J. Cell Sci. i i , 59-75 (1972)Printed in Great Britain

FINE-STRUCTURAL STUDIES ON

SPONTANEOUS AND INDUCED FUSION OF

HIGHER PLANT PROTOPLASTS

LYNDSEY A. WITHERS AND E. C. COCKINGDepartment of Botany, University Park, Nottingham NG7 zRD, England

SUMMARY

During the treatment of some plant tissues with cell-wall-degrading enzymes adjacent cellswithin the tissue fuse forming large multinucleate protoplasts. These have been termed spon-taneous fusion bodies. The symplastic nature of plant tissues suggests that the retention ofplasmodesmatal connexions might facilitate such spontaneous fusion. An electron-microscopicinvestigation of spontaneous fusion in tobacco-leaf and oat-root tissues has confirmed thissuggestion. Enzymic degradation of the walls removes constrictions on the plasmodesmata,permitting their expansion, and as a result mixing of the cytoplasms of the fusing protoplastscan then occur.

The fine structure of plasmodesmata and their relationship to the endoplasmic reticulum canbe more easily studied in plasmodesmata which are undergoing expansion. It has been observedthat the tubule which passes through the plasmodesma is in continuity with the endoplasmicreticulum membranes at either end. Models for plasmodesmatal structure are discussed in thelight of this observation.

The induced fusion of freely isolated protoplasts by sodium salts has been previously studiedusing the light microscope. Since it is difficult to follow the detailed mechanisms involved in theprocess, electron-microscopic methods have been employed in the present investigation. Itappears that sodium nitrate first induces protoplast adhesion. This occasionally involves pro-trusions from the plasmalemma, not unlike microvilli. Following adhesion membrane fusionoccurs, initially in localized regions, and then more generally. Eventually vacuolar fusion occursfacilitating complete cytoplasmic mixing.

These findings are compared with events occurring during animal cell fusion and are discussedin relationship to a recent theoretical model for membrane fusion.

INTRODUCTION

Techniques for the hybridization of plants have, in the past, been restricted tomanipulations of the normal processes of sexual reproduction. Although it is possibleto apply these widely to select desirable characteristics from parent plants and gradu-ally improve the hybrid, the technique meets its limit when the plants which onedesires to cross are sexually incompatible. In an attempt to overcome this difficulty thepossibility of somatic hybridization by means of the fusion of protoplasts isolatedfrom the somatic tissues of the plant is being investigated (Power, Cummins &Cocking, 1970).

The first report of induced, interspecific, animal cell fusion was made by Harris &Watkins in 1965, although spontaneous and induced intraspecific fusions had beenobserved long before this (see Harris, 1970). Parallel work in plant systems has notyet reached a comparable level of sophistication.

60 L. A. Withers and E. C. Cocking

The development of the technique of treating isolated protoplasts with salts, such assodium nitrate (Power et al. 1970), has enabled the fusion of naked plant cells to becarried out reproducibly. Little is as yet, however, known about the mechanismsinvolved in this induced plant cell fusion. Similarly the mechanisms involved in thetype of fusion, termed spontaneous fusion (Power et al. 1970) which occurs in planttissues when they are treated with a mixture of cell-wall- and middle-lamella-digesting enzymes are also poorly understood. In both cases the detailed interactionsof the fusing protoplasts occur at a level beyond the resolution of the light microscope;consequently an electron-microscopic study has been carried out in an attempt toelucidate the mechanisms involved.

MATERIALS AND METHODS

Spontaneous fusion in the Avena root tip

Seeds of Avena sativa cv. Victory were surface-sterilized using 50 % (v/v) sulphuric acid andgerminated aseptically at 20 °C in the dark. After 3 days the terminal 2 mm of the primaryroots were excised and incubated in a mixture of Millipore-filtered 10% (w/v) cellulase (Ono-zuka P 1500) and 5 % (w/v) Macerozyme (both from Kinki Yakult Manuf. Co., Nishinomiya,Japan) in 20 % sucrose, at 20 °C, for lengths of time varying from 3 to 36 h. The incubationswere terminated by washing the material twice with 20 % sucrose and fixing in 6 % glutaralde-hyde in 0-025 M phosphate buffer (pH 7) overnight at 4 °C. Tissue completely untreated andtissue plasmolysed for 2 h in 20 % sucrose were also prepared as controls.

Spontaneous fusion in the tobacco leaf

Pieces of tobacco leaf taken from 50- to 60-day-old Nicotiana tabacum cv. Xanthi nc plantsfrom which the lower epidermis had been removed (Power & Cocking, 1970), were incubatedin a mixture of 5 % (w/v) cellulase (Onozuka P 1500) and 0-5 % (w/v) Macerozyme in 25 %sucrose, at 20 °C, for periods of 2 and 4 h. The incubations were terminated by twice washingthe leaf pieces in 25 % sucrose and fixing as described above. After 4 h digestion and gentlysquashing the enzyme-treated leaf pieces it was possible to release protoplasts. These werewashed by flotation in 25 % sucrose and fixed as above. Untreated and plasmolysed leaf pieceswere used as controls. In some incubations thorium dioxide ('Thorotrast' Fellows Testagar,Detroit, Michigan; British supplier: Martindales, Bayswater) was added in concentrations of2 - 6 % (w/v).

Induced fusion of freely isolated tobacco-leaf protoplasts

Pieces of tobacco leaf from which the lower epidermis had been removed were incubated for2 h in 25 % sucrose and then treated with the enzyme mixture, as above, for 4 h. The materialwas gently swirled at intervals during the treatment. The protoplasts thus released were washedtwice by flotation in 25 % sucrose, and then treated with sodium nitrate in the following2 ways:

(1) An equal volume of 0-55 M sodium nitrate was added to the protoplasts suspended in25 % sucrose giving a final sodium nitrate concentration of 0-275 M- Following immediatecentrifugation for 2 min at approximately 150^ the protoplasts were left compacted for 0-5 h.They were then washed with 25 % sucrose and fixed as described earlier.

(2) The sucrose in which the protoplasts were suspended was entirely replaced by o-8 Msodium nitrate. The protoplasts were immediately centrifuged for 5 min at approximatelyI5og. One preparation was fixed immediately, a second one being washed gently with 25%sucrose to remove the sodium nitrate and then left to allow the protoplasts to rise to the surfaceof the sucrose, before fixation.

A control preparation of untreated protoplasts was also prepared.

Fusing plant protoplasts 61

Preparation of material for electron microscopy

After glutaraldehyde fixation the material was washed 3 times with o-i M phosphate buffer(pH 7) containing 10 % sucrose, postfixed in 2 % osmium tetroxide in o-i M phosphate buffer(pH 7) plus 10% sucrose, and then dehydrated in a series of ethanols to 90% ethanol. Afterstaining with 1 % alcoholic uranyl acetate, followed by 2 changes of absolute ethanol, the mate-rial was finally embedded in a mixture of butyl and methyl methacrylates and styrene (4:3:3,v/v/v (stabilizers present), + 1 % (w/v) benzoyl peroxide) (modified from Mohr & Cocking,1968). Gold/silver sections cut on a Porter-Blum MT 2 Ultramicrotome were collected onFormvar-coated hexagonal grids (Athene Hexagonal type), post-stained with lead citrate andexamined in a GEC-AEI EM 6B electron microscope operating at 50 kV with Meek Modifica-tion (Meek, 1967).

RESULTS

Spontaneous fusion in the Avena root tip

Plasmolysis effects, plasmodesmatal relationships and cell-wall degradation. In theuntreated root (Fig. 1) plasmodesmata can be seen traversing the cell wall and it isevident that the endoplasmic reticulum is spatially related to the plasmodesmata, butthe relationship cannot be determined with clarity. Upon plasmolysis the plasmalemmawithdraws from the cell walls usually without damage. However, in some cases theplasmalemma is damaged and plasmodesmatal connexions are broken. There is agreater tendency for protoplasts to maintain contact via plasmodesmata across trans-verse walls than across longitudinal walls.

Enzyme treatment following plasmolysis results in the removal of the cellulosic cellwall and middle lamella. Fig. 2 shows the resulting cytoplasmic confluence whichoccurs when wall digestion is preceded by damage to the plasmalemma. Large massesof cytoplasm and organelles can be seen with the remains of the plasmalemma (arrowed)and the space left by the digestion of the cell wall still visible.

Plasmodesmatal ultrastructure and the relationship between plasmodesmata and theendoplasmic reticulum. The removal of the cell wall permits plasmodesmatal expansionwhich begins at the plasmodesma-protoplast junction and is followed by expansion ofthe entire plasmodesma. Fig. 3 shows plasmodesmata cut in semi-transverse section.A circular membrane can be seen within the ring of plasmalemma (Fig. 3, arrow).In longitudinal section (Fig. 4) the plasmodesma is seen to contain a 'desmotubule'(Robards, 1968) which joins the endoplasmic reticulum at either end.

In Figs. 5 and 6 plasmodesmatal expansion is progressing. The spatial relationshipof plasmodesmata and endoplasmic reticulum can be observed (Fig. 6, arrows). Insome cases (Fig. 5, arrow) the desmotubule remains unexpanded and close to one sideof the expanded plasmodesma. This explains why the desmotubule does not appearin these sections as frequently as it does in sections of unexpanded plasmodesma.Occasionally wider structures are seen in expanded plasmodesmata (e.g. Fig. 9,arrows) but these are almost certainly displaced endoplasmic reticulum elements. InFig. 7 two adjacent plasmodesmata are seen whose desmotubules connect on one sidewith the same endoplasmic reticulum region.

Plasmodesmatal expansion and protoplast fusion. The first sign of cytoplasmic con-

62 L.A. Withers and E. C. Cocking

tinuity being established between protoplasts is the appearance of ribosomes in theexpanded plasmodesmata (Fig. 5). Further expansion of plasmodesmata enablesorganelle movement to occur (Figs. 8-10). It is difficult to determine the fate of thecontinuum of membrane which was in contact with the digested wall and which alsolined the plasmodesmatal canals. Possibly small vesicles which undergo movementinto the main cytoplasmic body are formed (Fig. 8).

In tissue left from the longest time in the enzyme incubation mixture (36 h) exten-sive fusion of protoplasts could be seen, but the general condition of the protoplastswas poor. In Fig. 10 a number of nuclear areas (damaged) can be seen within a massof fused cytoplasm. This plate illustrates the common observation that protoplasts tendto fuse across the transverse junctions (simple arrows) more readily than longitudinally.

Spontaneous fusion in the tobacco leaf

Plasmolysis effects. Observations on tobacco-leaf tissue treated similarly suggest acorresponding pattern of events leading to spontaneous fusion, although here thepicture is less complete.

Plasmolysis leads to a detaching of the plasmalemma from the cell wall and aninfolding of the plasmalemma to reduce its surface area. Thorium dioxide was includedin the enzyme incubation mixture in an attempt to trace the plasmalemma membranesoriginally separating the protoplasts during the following enzyme treatment. However,it was found that extensive uptake of thorium dioxide occurred during plasmolysisitself (Fig. 11). By pre-plasmolysing the tissue pieces in sucrose solution for 2 h beforethe enzyme—thorium dioxide incubation it was found possible to reduce greatly theextent of thorium dioxide uptake. Fig. 12 shows a protoplast isolated from tissue pre-plasmolysed in this way.

Cell-wall degradation, plasmodesmatal relationships, spontaneous fusion body release.After 2 h of enzyme treatment (no pre-plasmolysis) the cell walls were considerablydigested. Fig. 13 shows 2 protoplasts joined by an intact plasmodesma. Extending theenzyme treatment to 4 h and then squeezing the tissue released protoplasts some ofwhich were spontaneous-fusion bodies.

A number of the protoplasts released from the 4-h enzyme-incubated material wereseen to be connected by plasmodesmata (Fig. 14). As for the root, it can be seen thatthe endoplasmic reticulum is spatially related to the plasmodesmata (see also Fig. 13).Other protoplasts having broader cytoplasmic connexions have been observed (Figs. 15,16). In some cases endoplasmic reticulum could be seen in the region of the con-nexions, suggesting plasmodesma-endoplasmic reticulum relationships similar tothose seen in the root (Fig. 16). Here, however, the section does not pass through anystructure resembling a desmotubule. There is some suggestion, as in the root system,that small vesicles form from the plasmalemma membrane in the region of the digestedcell wall (Fig. 16).

Induced fusion of freely isolated tobacco-leaf protoplasts

By manipulation of isolation conditions it is possible to produce a high proportionof uninucleate protoplasts (85—90%). Tobacco-leaf protoplasts suspended in high

Fusing plant protoplasts 63

concentration, in 25% sucrose, are seen to adhere to one another over restrictedregions of the plasmalemma. The adhesion appears to be fairly strong. In a few casesthe main protoplast bodies have separated leaving a connecting region of drawn-outcytoplasm still adhering. However, nothing suggesting later stages of fusion was seenin any untreated material.

Treatment of freely isolated protoplasts with sodium nitrate is found to induce theirfusion, producing very large fusion bodies. Treatment of tobacco-leaf protoplasts withsodium nitrate, alone and mixed with sucrose, induces the formation of fusion bodies.Various stages in the fusion can be observed. The protoplasts initially adhere to oneanother by direct plasmalemma contact (Fig. 17), or by the adhesion of the plasma-lemma protrusions which are produced in protoplasts treated with sodium nitrate(Fig. 18). In Fig. 17 the distortion of the protoplasts, due to the absence of largeorganelles in the adhering region, indicates the force of the adhesion.

Following contact and adhesion, it appears that the membranes fuse, firstly in smalllocalized areas (Fig. 19, arrows), then more extensively. This leads eventually to thesituation where the cytoplasms of the fusing protoplasts are in continuity.

Where protoplasts fuse in regions made up of thin cytoplasmic layers the followingpattern of events seems to occur. Fig. 20 shows a fusion body with four or five proto-plasts cut in one plane. Following sodium nitrate treatment (after 0-5 h), various stagesin the fusion process can be observed (Figs. 21, 22). In one region (Fig. 22), the plasma-lemmae of the 2 original protoplasts are still intact. However, in adjacent regions theplasmalemmae are not visible and the cytoplasm is completely continuous. The orga-nelles are beginning to move to the periphery of the fusion body leaving only mem-branes separated by a thin layer of cytoplasm. In Fig. 21 only the tonoplasts remain.The peripheral movement of the organelles continues and eventually the fusion ofadjacent vacuoles occurs by the breakdown of the thin cytoplasmic separating layer.It is only at this point when the vacuoles have fused and when the organelles havemoved to the periphery that complete cytoplasmic mixing can occur.

DISCUSSION

Harris (1970) uses the word 'spontaneous' to describe a type of hybridization foundto occur in some animal cell cultures without the stimulus of an inducing agent such asan inactivated virus. However, in work on the fusion of plant protoplasts 'spontaneousfusion' is used in a different context (Power et al. 1970). Plant spontaneous fusionbodies form as a result of enzymic degradation of cell walls in tissues and are thereforestrictly intraspecific. Spontaneous animal cell fusion may, however, be either inter-specific or intraspecific.

In the plant kingdom spontaneous fusion has been found to occur in a number oftissues such as soybean callus (Miller, Gamborg, Keller & Kao, 1971), oat root tip(Power et al. 1970) and tobacco leaf (Power, Frearson & Cocking, 1971). It wouldseem likely that spontaneous fusion in plant tissues is mediated by the retention ofplasmodesmatal connexions during the enzyme treatment. It should be noted thatthe efficacy of plasmodesmata as physiological connexions within the plant tissuesymplasm is already well recognized (Arisz, 1969).

64 L. A. Withers and E. C. Cocking

During enzyme treatment of particularly the Avena root tissue one of the majortransformations is the appearance of broad connexions between many protoplasts anda reduction in the proportion of plasmodesmata. These connexions range from cyto-plasmic channels, containing ribosomes, to much broader connexions containingendoplasmic reticulum elements and mitochondria (Figs. 5-10). It would seem likelythat plasmodesmatal expansion is involved. When using electron-microscopic evidencewhich can only illustrate a number of static stages one must be wary of drawing toodefinite conclusions relating to such a dynamic situation (Gunning, 1970). Additionalevidence can, however, be brought in to support these conclusions.

Avena root tip and tobacco leaf differ greatly in their organization. In the leaf thereare large air spaces and fewer cells in the thickness of tissue involved compared with theroot. Consequently the total area of contact between cells is lower in the leaf than in theroot. These differences are reflected in differences in the size and multiplicity of spon-taneous fusion bodies forming from the 2 tissues; they point to a dependence on area ofintercellular contact prior to enzyme treatment. In the root there is a pattern of fusionbody formation which tends to result in protoplasts fusing in longitudinal rows moreoften than laterally (Fig. 10). This finding may be related to the observations that insome root tissues there is a higher density of plasmodesmata on transverse walls thanlongitudinal ones (Juniper & Barlow, 1969) and, as observed in the present study, thatprotoplasts tend to maintain contact more across transverse walls when the tissue isplasmolysed.

If plasmodesmatal expansion occurs, this will involve considerable redistribution ofmembrane material, but no actual fusion of membranes is necessitated. The relation-ships of the membranes are of interest since during wall degradation and plasmodes-matal expansion the plasmalemma between the fusing protoplasts appears to becomeenclosed within the main cytoplasmic mass. It would appear from the studies usingthorium dioxide that extensive uptake from the plasmolyticum occurs during plasmo-lysis, probably by large-scale membrane invagination. This reduces the surface area ofthe protoplasts and results in the conversion of some of the plasmalemma into internalvesicles. In view of this extensive uptake it would seem advantageous to carry outpre-plasmolysis in a suitable plasmolyticum before subjecting the tissue to digestionin a possibly harmful cell-wall-degrading enzyme mixture (Hall & Wood, 1970).

An interesting outcome of the present investigation into spontaneous fusion is thatit provides a new approach to the study of plasmodesmatal ultrastructure. For sometime there has been disagreement over the detailed structure of plasmodesmata. Manyof the structures proposed have lacked satisfactory supporting evidence partly due tovariations in the tissues and difficulties in section orientation and electron-microscope-image interpretations (see Helder & Boerma, 1969). It is generally accepted that theplasmalemma passes through the plasmodesmata. This plasmalemma has been des-cribed as structurally similar to the plasmalemma enclosing the main cytoplasmic mass(Robards, 1968). Major disagreement arises over the nature and structure of the tubulepassing through the centre of the plasmodesmatal canal. The most common suggestionis that it is a strand of endoplasmic reticulum (Porter & Machado, 1969; Lope"z-Saez,Gimen6z-Martfn & Risueno, 1966; Frey-Wyssling & Miihlethaler, 1965). Continuity

Fusing plant protoplasts 65

between the tubule and the endoplasmic reticulum is seen in later stages of cell plateformation (Buvat, 1969), but no unequivocal pictorial evidence has been presented inthe case of differentiated plasmodesmata. Contrasting this are the suggestions ofO'Brien & Thimann (1967) that the tubule is more like a spindle fibre than an endo-plasmic reticulum element; and the initial proposal, now modified, of Robards (1968),in which the tubule termed the 'desmotubule' is of a structure related to that of aspindle fibre completely lacking any continuity with the endoplasmic reticulum,although the latter passes very close to it.

More recently, a new interpretation has been put forward (Robards, 1971). Thedesmotubule is considered to be a microtubule-like structure resulting from a modu-lation of the proportion of lipid and protein components in a lipoprotein endoplasmicreticulum membrane. It is necessary to describe the membrane as having a micellarstructure (Lucy, 1970).

Robards (1968, 1971) and Helder & Boerma (1969) note that the points of junctionof the plasmodesmata cannot be visualized clearly and the latter illustrate the fact thatunless the section passes medianly through the plasmodesma the plasmalemma ap-pears to seal the ends of the canal. The same difficulty must therefore apply to visual-izing the endoplasmic reticulum-desmotubule relationships. By digesting away thecell wall the constrictions it imposes on the plasmodesmata are removed and thisappears to facilitate observation of the protoplast-plasmodesma junction. As theplasmodesmata expand the tubule does not appear to expand accordingly. However,such an expansion would be expected to occur, if, as suggested by Helder & Boerma(1969), the desmotubule assumes a micellar structure due to the constrictions imposedon it. A possible explanation for the apparent absence of desmotubule expansion isthat, when the wall is removed, the constrictions are removed firstly at the ends ofthe plasmodesmata. This progresses further as more wall is removed, but structuralchanges in the desmotubule are not as rapid.

If one accepts that the desmotubule is a membranous structure in micellar form thepast difficulties in visualizing its relationship to the endoplasmic reticulum may be dueto a dynamic situation existing. This dynamic situation could be such that the endo-plasmic reticulum is generally in a bilayer form, but that near the desmotubule it is ina micellar form in dynamic equilibrium with the desmotubule. The removal of thewall could then alter the balance of the equilibrium favouring the joining of the desmo-tubule to the endoplasmic reticulum.

The additional possibility still exists of a desmotubule of microtubule origin attach-ing to endoplasmic reticulum membrane at either end of the plasmodesma. Althoughno pictorial evidence exists, there are some suggestions of microtubule-membraneassociations in plant cells (Burgess, 1970; Pickett-Heaps & Northcote, 1966).

Work on the induced fusion of animal cells has thrown considerable light on theconditions conducive to fusion in terms of temperature, ionic requirements andinducers. It seems that fusion is an energy-requiring process involving oxidativephosphorylation. The surface structure of the animal cells is also of importance, thepresence of microvilli leading to more extensive fusion (Harris, 1970).

It is thought from the present work that induced protoplast fusion begins with

66 L. A. Withers and E. C. Cocking

contact, followed by adhesion. This adhesion is quite distinct in extent and force fromassociations of untreated control protoplasts. The adhesion of protoplasts treated withsodium nitrate does not appear to be dependent on centrifugation although this facili-tates contact. The actual mechanism of action of the salt is undetermined. Frequentlyit results in the formation of surface protrusions. However, it is observed that theseprotrusions are not essential to fusion and therefore the salt must have other fusion-inducing effects. Similar surface protrusions have been observed in protoplasts treatedwith certain growth substances (Cocking, 1968). Whether the protrusions merelyhave a physical action, analogous to microvilli of animal cells which facilitate greatercontact with the fusion inducer (Harris, 1970), or whether they are associated withlocalized membrane modifications conducive to fusion is not known.

The theoretical membrane fusion model of Poste & Allison (1971) necessitates aninitial membrane separation of 1 nm for contact and adhesion to occur. The radius ofcurvature of the membrane-enclosed mass influences this separation. This possiblypartly explains the effect of animal cell microvilli and plant protoplast surface protru-sions. The model suggests that differences in chemical composition, structural organ-ization and surface charge distribution give some degree of specificity to the membranefusion reaction. Accordingly, the nature of the medium used, particularly its ioniccontent and pH, must be taken into consideration. In this context too the nature of thesurface coating of animal cells and plant protoplasts is of importance. Animal cellshave a glycocalyx, thought to be involved in the fusion reaction, and there is somesuggestion (Roland & Vian, 1971) that plant cells have an analogous coating. There isalso some evidence for the presence of glycoprotein on the outer surface of some isola-ted plant protoplasts (Mayo & Cocking, 1969).

The fate of fusing membranes after cytoplasmic coalescence has yet to be determined.They possibly break down into small structureless lipoprotein units; or they maymaintain their integrity as lamellae passing into the cytoplasm as small vesicles, as isfound in some cases with fusing animal cells (Harris, 1970).

Due to the presence of some bi- and multinucleate protoplasts in the preparationsused for induced-fusion studies there arises the possibility of some ambiguity in theinterpretation of the mode of formation of the fusion bodies observed. It should benoted, however, that the appearance of protoplasts in the adhesion stage of inducedfusion is quite different from any stage of spontaneous fusion. The areas of contactbetween such protoplasts are greater and cause considerably more distortion than anyconnexions between spontaneously fusing protoplasts or the occasionally observedadhering protoplasts found in control preparations. Moreover, induced-fusion bodiesare larger and occur more frequently than spontaneous fusion bodies.

This work has involved the spontaneous fusion of highly vacuolated and also rela-tively less highly vacuolated protoplasts and the induced fusion of highly vacuolatedprotoplasts, each in its way having some relevance to the objective of producing asomatic hybrid plant (Withers, Power & Cocking, 1971). The process of interspecifichybridization naturally excludes the use of spontaneous fusion. The value of spon-taneous fusion lies in the fact that the products, multinucleate protoplasts, can bereadily cultured and their growth, wall regeneration and division observed (Power

Fusing plant protoplasts 67

et al. 1971). Any damaging effects which fusion-inducing agents might have areeliminated since no such agent is required.

In the study of induced fusion it has been possible to compare the patterns of plantprotoplast fusion with animal cell fusion and some common features have been ob-served. If the subsequent events follow a similar pattern the general study of plantprotoplast fusion could yield a wealth of information on the biology of plant cells inaddition to the primary aim of producing somatic plant hybrids.

L. A.W. thanks the Science Research Council for a Research Studentship. This work formspart of a project supported by a grant from the Agricultural Research Council.

REFERENCES

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BURGESS, J. (1970). Interactions between microtubules and the nuclear envelope during mitosisin a fern. Protoplasma 71, 77-89.

BUVAT, R. (1969). Plant Cells. London: World University Library.COCKING, E. C. (1968). The action of 3-indoleacetic acid on isolated protoplasts. In. Biochemistry

and Physiology of Plant Growth Substances (ed. F. Wightman & G. Setterfield). Proc. 6thInt. Conf. Plant Growth Substances, pp. 603-609. Ottawa: Runge Press.

FREY-WYSSUNC, A. & MOHLETHALER, K. (1965). Ultrastructural Plant Cytology. Amsterdam:Elsevier.

GUNNING, B. E. S. (1970). Lateral fusion of membranes in bacteroid-containing cells of legu-minous root nodules. J. Cell Sci. 7, 307-317.

HALL, J. A. & WOOD, R. K. S. (1970). Plant cells killed by soft rot parasites. Nature, Lond. 227,1266—1267.

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heterokaryons of mammalian cells from different species. Nature, Lond. 205, 640.HELDER, R. J. & BOKRMA, J. (1969). An electron microscopical study of the plasmodesmata in

the roots of young barley seedlings. Acta bot. neerl. 18, 99-107.JUNIPER, B. E. & BARLOW, P. W. (1969). The distribution of plasmodesmata in the root tip of

maize. Planta 89, 352-360.LOPEZ-SAEZ, J. F., GIMENEZ-MART/N, C. & RISUENO, M. C. (1966). Fine structure of the

plasmodesma. Protoplasma 61, 81-84.LUCY, J. A. (1970). The fusion of biological membranes. Nature, Lond. 227, 814-817.MAYO, M. A. & COCKING, E. C. (1969). Detection of pinocytic activity using selective staining

with phosphotungstic acid. Protoplasma 68, 231-236.MEEK, G. A. (1967). An improved operating method for the A.E.I. EM 6B electron microscope.

Jl R. microsc. Soc. 88, 419-429.MILLER, R. A., GAMBORG, O. L., KELLER, W. A. & KAO, K. N. (1971). Fusion and division of

nuclei in multinucleated soybean protoplasts. Can. J. Genet. Cytol. 13, 347-353.MOHR, W. P. & COCKING, E. C. (1968). A method for preparing highly vacuolated, senescent or

damaged plant tissues for ultrastructural study. J. Ultrastruct. Res. 21, 171-181.O'BRIEN, J. P. & THIMANN, K. V. (1967). Observations on the fine structure of the oat coleop-

tile. II. The parenchyma cells of the apex. Protoplasma 63, 417-442.PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Organization of microtubules and endo-

plasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, 109-120.PORTER, K. R. & MACHADO, R. D. (i960). Studies on the endoplasmic reticulum. IV. Its form

and distribution during mitosis in cells of onion root tip. J. biophys. biochem. Cytol. 7, 167-180.

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POSTE, G. & ALLISON, A. C. (1971). Membrane fusion reaction: A theory. J. theor. Biol. 32,165-184.

POWER, J. B. & COCKING, E. C. (1970). Isolation of leaf protoplasts: Macromolecule uptake andgrowth substance response. J. exp. Bot. 21 (66), 64-70.

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ABBREVIATIONS ON PLATES

chCIO

depderIm

chloroplastcell walldesmotubuleexpanding plasmodesmaendoplasmic reticulumER lamellaemitochondrion

n nucleuspd plasmodesmapi plasmalemmat tonoplastv vesicleva vacuole

Fig. 1. Untreated oat root tip. The wall separating the 2 protoplasts is traversed by anumber of plasmodesmata. Endoplasmic reticulum can be seen near the ends of theplasmodesmata. x 50000.Fig. 2. Oat root tip following plasmolysis and wall digestion. The plasmalemmae ofprotoplasts / and 2 are intact but that of protoplast 3 is considerably damaged, rem-nants of it being indicated (arrows) lying near the space left by cell-wall digestion,x 12500.

Fig. 3. Semi-transverse section of plasmodesmata in a region of oat root tip followingplasmolysis and wall digestion. The section passes through slightly expanded regionsof the plasmodesmata. An inner ring (arrow) can be seen within the plasmalemmawhich encloses the plasmodesma. x 100 000.Fig. 4. Specimen as Fig. 3. The section here, however, passes longitudinally throughthe plasmodesmata. Endoplasmic reticulum can be seen in proximity to the plasmo-desmata and it appears to be continuous with the desmotubules passing through theplasmodesmata. x 70000.Fig. 5. Oat root tip during enzyme digestion. Again the endoplasmic reticulum can beseen close to the plasmodesmata. A desmotubule is visible close to the plasmalemmaon one side of the expanding plasmodesma (arrow). The desmotubule itself does notappear to expand, x 90000.Fig. 6. Specimen as Fig. 5. Here the proximity of the endoplasmic reticulum to theplasmodesmata is very clear, x 60000.Fig. 7. Specimen as Fig. 5. Two plasmodesmata can be seen, the desmotubules of whichconnect to the same endoplasmic reticulum membranes at one end. x 130000.

Fusing plant protoplasts

70 L. A. Withers and E. C. Cockitig

Fig. 8. A later stage in the enzyme digestion of the oat root tip. Expanded plasmodes-mata are again evident. The organelles in the upper protoplast appear polarized, theirbeing grouped near the protoplasts' junction. The protoplast connexions are not yetsufficiently broad for the large organelles to enter them. Small vesicles, possibly formedfrom the plasmalemmae, are indicated (v). x 35000.Fig. 9. Montage, specimen as Fig. 8. Three protoplasts joined by broad cytoplasmicconnexions, some containing endoplasmic reticulum lamellae, others mitochondria.Inset shows a low-power view of the region, x 19000; inset, x 3500.Fig. 10. Montage, oat root tip following 36 h enzyme digestion. The longitudinal axisof the root is indicated (•<—>•). A fusion body, formed from a number of protoplastswhich have fused mainly across transverse junctions (arrows), can be seen. The proto-plasts are almost completely isolated from those in adjacent longitudinal rows. A num-ber of nuclear areas can be seen but it is clear that they have undergone considerablebreakdown, x 3500.

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72 L. A. Withers and E. C. Cocking

Fig. I I . Region of a tobacco protoplast released from leaf tissue incubated in anenzyme mixture containing thorium dioxide. Many small vesicles containing thismarker can be seen in the cytoplasm (arrows), x 20000.

Fig. 12. A tobacco-leaf protoplast released from leaf tissue pre-plasmolysed in sucrosesolution, before treatment with enzyme mixture containing thorium dioxide. Very fewtraces (arrows) of the latter can be seen in the cytoplasm, although the protoplast con-tains many small Vesicles, x 4500.

Fig. 13. Region of tobacco-leaf tissue following partial enzyme digestion. Twoprotoplasts, separated by a space, containing some wall remnants (arrows) can beseen. They are connected across this space by a plasmodesma. Endoplasmic reticulumis seen near one end of this plasmodesma. x 20000.

Fig. 14. Two tobacco-leaf protoplasts released from enzyme-digested leaf tissue. Theyare connected by plasmodesmata. x 3000.

Fig. 15. A low-power view of 2 tobacco-leaf protoplasts undergoing spontaneous fusion.A broad cytoplasmic bridge (arrow), possibly a result of plasmodesmatal expansion,joins the two. Small Vesicles (u), found in the connecting region, are possibly formedfrom the plasmalemmae of the 2 protoplasts, x 3000.

Fig. 16. Junction region of 2 tobacco-leaf protoplasts at a later stage of spontaneousfusion. A broad cytoplasmic bridge connects them. The disposition of the endoplasmicreticulum (arrows) suggests that here again the connexion is due to plasmodesmatalexpansion, x 10000.

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74 L. A. Withers and E. C. Cocking

Fig. 17. Tobacco-leaf protoplasts, treated with sodium nitrate, adhering where theirplasmalemmae are in contact. Protoplast / can undergo deformation, due to the absenceof large organelles near the adhering region, unlike in the corresponding region of pro-toplast 2. x 15000.Fig. 18. Junction region of 2 tobacco-leaf protoplasts treated with sodium nitrate. Peri-pheral cytoplasmic protrusions (arrows) aid in the adhesion of the protoplasts, x 6000.Fig. 19. Junction region of 2 tobacco-leaf protoplasts, treated as above. Followingadhesion the plasmalemmae are fusing, first in localized regions, leading to cyto-plasmic continuity (arrows). The line of fusion of the 2 protoplasts is still quite evident,x 40000.

Fig. 20. Montage, induced-fusion body formed from sodium nitrate-treated tobacco-leaf protoplasts. Various stages of the fusion have been reached in different regionsof the fusion body. The next 2 figures show higher-power views of the 2 regions indi-cated, x 1500.Fig. 21. Junction of protoplasts 2 and j . All traces of the plasmalemmae have gone andthe organelles are migrating to the peripheral regions of the fusion body. The vacuolesare separated only by a thin cytoplasmic layer and the 2 tonoplasts. x 3500.Fig. 22. Junction of protoplasts 3 and 4. Here the plasmalemmae still remain in part,although they have fused in adjacent regions (arrows). Organelle migration is againoccurring, x 3500.

75Fusing plant protoplasts

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