture. T h e y r e g e n e r a t e d cell walls b u t were n o t observed to divide.
L.C. Fowke et al. : Fine Structure of Fusion Products
261
Fig. 6. Light micrograph of fusion product in which pea and soybean nuclei have recently fused. The pea heterochromatin (arrow) is clearly recognizable in the nucleus, x 700. a Electron micrograph of the same fusion product as in Figure 6. The chromatin components of the pea (P) and soybean (S) nuclei can be distinguished in the hybrid nucleus. Note the pea chloroplasts (C) and soybean leucoplasts (L) in the cytoplasm, x 5100 Fig. 7. Light micrograph of two cells derived from a fusion product. • 600. a Electron micrograph of the same two cells as in Figure 7. The hybrid cytoplasm contains both pea chloroplasts (C) and soybean leucoplasts (L). The large nucleus (N) is lobed and contains patches of peripheral heterochromatin (arrows). • 4200
a) Nuclear Fusion: N u c l e a r fusion was o b s e r v e d in s o m e o f the fusion p r o d u c t s (Figs. 5, 5a). T h e fusing nuclei were c o n n e c t e d b y n u c l e a r e n v e l o p e channels o r b r i d g e s (Fig. 5 a) w h i c h v a r i e d in size a n d number. T h e b r i d g e s e x p a n d e d to a l l o w i n t e g r a t i o n o f the nuclei. I m m e d i a t e l y f o l l o w i n g fusion the pea heterochrom a t i n was easily r e c o g n i z a b l e w i t h i n the s y n k a r y o n
(Figs. 6, 6a). F i g u r e 6 a also illustrates the h y b r i d n a t u r e o f the c y t o p l a s m which c o n t a i n s b o t h soybean l e u c o p l a s t s a n d p e a chlorplasts.
b) Division: A f t e r one week in culture m a n y fusion p r o d u c t s h a d d i v i d e d to f o r m clusters c o n t a i n i n g from 2 to a p p r o x i m a t e l y 12 cells. F i g u r e s 7 a n d 7 a illustrate
262
L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 8. Light micrograph of a cell cluster derived from a fusion product, x 500. a Electron micrograph of the same cells shown in Figure 8. Serial sectioning revealed that all cells contained at least one pea chloroplast (C) and many soybean leucoplasts (L). Note the pseudocrystalline inclusions (arrows). x 2900
two cells derived f r o m a fusion product. Figure 7 a shows the hybrid cytoplasm with leucoplasts and chloroplasts f r o m the parental protoplasts. Each cell also contained an enlarged lobed nucleus with distinct patches of h e t e r o c h r o m a t i n at its periphery (Fig. 7a). This distribution of h e t e r o c h r o m a t i n was c o m m o n in nuclei of fusion products and their daughter cells. A g r o u p o f cells derived f r o m a fusion product
is shown in the Figures 8 and 8a. A l t h o u g h some cells appear to lack chloroplasts, serial sectioning revealed that each cell contained at least one pea chloroplast in addition to n u m e r o u s soybean leucoplasts. The n u m b e r o f chloroplasts per cell was apparently reduced with each cell division while the leucoplast population remained unchanged. Some o f the chloroplasts were n o r m a l while others exhibited swollen in-
L.C. Fowke et al. : Fine Structure of Fusion Products
263
Fig. 9. Light micrograph of multinucleate fusion product containing two soybean nuclei (single arrows) and one pea nucleus (double arrow). Note the chloroplasts clustered around the lobed nuclei, x 900. a Electron micrograph of the same multinucleate fusion product as in Figure 9, showing portions of two soybean (S) nuclei and one pea (P) nucleus. The cytoplasm contains both pea chloroplasts (C) and soybean leucoplasts (L). Note the presence of small vacuoles in addition to the large vacuoles illustrated in Figure 9. x 3900 Fig. 10. Light micrograph of multinucleate fusion product. Numerous pea chloroplasts (arrows) are present but a pea nucleus is not evident in this section. Note the occurrence of many tiny vacuoles in the cytoplasm, x 900
t e r n a l m e m b r a n e s . The l e u c o p l a s t s were essentially identical to t h o s e in c u l t u r e d s o y b e a n cells (cf Fig. 4). A single nucleus c o n t a i n i n g p e r i p h e r a l p a t c h e s o f h e t e r o c h r o m a t i n was p r e s e n t in each cell. The cells were b o u n d e d by thin loosely o r g a n i z e d cell walls. A few p l a s m o d e s m a t a were o b s e r v e d in the crosswalls b e t w e e n a d j a c e n t cells. P s e u d o c r y s t a l l i n e inclusions were p r e s e n t in s o m e cells (Fig. 8a) a n d the c y t o p l a s m c o n t a i n e d m a n y small vacuoles.
c) Multinucleates. M u l t i n u c l e a t e fusion p r o d u c t s were t y p i c a l l y large, spherical cells c o n t a i n i n g c h l o r o plasts clustered a r o u n d the nuclei (Figs. 9, 10). The n u m b e r o f nuclei u s u a l l y v a r i e d between 2 a n d 6 with s o y b e a n nuclei o u t n u m b e r i n g p e a nuclei. M o s t nuclei were highly l o b e d (Fig. 9), a l t h o u g h s o m e ret a i n e d a r e g u l a r s h a p e (Fig. 10). Tiny vacuoles were present in the c y t o p l a s m s u r r o u n d i n g the nuclei b u t their n u m b e r v a r i e d c o n s i d e r a b l y (Fig. 9 cf. Fig. 10).
264
L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 11. Electron micrograph of chloroplasts in a cultured multinucleate fusion product. They contain long stacks of intergranal lamellae (single arrow) and enlarged grana (double arrow), x 12,500 Fig. 12. Electron micrograph showing chloroplast (C) and pseudocrystalline inclusion (arrow) in a daughter cell from a fusion product. The internal membranes of the chloroplast are swollen, x 10,100 Fig. 13. Electron micrograph showing chloroplasts in a daughter cell from a fusion product. The internal membranes are disorganized. Note the cytoplasmic invaginations (arrows) at the margins of the chloroplasts, x 8300 Fig. 14. Electron micrograph showing a degenerating chloroplast (C) in a cultured multinucleate fusion product, x 15,800
L.C. Fowkeet ai. : Fine Structure of Fusion Products
d) Plastids." The structure of the leucoplasts in all fusion products and cell progeny seemed to remain unchanged during the culture period. In some cells derived from fusion products the chloroplasts showed very little change while in others there were distinct changes in these organelles. The chloroplast membranes were modified and formed long parallel stacks of intergranaI lamellae and fewer and comparatively larger grana (Fig. 11). Many chloroplasts were characterized by swollen internal membranes (Fig. 12). The chloroplasts of one cell cluster were considerably enlarged and the grana and stroma lamellae were completely disorganized (Fig. 13). The plastids in Figure 13 also contained cytoplasmic invaginations which were frequently observed in chloroplasts of fusion products (e.g. Figs. 5a, 7a). Total disintegration of chloroplasts was only observed in one fusion product (Fig. 14) in this study.
Discussion
Pea and soybean protoplasts treated with PEG exhibited tight adhesion over a wide surface area. Weber et al. (1976) have reported that complete wall removal is necessary to ensure extended adhesion of protoplasts. The scanning electron micrographs indicated the complete absence of cell walls on the pea and soybean protoplasts. The pattern of intermittant membrane contact during PEG agglutination as revealed by TEM is similar to that reported for agglutinated protoplasts of pea and Vicia (Fowke et al., 1975b). Membranes were not tightly adhering in a continuous pattern as reported for PEG agglutinated carrot protoplasts (Wallin et al., 1974). Both types of membrane contact were observed when tobacco protoplasts were agglutinated with PEG (Burgess and Flemming, 1974a). The procedures used for SEM proved satisfactory and preserved the majority of the agglutinated protoplasts in excellent condition. When agglutination with PEG was performed on glass coverslips all protoplasts adhered firmly to the glass throughout the preparative procedures for SEM. When PEG is not used, protoplasts can be attached to glass surfaces with poly-Llysine (Mazia et al., 1975) for SEM or replica studies (Williamson et al., 1976, 1977). Light microscope studies have demonstrated that nuclear fusion in plant heterokaryons can occur at interphase as well as during mitosis (pea-soybean, Constable et al., 1975; barley-carrot, Dudits etal., 1976; tobacco-soybean, Kao and Wetter, 1976). The TEM results have confirmed these observations and shown that the fusion process involves the formation of nuclear envelope bridges linking the two nuclei. Nu-
265 clear fusion in multinucleate soybean protoplasts also involved bridges but these were more uniform in size (Fowke et al., 1975a). The endoplasmic reticulum has been implicated in the movement of nuclei prior to fusion in some plant cells (Triemer and Brown, 1975). There was no evidence that strands of endoplasmic reticulum were involved in the links between the nuclei in the pea-soybean fusion products. After nuclear fusion was completed the pea heterochromatin was detected within the synkaryon by light and electron microscopy. The characteristic heterochromatin persisted in daughter cells from the fusion products suggesting that the nuclei are hybrids. The heterochromatin of tobacco is similarly retained in daughter cells of tobacco-soybean fusion products (Kao and Wetter, 1976). The cytoplasm of all fusion products was apparently hybrid in nature as evidenced by the presence of both soybean leucoplasts and pea chloroplasts. The leucoplasts were seemingly not affected by the hybrid cytoplasm. Their structure and distribution in the daughter cells of fusion products was similar to that in actively growing soybean cells. Soybean leucoplasts also remained unaffected by the hybrid cytoplasm in fusion products of sweet clover and soybean (Fowke et al., 1976). In contrast, the number of pea chloroplasts per cell in dividing pea-soybean fnsion products was considerably reduced and many exhibited structural abnormalities indicating that the chloroplasts may have ceased dividing and in some cases were degenerating. Similar developments were observed in chloroplasts of sweet clover-soybean fusion products (Fowke et al., 1976). The behaviour may reflect a type of cytoplasmic incompatibility or natural changes in chloroplasts as a result of the change from non-dividing to dividing cells in culture. It is not surprising that very little cell wall material was detected at the surface of fusion products with the TEM. The apparent lack of wall material on cultured protoplasts has been reported previously and is likely due to limitations in technical procedures (see Burgess and Flemming, 1974b; Willison and Cocking, 1975; Fowke et al., 1976). Wall fibrils were easily detected in cross-walls between divided cells. Plasmodesmata were also present in these walls. The significance of the pseudocrystalline inclusions in some fusion products is unknown. Such inclusions were not observed in pea or soybean protoplasts prior to fusion but are apparently common in the cytoplasm and nuclei of some plants (Wergin et al., 1970). We wish to thank Miss P.J. Rennie and Mr. J.W. Kirkpatrick for excellenttechnicalassistance.
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References
Burgess, J., Flemming, E.N. : Ultrastructural studies of the aggregation and fusion of plant protoplasts. Planta 118, 183-193 (1974a) Burgess, J., Flemming, E.N.: Ultrastructural observations of cell wall regeneration around isolated tobacco protoplasts. J. Cell Sci. 14, 439449 (1974b) Constabel, F.: Protoplast isolation. In: Plant tissue culture methods. Gamborg, O.L., Wetter, L.R. (eds.). Ottawa: National Research Council of Canada 1975 Constabel, F., Dudits, D., Gamborg, O.L., Kao, K.N.: Nuclear fusion in intergeneric heterokaryons. Canad. J. Bot. 53, 2092-2095 (1975) Constabel, F., Weber, G., Kirkpatrick, J.W., Pahl, K. : Cell division of intergeneric protoplast fusion products. Z. Pflanzenphysiol. 79, 1-7 (1976) Dudits, D., Kao, K.N., Constabel, F., Gamborg, O.L.: Fusion of carrot and barley protoplasts and division of heterokaryocytes. Canad. J. Genet. Cytol. 18, 263-269 (1976) Fowke, L.C. : Electron microscopy of protoplasts. In: Plant tissue culture methods. Gamborg, O.L., Wetter, L.R. (eds.). Ottawa: National Research Council of Canada 1975 Fowke, L.C., Bech-Hansen, C.W., Gamborg, O.L., Constabel, F. : Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean. J. Cell Sci. 18, 491-507 (1975a) Fowke, L.C., Rennie, P.J., Kirkpatrick, J W., Constabel, F. : Ultrastructural characteristics of intergeneric protoplast fusion. Canad. J. Bot. 53, 27~278 (1975b) Fowke, L.C., Reunie, P.J., Kirkpatrick, J.W., Constabel, F. : Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts. Planta 130, 3945 (1976) Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968) Kao, K.N., Constabel, F., Michayluk, M.R., Gamborg, O.L.:
L.C. Fowke et al. : Fine Structure of Fusion Products Plant protoplast fusion and growth of intergeneric hybrid cells, Planta 130, 215-227 (1974) Kao, K.N., Keller, W.A., Miller, R.A.: Cell division in newly formed cells from protoplasts of soybean. Exp. Cell Res. 62, 338-340 (1970) Kao, K.N., Wetter, L.R.: Advances in techniques of plant protoplast fusion and culture of heterokaryocytes. Proc. 1st Int. Cell Biol. Conf. Boston (1976) Kartha, K., Gamborg, O.L., Constabel, F., Kao, K.N.: Fusion of rapeseed and soybean protoplasts and subsequent division of heterokaryocytes. Canad. J. Bot. 52, 2435-2436 (1974) Mazia, D., Schatten, G., Sale, W.: Adhesion of cells to surfaces coated with polylysine. J. Cell Biol. 66, 198 200 (1975) Triemer, R.E., Brown, R.M.: The ultrastructure of fertilization in Chlamydomonas moewusii. Protoplasma 84, 315-325 (1975) Wallin, A., Glimelius, K., Eriksson, T. : The induction of aggregation and fusion of Daucus carota protoplasts by polyethylene glycol. Z. Pflanzenphysiol. 74, 64-80 (1974) Weber, G., Constabel, F., Williamson, F., Fowke, L.C., Gamborg, O.L.: Effect of preincubation of protoplasts on PEG-induced fusion of plant cells, Z. Pflanzenphysiol. 79, 459464 (1976) Wergin, W.P., Gruber, P,J., Newcomb, E.H. : Fine structural investigation of nuclear inclusions in plants. J. Ultrastruct. Res. 30, 533-557 (1970) Williamson, F.A., Fowke, L.C., Constabel, F.C., Gamborg, O.L.: Labelling of concanavalin A sites on the plasma membrane of soybean protoplasts. Protoplasma 89, 305-316 (1976) Williamson, F.A., Fowke, L.C., Weber, G., Constabel, F., Gamborg, O. : Microfibril deposition on cultured protoplasts of Vicia hajastana. Protoplasma 91, 213-220 (1977) Willison, J.H.M., Cocking, E.C.: Microfibril synthesis at the surfaces of isolated tobacco mesophyll protoplasts, a freeze-etch study. Protoplasma 84, 147 159 (t975) Withers, L.A., Cocking, E.C.: Fine-structural studies on spontaneous and induced fusion of higher plant protoplasts. J. Cell Sci. 11, 5%75 (1972) Recieved 27 January; accepted 18 April 1977
Planta 135, 257-266 (1977)
9 by Springer-Verlag 1977
Fine Structure of Fusion Products from Soybean Cell Culture and Pea Leaf Protoplasts L.C. Fowke* Department of Biology, University of Saskatchewan, Sakatoon, Saskatchewan S7NOW0, Canada
F. Constabel and O.L. Gamborg Prairie Regional Laboratory, National Research Council, Saskatoon, Sakatehewan S7NOW9, Canada
Abstract. Protoplasts from pea (Pisum sativum L.)
leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contact was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide. Key words: Cell culture plasts fusion.
Glycine
-
Pisum
-
Proto-
Introduction
Protoplasts of various plant genera have been fused by using polyethylene glycol (PEG) and the resulting fusion products regenerated cell walls and divided (Kao etal., 1974; Kartha etal., 1974; Constabel et al., 1975, 1976). A prerequisite for tight agglutination and subsequent fusion is the complete absence of cell wall microfibrils (Weber et al., 1976; Williamson et al., 1977). When intergeneric fusion products are cultured there is evidence of nuclear fusion. Fusion of inter* Supported by National Research Council of Canada, Grant A6304 Abbreviations." PEG polyethylene glycol ; SEM scanning electron microscopy; TEM=transmission electron microscopy
phase nuclei has been illustrated by light microscopy using a differential staining procedure (Constabel etal., 1975; Dudits etal., 1976; Kao and Wetter, 1976). Division has occurred in fusion products of protoplasts from widely different plant genera and there was no direct evidence of incomparability in the cell progeny from the fusion products (Constabel et al., 1976; Kao and Wetter, 1976). Some aspects of protoplast fusion have been studied by electron microscopy. These include intraspecific (Withers and Cocking, 1972; Burgess and Flemming, 1974a; Wallin et al., 1974) and also intergeneric (Fowke et al., 1975b) protoplast fusions. In a recent report, we described some of the ultrastructural changes occurring in cultured sweet clover-soybean fusion products (Fowke et al., 1976). In the present study the process of plasma membrane adhesion is examined by both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ultrastructural studies also include observations on cultured fusion products and daughter cells from fused pea and soybean protoplasts. The observations provide a possible structural basis for nuclear fusion and illustrate the hybrid nature of nuclei and cells arising from the fusion products.
Materials and Methods 1. Plant Material
Pea plants (Pisum sativum L. cv. Century) were grown under greenhouse conditions with 18 h light of a minimum of 2000 lx at the level of the plants provided by daylight and additional fluorescent lamps. Before pea leaves were used for protoplast isolation, shoot cuttings were kept in the dark at room temperature for 30 h. Soybean (Glycine max (L.) Merr. cv. Mandarin) cells were grown in 50 ml l-B5 medium (Gamborg et al., 1968) and cultured in Delong flasks on gyratory shakers (150rpm) under continuous light of 800 lx at about 28 ~ C. Protoplasts were isolated from 2 and 3 day old subcultures.
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2. Protoplast Isolation Pea leaflets were surface sterilized by immersion in 70% ethanol for 90-120 s. They were then given two washes in 0.55 M sorbitol (5 min each) at pH 5.8. The lower epidermis was removed and about 0.5 g tissue incubated with 10 ml of enzyme solution in 100 x 15 mm petri dishes. The enzyme solution contained 0.5% pectinase, 1.0% Driselase (Kyowa Hakko Kogyo Co., Tokyo, Japan) and 0.55 M sorbitol, pH 6.8. The petri dishes were sealed with Parafilm | wrapped in aluminum foil and placed on a gyratory shaker (30 rpm) at room temperature for 4-6 h. The protoplasts were filtered through an 85 pm stainless steel sieve to remove debris and then centrifuged at 100 g for 3 min. The supernatant was discarded and the sediment was washed in 10 ml 0.55 M sorbitol solution, pH 5.8, centrifuged and resuspended in 1 ml of sorbitol solution. The leaf protoplasts were then mixed with those from cultured soybean cells at a ratio of 1:2. The isolation of protoplasts from soybean cell cultures has been described previously (Kao et al., 1970; Constabel, 1975).
3. Protoplast Aggregation and Fusion Droplets of 0.2 ml of the protoplast suspension were transferred to 60 x 15 mm plastic petri dishes or glass coverslips (for SEM). After the protoplasts had settled, three 0.1-0.2 ml drops of PEG solution were gently added. To prepare the PEG solution, 5g of PEG 1540 (Baker pure) were dissolved in 10 ml of distilled water containing 10 mM CaClz2H20, 0.7 m M KH2PO 4 and 0.1 M glucose (pH 5.8). After 10 to 15 rain the PEG was slowly diluted by dropwise addition o f rinse solution at intervals of several minutes. The rinse solution contained 0.3 M glucose, 0.2 M sorbitol, pH 9.0. About 15 rain after the dilution had begun, the petri dishes were flooded with a thin layer of the rinse solution. After a further 20 min the supernatant was discarded and the protoplasts adhering to the petri dishes were covered with a thin layer of culture medium. The medium contained mineral salts and vitamins after Gamborg et al. (1968), 20 mg/ml sucrose, 36 mg/ml sorbitol, 36 mg/ml mannitol, 0.25mg/ml CaH4(PO4)2-H20 , 0.3mg/ml L-glutamine, 1 mg/ml casein hydrolysate (N-Z-amine, Sheffield Chemicals, Norwich, N.Y.), 1 mg/ml ribose, 1 rag/1 2,4-D, 2 rag/1 kinetin, pH 5.8. The protoplasts were then cultured for 7 days at 26 ~ C with continuous low intensity light (200-300 lx). Samples for electron microscopy were fixed 1-2 rain after dilution of the PEG with rinse solution and after one week of culture.
4. Microscopy Samples for TEM and SEM were fixed and dehydrated together. The methods have been reported in detail (Fowke, 1975) and are only briefly outlined below. Samples were fixed in 1% glutaraldehyde for 1 h followed by 3% glutaraldehyde for 2-3 h at room temperature. The glutaraldehyde was prepared in culture medium or 0.55 M sorbitol (pH 5.8). The protoplasts or cells adhering to the culture dishes were removed by a stream of fixative from a fine bore hypodermic needle and fixed along with those floating in the culture medium. They were washed in 0.05 M sodium phosphate buffer (0 ~ C) for 2 h (with 3 changes) and then postfixed overnight in 1% osmium tetroxide in the same buffer (0 ~ C). After a brief wash they were slowly dehydrated in ethanol (0 ~ C).
a) Scanning Electron Microscopy (SEM) : The dehydrated proto plasts were brought to room temperature and transferred to n-amyl acetate by 25% increments. They were critical point dried, coated with gold and examined in a Cambridge scanning electron microscope.
L.C. Fowke et al. : Fine Structure of Fusion Products
b) Transmission Electron Microscopy (TEM) : The dehydrated samples were transferred gradually to propylene oxide (0 ~ (2) and infiltrated with Araldite at room temperature. They were embedded in a thin layer of Araldite in glass petri dishes previously coated with a release agent (MS 122, Miller-Stephenson Chemical Co. Ltd., 156 Front St., Toronto, Canada). Silver sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, mounted on coated grids and examined in a Philips 300 electron microscope.
c) Light Microscopy: Thick sections (0.5-1.0 gm) were cut on glass knives, mounted on slides, stained with toluidine blue and examined with a Zeiss light microscope.
Results
1. Agglutination Protoplasts fixed 1-2 rain after commencing dilution of the P E G were agglutinated over large areas of their surface (Figs. 1-3). Groups of protoplasts, usually containing both pea and soybean protoplasts, and single pea-soybean pairs (Fig. 2) were observed. The pea protoplast in Figure 2 is recognizable by the outlines of the chloroplasts against the inner surface of the plasma membrane. Some protoplasts lost their outer membranes or were completely disrupted when prepared for SEM. The plasma membrane has been lost from one protoplast in Figure 1 exposing the peripheral chloroplasts. Intact pea and soybean protoplasts appear to be completely free of cellulose microfibrils. TEM revealed that tight adhesion of the plasma membranes was discontinuous along the agglutinated surfaces (Fig. 3). Considerable variation was noted in the thickness of cytoplasm adjacent to adhering membranes.
2. Cultured Soybean and Pea Protoplasts During the culture period soybean protoplasts regenerated cell walls, divided and formed cell clusters. Thus the nucleus and cytoplasmic organelles, particularly the leucoplasts, in co-cultured soybean cells and fusion products could be directly compared. The soybean cells were characterized by numerous leucoplasts containing small spherica! starch grains and very few lamellae (Fig. 4). The nuclei were relatively isodiametric and contained very little heterochromatin. It was not possible to compare the structure of co-cultured pea cells because pea protoplasts deteriorated under the culture conditions. Organelles in the fusion products derived from the pea protoplasts (nucleus, chloroplasts) were recognized by comparison with those in freshly prepared pea protoplasts. The nuclei of pea protoplasts were smaller than those in
L.C. Fowke et al. : Fine Structure of Fusion Products
259
Fig. 1". Scanning electron micrograph showing agglutinated soybean (5) protoplasts. The pea (P) protoplast has lost its plasma m e m b r a n e revealing n u m e r o u s chloroplasts (arrows), x 1800 Fig. 2. Scanning electron micrograph showing agglutinated pea (P) and soybean (5) protoplasts. Note the outlines of the chloroplasts under the plasma m e m b r a n e of the pea protoplast, x 1800 Fig. 3. Electron micrograph showing agglutination between pea (P) and soybean (5) protoplasts. Areas of tight adhesion (arrows) are separated by gaps. • 7200
Abbreviations used on Figures."
C = chloroplast ; L = leucoplast ; M = mitochondrion ; N ~ nucleus ; N u = nucleolus ; P = pea; S = soybean ; V = vacuole. The figures in series (e.g. 5 and 5a) represent micrographs of the same fusion products
260
L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 4. Electron micrograph of regenerated soybean cells containing numerous leucoplasts (arrows). Note the small spherical starch granules in the plastids, x 1800 Fig. 5. Light micrograph of fusion product containing a soybean nucleus (single arrow) and a pea nucleus (double arrow), x700. a Electron micrograph of the same fusion product as in Figure 5. The heterochromatic pea (P) nucleus is linked to the soybean (S) nucleus by a narrow nuclear envelope bridge (arrow). x 8300
s o y b e a n a n d were densely h e t e r o c h r o m a t i c . T h e clor o p l a s t s c o n t a i n e d n u m e r o u s g r a n a a n d l a c k e d starch grains (Fig. 3).
3. Cultured Fusion Products C u l t u r e d fusion p r o d u c t s were r e c o g n i z e d b y the coo c c u r r e n c e of green c h l o r o p l a s t s a n d s o y b e a n leuco-
plasts. F u s i o n p r o d u c t s r e g e n e r a t e d cell walls a n d div i d e d to f o r m aggregates of cells. D i v i s i o n figures were o b s e r v e d w i t h the light m i c r o s c o p e . S o m e single cell m u l t i n u c l e a t e fusion p r o d u c t s c o n t a i n i n g b o t h p e a a n d s o y b e a n nuclei survived the 7 d a y s in culture. T h e y r e g e n e r a t e d cell walls b u t were n o t observed to divide.
L.C. Fowke et al. : Fine Structure of Fusion Products
261
Fig. 6. Light micrograph of fusion product in which pea and soybean nuclei have recently fused. The pea heterochromatin (arrow) is clearly recognizable in the nucleus, x 700. a Electron micrograph of the same fusion product as in Figure 6. The chromatin components of the pea (P) and soybean (S) nuclei can be distinguished in the hybrid nucleus. Note the pea chloroplasts (C) and soybean leucoplasts (L) in the cytoplasm, x 5100 Fig. 7. Light micrograph of two cells derived from a fusion product. • 600. a Electron micrograph of the same two cells as in Figure 7. The hybrid cytoplasm contains both pea chloroplasts (C) and soybean leucoplasts (L). The large nucleus (N) is lobed and contains patches of peripheral heterochromatin (arrows). • 4200
a) Nuclear Fusion: N u c l e a r fusion was o b s e r v e d in s o m e o f the fusion p r o d u c t s (Figs. 5, 5a). T h e fusing nuclei were c o n n e c t e d b y n u c l e a r e n v e l o p e channels o r b r i d g e s (Fig. 5 a) w h i c h v a r i e d in size a n d number. T h e b r i d g e s e x p a n d e d to a l l o w i n t e g r a t i o n o f the nuclei. I m m e d i a t e l y f o l l o w i n g fusion the pea heterochrom a t i n was easily r e c o g n i z a b l e w i t h i n the s y n k a r y o n
(Figs. 6, 6a). F i g u r e 6 a also illustrates the h y b r i d n a t u r e o f the c y t o p l a s m which c o n t a i n s b o t h soybean l e u c o p l a s t s a n d p e a chlorplasts.
b) Division: A f t e r one week in culture m a n y fusion p r o d u c t s h a d d i v i d e d to f o r m clusters c o n t a i n i n g from 2 to a p p r o x i m a t e l y 12 cells. F i g u r e s 7 a n d 7 a illustrate
262
L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 8. Light micrograph of a cell cluster derived from a fusion product, x 500. a Electron micrograph of the same cells shown in Figure 8. Serial sectioning revealed that all cells contained at least one pea chloroplast (C) and many soybean leucoplasts (L). Note the pseudocrystalline inclusions (arrows). x 2900
two cells derived f r o m a fusion product. Figure 7 a shows the hybrid cytoplasm with leucoplasts and chloroplasts f r o m the parental protoplasts. Each cell also contained an enlarged lobed nucleus with distinct patches of h e t e r o c h r o m a t i n at its periphery (Fig. 7a). This distribution of h e t e r o c h r o m a t i n was c o m m o n in nuclei of fusion products and their daughter cells. A g r o u p o f cells derived f r o m a fusion product
is shown in the Figures 8 and 8a. A l t h o u g h some cells appear to lack chloroplasts, serial sectioning revealed that each cell contained at least one pea chloroplast in addition to n u m e r o u s soybean leucoplasts. The n u m b e r o f chloroplasts per cell was apparently reduced with each cell division while the leucoplast population remained unchanged. Some o f the chloroplasts were n o r m a l while others exhibited swollen in-
L.C. Fowke et al. : Fine Structure of Fusion Products
263
Fig. 9. Light micrograph of multinucleate fusion product containing two soybean nuclei (single arrows) and one pea nucleus (double arrow). Note the chloroplasts clustered around the lobed nuclei, x 900. a Electron micrograph of the same multinucleate fusion product as in Figure 9, showing portions of two soybean (S) nuclei and one pea (P) nucleus. The cytoplasm contains both pea chloroplasts (C) and soybean leucoplasts (L). Note the presence of small vacuoles in addition to the large vacuoles illustrated in Figure 9. x 3900 Fig. 10. Light micrograph of multinucleate fusion product. Numerous pea chloroplasts (arrows) are present but a pea nucleus is not evident in this section. Note the occurrence of many tiny vacuoles in the cytoplasm, x 900
t e r n a l m e m b r a n e s . The l e u c o p l a s t s were essentially identical to t h o s e in c u l t u r e d s o y b e a n cells (cf Fig. 4). A single nucleus c o n t a i n i n g p e r i p h e r a l p a t c h e s o f h e t e r o c h r o m a t i n was p r e s e n t in each cell. The cells were b o u n d e d by thin loosely o r g a n i z e d cell walls. A few p l a s m o d e s m a t a were o b s e r v e d in the crosswalls b e t w e e n a d j a c e n t cells. P s e u d o c r y s t a l l i n e inclusions were p r e s e n t in s o m e cells (Fig. 8a) a n d the c y t o p l a s m c o n t a i n e d m a n y small vacuoles.
c) Multinucleates. M u l t i n u c l e a t e fusion p r o d u c t s were t y p i c a l l y large, spherical cells c o n t a i n i n g c h l o r o plasts clustered a r o u n d the nuclei (Figs. 9, 10). The n u m b e r o f nuclei u s u a l l y v a r i e d between 2 a n d 6 with s o y b e a n nuclei o u t n u m b e r i n g p e a nuclei. M o s t nuclei were highly l o b e d (Fig. 9), a l t h o u g h s o m e ret a i n e d a r e g u l a r s h a p e (Fig. 10). Tiny vacuoles were present in the c y t o p l a s m s u r r o u n d i n g the nuclei b u t their n u m b e r v a r i e d c o n s i d e r a b l y (Fig. 9 cf. Fig. 10).
264
L.C. Fowke et al. : Fine Structure of Fusion Products
Fig. 11. Electron micrograph of chloroplasts in a cultured multinucleate fusion product. They contain long stacks of intergranal lamellae (single arrow) and enlarged grana (double arrow), x 12,500 Fig. 12. Electron micrograph showing chloroplast (C) and pseudocrystalline inclusion (arrow) in a daughter cell from a fusion product. The internal membranes of the chloroplast are swollen, x 10,100 Fig. 13. Electron micrograph showing chloroplasts in a daughter cell from a fusion product. The internal membranes are disorganized. Note the cytoplasmic invaginations (arrows) at the margins of the chloroplasts, x 8300 Fig. 14. Electron micrograph showing a degenerating chloroplast (C) in a cultured multinucleate fusion product, x 15,800
L.C. Fowkeet ai. : Fine Structure of Fusion Products
d) Plastids." The structure of the leucoplasts in all fusion products and cell progeny seemed to remain unchanged during the culture period. In some cells derived from fusion products the chloroplasts showed very little change while in others there were distinct changes in these organelles. The chloroplast membranes were modified and formed long parallel stacks of intergranaI lamellae and fewer and comparatively larger grana (Fig. 11). Many chloroplasts were characterized by swollen internal membranes (Fig. 12). The chloroplasts of one cell cluster were considerably enlarged and the grana and stroma lamellae were completely disorganized (Fig. 13). The plastids in Figure 13 also contained cytoplasmic invaginations which were frequently observed in chloroplasts of fusion products (e.g. Figs. 5a, 7a). Total disintegration of chloroplasts was only observed in one fusion product (Fig. 14) in this study.
Discussion
Pea and soybean protoplasts treated with PEG exhibited tight adhesion over a wide surface area. Weber et al. (1976) have reported that complete wall removal is necessary to ensure extended adhesion of protoplasts. The scanning electron micrographs indicated the complete absence of cell walls on the pea and soybean protoplasts. The pattern of intermittant membrane contact during PEG agglutination as revealed by TEM is similar to that reported for agglutinated protoplasts of pea and Vicia (Fowke et al., 1975b). Membranes were not tightly adhering in a continuous pattern as reported for PEG agglutinated carrot protoplasts (Wallin et al., 1974). Both types of membrane contact were observed when tobacco protoplasts were agglutinated with PEG (Burgess and Flemming, 1974a). The procedures used for SEM proved satisfactory and preserved the majority of the agglutinated protoplasts in excellent condition. When agglutination with PEG was performed on glass coverslips all protoplasts adhered firmly to the glass throughout the preparative procedures for SEM. When PEG is not used, protoplasts can be attached to glass surfaces with poly-Llysine (Mazia et al., 1975) for SEM or replica studies (Williamson et al., 1976, 1977). Light microscope studies have demonstrated that nuclear fusion in plant heterokaryons can occur at interphase as well as during mitosis (pea-soybean, Constable et al., 1975; barley-carrot, Dudits etal., 1976; tobacco-soybean, Kao and Wetter, 1976). The TEM results have confirmed these observations and shown that the fusion process involves the formation of nuclear envelope bridges linking the two nuclei. Nu-
265 clear fusion in multinucleate soybean protoplasts also involved bridges but these were more uniform in size (Fowke et al., 1975a). The endoplasmic reticulum has been implicated in the movement of nuclei prior to fusion in some plant cells (Triemer and Brown, 1975). There was no evidence that strands of endoplasmic reticulum were involved in the links between the nuclei in the pea-soybean fusion products. After nuclear fusion was completed the pea heterochromatin was detected within the synkaryon by light and electron microscopy. The characteristic heterochromatin persisted in daughter cells from the fusion products suggesting that the nuclei are hybrids. The heterochromatin of tobacco is similarly retained in daughter cells of tobacco-soybean fusion products (Kao and Wetter, 1976). The cytoplasm of all fusion products was apparently hybrid in nature as evidenced by the presence of both soybean leucoplasts and pea chloroplasts. The leucoplasts were seemingly not affected by the hybrid cytoplasm. Their structure and distribution in the daughter cells of fusion products was similar to that in actively growing soybean cells. Soybean leucoplasts also remained unaffected by the hybrid cytoplasm in fusion products of sweet clover and soybean (Fowke et al., 1976). In contrast, the number of pea chloroplasts per cell in dividing pea-soybean fnsion products was considerably reduced and many exhibited structural abnormalities indicating that the chloroplasts may have ceased dividing and in some cases were degenerating. Similar developments were observed in chloroplasts of sweet clover-soybean fusion products (Fowke et al., 1976). The behaviour may reflect a type of cytoplasmic incompatibility or natural changes in chloroplasts as a result of the change from non-dividing to dividing cells in culture. It is not surprising that very little cell wall material was detected at the surface of fusion products with the TEM. The apparent lack of wall material on cultured protoplasts has been reported previously and is likely due to limitations in technical procedures (see Burgess and Flemming, 1974b; Willison and Cocking, 1975; Fowke et al., 1976). Wall fibrils were easily detected in cross-walls between divided cells. Plasmodesmata were also present in these walls. The significance of the pseudocrystalline inclusions in some fusion products is unknown. Such inclusions were not observed in pea or soybean protoplasts prior to fusion but are apparently common in the cytoplasm and nuclei of some plants (Wergin et al., 1970). We wish to thank Miss P.J. Rennie and Mr. J.W. Kirkpatrick for excellenttechnicalassistance.
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References
Burgess, J., Flemming, E.N. : Ultrastructural studies of the aggregation and fusion of plant protoplasts. Planta 118, 183-193 (1974a) Burgess, J., Flemming, E.N.: Ultrastructural observations of cell wall regeneration around isolated tobacco protoplasts. J. Cell Sci. 14, 439449 (1974b) Constabel, F.: Protoplast isolation. In: Plant tissue culture methods. Gamborg, O.L., Wetter, L.R. (eds.). Ottawa: National Research Council of Canada 1975 Constabel, F., Dudits, D., Gamborg, O.L., Kao, K.N.: Nuclear fusion in intergeneric heterokaryons. Canad. J. Bot. 53, 2092-2095 (1975) Constabel, F., Weber, G., Kirkpatrick, J.W., Pahl, K. : Cell division of intergeneric protoplast fusion products. Z. Pflanzenphysiol. 79, 1-7 (1976) Dudits, D., Kao, K.N., Constabel, F., Gamborg, O.L.: Fusion of carrot and barley protoplasts and division of heterokaryocytes. Canad. J. Genet. Cytol. 18, 263-269 (1976) Fowke, L.C. : Electron microscopy of protoplasts. In: Plant tissue culture methods. Gamborg, O.L., Wetter, L.R. (eds.). Ottawa: National Research Council of Canada 1975 Fowke, L.C., Bech-Hansen, C.W., Gamborg, O.L., Constabel, F. : Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean. J. Cell Sci. 18, 491-507 (1975a) Fowke, L.C., Rennie, P.J., Kirkpatrick, J W., Constabel, F. : Ultrastructural characteristics of intergeneric protoplast fusion. Canad. J. Bot. 53, 27~278 (1975b) Fowke, L.C., Reunie, P.J., Kirkpatrick, J.W., Constabel, F. : Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts. Planta 130, 3945 (1976) Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968) Kao, K.N., Constabel, F., Michayluk, M.R., Gamborg, O.L.:
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