Planta

Planta (1985)165:217-224

9 Springer-Verlag 1985

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana George W. Bates Department of Biological Science, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA

Abstract. The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1 000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5.10 s protoplasts (0.5 ml of cells at 106/ml) can be fused in 5-7 rain with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.

Key words: Electrical fusion - Heterokaryon Nicotiana (protoplast fusion) - Protoplast fusion.

Introduction

Success in plant somatic hybridization depends on the availability of a suitable technique for protoplast fusion. An ideal technique would allow the rapid production of large numbers of binucleate heterokaryons by a simple protocol. Polyethylene glycol (PEG), the most widely used fusion agent, is an effective, but not ideal, fusogen. Abbreviations: AC = alternating current; DC = direct current; PEG = polyethylene glycol

Protoplasts exposed to PEG are generally reported to undergo only 1-10% random fusions and in addition require extensive washing prior to culture in order to remove the fusogenic agent. Higher fusion efticiencies can be obtained by increasing the PEG concentration (Kao and Michayluk 1974) but this leads to a reduction in cell viability (Kao 1981). Indeed, PEG is quite toxic to mesophyll protoplasts of certain species (Kartha et al. 1974; Kao and Michayluk 1974; Constabel et al. 1975b) as well as to isolated organelles (Benbadis and deVirville 1982). Protoplast fusion in high-strength electric fields (Zimmermann and Vienken 1982) has attracted interest as an alternative to the use of PEG. Senda et al. (1979) first reported that protoplasts could be induced to fuse by a short direct-current (DC) electric pulse. In their experiments, cell-to-cell contact was established by physically pushing two cells together with microelectrodes; thus, yields were restricted to single heterokaryons. Zimmermann and Scheurich (1981), taking advantage of the phenomenon of dielectrophoresis (Pohl 1978), greatly improved this technique by showing that groups of protoplasts could be fused electrically. In dielectrophoresis cells are exposed to a rapidly oscillating, non-uniform, alternating-current (AC) electric field. This creates transient dipoles on the cell's surfaces which causes the cells to become stacked in chains ("pearl chains") on the electrodes. Once membrane contact is established, the protoplasts can be fused by application of one or more DC pulses. Electrical fusion has been reported to be rapid, highly efficient and effective with a wide variety of protoplast types (Zimmermann and Scheurich 1981; Bates et al. 1983; Zachrisson and Bornman 1984; Watts and King 1984). One study even demonstrates the preferential formation of binu-

218

cleate heterokaryons by electrical fusion (Vienken and Zimmermann 1982). However, serious questions have been raised about the viability of electrically fused protoplasts (Davey and Kumar 1983; see also the discussion following Zimmermann et al. 1984). Recently, Bates and Hasenkampf (1985) have succeeded in regenerating somatic hybrids following electrical fusion of tobacco protoplasts. These experiments indicate no reduction in the viability of electrically fused protoplasts beyond that which might be expected from the genetic imbalances characteristic of heterokaryons. Reports that electrically fused yeast (Halfmann etal. 1983) and mammalian cells (Bischoff et al. 1982; Finaz et al. 1984) also remain viable strengthen the argument that electrical fusion is not cytotoxic. Disadvantages to electrical protoplast fusion are that the methodology is technically demanding and is restricted to small numbers of cells. These problems, which arise largely from the design of the fusion equipment, have greatly limited the application of this technique to somatic hybridization studies with plant and animal cells. In addition, detailed quantitative information describing the electrical-fusion products is lacking. In this paper I describe a quantitative fusion index for determining the effects of various electric fields and different fusion-chamber designs on the formation of protoplast heterokaryons. This work has led to the development of a fusion protocol which is wellsuited to cell-culture applications and which yields large numbers of heterokaryons rapidly and in a simple and direct manner.

G.W. Bates: Electrical fusion of protoplasts in Nicotiana transfers this callus was used to initiate suspension cultures. The suspension cultures were maintained at 25~ on liquid MS medium containing l rag/1 2,4-D under light from fluorescent lamps (5 gmol photons m 2 s ~, continuous illumination), with shaking at 125 rpm. After six weekly transfers the culture was ready for protoplast isolations. Thereafter, it was maintained on a 4-d transfer schedule.

Protoplast isolations. Suspension-culture cells of N. plumbaginifolia were washed once with a solution containing 1 mM KNO3+0.2mM KH2PO4+lmM CaC12+lgM K I + 0 . 4 M mannitol + 3 mM 2-(N-morpholino)ethanesulfonicacid (Mes), adjusted to pH 5.7 with NaOH (Shekhawat and Gatston 1983). The packed cells were then resuspended in fresh wash medium to which 1% CELF cellulase (Worthington Diagnostics, Freehold, N.J., USA) and 0.1% Pectolyase Y-23 (Seishin Pharmaceutical Co., Tokyo, Japan) had been added. The pH of this medium was adjusted to pH 5.5 with NaOH and the medium was sterilized by filtration. Digestion was carried out in the dark at 27~ for 3-4 h with manual agitation at 30-rain intervals. The protoplasts were purified by passage through a 62gm-mesh nylon screen, subsequently pelleted (100 g for 3 min), and washed twice with wash medium and then once with 0.4 M mannitol. The final protoplast pellet was resuspended in 0.4 M mannitol, and the protoplast density was determined with a hemacytometer. Nicotiana tabacum mesophyll protoplasts were isolated as follows: half-expanded leaves from 2- to 4-month-old, vegetative plants were surface sterilized as described above for the N. plumbaginifolia stern explants. Portions of the lower epidermis were removed with fine forceps. Sections of peeled leaves were cut out and placed face down in the enzyme mixture. Digestion was carried out at 27~ in darkness with manual agitation every 30 rain. After 1 h, undigested leaf material was removed and digestion was continued for another 1-2 h. Protoplasts were purified by sequential passage through a 62~tm-mesh nylon screen and Miracloth (Calbiochem-Behring Corp., La Jolla, Cal., USA). The protoplasts were then pelleted, washed, and counted in the same fashion as the suspension-cell protoplasts. Production of the two protoplast types was synchronized by starting digestion of the N. tabacum leaf material 1 h after the suspension culture cells.

Fusion equipment and procedure. Electrical fusion is carried out

Materials and methods Plants. Seeds of Nicotianatabacum L. cv. Xanthi and N. plumbaginifolia Viviani were obtained from the Tobacco Laboratory, Plant Genetics and Germplasm Institute, U.S. Department of Agriculture, Beltsville, Md. The plants were grown either in the laboratory, at room temperature, under a mixture of light from fluorescent lamps ("cool white", General Electric, Cleveland, O., USA) and natural light (100-150 gmol photons m-2 s-I; 14 h light: 10 h dark) or in the greenhouse, at 24-32~ C without supplemental lighting; all plants were watered regularly and fertilized biweekly with an all-purpose fertilizer (6-6-6, "garden favorite"; Florida Seed and Feed Co., Ocala, Fla., USA). Callus was initiated from whole explants ofN. plumbaginifolia stems (third to sixth internodes). The explants were surfacesterilized by a 6-rain treatment with 0.5% NaOC1 prepared from commercial bleach, with 0.25% polyethylensorbitan monooleate (Tween 80; Sigma Chemical. Co., St. Louis, Mo., USA) added, rinsed three times with sterile water, and cultured on solid MS medium (Murashige and Skoog 1962) containing 2rag/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 rag/1 kinetin (N6-furfurylaminopurine). After several monthly

in a small chamber, the design of which is crucial to successful fusions. The simplest design is exemplified by the D.E.P. Systems (Metamora, Mich., USA) Open Fusion Slide which consists of two platinum plates glued parallel to each other on a glass slide. A slot remains between the electrodes in which dielectrophoresis and fusion take place. The number of protoplasts which can be fused is dependent upon the size of this gap. Since protoplast culture normally requires large numbers of cells, it would be advantageous to make the gap between the electrodes as large as possible. However, as the distance between the electrodes increases the voltage required to produce dielectrophoresis also increases; this leads to protoplast lysis because of heat generation and electromechanical stretching of the cells. The electric fields necessary to induce protoplast fusion were generated by a Zimmermann CelI Fusion Power Supply (GCA Corp., Chicago, Ill., USA). Standard electrical parameters were: AC field, 600 kilocycles/s, 150V/cm; DC field, 1000 V/cm square-wave pulses 50 gs in duration. Unless stated otherwise the fusion chamber used was a D.E.P. Systems Open Fusion Slide No. FCO 2050S (internal volume 5-10 ~1). For fusion the two types of protoplasts were suspended at 5-105-106 cells/ml in 0.4 M mannitol, mixed together in equal amounts and pipetted

G.W. Bates: Electrical fusion of protoplasts in Nicotiana

219

a Total

~,

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....

4/

500

/

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i

1500

i

2000

VOLTS/cm (EC.) Fig. 1. Live heterokaryon resulting from the electrical fusion of a single N. plumbaginifolia suspension-culture protoplast with two N. tabacum mesophyll protoplasts. Arrows indicate positions of the mesophyll protoplasts. X 610; bar= 20 gm

into the fusion chamber. After fusion the protoplasts were transferred to plastic Petri dishes (60 mm diameter, 15 mm high; Falcon, Oxnard, Cal., USA) for fixation or culture. All manipulations during fusion were carried out in a laminar-flow hood. Fusion chambers were sterilized either by autoclaving or by alcohol rinses.

I

i i blll - Binucleate Multinucleate -

2 O

Fixation, staining, and calculation of fusion index. Fusion products were diluted 1 : 1 with a modified K3 culture medium (Nagy and Maliga 1976) and allowed 1 h to coalesce. They were then placed in fixative for 12-24 h and stained with modified carbol-fuchsin as described by Kao (1975a). The fixative was prepared by combining one part of a 0.8 M solution of sorbitol with three parts of acetic alcohol (acetic acid-95 % ethanol, 1:3, v/v). Although individual heterokaryons can be identified in live material (Fig. 1), an accurate quantitative analysis of fusion events is most readily done with fixed and stained cells. In this study nuclear staining was chosen over fluorescent labelling because nuclear staining allows one to distinguish heterokaryons from cybrids (fusions between an intact protoplast and an anucleate protoplast fragment) and also permits determination of the number of nuclei per cell. Fixed suspension-cell protoplasts have a clear cytoplasm. Mesophyll protoplasts, as a consequence of their chloroplasts, have a granular cytoplasm. Cells with a hybrid cytoplasm were detected by the presence of distinct zones of both parental cytoplasms. Fusion percentages were calculated from the total number of cells recovered after fusion which contained two or more nuclei. Binucleate and multinucleate cells with hybrid cytoplasms were the only cells scored as heterokaryons. This leads to a conservative estimate of the percentage of heterokaryons because the granular aspect of the mesophyll cytoplasm is sometimes lost during fixation and staining because of destruction of the chloroplasts. Fusion induced with PEG. Nicotiana tabacum mesophyll protoplasts and N. plumbaginifolia suspension-cell protoplasts were both suspended at 2-105 cells/ml in 0.5 M glucose, 3.5 mM CaCI2, 0.7 mM KH2PO4, pH 5.9. Equal volumes of the two protoplast suspensions were mixed together and fused with PEG 8000 (Sigma) following the method of Kao (1975b).

0

500 I000 2000 VOLTS/cm(D.C.)

Fig. 2a, b. Characteristics of fusion as the DC voltage is varied. A mixed population of N. tabacum and N. plumbaginifolia protopIasts was introduced into the D.E.P. Systems fusion chamber and aligned in an AC field of 150 V/cm, 600 kilocycles/s. After 90 s, two DC pulses (each 50 gs long) of the voltage indicated in the graph were applied. The AC field was damped out over a period of 60 s and the protoplasts were removed from the chamber and placed in culture medium. One hour later they were fixed, stained and the extent and types of fusions determined, a Overall fusion and heterokaryon formation; b proportions of bi- and multinucleate cells

Results Yields of fusion products as a function of voltage. All fusion techniques generate both binucleate and m u l t i n u c l e a t e cells. S i n c e b i n u c l e a t e h e t e r o k a r y o n s should be more viable than rnultinucleate ones, conditions were sought for the maximal production of binucleate heterokaryons using the D.E.P. fusion chamber. F i g u r e 2 s h o w s t h e effect o f v a r y i n g t h e D C v o l t a g e (while h o l d i n g t h e A C v o l t a g e c o n s t a n t ) o n t h e o v e r a l l f u s i o n efficiency, t h e f r e q u e n c y o f heterokaryon formation, and the relative numbers of binucleate and multinucleate fusion bodies. T h e r e is a t h r e s h o l d f o r o v e r a l l f u s i o n a n d h e t e r o k a r y o n f o r m a t i o n b e t w e e n 500 a n d 1000 V/ c m D C ( F i g , 2 a , b). I n c r e a s i n g t h e v o l t a g e b e y o n d

G.W. Bates: Electrical fusion of protoplasts in Nicotiana

220

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I I I00 150 VOLTS/cm (A.C.)

200

Fig. 3. The effect of AC field strength on overall fusion and heterokaryon production in a mixed population of N. tabacum and N. plumbaginifolia protoplasts. All methods were the same as described for Fig. 2 except that the AC voltage was varied as indicated in the graph while the DC pulses used for fusion were always 1000 V/cm

1000 V/cm does not increase the efficiency of fusion or yield more heterokaryons. However, DC pulses above 1 000 V/cm do increase the proportion of multinucleate cells at the expense of the binucleate ones. The homokaryotic fusions observed at 0 V are the result of spontaneous fusions which occur during protoplast isolation; the heterokaryons identified at 0 V probably indicate the level of uncertainty in distinguishing homokaryotic and heterokaryotic fusions in our assay. In an analogous experiment the AC voltage was varied while the DC voltage was kept constant (Fig. 3). In this case a clear optimum voltage (150V/cm AC) for fusion and heterokaryon production was obtained. No threshold for fusion is observed in this experiment because almost any applied AC voltage will produce some dielectrophoretic cell-to-cell contacts. Above 150V/cm (AC) the efficiency of fusion decreased because of cell lysis. These experiments show the optimum fields for fusion to be 150 V/cm AC and 1000 V/cm DC. As would be expected from random fusions in a mixed population of cells, heterokaryon formation always parallels overall fusion.

Effect of protoplast density on fusion. Fusing protoplasts at low cell densities tends to shorten the pearl chains (Zimmermann and Scheurich 1981) and therefore might be expected to favor two-cell fusions over multicellular fusions. When fusion was

compared at 106 and l05 cells/ml, a slight increase in the proportion of binucleate cells formed was observed at the lower density. However, at the lower density the overall percentage of fusion was also cut in half, probably because many protoplasts failed to find a fusion partner. The net result was a 20-fold reduction in the total number of protoplasts fused. These results, while preliminary, indicate that the production of binucleate heterokaryons, in absolute terms, can be maximized by keeping the cell density high.

"True" versus "apparent" maximum fusion efficiencies. The D.E.P. Systems fusion chamber was used for all the experiments described thus far. Using optimal electrical settings an average of 15.4% fusion, with a total of 6.2% heterokaryons and 2.9% binucleate heterokaryons, was obtained. This chamber, which it would be more accurate to call a fusion slide, holds 5-10 pl of protoplast suspension between its electrodes. In a typical fusion one drop of protoplasts (about 50 gl) is placed on the fusion slide and an unknown portion of these protoplasts settle into the gap between the electrodes. Because the AC field draws protoplasts into the center of the chamber their distribution is not entirely random. However, there is always a substantial number of protoplasts which remain outside the electric fields and hence do not participate in the fusion. Thus, among the protoplasts recovered from the chamber, the proportion of fused cells is lower than the percentage of cells between the electrodes which undergo fusion. To get a better estimate of the true efficiency of fusion, an experiment was done such that the only protoplasts considered were those aggregated in the electric field. Protoplasts from N. plumbaginifolia suspension-culture cells were introduced into the chamber at low cell density (5.104/ml) and exposed to a 150 V/cm AC field. Two 1000 V/cm DC pulses were given and the proportion of those protoplasts aligned in chains which underwent fusion was determined by microscopic observation. The result, 35%, ignores single, unaligned protoplasts and therefore reflects the maximum fusion efficiency for these cells. Whether mesophyll-mesophyll or mesophyllsuspension cell fusions would occur with the same efficiency was not studied. However, in all fusions between N. tabacum and N. plumbaginifolia roughly one-half of the fusion products proved to be heterokaryons. This indicates that the efficiencies of hetero- and homofusions are similar.

G.W. Bates: Electrical fusion of protoplasts in Nicotiana

221

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Homokaryons

9

Heterokaryons

ELECTRICALLY FUSED

-I Fig. 4. Photograph of the "mass" fusion chamber. The chamber consists of a polycarbonate plastic slide on which are mounted parallel gold-palladium wire (60:40 Au:Pd, 0.2 mm diameter) electrodes. The gap between neighboring wires is 0.5 ram. A polycarbonate collar placed over the electrodes forms a well to hold the protoplasts. Protoplasts are introduced into and removed from the chamber using Pasteur pipettes. The slide is 2.8 cm in diameter

A fusion chamber which exposes all the protoplasts to the effective portions of the electric fields should give a fusion percentage approaching the maximum of 35%. This is more than twice that ordinarily obtained with the D.E.P. chamber.

Improvement of Jusion-chamber design. The preceeding considerations indicate that chamber design is crucial to obtaining satisfactory results in electrical fusion. Consequently, several chamber designs in addition to the D.E,P. chamber were examined. The goals were to develop a chamber which would maximize both the fusion percentage and the yield of recoverable fusion products. This work also provides a useful comparison using the same protoplast system of several substantially different chamber designs. Three chambers were built and tested. One of them proved more effective than the D.E.P. chamber; the others were considerably less effective. Results obtained with the successful chamber are described below, following which the advantages and disadvantages of each chamber will be discussed. The chamber shown in Fig. 4 consists of an interdigitated network of gold wires mounted on a polycarbonate plastic slide. Each wire is 0.5 mm from its neighbors and of opposite electrical polarity. A polycarbonate collar placed over the wires produces a well which holds the protoplasts. This "mass" chamber holds 300-500 gl of cell suspension. The optimal electric fields obtained with this chamber are the same as for the D.E.P. chamber because the distance between the electrodes is the same in both designs. Figure 5 indicates the types of fusions typically produced by this mass chamber. In this experiment there was an

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5 6 7 B NUMBER OF NUCLEI PER CELL

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Fig. 5. Histogram of the types of fusions produced in the mass fusion chamber. A 0.3-ml suspension containing equal numbers of N. tabacum and N. plumbaginifolia protoplasts was placed in the chamber. An AC field of 75 V/cm, 600 kilocycles/s was applied for 4.5 min, then the AC field was increased to 150 V/cm for 30 s after which two DC pulses (1000 V/cm, 50 ps long) were applied. The AC field was damped to 0 V over a period of 60 s. Protoplasts were removed from the chamber, mixed with an equal volume of culture medium, and left to stand 1 h. They were then fixed, stained with carbol-fuchsin and the distribution of fusions was determined. Open bars, homokaryotic fusions; solid bars, heterokaryotic fusions

overall fusion efficiency of 34% (compared with 7% in the control). The fusion products contained from 2 to 30 nuclei; however, 60% were either bi- or trinucleate. A similar spread in nuclear constitution was also observed among fusion products recovered from the D.E.P. chamber. The mass chamber appears simply to increase the efficiency of fusion by reducing the number of protoplasts which are not exposed to the appropriate electric fields. Table 1 contains a quantitative comparison of the average results obtained with the D.E.P. and mass chambers and with fusions induced by PEG. The mass chamber yields significantly greater percentages of fusion products for all categories including the binucleate heterokaryons. In addition, the percentage of overall fusion obtained with the mass chamber was very close to the maximum obtained earlier for N. plumbaginifolia suspensioncell protoplasts. This indicates that nearly all the protoplasts are exposed to the fusogenic electric fields. In addition to the mass chamber two other chamber designs were tried. One was a "flowthrough" chamber which consisted of a small chamber, enclosed by a coverslip, through which protoplasts were pumped by means of syringes and

222

G.W. Bates: Electrical fusion of protoplasts in Nicotiana

Table 1. Percentages and types of fusions observed using different techniques. Values are given as the percentage of cells which contain more than one nucleus. The PEG fusion procedure is described in Materials and methods. The row marked Control represents N. tabacum and N. plumbaginifolia protoplasts which were mixed together and then fixed and stained without first being subjected to electric fields or PEG treatment. "n" is the number of trials, each percentage is followed by the SE. Fusions (%) Total

Heterokaryons

Binucleate heterokaryons

Mass Chamber (n = 4)

36.8 • 2.1

19.5 4- 1.0

5.4 • 0.8

D.E.P. Chamber (n = 6)

15.4 m 2.3

6.2 • 0.1

2.9 • 0.9

PEG treated

17.0 • 1.0

10.0 • 0.5

3.1 :t_ 1.0

6.8 • 1.3

0.2 2= 0.1

0.2 • 0.1

(~ =

2)

Control

(~ = 5) Table 2. Comparison of some of the properties of different fusion-chamber designs. The mass chamber is shown and described in Fig. 4. The flow-through chamber has two parallel wires mounted on a glass or plexiglass slide and enclosed by a coverslip. Protoplasts are introduced into and removed from this chamber by means of plastic tubing and syringes. The trough-like chamber is a plexiglass slot with two parallel plates serving as electrodes Chamber

Advantages

Disadvantages

Mass

Easy to use Holds large numbers of cells Most cells exposed to appropriate electric fields

Poor visibility during fusion

Flow-through

High quality Operation curemicroscopic observabersome tions possible Many cells not during fusion exposed to appropriate fields

Trough-like

Easy to use Holds large numbers of cells All cells exposed to electric fields

Dielectrophoresis poor Overheating Poor visibility during fusion

plastic tubing. The electrodes in this chamber were gold wires (0.13 mm thick) strung along the chamber walls leaving a gap of 0.5 mm in which fusion took place. The effective internal volume of the chamber was about 20gl. Flow-through chambers with both single and multiple inlet ports were tested. Yields of recoverable fusion products were generally 1% or less.

The other chamber design tested consisted of an open, rectangular plexiglass trough. The inner faces of its two long side walls were lined with stainless steel plates (0.13 mm thick) leaving an electrode gap of 1 mm and a chamber volume of 0.25ml. However, AC fields large enough to produce dielectrophoresis in this chamber invariably led to overheating of the medium and cell death. Table 2 compares the advantages and disadvantages of the mass, flow-through and trough-like chambers described above.

Discussion

This study provides a detailed, quantitative assessment of the types of fusions induced by electric fields. By determining optimal electrical parameters and improving the design of the fusion chamber, batches of up to 5.105 protoplasts have been fused with yields of nearly 40%. Using similar procedures Bates and Hasenkampf (1985) have recovered viable somatic hybrids between Nicotiana tabacum and N. plumbaginiJblia. They found that 30% of the heterokaryons formed by electrical fusion were capable of cell divisions; unfused control cells had plating efficiencies of 50%. Considering that 40% of the heterokaryons probably have more than three nuclei (Fig. 5), and thus a reduced morphogenetic potential, a plating efficiency of 30% indicates good heterokaryon viability. Comparable viabilities were recently reported for PEG-induced fusions between Brass• and Nicotiana protoplasts (Menczel and Wolfe 1984). The available reports on the frequency of multinucleate cells following fusion by PEG (Kao 1977; Constabel et al. 1975a) indicate that binucleate heterokaryons comprise about one-third of the total heterokaryons formed. The proportion I found for electrically fused tobacco protoplasts was roughly similar. Electrical fusion differs from PEG in the formation of some large fusion bodies containing 10-30 nuclei, but these highly multinucleate fusion products present no difficulty in culture since they die rapidly. The fusion percentage I obtained with the mass chamber is better than that generally reported for PEG but is considerably less than the 100% reported by Zimmermann and Scheurich (1981) for the electrical fusion of Vicia mesophyll protoplasts. One reason for this discrepancy is undoubtedly how fusion efficiency is assessed. Zimmermann and Scheurich (1981) appear to have made microscopic

G.W. Bates: Electrical fusion of protoplasts in Nicotiana

observations of the fusions undergone by protoplasts in the center of their chamber where the electric fields were maximal, while I determined fusion percentages from the cells which were recovered from the chamber and available for culture. It is, of course, also possible that protoplasts of different species and cell types exhibit different maximal electrical fusion percentages. My data provide no evidence for the preferential production of either homo- or heterokaryons; fusion appears to be random. Vienken and Zimmermann (1982) reported preferential formation of heterokaryons of animal cells when a flow-through chamber was used. Using my flow-through chamber (Table 2) it was very difficult to control the flow of protoplasts into and through the chamber. Thus, the percentage of fusion products among the protoplasts recovered from the chamber was very low, indicating that procedures leading to preferential heterokaryon production would also result in a drastic cut in the yields of fusion products. High yields, that is large numbers, of fusion products are very important to cell-culture applications. Achieving sufficient yields has been a serious drawback to electrical fusion. The mass chamber described here not only gives efficient fusion but holds large numbers of protoplasts as well (up to 5-105 if they are suspended at 106/ml). Two other groups of workers have also developed fusion chambers which accomodate substantial numbers of cells (Watts and King 1984; Zachrisson and Bornman 1984). In both cases the chamber employed had flat-plate electrodes similar to the trough-like chamber I used (Table 2). Flat plates produce a uniform electric field and efficient dielectrophoresis requires a non-uniform AC field (Pohl 1978) like that produced by wire electrodes. When the AC field is large, some dielectrophoresis still occurs between flat-plate electrodes because the protoplasts create local distortions in the electric field. However, my results with flat-plate electrodes indicate that large-capacity chambers using this design will require a heat-dissipating system. The electric fields I found optimal for the fusion of Nicotiana protoplasts may not be optimal for the fusion of protoplasts of all other species. Zachrisson and Bornman (1984) and VerhoekK6hler et al. (1983) fused protoplasts of Brassica and Avena, respectively, with electric fields very close to those I used (120 V/cm AC, 950 V/cm DC; 200 V/cm AC, 750 V/cm DC, respectively). On the other hand, Zimmermann and Scheurich (198l)

223

fused ViciaJaba mesophyll protoplasts using considerably weaker fields (60V/cm, AC, and 375 V/cm, DC). In all of these cases fusion was efficient but it is not certain that the fields used were optimal. Nonetheless, the DC pulse used by Zimmermann and Scheurich (1981) to fuse Vicia protoplasts is below the threshold I found for fusion of Nicotiana protoplasts. This may in part be the result of differences in membrane composition but it could also reflect differences in cell size since the DC voltage at which fusion occurs is inversely proportional to the cell's radius (Jeltsch and Zimmermann 1979). This work was supported by a U.S. Department of Agriculture Competetive Research Grant. 1 also wish to thank Dr. James Pallas, Southern Piedmont Conservation Research Center, Watkinsville, Ga., USA for loan of the Zimmermann Cell Fusion Power Supply.

References Bates, G.W., Gaynor, J.J., Shekhawat, N.S. (1983) Fusion of plant protoplasts by electric fields. Plant Physiol. 72, 11101113 Bates, G.W., Hasenkampf, C. (1985) Culture of plant somatic hybrids following electrical fusion. Theor. Appl. Genet. 70, 227 233 Benbadis, A., deVirville, J.D. (1982) Effects of polyethylene glycol treatment used for protoplast fusion and organelle transplantation on the functional and structural integrity of mitochondria isolated from spinach leaves. Plant Sci. Lett. 26, 257-264 Bischoff, R., Eisert, R.M., Schedel, I., Vienken, J,, Zimmermann, U. (1982) Human hybridoma cells by electro-fusion. FEBS Lett. 147, 64-68 Constabel, F., Dudits, D., Gamborg, O.L., Kao, K.N. (]975a) Nuclear fusion in intergeneric heterokaryons, a note. Can. J. Bot. 53, 2092-2095 Constabel, F., Kirkpatrick, J.W., Kao, K.N., Kartha, K_K. (1975 b) The effect of canavanine on the growth of cells from suspension cultures and on intergeneric heterokaryocytes of canavanine sensitive and tolerant plants. Biochem. Physiol. Pflanz. 168, 319-325 Davey, M.R., Kumar, A. (1983) Higher plant protoplasts retrospect and prospect. Int. Rev. Cytol., Suppl. No. 16, 219 299 Finaz, C., Lefevre, A., Teissie, J. (1984) Electrofusion: A new, highly efficient technique for generating somatic cell hybrids. Exp. Cell Res. 150, 477~482 Halfmann, H.J., Emeis, C.C., Zimmermann, U. (1983) Electrofusion of haploid Saccharomyces yeast cells of identical mating type. Arch. Microbiol. 134, 1~4 Jeltsch, E., Zimmermann, U. (1979) Particles in a homogeneous electrical field: a model for the electrical breakdown of living cells in a Coulter Counter. J. Electroanal. Chem. 104, 349-384 Kao, K.N. (1975a) A method for fusion of plant protoplasts with potyethylene glycoI. In: PIant tissue culture methods, pp. 22-27, Gamborg, O.L., Wetter, L.R., eds. Prairie Research Laboratory~ Saskatoon~ Sask., Canada Kao, K.N. (1975b) A nuclear staining method for plant protoplasts. In: Plant tissue culture methods, pp. 60-62,

224 Gamborg, O.L., Wetter, L.R., eds. Prairie Research Laboratory, Saskatoon, Sask., Canada Kao, K.N. (1977) Chromosomal behaviour in somatic hybrids of soybean-Nicotiana glauca. Mol. Gen. Genet. 150, 225-230 Kao, K.N. (1981) Plant protoplast fusion and somatic hybrids. In: Plant tissue culture. The Pitman Int. Ser. in Appl. Biol., Proc. Beijing Symp., pp. 331-339, Han, H., ed. Pitman, London Kao, K.N., Michayluk, M.R. (1974) A method for high frequency intergeneric fusion of plant protoplasts. Planta 115, 355-367 Kartha, K.K., Gamborg, O.L., Constabel, F., Kao, K.N. (1974) Fusion of rapeseed and soybean protoplasts and subsequent division of heterokaryocytes. Can. J. Bot. 52, 2435-2436 Menczel, L., Wolfe, K. (1983) High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH. Plant Cell Rep. 3, 196-198 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 Nagy, J.I., Maliga, P. (1976) Callus induction and plant regeneration from protoplasts of Nicotiana sylvestris. Z. Pflanzenphysiol. 78, 453-455 Pohl, H.A. (1978) Dielectrophoresis. Cambridge University Press, Cambridge. Senda, M., Takeda, J., Abe, S., Nakamura, T. (1979) Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol. 20, 1441-1443

G.W. Bates: Electrical fusion of protoplasts in Nicotiana Shekhawat, N.S., Galston, A.W. (1983) Mesophyll protoplasts of fenugreek (Trigonellafoenumgraeeum):isolation, culture, and shoot regeneration. Plant Cell Rep. 2, 119-121 Verhoek-K6hler, B., Hampp, R., Ziegler, H., Zimmermann, U. (1983) Electro-fusion of mesophyll protoplasts of Avena sat# va. Determination of the cellular adenylate-level of hybrids and its influence on the fusion process. Planta 158, 199-204 Vienken, J., Zimmermann, U. (1982) Electric field-induced fusion: electro-hydraulic procedure for production of heterokaryon cells in high yield. FEBS Lett. 137, 11-13 Watts, J.W., King, J.M. (1984) A simple method for large-scale electrofusion and culture of plant protoplasts. Biosci. Rep. 4, 335 342 Zachrisson, A., Bornman, C.H. (1984) Application of electric field fusion in plant tissue culture. Physiol. Plant. 61, 31d~320 Zimmermann, U., Scheurich, P. (1981) High frequency fusion of plant protoplasts by electric fields. Planta 151, 26-32 Zimmermann, U., Vienken, J. (1982) Electric field-induced cellto-cell fusion. J. Membr. Biol. 67, 165-182 Zimmermann, U., Vienken, J., Pilwat, G., Arnold, W.M. (1984) Electro-fusion of cells: principals and potential for the future. In: Cell fusion (Ciba Foundation Syrup. 103), pp. 60-85, Evered, D., Whelan, J., eds. Pitman, London

Received 26 November 1984; accepted 11 March 1985