Planta
Planta (1989) 178:207-214
9 Springer-Verlag1989
Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans Paul T. Lynch*, Susan Isaac **, and Hamish A. Collin Department of Genetics and Microbiology, Nicholson Building, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK
Abstract. A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from each species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion. Key words: Apium (protoplast fusion) - Aspergillus (protoplast fusion) - Electrofusion (protoplasts) Protoplast fusion
Introduction
The use of high-strength electrical fields combined with the electrically induced formation of chains of cells (pearl chains) has been used to stimulate cell fusion (Zimmermann and Scheurich 1981). Such techniques offer more controllable and more efficient methods for protoplast fusion than chemi* Present address: Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK ** To whom correspondence should be addressed Abbreviations: AC = alternating current; DC ~ direct current
cal treatments. The use of electrofusion techniques avoids the need for toxic chemical stimulants and excessive washing of cells (Bates 1985). Chemical agents induce random aggregation and fusion events (Watts and King 1984). Electrofusion, however, combined with micromanipulation of electrodes, permits parentage of heterokaryons to be precisely defined (Koop and Schweiger 1985; Morikawa et al. 1988). Electrofusion can also be monitored microscopically, which aids determination of the electrical parameters required for heterokaryon formation and permits the observation of fusion events (Zachrisson and Bornman 1986). Additionally Arnold and Zimmermann (1984) have reported that zones of membrane disturbance can be restricted to zones of membrane contact, whereas chemical treatments affect the whole protoplast plasmelemma. Reduced membrane disturbance may maintain protoplast viability. Chemical treatments usually result in fusion frequencies of 1-5% (Zachrisson and Bornman 1986). Electrofusion can result in fusion frequencies, between plant protoplasts, in excess of 50% (Bates and Hasenkampf 1985; Watts and King 1984). Electrofusion is a two-stage process. Initially, the subjection of protoplasts to a non-uniform electrical field causes polarized protoplasts to move towards regions of higher field strength and the formation of pearl chains results (Arnold and Zimmermann 1984). This effect is termed dielectrophoresis (Pohl 1978). Subsequent application of short DC (direct current) pulses causes breakdown of aligned membranes and protoplast fusion results. After fusion, protoplasts round up into spheres (Zimmermann et al. 1985). The technique has been used successfully to fuse a range of protoplast types e.g. fl-lymphocytes and myeloma cells, to produce hybridoma cells (Zimmermann and Vienken 1982); Friend cells with Petunia protoplasts
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(Salhani et al. 1985); plant protoplasts (Morikawa et al. 1987; Terada et al. 1987); yeast protoplasts (Emeis 1987) and protoplasts from filamentous fungi (Kunkel et al. 1987). The technique of electrofusion offers the possibility for examination of physiological interactions between different cell types. Providing, for example, opportunities for detailed measurements of short-term metabolic interactions between plant and fungal cells. Differences in cell fusability have been related to differences in size, membrane characteristics and cytoplasmic properties between cells and protoplasts which control their electrical characteristics (Zimmermann 1982). To maximise fusion frequency and to preserve viability, electrofusion parameters must be carefully assessed for each protoplast type. The aim of this work was to develop a method for the electrofusion of higher-plant protoplasts (celery) and protoplasts from a filamentous fungus (Aspergiltus nidulans), to assess the fate of fused products and to determine the effects on viability. In the first instance, independent determinations of electrical parameters were made for each species. Subsequently, a method was developed for fusion between the protoplast types. The effects of electrofusion on treated protoplasts, during a 24-h incubation were assessed.
Liberation of protoplasts from celery cells. Protoplasts were iso-
Materials and methods Initiation of celery cultures. Celery seed (Apium graveolens Linn.) cv. French Dimant (Thompson and Morgan, Ipswich, Norfolk, UK) was surface sterilised by immersion in 20% Domestos (Lever Bros., Port Sunlight, Merseyside, UK) solution for 8 min and subsequently washed in three changes of sterile distilled water. Seeds were transferred on to germination medium; Murashige and Skoog (1962) salts (MS) 4.71 g-l-1 (Flow Laboratories, Irvine, UK), supplemented with 30 g. 1-1 sucrose and solidified with Bacto Agar 10 g.1-1 (Difco, Detroit, Mich, USA) in glass Universal vials. Cultures were maintained under continuous light at 4 ~ for 48 h and subsequently at room temperature. Sterility and germination were assessed 30 d after sowing. Germination occurred within three to four weeks. When seedlings had developed four to six leaves (six to eight weeks after sowing), sections of petiole (1 cm) were excised and ptaced on to medium for callus initiation; MS salts 4.71 g. 1-1 supplemented with 30g.1 i sucrose, 10g.1 -~ agar, 0.25 rag. 1 1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.6 rag.l-1 N6_furfurylaminopurine (kinetin). Cultures were maintained at 20 ~ C under a 12-h light/dark cycle. Fast growing, friable callus was selected 37 d after initiation and subcultured monthly. Celery suspension cultures were prepared by inoculating 50 ml of MS medium supplemented with 30 g-1 1 sucrose, 0.25 rag. 1-x 2,4-D, 0.5 rag.l-1 kinetin, with approximately 5 g friable undifferentiated callus. Cultures were grown under a 12-h light/dark cycle at 20 ~ C with shaking (100 r.p.m.). Cultures were continuously selected for fast-growing, finely dispersed clones and subcultured at weekly intervals by inoculating 10 ml of a 7-d-old culture into 50 m! fresh medium.
lated from 3-d-old suspension cultures (late lag phase), using a modified method of Orton (1984). Protoplast isolation mixture was composed of 0.6 M mannitol with 0.1% Pectolyase Y23 (Seishin Pharmaceutical Co., Japan) and 2% Cellulysin (Cambridge Bioscience, Cambridge, UK). Incubations of 16 h (overnight) with shaking (40 r.p.m.) at 30 ~ C resulted in a protoplast yield of 9.2.103_+0.81 . 103.rag - t cell dry weight. After harvest, protoplasts were resuspended in 0.6 M mannitol plus 0.5 mM CaCI~-2H20.
Fungal culture. Aspergillus nidulans. B D U N 33 (University of Nottingham collection) was maintained on 1% (w/v) malt extract (Oxoid, Basingstoke, UK) solidified with 1.5% (w/v) agar (Difco). Cultures grown on agar slopes in 300-ml medical flat bottles at 37 ~ C for 2 d were used as sources of conidia.
Liberation offungalprotoplasts. Protoplasts were liberated from A. nidulans mycelium grown in Iiquid culture (18 h) by the method of Isaac and Gokhale (1982). A final concentration of 0.2 M phosphate buffer pH 5.8 plus 0.4 M mannitol was used as buffer/stabiliser with Novozym 234 (Novo Industri, Copenhagen, Denmark) at 1 mg.m1-1. Lyric incubation was with gentle shaking at 30 ~ C for 3 h. Protoplasts were harvested as described by Peberdy and Isaac (1976) and resuspended in 0.6 M mannitol plus 0.5 mM CaCI2.2H20.
Nuclear staining. Nuclei of protoplasts were observed microscopically using the method of L i n n e t al. (1977). A solution of DAPI (4,6-diaminido-2-phenylindole; Sigma, Poole, Dorset, UK) was prepared in 0.1 M (Tris) 2-amino-2-(hydroxymethyl)1,3-propanediol-HC1 buffer pH7.0, plus 0.1 M NaC1 and 0.01 M ethylenediaminetetraacetic acid (EDTA). Protoplast samples were fixed in osmotically stabilised 2% gluteraldehyde solution buffered with 0.1 M Tris-HC1 buffer pH 7.0 for 30 rain at room temperature. Samples were resuspended in buffer/stabiliser solution. A drop of protoplast suspension was mixed with DAPI (diIuted to give 300 ng.mI ~ on the slide) and allowed to stand for 5 rain to allow uptake of the stain. Observations were made under ultraviolet illumination.
Eleetrofusion apparatus. Electric fields were generated by a Zimmermann Cell Fusion TM System (GCA Corporation, Chicago, Ill., USA), described by Zimmermann and Scheurich (1981). Two types of fusion chamber (supplied by GCA Corporation) were used. A flat fusion chamber (7 gl capacity), similar to that described by Pilwat et al. (1981) and a helical chamber (200 gl capacity) of similar construction to that described by Zimmermann and Vienken (1984).
Eleetrofusion of celery protoplasts. Alignment and fusion parameters for celery protoplasts (1-103 protoplasts-ml z) were determined in a 100-gin flat fusion chamber, and used to fuse celery protoplasts unless otherwise stated. Protoplasts were aligned in a 1.5 MHz, 200 V. c m - 1 electric field and the number of aligned protoplasts assessed. After approx. 45 s, protoplasts were fused using two 150 V. cm 1 fusion pulses of 99 gs duration, with a 1-s period between pulses. After the second pulse the AC (alternating current) field was smoothly damped to zero V over a 30-s period. The number of adhering protoplast pairs (membrane contact areas) which fused, was determined (fusion events) by microscopic observation. For larger volumes a helical fusion chamber was used.
Electrofusion offungat protoplasts. Fusion parameters were determined in a 100-1al fusion chamber, loaded with 1- 103 proto-
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plasts.m1-1. Protoplasts were aligned in a 1.0 MHz, 1000 V. cm- 1 electric field for about 45 s. Fusion was achieved by two, 500 V.cm -1 DC pulses of 50 Ixs duration with a 0.5-s period between pulses. After the second pulse, the AC field was smoothly damped to zero V over a 30-s period, Aignment and fusion characteristics were assessed as described for celery protoplast electrofusion.
Table 1. Electrofusion parameters used to stimulate maximum
Electrofusion of celery andfungal protoplasts. Immediately prior to electrofusion, celery protoplasts were incubated for up to 1 h at 30~ with shaking in 0.6 M mannitol and 0.5 mM CaClz.2HzO plus 1 mg.ml 1 Pronase E (protease, Type XXV; Sigma) after which protoplasts were washed and resuspended in 0.6 M mannitol plus 0.5 mM CaClz.2HzO. A suspension of equal numbers of both protoplast type (1.103 protoplasts. m1-1) were used in 100-pm flat fusion chambers. Protoplasts were aligned in 3.0MHz, 400V-cm 1 electric fields. The numbers of celery protoplasts associated with A. niduIans protoplasts was assessed by microscopic observation. Protoplasts were fused by two 250 V. cm-1 fusion pulses of 75 gs duration, with a 1-s period between pulses. The AC field was damped to zero V over a 30-s period after fusion, during which time the number of celery protoplasts which fused with A. nidulans protoplasts was determined. Celery and fungal protoplasts were fused using these parameters unless otherwise stated. Larger volumes were fused in a helical chamber.
Alignment frequency 1.5 (MHz) Alignment voltage 200 (V.cm -1) Fusion voltage t 50 (V.cm- 1) Number of DC pulses 2 Pulse duration 99 (~s) Time between pulses 1.0 (s)
Protoplast incubation and viability afterfusion. Protoplasts were incubated in 0.6 M mannitol with gentle shaking, at 20~ C in a 12-h light/dark cycle for up to 24 h. It was anticipated that short-term changes would be likely to occur during this time. Viability was measured by the ability of protoplasts to exclude Evans' Blue vital dye (Gaff and Okong-Ogola 1971). Assessments were made at intervals throughout incubation. Respiration rates. Protoplast oxygen consumption was measured at intervals for 24 h after fusion using a Rank Oxygen Electrode (Rank Brothers, Cambridge, UK) calibrated at 25~ C. To avoid sedimentation of protoplasts a magnetic stirrer was used at constant speed.
Results
Electric-field treatment of celery protoplasts. T h e a p p l i c a t i o n o f h e t e r o g e n e o u s (AC) electrical fields resulted in a l i g n m e n t o f celery p r o t o p l a s t s into pearl chains o f various lengths. Chains f o r m e d parallel to electric fields a n d tended to associate with one or b o t h electrodes. P a r a m e t e r s were selected to f a v o u r the f o r m a t i o n o f p r o t o p l a s t pairs. A m i n i m u m f r e q u e n c y o f 0.1 M H z was required to induce chain f o r m a t i o n . The m a x i m u m n u m b e r o f celery p r o t o p l a s t s f o r m e d pearl chains at 2.0 M H z , b u t p r o t o p l a s t p a i r association was m a x i m u m in 1.25 M H z . M o s t p r o t o p l a s t s f o r m e d chains in a field strength o f 200 V. c m - 1, a n d m o s t p r o t o p l a s t pairing o c c u r r e d at the s a m e field strength. A t high field strength, p r o t o p l s t a l i g n m e n t was very rapid, p r o t o p l a s t s s o m e t i m e s b e c a m e distorted a n d occasionally r u p t u r e d .
fusion between protoplasts Celery/ celery protoplast fusion
A. nidulans/ A. nidulans protoplast fusion 1.0
Celery/ A. nidulans protoplast fusion 3.0
1000
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2 50
2 75
0.5
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D i r e c t - c u r r e n t pulses resulted in fusion o f celery p r o t o p l a s t s at points o f p l a s m a - m e m b r a n e contact. Fusion f r e q u e n c y was d e p e n d a n t on the strength o f D C pulses, the m a x i m u m n u m b e r o f fusion events (83.2_+6.8%, m e a n + S E ) o c c u r r e d after 150 V. c m - 1 pulses. F u r t h e r increases in D C pulse strength resulted in m o v e m e n t o f p r o t o p l a s t s during pulse application, a n d also lysis o f s o m e larger p r o t o p l a s t s . After electrofusion, p r o t o p l a s t s r o u n d e d u p into discrete spheres. P r o t o p l a s t viability was m o n i t o r e d for 24 h after electrofusion. D u r i n g the fusion process the n u m b e r o f viable p r o t o p l a s t s was reduced f r o m 96.7 +_ 1.2% to 67.1 + 1.2%. After fusion, viability decreased b y only a further 7 % during 24 h incubation. The electrofusion process g e n e r a t e d 2 4 % multinucleate celery p r o t o p l a s t s . D u r i n g the subseq u e n t 24 h large, multinucleate p r o t o p l a s t s were preferentially lost.
Electrofusion of A. nidulans protoplasts. The p r o t o plasts o f A. nidulans f o r m e d pearl chains in an A C field. A n a l i g n m e n t field f r e q u e n c y o f 3.0 M H z caused all p r o t o p l a s t s to f o r m pearl chains but m a x i m u m p a i r f o r m a t i o n o c c u r r e d at 1.0 M H z . A m i n i m u m field strength was required to stim u l a t e p e a r l - c h a i n f o r m a t i o n . A b o v e 100 V. c m 1 the total n u m b e r o f p r o t o p l a s t s in pearl chains rose. In excess o f this, b o t h p r o t o p l a s t lysis a n d r o t a t i o n occurred. A l i g n m e n t voltages required were m u c h higher t h a n for a l i g n m e n t o f celery p r o toplasts (Table 1). Pulses o f D C current resulted in the fusion o f A. nidulans p r o t o p l a s t s at points o f m e m b r a n e contact. A p p l i c a t i o n o f D C pulses caused m o v e m e n t o f p r o t o p l a s t s . Fusion voltages were m u c h higher t h a n required for celery p r o t o p l a s t fusion (Ta-
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80
~
60
u~ m
Fig. 1. Pearl chains of celery (CP) and A. nidulans (FP) protoplasts; AC field 3.0 MHz, 400 V . c m - L Bar represents 10 gm !
ble 1). Maximum fusion events (/7.2_+ 2.0%) were observed after two DC pulses, a reduced number of events were observed after pulses in excess of 50 gs. Electric-field treatment of celery and A. nidulans protoplast m&tures. In an AC field, celery and A. nidulans protoplasts formed pearl chains which contained both protoplast types (Fig. 1). In general, celery protoplasts adhered to electrodes and A. nidulans protoplasts associated with the ends of celery protoplast pearl chains. Maximum plantfungal associations occurred in a 3.0 MHz alignment field (Fig. 2). Increasing the alignment field strength, up to 400 V. c m - ~, resulted in an increase in the frequency of celery/A, nidulans protoplast association (Fig. 3). Optimum alignment voltage was intermediate between values for the two protoplast types (Table 1). Treatment with DC pulses resulted in fusion between celery and A. nidulans protoplasts (Fig. 4). Protoplasts gradually rounded up after fusion. The presence of C a 2 + in the medium increased celery protoplast stability but the voltage required to stimulate fusion caused celery protoplasts to rupture (Fig. 5), in some cases immediately after fusion with A. nidulans protoplasts. A 30-min incubation with Pronase E stabilised celery protoplasts which were subjected to higher DC voltages (Fig. 5c). Longer and shorter Pronase-E treatments were not as successful (Fig. 5b, d). Additionally, after Pronase-E treatment, a lower voltage was required for celery/A, nidulans protoplast fusion. Small, cytoplasmically dense A. nidulans protoplasts fused with celery protoplasts at a higher frequency than the larger, more vacuolate protoplasts. By direct microscopic examination, in excess of 40% of celery protoplasts were observed to fuse with A. nidulans protoplasts. Some celery/celery protoplast fusions also occurred under these electrical treatments.
I
1
I
I
3
I
5
Alignment frequency (MHz) Fig. 2. Effect of alignment frequency on the number of celery protoplasts observed with A. nidulans protoplasts adhering. Field strength 400 V. c m - 1. Values are means • SE of three replicates
t~
"5
80
'~
60
20 I
100
I
I
I
300 Field strength (V.cm-1)
I
500
Fig. 3. Effect of electrical field strength on the number of celery protoplasts observed with A. nidulans protoplasts adhering. Alignment frequency 3.0 MHz. Values are means_+ SE of three replicates
During the fusion process the number of viable celery protoplasts fell from 96.7_+ 1.2% to 58.0_+ 1.2%. Following fusion with A. nidulans protoplasts, viability decreased by a further 9% after 24 h incubation. The numbers of multinucleate protoplasts decreased during this time. The respiration rates of protoplast samples were measured during 24 h incubation (Fig. 6). Respiration rates of celery protoplasts maintained in 0.6 M mannitol declined slightly over 24 h. Pronase-E treatment of celery protoplasts, in the ab-
P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
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(a) 100
50
!
(b)
100
50 v
O*
(c)
100
50 Fig. 4a-e. Celery/A. nidulans heterokaryons after fusion treatment. a Celery and A. nidulans protoplasts in a pearl chain (AC field), b Celery and A. nidulans protoplasts fused immediately following fusion pulses; alignment field present, e Heterokaryons rounded up 5 min after fusion pulses. Bars represent 10 ~xm
sence of A. nidulans protoplasts, slightly depressed respiration initially but after 24 h incubation respiration rates were similar to those for untreated, isolated protoplasts. After electrofusion, protoplast respiration rates were depressed but levels did not fall below those of untreated isolated protoplasts after 24 h incubation. After electrofusion with A. nidulans protoplasts, celery protoplasts showed elevated respiration rates for the first 3 h after fusion. Subsequently, however, values were comparable with other treatments.
(d)
100
50
0
200 400 Fusion voltage (V. cm-1)
Discussion
Fig. $a-d. Effect of fusion voltage on celery/A, nidulans protoplast fusion events ( u - - u ) and celery protoplast lysis (,~--A). Celery protoplasts treated with Pronase E (1 mg-ml 1) for a zero time; b 15 min; e 30 min; d 60 min. Values are means + SE of three replicates
Successful electrofusion of celery protoplasts required similar paramters to those required for fusion of Nicotiana mesophyll protoplasts (Bates 1985). Fusion frequencies were similar to those reported for N. tabacum cultured cells (Kohn et al.
1985), but lower than the 100% fusion reported for Vinca protoplast fusion (Zimmermann and Scheurich 1981). The source of protoplasts has a marked influence over fusion characteristics and frequencies (Gaynor 1986; Zachrisson and Born-
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5
~,~
::I.
2
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10
15
20
25
Incubation period (h)
Fig. 6. Oxygenconsumption,during 24 h incubation, of isolated celery protoplasts (A--A); celeryprotoplasts after Pronase-E treatment (o--o); celeryprotoplasts after electrofusiontreatment (D--m); celery protoplasts after electrofusion with A. nidulans protoplasts (o--o). Values are means of two replicates, bar marker indicates data range mann 1986). However, technical considerations make direct numerical comparisons with published results difficult (Bates et al. 1987). The alignment and fusion of A. nidulans protoplasts required stronger electric fields than for celery protoplasts. The requirement for higher voltages has been reported for P. chrysogenum protoplasts (Kunkel et al. 1987) and N. crassa slime cells (Fikus et al. 1985). The different electrical properties of yeast and fungal protoplasts may relate to their small size, in comparison with plant protoplasts. Protoplasts released from A. nidulans by lytic digestion of mycelium were morphologically heterogeneous. Small, dense, vesicular protoplasts have been reported to originate from apical regions of hyphae while larger, more vacuolate protoplasts arise from older regions (Isaac 1985). Small, dense protoplasts fused more readily with celery protoplasts. The reason for this is not clear but a similar observation has been made for chemically stimulated (polyethyleneglycol) celery/A, nidulans protoplast fusions (Lynch 1987). Rotation of both celery and A. niduIans protoplasts occurred in electrical fields. Any such movements reduce the possibility of fusion because in-
sufficient membrane contact occurs (Arnold and Zimmermann 1984). The use of electrical conditions which caused rotation were avoided. Celery protoplast lysis during electrofusion with A. nidulans protoplasts probably occurred as a result of the marked size differences between the protoplasts and the inequality of the electrical fields required to induce reversible membrane breakdown (Zimmermann and Urnovitz 1987). The presence of Ca z + and Pronase-E treatment, prior to electrofusion, reduced protoplast lysis. Similar treatment was successful for other cells of different sizes (Salhani et al. 1985) and has been related to the extension of lipid domains on the plasma membrane as a result of membrane-protein hydrolysis by Pronase E (Zimmermann and Vienken 1982; Rusin and McCarthy 1986). Calcium may alter membrane resealing times and therefore increase protoplast stability (Abe and Takeda 1986). After Pronase-E treatment, celery protoplasts fused with A. nidulans protoplasts in weaker electrical fields. Similar elicitation of electrofusion of plant protoplasts has been observed (Nea et al. 1987; Nea and Bates 1987). Substantial electrical current flow through protoplasts results in damage to organelles and eventually to deterioration (Zimmermann et al. 1980). This may account for differences in protoplast viability measured after electrical treatments. Celery protoplast viability may have been affected by the loss of ions as a result of membrane breakdown (Zimmermann et al. 1980). However, Hahn-Hagerdal et al. (1986) showed that electrofusion of Brassica napus protoplasts resulted in less leakage of amino acids, protein and D N A than from protoplasts treated with polyethyleneglycol. Subjection to electrical fields may not necessarily reduce cell viability (Zimmermann and Vienken 1984) and has been reported to be stimulatory (Rech et al. 1987; 1988). Some reduction in viability may have been the result of mechanical rupture due to shearing effects between the electrode assembly and the receptacle of the helical fusion chamber. Respiration in isolated protoplasts occurs through normal biochemical pathways, as shown by the use of a range of metabolic inhibitors, although increased respiration rates over source material have been measured (Taylor and Hall 1976). Increased respiration after electrofusion between celery and A. nidulans protoplasts may have been the consequence of an additive effect of the fungal mitochondria, and-or an effect of mixing fungal and plant cytoplasm. The subsequent decrease in respiration rate thereafter, to that of both treated or untreated celery protoplasts alone, may have
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b e e n c a u s e d b y the i n a c t i v a t i o n o f f u n g a l p r o t o plasts f o l l o w i n g c y t o p l a s m i c mixing. Electrical t r e a t m e n t h a s b e e n s h o w n to i n d u c e f u s i o n b e t w e e n celery a n d A . nidulans p r o t o p l a s t s . F u s i o n p r o d u c t s r e m a i n e d viable d u r i n g e x t e n d e d i n c u b a t i o n a n d exhibited an elevated r e s p i r a t i o n rate f o r 3 h after electrofusion. Since e l e c t r o f u s i o n p r o d u c e s g r e a t e r n u m b e r s o f fused p r o d u c t s t h a n c h e m i c a l s t i m u l a t i o n ( L y n c h 1987), it m a y p r o v i d e a useful s y s t e m f o r f u r t h e r p h y s i o l o g i c a l a n d m o r p h o l o g i c a l e x a m i n a t i o n o f the i n t e r a c t i o n s b e t w e e n plant and fungal cytoplasm, and additionally may p r o v i d e a m e a n s f o r the t r a n s f e r o f genetic c h a r a c ters f r o m f u n g a l to p l a n t p r o t o p l a s t s .
of protoplasts of Penicillium chrysogenum. Studia Biophys. 119, 35-356 Linn, M.S., Comings, D.E., Alfi, O.S. (1977) Optical studies of the interaction of 4,6-diamidino-2-phenylindole with DNA and metaphase chromosomes. Chromosoma 60, 1520 Lynch, P.T. (1987) The uptake of fungal protoplasts by plant protoplasts. Ph.D. Thesis, University of Liverpool, Liverpool, UK Morikawa, H., Kumashiro, T., Kusakari, K., Iida, A., Hirai, A., Yamada, Y. (1987) Interspecific hybrid plant formation by electrofusion in Nicotiana. Theor. Appl. Genet. 75, 1-4 Morikawa, H., Hayashi, Y., Hirabayashi, Y., Asada, M., Yamada, Y. (1988) Cellular and vacuolar fusion of protoplasts electrofused using platinum microelectrodes. Plant Cell Physiol. 29, 189-193 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497 Nea, L.J., Bates, G.W. (1987) Factors affecting protoplast electrofusion efficiency. Plant Cell Rep. 6, 337 340 Nea, L.J., Bates, G.W., Gilmer, P.J. (1987) Facilitation of electrofusion of plant protoplasts by membrane active agents. Biochim. Biophys. Acta 897, 293 301 Orton, T.J. (1984) Celery. In: Handbook of plant cell culture, pp. 243 267, Sharp, W.R., Evans, D.A., Ammirato, P.V., Yamada, Y. eds., Collier Macmillan, London Peberdy, J.F., Isaac, S. (1976) An improved procedure for the isolation of protoplasts from Aspergillus nidulans. Microbios Lett. 3, 7-9 Pilwat, G., Zimmermann, U., Richter, H.P. (1981) Giant culture cells by electric field induced fusion. FEBS Letts. 133, 169 174 Pohl, H.A. (1978) Dielectrophoresis; the behaviour of neutral matter in non-uniform electric fields. Cambridge University Press, Cambridge Rech, E.L., Ochatt, S.J., Chand, P.K., Power, J.B., Davey, M.R. (1987) Electroenhancement of division of plant protoplast-derived cells. Protoplasma 141, 169-176 Rech, E.L., Ochatt, S.J., Chand, P.K., Davey, M.R., Mulligan, B.J., Power, J.B. (1988) Electroporation increases DNA synthesis in cultured plant protoplasts. Biotechnology 6, 1091 1094 Rusin, S.E., McCarthy, S.C. (1986)The effect of chemical facilitators on the frequency of electrofusion of tobacco mesophyll protoplasts. Plant Cell Rep. g, 342 345 Salhani, N., Vienken, J., Zimmermann, U., Ward, M., Davey, M.R., Clothier, R.M., Balls, M., Cocking, E.C., Lucy, J.A. (1985) Haemaglobin synthesis and cell wall regeneration by electric field-induced interkingdom heterokaryons. Protoplasma 126, 30 35 Taylor, A.R.D., Hall, J.L. (1976) Some physiological properties of protoplasts isolated from maize and tobacco tissues. J. Exp. Bot. 27, 383-391 Terada, R., Kyozuka, J., Nishibayashi, S., Shimamoto, K. (1987) Plantlet regeneration from somatic hybrids of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola Vasing). Mol. Gen. Genet. 210, 39-43 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. (1986) Electromanipulation of plant protoplasts. Physiol. Plant. 67, 507-516 Zimmermann, U. (1982) Electrical field induced fusion of biological cells. Biochim. Biophys. Acta 694, 227-277 Zimmermann, U., Scheurich, P. (1981) High frequency fusion of plant protoplasts by electrical fields. Planta 151, 26-32
References Abe, S., Takeda, J. (1986) Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts. Plant Physiol. 81, 1151 1155 Arnold, W.M., Zimmermann, U. (1984) Electric field induced fusion and rotation of cells. In: Biological membranes, vol. 5, pp. 389-454, Chapman, D. ed. Academic Press, London Bates, G.W. (1985) Electrical fusion for optimal formation of protoplasts heterokaryons in Nicotiana. Planta 165,217-224 Bates, G.W., Hasenkampf, C.A. (1985) Culture of plant somatic hybrids following electrical fusion. Theor. Appl. Genet. 70, 227-233 Bates, G.W., Saunders, J.A., Sowers, A.E. (1987) Electrofusion: principles and applications. In: Cell fusion, pp. 367-395, Sowers, A.E. ed. Plenum Press, New York Emeis, C.C. (1987) Intergeneric hybridization of yeasts by electrofusion. Studia Biophys. 119, 31-34 Fikus, M., Gresiuk, E., Marszalek, P., Rozycki, S., Zielinski, J. (1985) Electrofusion of Neurospora crassa slime cells. FEMS Microbiol. Letts. 27, 123 127 Gaff, D.F., Okong-Ogola, O. (1971) The use of non-permeating pigments for testing the survival of cells. J. Exp. Bot. 22, 756 758 Gaynor, J.J. (1986) Electrofusion of plant protoplasts. In: Handbook of plant cell culture, vol. 4, pp. 149-171, Evans, D.A., Sharp, W.R., Ammirato, P.V., eds. Collier MacMillan, London Hahn-Hagerdal, B., Hosono, K., Zachrisson, A., Bornman, C.H. (1986) PEG and electric field treatment of plant protoplasts : characterisation of some membrane properties. Physiol. Plant. 67, 359-364 Isaac, S. (1985) Metabolic properties of protoplasts. In: Fungal protoplasts, applications in biochemistry and genetics, pp. 171 188, Peberdy, J.F., Ferenczy, L. eds., Marcel Dekker, New York Isaac, S., Gokhale, A.V. (1982) Autolysis: a tool for protoplast production from Aspergillus nidulans. Trans. Brit. Mycol. Soc. 78, 389-394 Kohn, H., Scheider, R., Scheider, O. (1985) Somatic hybrids in tobacco mediated by electrofusion. Plant Sci. 38, 121-128 Koop, H.U., Schweiger, H.G. (1985) Regeneration of plants after electrofusion of selected pairs of protoplasts. Eur. J. Cell Biol. 39, 46-49 Kunkel, W., Groth, I., Jacob, H.-E., Risch, S., Harnisch, M., May, R., Berg, H., Katenkamp, U. (1987) Electrofusion
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Zimmermann, U., Vienken, J. (1982) Electric field induced cellto-cell fusion. J. Membr. Biol. 67, 165-182 Zimmermann, U., Vienken, J. (1984) Electrofusion of cells. In: Hybridoma technology in agricultural and veterinary research, pp. 173-199, Stern, N.J., Gamble, H.R. eds., Rowman and Allanheld Towtowa, USA Zimmermann, U., Urnovitz, H.B. (1987) Principles of electrofusion and electropermeabilization. Methods Enzymol. 151, 194~221 Zimmermann, U., Vienken, J., Pilwat, G. (1980) Development
of drug carrier systems: electric field induced effects in cell membranes. Bioelectrochem. Bioenerg. 7, 553-574 Zimmermann, U., Vienken, J., Halfman, J., Emeis, C.C. (/985) Electrofusion: a novel hybridization technique. In: Advances in biotechnological processes, pp. 79 150. Mizrahi, A., Van Wezel, A.L. eds, Alan R. Liss Inc., New York
Received 24 November 1988; accepted I February 1989
Planta (1989) 178:207-214
9 Springer-Verlag1989
Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans Paul T. Lynch*, Susan Isaac **, and Hamish A. Collin Department of Genetics and Microbiology, Nicholson Building, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK
Abstract. A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from each species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion. Key words: Apium (protoplast fusion) - Aspergillus (protoplast fusion) - Electrofusion (protoplasts) Protoplast fusion
Introduction
The use of high-strength electrical fields combined with the electrically induced formation of chains of cells (pearl chains) has been used to stimulate cell fusion (Zimmermann and Scheurich 1981). Such techniques offer more controllable and more efficient methods for protoplast fusion than chemi* Present address: Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK ** To whom correspondence should be addressed Abbreviations: AC = alternating current; DC ~ direct current
cal treatments. The use of electrofusion techniques avoids the need for toxic chemical stimulants and excessive washing of cells (Bates 1985). Chemical agents induce random aggregation and fusion events (Watts and King 1984). Electrofusion, however, combined with micromanipulation of electrodes, permits parentage of heterokaryons to be precisely defined (Koop and Schweiger 1985; Morikawa et al. 1988). Electrofusion can also be monitored microscopically, which aids determination of the electrical parameters required for heterokaryon formation and permits the observation of fusion events (Zachrisson and Bornman 1986). Additionally Arnold and Zimmermann (1984) have reported that zones of membrane disturbance can be restricted to zones of membrane contact, whereas chemical treatments affect the whole protoplast plasmelemma. Reduced membrane disturbance may maintain protoplast viability. Chemical treatments usually result in fusion frequencies of 1-5% (Zachrisson and Bornman 1986). Electrofusion can result in fusion frequencies, between plant protoplasts, in excess of 50% (Bates and Hasenkampf 1985; Watts and King 1984). Electrofusion is a two-stage process. Initially, the subjection of protoplasts to a non-uniform electrical field causes polarized protoplasts to move towards regions of higher field strength and the formation of pearl chains results (Arnold and Zimmermann 1984). This effect is termed dielectrophoresis (Pohl 1978). Subsequent application of short DC (direct current) pulses causes breakdown of aligned membranes and protoplast fusion results. After fusion, protoplasts round up into spheres (Zimmermann et al. 1985). The technique has been used successfully to fuse a range of protoplast types e.g. fl-lymphocytes and myeloma cells, to produce hybridoma cells (Zimmermann and Vienken 1982); Friend cells with Petunia protoplasts
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P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
(Salhani et al. 1985); plant protoplasts (Morikawa et al. 1987; Terada et al. 1987); yeast protoplasts (Emeis 1987) and protoplasts from filamentous fungi (Kunkel et al. 1987). The technique of electrofusion offers the possibility for examination of physiological interactions between different cell types. Providing, for example, opportunities for detailed measurements of short-term metabolic interactions between plant and fungal cells. Differences in cell fusability have been related to differences in size, membrane characteristics and cytoplasmic properties between cells and protoplasts which control their electrical characteristics (Zimmermann 1982). To maximise fusion frequency and to preserve viability, electrofusion parameters must be carefully assessed for each protoplast type. The aim of this work was to develop a method for the electrofusion of higher-plant protoplasts (celery) and protoplasts from a filamentous fungus (Aspergiltus nidulans), to assess the fate of fused products and to determine the effects on viability. In the first instance, independent determinations of electrical parameters were made for each species. Subsequently, a method was developed for fusion between the protoplast types. The effects of electrofusion on treated protoplasts, during a 24-h incubation were assessed.
Liberation of protoplasts from celery cells. Protoplasts were iso-
Materials and methods Initiation of celery cultures. Celery seed (Apium graveolens Linn.) cv. French Dimant (Thompson and Morgan, Ipswich, Norfolk, UK) was surface sterilised by immersion in 20% Domestos (Lever Bros., Port Sunlight, Merseyside, UK) solution for 8 min and subsequently washed in three changes of sterile distilled water. Seeds were transferred on to germination medium; Murashige and Skoog (1962) salts (MS) 4.71 g-l-1 (Flow Laboratories, Irvine, UK), supplemented with 30 g. 1-1 sucrose and solidified with Bacto Agar 10 g.1-1 (Difco, Detroit, Mich, USA) in glass Universal vials. Cultures were maintained under continuous light at 4 ~ for 48 h and subsequently at room temperature. Sterility and germination were assessed 30 d after sowing. Germination occurred within three to four weeks. When seedlings had developed four to six leaves (six to eight weeks after sowing), sections of petiole (1 cm) were excised and ptaced on to medium for callus initiation; MS salts 4.71 g. 1-1 supplemented with 30g.1 i sucrose, 10g.1 -~ agar, 0.25 rag. 1 1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.6 rag.l-1 N6_furfurylaminopurine (kinetin). Cultures were maintained at 20 ~ C under a 12-h light/dark cycle. Fast growing, friable callus was selected 37 d after initiation and subcultured monthly. Celery suspension cultures were prepared by inoculating 50 ml of MS medium supplemented with 30 g-1 1 sucrose, 0.25 rag. 1-x 2,4-D, 0.5 rag.l-1 kinetin, with approximately 5 g friable undifferentiated callus. Cultures were grown under a 12-h light/dark cycle at 20 ~ C with shaking (100 r.p.m.). Cultures were continuously selected for fast-growing, finely dispersed clones and subcultured at weekly intervals by inoculating 10 ml of a 7-d-old culture into 50 m! fresh medium.
lated from 3-d-old suspension cultures (late lag phase), using a modified method of Orton (1984). Protoplast isolation mixture was composed of 0.6 M mannitol with 0.1% Pectolyase Y23 (Seishin Pharmaceutical Co., Japan) and 2% Cellulysin (Cambridge Bioscience, Cambridge, UK). Incubations of 16 h (overnight) with shaking (40 r.p.m.) at 30 ~ C resulted in a protoplast yield of 9.2.103_+0.81 . 103.rag - t cell dry weight. After harvest, protoplasts were resuspended in 0.6 M mannitol plus 0.5 mM CaCI~-2H20.
Fungal culture. Aspergillus nidulans. B D U N 33 (University of Nottingham collection) was maintained on 1% (w/v) malt extract (Oxoid, Basingstoke, UK) solidified with 1.5% (w/v) agar (Difco). Cultures grown on agar slopes in 300-ml medical flat bottles at 37 ~ C for 2 d were used as sources of conidia.
Liberation offungalprotoplasts. Protoplasts were liberated from A. nidulans mycelium grown in Iiquid culture (18 h) by the method of Isaac and Gokhale (1982). A final concentration of 0.2 M phosphate buffer pH 5.8 plus 0.4 M mannitol was used as buffer/stabiliser with Novozym 234 (Novo Industri, Copenhagen, Denmark) at 1 mg.m1-1. Lyric incubation was with gentle shaking at 30 ~ C for 3 h. Protoplasts were harvested as described by Peberdy and Isaac (1976) and resuspended in 0.6 M mannitol plus 0.5 mM CaCI2.2H20.
Nuclear staining. Nuclei of protoplasts were observed microscopically using the method of L i n n e t al. (1977). A solution of DAPI (4,6-diaminido-2-phenylindole; Sigma, Poole, Dorset, UK) was prepared in 0.1 M (Tris) 2-amino-2-(hydroxymethyl)1,3-propanediol-HC1 buffer pH7.0, plus 0.1 M NaC1 and 0.01 M ethylenediaminetetraacetic acid (EDTA). Protoplast samples were fixed in osmotically stabilised 2% gluteraldehyde solution buffered with 0.1 M Tris-HC1 buffer pH 7.0 for 30 rain at room temperature. Samples were resuspended in buffer/stabiliser solution. A drop of protoplast suspension was mixed with DAPI (diIuted to give 300 ng.mI ~ on the slide) and allowed to stand for 5 rain to allow uptake of the stain. Observations were made under ultraviolet illumination.
Eleetrofusion apparatus. Electric fields were generated by a Zimmermann Cell Fusion TM System (GCA Corporation, Chicago, Ill., USA), described by Zimmermann and Scheurich (1981). Two types of fusion chamber (supplied by GCA Corporation) were used. A flat fusion chamber (7 gl capacity), similar to that described by Pilwat et al. (1981) and a helical chamber (200 gl capacity) of similar construction to that described by Zimmermann and Vienken (1984).
Eleetrofusion of celery protoplasts. Alignment and fusion parameters for celery protoplasts (1-103 protoplasts-ml z) were determined in a 100-gin flat fusion chamber, and used to fuse celery protoplasts unless otherwise stated. Protoplasts were aligned in a 1.5 MHz, 200 V. c m - 1 electric field and the number of aligned protoplasts assessed. After approx. 45 s, protoplasts were fused using two 150 V. cm 1 fusion pulses of 99 gs duration, with a 1-s period between pulses. After the second pulse the AC (alternating current) field was smoothly damped to zero V over a 30-s period. The number of adhering protoplast pairs (membrane contact areas) which fused, was determined (fusion events) by microscopic observation. For larger volumes a helical fusion chamber was used.
Electrofusion offungat protoplasts. Fusion parameters were determined in a 100-1al fusion chamber, loaded with 1- 103 proto-
P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
209
plasts.m1-1. Protoplasts were aligned in a 1.0 MHz, 1000 V. cm- 1 electric field for about 45 s. Fusion was achieved by two, 500 V.cm -1 DC pulses of 50 Ixs duration with a 0.5-s period between pulses. After the second pulse, the AC field was smoothly damped to zero V over a 30-s period, Aignment and fusion characteristics were assessed as described for celery protoplast electrofusion.
Table 1. Electrofusion parameters used to stimulate maximum
Electrofusion of celery andfungal protoplasts. Immediately prior to electrofusion, celery protoplasts were incubated for up to 1 h at 30~ with shaking in 0.6 M mannitol and 0.5 mM CaClz.2HzO plus 1 mg.ml 1 Pronase E (protease, Type XXV; Sigma) after which protoplasts were washed and resuspended in 0.6 M mannitol plus 0.5 mM CaClz.2HzO. A suspension of equal numbers of both protoplast type (1.103 protoplasts. m1-1) were used in 100-pm flat fusion chambers. Protoplasts were aligned in 3.0MHz, 400V-cm 1 electric fields. The numbers of celery protoplasts associated with A. niduIans protoplasts was assessed by microscopic observation. Protoplasts were fused by two 250 V. cm-1 fusion pulses of 75 gs duration, with a 1-s period between pulses. The AC field was damped to zero V over a 30-s period after fusion, during which time the number of celery protoplasts which fused with A. nidulans protoplasts was determined. Celery and fungal protoplasts were fused using these parameters unless otherwise stated. Larger volumes were fused in a helical chamber.
Alignment frequency 1.5 (MHz) Alignment voltage 200 (V.cm -1) Fusion voltage t 50 (V.cm- 1) Number of DC pulses 2 Pulse duration 99 (~s) Time between pulses 1.0 (s)
Protoplast incubation and viability afterfusion. Protoplasts were incubated in 0.6 M mannitol with gentle shaking, at 20~ C in a 12-h light/dark cycle for up to 24 h. It was anticipated that short-term changes would be likely to occur during this time. Viability was measured by the ability of protoplasts to exclude Evans' Blue vital dye (Gaff and Okong-Ogola 1971). Assessments were made at intervals throughout incubation. Respiration rates. Protoplast oxygen consumption was measured at intervals for 24 h after fusion using a Rank Oxygen Electrode (Rank Brothers, Cambridge, UK) calibrated at 25~ C. To avoid sedimentation of protoplasts a magnetic stirrer was used at constant speed.
Results
Electric-field treatment of celery protoplasts. T h e a p p l i c a t i o n o f h e t e r o g e n e o u s (AC) electrical fields resulted in a l i g n m e n t o f celery p r o t o p l a s t s into pearl chains o f various lengths. Chains f o r m e d parallel to electric fields a n d tended to associate with one or b o t h electrodes. P a r a m e t e r s were selected to f a v o u r the f o r m a t i o n o f p r o t o p l a s t pairs. A m i n i m u m f r e q u e n c y o f 0.1 M H z was required to induce chain f o r m a t i o n . The m a x i m u m n u m b e r o f celery p r o t o p l a s t s f o r m e d pearl chains at 2.0 M H z , b u t p r o t o p l a s t p a i r association was m a x i m u m in 1.25 M H z . M o s t p r o t o p l a s t s f o r m e d chains in a field strength o f 200 V. c m - 1, a n d m o s t p r o t o p l a s t pairing o c c u r r e d at the s a m e field strength. A t high field strength, p r o t o p l s t a l i g n m e n t was very rapid, p r o t o p l a s t s s o m e t i m e s b e c a m e distorted a n d occasionally r u p t u r e d .
fusion between protoplasts Celery/ celery protoplast fusion
A. nidulans/ A. nidulans protoplast fusion 1.0
Celery/ A. nidulans protoplast fusion 3.0
1000
400
500
250
2 50
2 75
0.5
1.0
D i r e c t - c u r r e n t pulses resulted in fusion o f celery p r o t o p l a s t s at points o f p l a s m a - m e m b r a n e contact. Fusion f r e q u e n c y was d e p e n d a n t on the strength o f D C pulses, the m a x i m u m n u m b e r o f fusion events (83.2_+6.8%, m e a n + S E ) o c c u r r e d after 150 V. c m - 1 pulses. F u r t h e r increases in D C pulse strength resulted in m o v e m e n t o f p r o t o p l a s t s during pulse application, a n d also lysis o f s o m e larger p r o t o p l a s t s . After electrofusion, p r o t o p l a s t s r o u n d e d u p into discrete spheres. P r o t o p l a s t viability was m o n i t o r e d for 24 h after electrofusion. D u r i n g the fusion process the n u m b e r o f viable p r o t o p l a s t s was reduced f r o m 96.7 +_ 1.2% to 67.1 + 1.2%. After fusion, viability decreased b y only a further 7 % during 24 h incubation. The electrofusion process g e n e r a t e d 2 4 % multinucleate celery p r o t o p l a s t s . D u r i n g the subseq u e n t 24 h large, multinucleate p r o t o p l a s t s were preferentially lost.
Electrofusion of A. nidulans protoplasts. The p r o t o plasts o f A. nidulans f o r m e d pearl chains in an A C field. A n a l i g n m e n t field f r e q u e n c y o f 3.0 M H z caused all p r o t o p l a s t s to f o r m pearl chains but m a x i m u m p a i r f o r m a t i o n o c c u r r e d at 1.0 M H z . A m i n i m u m field strength was required to stim u l a t e p e a r l - c h a i n f o r m a t i o n . A b o v e 100 V. c m 1 the total n u m b e r o f p r o t o p l a s t s in pearl chains rose. In excess o f this, b o t h p r o t o p l a s t lysis a n d r o t a t i o n occurred. A l i g n m e n t voltages required were m u c h higher t h a n for a l i g n m e n t o f celery p r o toplasts (Table 1). Pulses o f D C current resulted in the fusion o f A. nidulans p r o t o p l a s t s at points o f m e m b r a n e contact. A p p l i c a t i o n o f D C pulses caused m o v e m e n t o f p r o t o p l a s t s . Fusion voltages were m u c h higher t h a n required for celery p r o t o p l a s t fusion (Ta-
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P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
80
~
60
u~ m
Fig. 1. Pearl chains of celery (CP) and A. nidulans (FP) protoplasts; AC field 3.0 MHz, 400 V . c m - L Bar represents 10 gm !
ble 1). Maximum fusion events (/7.2_+ 2.0%) were observed after two DC pulses, a reduced number of events were observed after pulses in excess of 50 gs. Electric-field treatment of celery and A. nidulans protoplast m&tures. In an AC field, celery and A. nidulans protoplasts formed pearl chains which contained both protoplast types (Fig. 1). In general, celery protoplasts adhered to electrodes and A. nidulans protoplasts associated with the ends of celery protoplast pearl chains. Maximum plantfungal associations occurred in a 3.0 MHz alignment field (Fig. 2). Increasing the alignment field strength, up to 400 V. c m - ~, resulted in an increase in the frequency of celery/A, nidulans protoplast association (Fig. 3). Optimum alignment voltage was intermediate between values for the two protoplast types (Table 1). Treatment with DC pulses resulted in fusion between celery and A. nidulans protoplasts (Fig. 4). Protoplasts gradually rounded up after fusion. The presence of C a 2 + in the medium increased celery protoplast stability but the voltage required to stimulate fusion caused celery protoplasts to rupture (Fig. 5), in some cases immediately after fusion with A. nidulans protoplasts. A 30-min incubation with Pronase E stabilised celery protoplasts which were subjected to higher DC voltages (Fig. 5c). Longer and shorter Pronase-E treatments were not as successful (Fig. 5b, d). Additionally, after Pronase-E treatment, a lower voltage was required for celery/A, nidulans protoplast fusion. Small, cytoplasmically dense A. nidulans protoplasts fused with celery protoplasts at a higher frequency than the larger, more vacuolate protoplasts. By direct microscopic examination, in excess of 40% of celery protoplasts were observed to fuse with A. nidulans protoplasts. Some celery/celery protoplast fusions also occurred under these electrical treatments.
I
1
I
I
3
I
5
Alignment frequency (MHz) Fig. 2. Effect of alignment frequency on the number of celery protoplasts observed with A. nidulans protoplasts adhering. Field strength 400 V. c m - 1. Values are means • SE of three replicates
t~
"5
80
'~
60
20 I
100
I
I
I
300 Field strength (V.cm-1)
I
500
Fig. 3. Effect of electrical field strength on the number of celery protoplasts observed with A. nidulans protoplasts adhering. Alignment frequency 3.0 MHz. Values are means_+ SE of three replicates
During the fusion process the number of viable celery protoplasts fell from 96.7_+ 1.2% to 58.0_+ 1.2%. Following fusion with A. nidulans protoplasts, viability decreased by a further 9% after 24 h incubation. The numbers of multinucleate protoplasts decreased during this time. The respiration rates of protoplast samples were measured during 24 h incubation (Fig. 6). Respiration rates of celery protoplasts maintained in 0.6 M mannitol declined slightly over 24 h. Pronase-E treatment of celery protoplasts, in the ab-
P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
211
(a) 100
50
!
(b)
100
50 v
O*
(c)
100
50 Fig. 4a-e. Celery/A. nidulans heterokaryons after fusion treatment. a Celery and A. nidulans protoplasts in a pearl chain (AC field), b Celery and A. nidulans protoplasts fused immediately following fusion pulses; alignment field present, e Heterokaryons rounded up 5 min after fusion pulses. Bars represent 10 ~xm
sence of A. nidulans protoplasts, slightly depressed respiration initially but after 24 h incubation respiration rates were similar to those for untreated, isolated protoplasts. After electrofusion, protoplast respiration rates were depressed but levels did not fall below those of untreated isolated protoplasts after 24 h incubation. After electrofusion with A. nidulans protoplasts, celery protoplasts showed elevated respiration rates for the first 3 h after fusion. Subsequently, however, values were comparable with other treatments.
(d)
100
50
0
200 400 Fusion voltage (V. cm-1)
Discussion
Fig. $a-d. Effect of fusion voltage on celery/A, nidulans protoplast fusion events ( u - - u ) and celery protoplast lysis (,~--A). Celery protoplasts treated with Pronase E (1 mg-ml 1) for a zero time; b 15 min; e 30 min; d 60 min. Values are means + SE of three replicates
Successful electrofusion of celery protoplasts required similar paramters to those required for fusion of Nicotiana mesophyll protoplasts (Bates 1985). Fusion frequencies were similar to those reported for N. tabacum cultured cells (Kohn et al.
1985), but lower than the 100% fusion reported for Vinca protoplast fusion (Zimmermann and Scheurich 1981). The source of protoplasts has a marked influence over fusion characteristics and frequencies (Gaynor 1986; Zachrisson and Born-
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P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
5
~,~
::I.
2
1
'
0
5
I
I
I
I
10
15
20
25
Incubation period (h)
Fig. 6. Oxygenconsumption,during 24 h incubation, of isolated celery protoplasts (A--A); celeryprotoplasts after Pronase-E treatment (o--o); celeryprotoplasts after electrofusiontreatment (D--m); celery protoplasts after electrofusion with A. nidulans protoplasts (o--o). Values are means of two replicates, bar marker indicates data range mann 1986). However, technical considerations make direct numerical comparisons with published results difficult (Bates et al. 1987). The alignment and fusion of A. nidulans protoplasts required stronger electric fields than for celery protoplasts. The requirement for higher voltages has been reported for P. chrysogenum protoplasts (Kunkel et al. 1987) and N. crassa slime cells (Fikus et al. 1985). The different electrical properties of yeast and fungal protoplasts may relate to their small size, in comparison with plant protoplasts. Protoplasts released from A. nidulans by lytic digestion of mycelium were morphologically heterogeneous. Small, dense, vesicular protoplasts have been reported to originate from apical regions of hyphae while larger, more vacuolate protoplasts arise from older regions (Isaac 1985). Small, dense protoplasts fused more readily with celery protoplasts. The reason for this is not clear but a similar observation has been made for chemically stimulated (polyethyleneglycol) celery/A, nidulans protoplast fusions (Lynch 1987). Rotation of both celery and A. niduIans protoplasts occurred in electrical fields. Any such movements reduce the possibility of fusion because in-
sufficient membrane contact occurs (Arnold and Zimmermann 1984). The use of electrical conditions which caused rotation were avoided. Celery protoplast lysis during electrofusion with A. nidulans protoplasts probably occurred as a result of the marked size differences between the protoplasts and the inequality of the electrical fields required to induce reversible membrane breakdown (Zimmermann and Urnovitz 1987). The presence of Ca z + and Pronase-E treatment, prior to electrofusion, reduced protoplast lysis. Similar treatment was successful for other cells of different sizes (Salhani et al. 1985) and has been related to the extension of lipid domains on the plasma membrane as a result of membrane-protein hydrolysis by Pronase E (Zimmermann and Vienken 1982; Rusin and McCarthy 1986). Calcium may alter membrane resealing times and therefore increase protoplast stability (Abe and Takeda 1986). After Pronase-E treatment, celery protoplasts fused with A. nidulans protoplasts in weaker electrical fields. Similar elicitation of electrofusion of plant protoplasts has been observed (Nea et al. 1987; Nea and Bates 1987). Substantial electrical current flow through protoplasts results in damage to organelles and eventually to deterioration (Zimmermann et al. 1980). This may account for differences in protoplast viability measured after electrical treatments. Celery protoplast viability may have been affected by the loss of ions as a result of membrane breakdown (Zimmermann et al. 1980). However, Hahn-Hagerdal et al. (1986) showed that electrofusion of Brassica napus protoplasts resulted in less leakage of amino acids, protein and D N A than from protoplasts treated with polyethyleneglycol. Subjection to electrical fields may not necessarily reduce cell viability (Zimmermann and Vienken 1984) and has been reported to be stimulatory (Rech et al. 1987; 1988). Some reduction in viability may have been the result of mechanical rupture due to shearing effects between the electrode assembly and the receptacle of the helical fusion chamber. Respiration in isolated protoplasts occurs through normal biochemical pathways, as shown by the use of a range of metabolic inhibitors, although increased respiration rates over source material have been measured (Taylor and Hall 1976). Increased respiration after electrofusion between celery and A. nidulans protoplasts may have been the consequence of an additive effect of the fungal mitochondria, and-or an effect of mixing fungal and plant cytoplasm. The subsequent decrease in respiration rate thereafter, to that of both treated or untreated celery protoplasts alone, may have
P.T. Lynch et al. : Electrofusion of plant and fungal protoplasts
213
b e e n c a u s e d b y the i n a c t i v a t i o n o f f u n g a l p r o t o plasts f o l l o w i n g c y t o p l a s m i c mixing. Electrical t r e a t m e n t h a s b e e n s h o w n to i n d u c e f u s i o n b e t w e e n celery a n d A . nidulans p r o t o p l a s t s . F u s i o n p r o d u c t s r e m a i n e d viable d u r i n g e x t e n d e d i n c u b a t i o n a n d exhibited an elevated r e s p i r a t i o n rate f o r 3 h after electrofusion. Since e l e c t r o f u s i o n p r o d u c e s g r e a t e r n u m b e r s o f fused p r o d u c t s t h a n c h e m i c a l s t i m u l a t i o n ( L y n c h 1987), it m a y p r o v i d e a useful s y s t e m f o r f u r t h e r p h y s i o l o g i c a l a n d m o r p h o l o g i c a l e x a m i n a t i o n o f the i n t e r a c t i o n s b e t w e e n plant and fungal cytoplasm, and additionally may p r o v i d e a m e a n s f o r the t r a n s f e r o f genetic c h a r a c ters f r o m f u n g a l to p l a n t p r o t o p l a s t s .
of protoplasts of Penicillium chrysogenum. Studia Biophys. 119, 35-356 Linn, M.S., Comings, D.E., Alfi, O.S. (1977) Optical studies of the interaction of 4,6-diamidino-2-phenylindole with DNA and metaphase chromosomes. Chromosoma 60, 1520 Lynch, P.T. (1987) The uptake of fungal protoplasts by plant protoplasts. Ph.D. Thesis, University of Liverpool, Liverpool, UK Morikawa, H., Kumashiro, T., Kusakari, K., Iida, A., Hirai, A., Yamada, Y. (1987) Interspecific hybrid plant formation by electrofusion in Nicotiana. Theor. Appl. Genet. 75, 1-4 Morikawa, H., Hayashi, Y., Hirabayashi, Y., Asada, M., Yamada, Y. (1988) Cellular and vacuolar fusion of protoplasts electrofused using platinum microelectrodes. Plant Cell Physiol. 29, 189-193 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497 Nea, L.J., Bates, G.W. (1987) Factors affecting protoplast electrofusion efficiency. Plant Cell Rep. 6, 337 340 Nea, L.J., Bates, G.W., Gilmer, P.J. (1987) Facilitation of electrofusion of plant protoplasts by membrane active agents. Biochim. Biophys. Acta 897, 293 301 Orton, T.J. (1984) Celery. In: Handbook of plant cell culture, pp. 243 267, Sharp, W.R., Evans, D.A., Ammirato, P.V., Yamada, Y. eds., Collier Macmillan, London Peberdy, J.F., Isaac, S. (1976) An improved procedure for the isolation of protoplasts from Aspergillus nidulans. Microbios Lett. 3, 7-9 Pilwat, G., Zimmermann, U., Richter, H.P. (1981) Giant culture cells by electric field induced fusion. FEBS Letts. 133, 169 174 Pohl, H.A. (1978) Dielectrophoresis; the behaviour of neutral matter in non-uniform electric fields. Cambridge University Press, Cambridge Rech, E.L., Ochatt, S.J., Chand, P.K., Power, J.B., Davey, M.R. (1987) Electroenhancement of division of plant protoplast-derived cells. Protoplasma 141, 169-176 Rech, E.L., Ochatt, S.J., Chand, P.K., Davey, M.R., Mulligan, B.J., Power, J.B. (1988) Electroporation increases DNA synthesis in cultured plant protoplasts. Biotechnology 6, 1091 1094 Rusin, S.E., McCarthy, S.C. (1986)The effect of chemical facilitators on the frequency of electrofusion of tobacco mesophyll protoplasts. Plant Cell Rep. g, 342 345 Salhani, N., Vienken, J., Zimmermann, U., Ward, M., Davey, M.R., Clothier, R.M., Balls, M., Cocking, E.C., Lucy, J.A. (1985) Haemaglobin synthesis and cell wall regeneration by electric field-induced interkingdom heterokaryons. Protoplasma 126, 30 35 Taylor, A.R.D., Hall, J.L. (1976) Some physiological properties of protoplasts isolated from maize and tobacco tissues. J. Exp. Bot. 27, 383-391 Terada, R., Kyozuka, J., Nishibayashi, S., Shimamoto, K. (1987) Plantlet regeneration from somatic hybrids of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola Vasing). Mol. Gen. Genet. 210, 39-43 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. (1986) Electromanipulation of plant protoplasts. Physiol. Plant. 67, 507-516 Zimmermann, U. (1982) Electrical field induced fusion of biological cells. Biochim. Biophys. Acta 694, 227-277 Zimmermann, U., Scheurich, P. (1981) High frequency fusion of plant protoplasts by electrical fields. Planta 151, 26-32
References Abe, S., Takeda, J. (1986) Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts. Plant Physiol. 81, 1151 1155 Arnold, W.M., Zimmermann, U. (1984) Electric field induced fusion and rotation of cells. In: Biological membranes, vol. 5, pp. 389-454, Chapman, D. ed. Academic Press, London Bates, G.W. (1985) Electrical fusion for optimal formation of protoplasts heterokaryons in Nicotiana. Planta 165,217-224 Bates, G.W., Hasenkampf, C.A. (1985) Culture of plant somatic hybrids following electrical fusion. Theor. Appl. Genet. 70, 227-233 Bates, G.W., Saunders, J.A., Sowers, A.E. (1987) Electrofusion: principles and applications. In: Cell fusion, pp. 367-395, Sowers, A.E. ed. Plenum Press, New York Emeis, C.C. (1987) Intergeneric hybridization of yeasts by electrofusion. Studia Biophys. 119, 31-34 Fikus, M., Gresiuk, E., Marszalek, P., Rozycki, S., Zielinski, J. (1985) Electrofusion of Neurospora crassa slime cells. FEMS Microbiol. Letts. 27, 123 127 Gaff, D.F., Okong-Ogola, O. (1971) The use of non-permeating pigments for testing the survival of cells. J. Exp. Bot. 22, 756 758 Gaynor, J.J. (1986) Electrofusion of plant protoplasts. In: Handbook of plant cell culture, vol. 4, pp. 149-171, Evans, D.A., Sharp, W.R., Ammirato, P.V., eds. Collier MacMillan, London Hahn-Hagerdal, B., Hosono, K., Zachrisson, A., Bornman, C.H. (1986) PEG and electric field treatment of plant protoplasts : characterisation of some membrane properties. Physiol. Plant. 67, 359-364 Isaac, S. (1985) Metabolic properties of protoplasts. In: Fungal protoplasts, applications in biochemistry and genetics, pp. 171 188, Peberdy, J.F., Ferenczy, L. eds., Marcel Dekker, New York Isaac, S., Gokhale, A.V. (1982) Autolysis: a tool for protoplast production from Aspergillus nidulans. Trans. Brit. Mycol. Soc. 78, 389-394 Kohn, H., Scheider, R., Scheider, O. (1985) Somatic hybrids in tobacco mediated by electrofusion. Plant Sci. 38, 121-128 Koop, H.U., Schweiger, H.G. (1985) Regeneration of plants after electrofusion of selected pairs of protoplasts. Eur. J. Cell Biol. 39, 46-49 Kunkel, W., Groth, I., Jacob, H.-E., Risch, S., Harnisch, M., May, R., Berg, H., Katenkamp, U. (1987) Electrofusion
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Zimmermann, U., Vienken, J. (1982) Electric field induced cellto-cell fusion. J. Membr. Biol. 67, 165-182 Zimmermann, U., Vienken, J. (1984) Electrofusion of cells. In: Hybridoma technology in agricultural and veterinary research, pp. 173-199, Stern, N.J., Gamble, H.R. eds., Rowman and Allanheld Towtowa, USA Zimmermann, U., Urnovitz, H.B. (1987) Principles of electrofusion and electropermeabilization. Methods Enzymol. 151, 194~221 Zimmermann, U., Vienken, J., Pilwat, G. (1980) Development
of drug carrier systems: electric field induced effects in cell membranes. Bioelectrochem. Bioenerg. 7, 553-574 Zimmermann, U., Vienken, J., Halfman, J., Emeis, C.C. (/985) Electrofusion: a novel hybridization technique. In: Advances in biotechnological processes, pp. 79 150. Mizrahi, A., Van Wezel, A.L. eds, Alan R. Liss Inc., New York
Received 24 November 1988; accepted I February 1989
