Fusion of Plant Protoplasts by Electric Fields - NCBI

Plant Physiol. (1983) 72, 1110-1113 0032-0889/83/72/11 10/04/$00.$0/0

Fusion of Plant Protoplasts by Electric Fields Received for publication February 8, 1983 and in revised form April 29, 1983

GEORGE W. BATES', JOHN J. GAYNOR2, AND NARPAT S. SHEKHAWAT Department of Biology, Yale University, New Haven, Connecticut 06511 ABSTRACT

MATERIALS AND METHODS

The electrical fusion technique of Zimmermann and Scheurich (1981 Planta 151: 26-32) has been used to fuse mesophyll protoplasts of Avena, Zea, Vigna, Petunia, and Amaranthus. Electrical fusion proves to be a simple, effective, and general fusion technique that can be controlled to form either dikaryons or large multinucleate fusion bodies. In addition, we show that Vigna mesophyll protoplasts that are subjected to the electrical fields used in this technique are viable in culture. The construction of the fusion chambers, necessary electrical equipment, and the fusion protocol are described in sufficient detail for reproduction of the technique.

Fusion Chamber. We used the flow-through chamber diagrammed in Figure 1. It was built of two pieces of glass glued onto a glass microscope slide with epoxy cement. Two metal rods, which served as electrodes, were glued between these pieces of glass so that an even slot 500 ,um wide remained in the center of the chamber. Care was taken to keep this central slot free of glue. The ends of the chamber were fitted with polyethylene tubing to make an inlet and an outlet port. The entire chamber was covered with a cover slip and sealed with epoxy cement. Several different types of metal were tried as electrodes; gold, platinum-iridium, and silver all worked equally well. The crucial factor in making a successful chamber proved to be getting the electrodes as nearly

parallel as possible. The fusion of protoplasts has become an important tool in plant somatic cell genetics, especially for the production of interspecific hybrids (10). Recently, the list of methods available for protoplast fusion has been expanded by the development of techniques for fusing cells electrically (8, 14). The electrical technique uses a short DC pulse, of sufficient voltage to cause reversible membrane breakdown, to fuse the protoplasts. Obviously a prerequisite for fusion is intimate contact between the membranes of adjacent cells. Senda et al. (8) provided membrane contact by pushing two protoplasts together with the microelectrodes used to give the fusogenic electrical pulse. Although this approach was effective, its usefulness is limited by the small number of cells that can be fused at one time. An elegant improvement on this technique, developed by Zimmermann and coworkers (13, 14), now makes possible the simultaneous fusion of large batches of protoplasts by electrical fields. Zimmermann's method appears to have several important advantages over the standard PEG technique for protoplast fusion. (a) Fusion is rapid, highly synchronous, and usually complete within 15 min. (b) The yield of fusion products can be very high (50-80%). (c) Electrically induced protoplast fusion does not require any chemical treatment. (d) The electrical fusion technique affords a greater degree of control over the number of cells fusing and possibly even the types of fusions produced (i.e. heterokaryons as opposed to homokaryons) than does PEG. Despite the outstanding attributes of Zimmermann's technique, it has not been shown that protoplasts are still capable of growth and cell division after being subjected to the strong electrical fields required for fusion. Clearly, this is an extremely important point if the technique is to be applied to somatic cell genetics. We report here that the protoplast fusion technique of Zimmerman and Scheurich (13) is reproducible and relatively simple. We also show that Vigna protoplasts that have been subjected to the electrical fusion technique remain capable of cell division in culture. ' Present address: Department of Biological Science, Florida State University, Tallahassee, FL 32306. 2 Present address: Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10021.

Electronics. AC fields were provided by a Hewlett Packard oscillator model 200CD. A Grass Medical Instruments (Quincy, MA) stimulator model S4 was used to give the DC pulses. These instruments were arranged in parallel with each other and the fusion chamber. The performance of the stimulator and oscillator were monitored on an oscilloscope. Isolation of Oat and Corn Protoplasts. The upper epidermis of leaves from light-grown, 6-d-old oat seedlings (A vena sativa, cv Victory) and 8-d-old corn seedlings (Zea mays, cv Bear Hybrid) were removed with fine forceps. The peeled leaves were floated, upside down, on a solution containing 2% (w/v) Cellulysin (Calbiochem), 0.5 M mannitol, 3 mm CaCl2, 1 mm KCl, and 3 mm Mes (morpholinoethanesulfonic acid) at pH 5.6. Digestion was complete after 3 h at 30'C in the dark. The protoplasts were filtered through a nylon screen (pore diameter 80 ,um), layered onto a 17% (w/v) sucrose pad, and centrifuged 10 min at 100g. The protoplasts at the interface were collected, resuspended in 12 ml of 0.5 M mannitol and centrifuged for 3 min at 70g. The pellet was washed once with 0.5 M mannitol. Isolation of Amaranthds Protoplasts. Primary leaves from 7-dold plants of Amaranthus cruentus (strain R102/104) were cut into 1-mm-thick strips, washed briefly with 0.6 M mannitol, and infiltrated 4 min under vacuum with 1% (w/v) Cellulysin, 0.5% (w/v) Pectolyase Y-23, 0.6 M mannitol, 5 mm CaCl2, and 5 mm Mes at

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FIG. 1. Diagram of the fusion chamber; the drawing is not necessarily to scale. The inter-electrode distance, which is the critical dimension, is 500 pm. + and - designate the two electrodes, and the arrows indicate the

path of the protoplasts through the chamber.

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FUSION OF PROTOPLASTS BY ELECTRIC FIELDS

pH 5.8. Tissue slices were gently shaken, in the dark, at 30C for 4 h. The protoplasts were passed through an 80-,tm nylon screen and purified on an isoosmotic sucrose gradient as described by Harms and Potrykus (2). The isolated protoplasts were resuspended in 0.5 M mannitol. Isolation of Petunia Protoplasts. The upper surfaces of leaves of Petunia axillaris were removed and the leaves were floated on 2% (w/v) Cellulysin, 1% (w/v) Pectinol AC (Rohm and Haas Co.), 0.5 M mannitol, 3 mm CaCl2, 1 mm KCl, and 3 mm Mes at pH 5.7. Digestion took 4 h at 30'C. The Petunia protoplasts were recovered and purified by the same methods used for oats and corn. In early experiments, the pectinase was omitted from the digestion medium. Isolation and Culture of Vigna Protoplasts. Mesophyll protoplasts were isolated from Vigna aconitifolia 'Jadia' as described by Shekhawat and Galston (9). Briefly, primary leaves of 14-d-old plants were surface sterilized for 5 min in 5% (w/v) commercial bleach containing a few drops of Tween-80. After 5 to 6 washings in sterile distilled H20, the lower epidermis was removed and the leaves were floated on desalted 1% (w/v) Driselase (Kyowa Hakko, Co., Ltd., Japan), 9% (w/v) mannitol, salts (KH2PO4, 27.2; KNO3, 101; CaCl2 2H20, 148; MgSO4.7H20, 240; and KI, 0.16 mg/l), and 3 mM Mes, at pH 5.8. This solution was filter sterilized. After a 5-h digestion at 30°C the protoplasts were passed through a 55-,um nylon mesh and centrifuged for 5 min at 100g. The protoplast pellet was resuspended in 9% mannitol and recentrifuged. The final pellet was resuspended in 9% mannitol layered over 2 ml of a 21% (w/v) sucrose solution and centrifuged at lOOg for 5 min. The protoplasts at the interface were resuspended in 9% mannitol. After fusion, the protoplasts were concentrated by centrifugation and cultured in 25-,ul sitting drops on the bottoms of 60 x 15 mm plastic Petri dishes. The culture dishes were sealed with parafilm and maintained at 26°C under day light fluorescent tubes at a Photon Flux Density of 30 to 50 ,iE/m2 -s. The culture medium contained Murashige and Skoog (6) macro- and micronutrients with KM vitamins and organic acids (4); CaCl2.2H20, 900 mg/l; sucrose, 30 g/l; L-glutamine and L-asparagine (2 mm each); arginine, 10 mg/l; mannitol, 45 g/l; glucose, 30 g/l; ribose, 500 mg/l; and xylose, 300 mg/l. Hormones were supplied as 0.5 mg/l each of 2,4-D, naphthaleneacetic acid, BA, Zeatin, and GA3. The pH of the culture medium was adjusted to 5.8 before filter sterilization. RESULTS The electrical fusion of protoplasts is a two-step process. First, the protoplasts are subjected to a high-frequency AC field (>100 kHz), which draws the protoplasts towards regions of higher field strength, that is toward the electrodes. This phenomenon, called dielectrophoresis, has been described in detail by Pohl (7). Dielectrophoresis requires a medium of low conductivity. Thus, compared with the medium, the protoplasts are a highly conductive electrical path and the poles of the protoplasts also become local regions of high field strength. Consequently, the protoplasts are attracted to each other as well as to the electrodes and become aligned in 'pearl chains' along the lines of force of the AC field (Fig. 2). Once cell contact has been established, the second step is to superimpose a DC pulse (duration 2-100 ,us) of sufficient magnitude (about 1 kv/cm) to produce reversible breakdown of the cell membrane (1, 5). This procedure causes the fusion of neighboring protoplasts within the pearl chains. Cell lysis rather than fusion results if the DC pulse is too long or too large (13). Oat-Oat Fusions. Initial experiments were carried out with oat protoplasts suspended in 0.5 M mannitol (conductivity, mesoph7ll 4 x 10 mho/cm). A small number of protoplasts were introduced into the chamber and an AC field of 10 v (peak to peak) at 500 kHz was applied. Chains of protoplasts form on each electrode in

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FIG. 2. Examples of pearl chains. Oat mesophyll protoplasts aligned in an AC field (500 kHz, 100 v/cm) at a low density. Under these conditions, pearl chains two to three cells long predominate.

about 10 s (Fig. 2). In all of the experiments discussed in this paper, an AC frequency of 500 kHz was used. At lower frequencies, especially those below 100 kHz, dielectrophoresis still occurs, but many of the protoplasts also spin. This spinning motion breaks membrane contact and prevents fusion. Pearl chains were found to form in AC fields of 5 to 15 v (50150 v/cm). The AC field strength and the number of cells allowed into the chamber control the lengths of the pearl chains. Pearl chains two to three cells long predominate when the density of cells and the AC field strength are kept low (Fig. 2). Fusion, however, is most efficient when the AC field strength is fairly high because of the increased area of contact between the protoplasts. Therefore, in order to develop and fuse short chains of protoplasts, a low-strength AC field was used to form the pearl chains, then the chamber was gently flushed with mannitol to remove any unaligned cells, and the AC field was increased in strength. We found that dielectrophoresis was not prevented by the presence of 0.1 mm CaCl2 in the fusion medium; however, higher concentrations of salt were inhibitory. Inclusion of 0.1 mm CaCl2 seemed to have no effect on the fusion rate, so it was therefore generally omitted from the medium. However, in an effort to keep the cells viable and to reduce lysis, calcium was supplied during protoplast isolation. Once pearl chains had formed, the oat protoplasts were fused by application of a DC square wave. We found that DC pulses of 10 to 50 ,is duration and 35 v (700 v/cm) were effective. The precise DC voltage required for fusion varies somewhat between experiments and appears to be a function of the number of cells in the chamber. Our general procedure was to give a DC pulse of 25 v, and if no fusion occurred the voltage was increased stepwise by 5 v until fusion was observed. Because applying trains of DC pulses causes cell lysis and electrolysis, we allowed 30 s between successive pulses. When the critical voltage for fusion is reached, the cells elongate immediately following the pulse and flatten at their points of contact (Fig. 3b). Fusion of the plasma membranes is probably immediate, but actual coalescence of the protoplasts takes 2 to 5 min for oats. After fusion, the AC field strength was reduced because this procedure was found to shorten the time required for the fusion product to round up as a single protoplast

(Fig. 3d).

The percentage of protoplasts in the chamber that fuse depends on the uniformity of the lengths of the pearl chains. Cells in longer chains undergo fusion at lower DC voltages than cells in short chains. This problem can be largely overcome by giving several DC pulses at 30- to 60-s intervals. However, as already mentioned this procedure causes some cell lysis. We found that 2 or 3 DC

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FIG. 4. Fusion sequence of corn and oat mesophyll protoplasts. The oat protoplast is the darker of the two because of staining with neutral red. The corn protoplast was unstained. In 'a' the protoplasts are aligned in the AC field (500 kHz, 100 v/cm). b to f are at various times following the DC pulse (20 ps, 700 v/cm): (b) 5 s; (c) 30 s; (d) 2 min, (e) 5 min, (f) 10 min. Just before the photograph in 'e' was taken, the AC field was disconnected.

with the dicots with precisely the same settings and approaches that have been described for oat protoplasts. However, the dicot FIG. 3. Fusion sequence of two oat mesophyll protoplasts. These pro- protoplasts were more resistant to electrical fusion than the montoplasts were aligned in an AC field of 70 v/cm at 500 kHz (a). Then a ocots were. DC pulses of 10 to 20 ps duration, which were very DC square wave (15 Us, 800 v/cm) was applied. Photographs were taken efficient in fusing oat and corn protoplasts, rarely produced any at the following intervals after the DC pulse: (b) 5 s; (c) 75 s; (d) 135 s. Jusi dicot fusions. Moreover, a greater DC voltage was required for before the photograph in 'd' was taken, the AC field was disconnected. fusing the dicot protoplasts than for the monocot protoplasts. The optimal settings of the DC pulse for fusing the dicot protoplasts pulses of sufficient voltage to cause fusion could be given safely proved to be 50 ,is and 50 to 60 v (1.0-1.2 kv/cm). Even with and maximized the number of protoplasts fused. We have not these settings the rates of dicot fusion were lower than those seen tried to quantify the percentage of cells fused, but the reports of for the monocots. We found, however, that the fusion of dicot 50 to 80% fusion rates (13, 14) appear to be justified as long as the protoplasts was greatly enhanced if a pectinase was included pearl chains are quite uniform in length. during protoplast isolation. Possibly residual cell wall material or If a large number of protoplasts are introduced into the cham- cell surface determinants were blocking fusion. ber, the pearl chains can become long enough to bridge the gap Vigna protoplasts were subjected to electrical fusion, recovered between the electrodes. Under these conditions, electrical fusion from the fusion chamber and cultured in microdrops. Cell wall results in the production of giant cells. We have recovered oat synthesis and cell division were monitored by staining of the fusion products with as many as 25 nuclei. protoplasts with Calcofluor White ST (American Cyanamid Following fusion, we disconnected the AC field and collected Corp.). Immediately after recovery from the fusion chamber the the protoplasts by flushing the chamber with fresh medium. The protoplasts lacked any fluorescence when stained with Calcofluor unfused protoplasts do not stick to each other in the absence of White. After 3 d in culture, the protoplasts had reformed cell walls the AC field. However, some protoplasts do adhere to the elec- and some dividing cells were observed. After 5 d in culture, about trodes. Most ofthe fusion products can be recovered if the medium 50%1o of the cultured cells had undergone at least one division. Cell is gently forced back and forth in the chamber a few times. wall biosynthesis and the cell division rates of electrically treated Corn-Oat Fusions. Mesophyll protoplasts prepared from oat cells were identical to those of control cells, which were cultured and corn leaves were readily fused with each other. So that the directly (9) or were passed through the chamber (without electrical two types of protoplasts could be distinguished, the oat protoplasts stimulation) and then cultured. were stained with neutral red for 5 min (and washed) prior to fusion. Inasmuch as our chamber contains only a single inlet, the DISCUSSION two types of protoplasts had to be mixed before they were introduced into the chamber. Under these conditions, we had no Our experiments support the observations of Zimmermann and control over whether homokaryons or heterokaryons were pro- his co-workers to a large degree. We find that electrically induced duced. However, the experiment clearly shows that heterokaryons cell fusion is a rapid, simple, and controllable process, which can be produced by electrical fusion. Figure 4 shows a time works with a wide range of plant species. Moreover, with monocots sequence of electrically stimulated fusion of a corn and an oat we were able to obtain very high rates of fusion. The success we had with culturing the Vigna protoplasts indiprotoplast. The specific frequency and voltages required for dielectrophoresis and fusion were exactly the same for corn and oat cates that viable protoplasts can be recovered following applicaprotoplasts. Coalescence of the protoplasts took longer in this tion ofthe electrical fusion technique. Because no selection scheme experiment than in that shown in Figure 2. This range of fusion was applied after fusion we do not know whether any of the times probably reflects some difference between the protoplast dividing Vigna protoplasts were actually fusion products. Howpreparations used in these experiments. ever, the dividing cells were subjected to all of the electrical fields Fusion of Dicot Protoplasts. We have successfully fused proto- and manipulations required for production, fusion, and recovery plasts from several species of dicots by the electrical technique, of the protoplasts under sterile conditions. Therefore, it is highly including those from Amaranthus, Vigna, and Petunia. All of these likely that some of the fusion products are also viable. were homokaryotic fusions. Dielectrophoresis occurred readily We encountered two problems with the electrical fusion tech-

FUSION OF PROTOPLASTS BY ELECTRIC FIELDS

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nique: (a) the protoplasts often adhered to the electrodes and (b) two types of cells into the chamber. Use of this approach could be important to the field of plant somatic cell genetics as it might dicots were more difficult to fuse than monocots. The protoplasts stuck to all of the metals we tried as electrodes make possible the recovery of large numbers ofheterokaryons and (i.e. gold, silver, and platinum-irridium). Moreover, the longer the thereby reduce the need for a stringent post-fusion selection protoplasts were in contact with the electrodes the more strongly procedure. they became attached. While most of the adhering protoplasts Acknowledgments-We wish to thank Drs. Philip Applewhite and Timoth Goldcould be recovered if the medium was forced back and forth in smith for generously lending electrical equipment and Drs. Arthur Galston and Mary the chamber, this procedure does break some cells. Although Helen Goldsmith for their advice and support. N. S. S. thanks the Ministry of flushing the chamber with 200 mm KCI did not release the Education and Culture, Government of India, New Delhi, for granting National protoplasts, it is likely that the initial interaction of the cells with Scholarship for Study Abroad, 1980-81. G. W. B. thanks the Whitehall Foundation the electrodes is electrostatic and is probably followed by a for support. progressive molding of the plasma membrane to the electrode's LITERATURE CITED surface. It should be possible to reduce adhesion by placement of U ZIMMERMANN 1981 High electric field effects on the cell membranes 1. BENz R, an inert membrane filter between the protoplasts and the elecof Halicystis parvula A charge pulse study. Planta 152: 314-318 trodes. CT, T POTRYKUS 1978 Fractionation of plant protoplast types by isoThe observation that adding a pectinase to the protoplast iso- 2. HARMs osmotic density gradient centrifugation. Theor Appl Genet 53: 57-63 lation medium makes dicot protoplasts easier to fuse indicates that 3. KAo KN, F CONSTABEL, MR MICHAYLUK, OL GAMBORG 1974 Plant protoplast fusion and growth of intergeneric hybrid cells. Planta 120: 215-227 part of the difficulty in fusing these protoplasts is related to 4. KAo KN, MR MICHAYLUK 1975 Nutritional requirements for growth of Vicia A observation was walls. of their cell similar incomplete removal hajastana cells and protoplasts at a very low population density in liquid media. made by Kao et al. (3) during PEG-induced fusion of dicot Planta 126: 105-109 protoplasts. Because cell fusion requires very close approach of 5. KINOSITA K, TY TSONG 1977 Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature 268: 438-440 the lipid phases of the two cell membranes, any exposed memT, F SKOOG 1962 A revised medium for rapid growth and bio assays brane proteins or residual wall material adhering to the membrane 6. MURASHIGE with tobacco tissue cultures. Physiol Plant 15: 473-497 could prevent fusion. Consistent with this view is the report by 7. PoHL HA 1978 Dielectrophoresis. Cambridge University Press, Cambridge Zimmermann et al. (12) that pretreating animal cells with pronase 8. SENDA M, J TAKEDA, S ABa, T NAKAMURA 1979 Induction of cell fusion of plant by electrical stimulation. Plant Cell Physiol 20: 1441-1443 protoplasts greatly increases the yield of electrically induced fusion products. NS, AW GALSTON 1983 Isolation, culture and regeneration of moth Undoubtedly, the fusion rates of the dicot protoplasts can be 9. SHEKHAWAT bean ( Vigna aconitifolia) leaf protoplasts. Plant Sci Lett In press improved even further with similar treatments. 10. VASIL IK, V VASIL 1980 Isolation and culture of protoplasts. Int Rev Cytol I IB: Future work needs to address the problem of identifying and 1-19 recovering heterokaryons. This is, of course, the same problem 1 1. VIENKEN J, U ZIMMERMANN 1982 Electric field-induced fusion: electro-hydraulic procedure for production of heterokaryon cells in high yield. FEBS Lett 137: that arises when any technique is used to fuse protoplasts. How11-13 ever, electrically stimulated fusion may simplify this problem by 12. ZIMMERMAN U, G PILWAT, H-P RICHTER 1981 Electric-field-stimulated fusion: increased field stability of cells induced by pronase. Naturwissenschaften 68: making possible high yields of heterokaryons. Vienken and Zim577-579 merman (11) reported that viable heterokaryons of lymphocytes U, P SCHEURICH 1981 High frequency fusion of plant protoplasts and murine myeloma cells could be produced by electrical fusion 13. ZIMMERMAN by electric fields. Planta 151: 26-32 with yields of 60 to 80%o. This result required a fusion chamber 14. ZIMMERMANN U, J VIENKEN 1982 Electric field-induced cell-to-cell fusion. J Membr Biol 67: 165-182 with multiple inlet ports to allow sequential introduction of the