Planta
Planta (1988) 175:396-402
9 Springer-Verlag 1988
Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops M.R. Thomas* and R.J. Rose** Department of Biological Sciences, University of Newcastle, N.S.W. 2308, Australia
Abstract. Protoplasts isolated from Nicotiana tabacure L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures. Heterokaryons obtained were embedded in 20-~tl drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development. The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed. * Present address: Department of Plant Pathology, Kansas State University, Throckmorton Hall, Manhattan, KS 66506, USA ** To whom correspondence should be addressed Abbreviations: BAP=N6-benzylaminopurine; BM=basal me-
dium; 2,4-D = 2,4-dichlorophenoxyacetic acid; NAA = 1-naphthalene acetic acid; PEG =polyethylene glycol; PKM =modified Kao (1977) medium for protoplast culture
Key words: Heterokaryon enrichment - Nicotiana (protoplasts) - Protoplast fusion.
Introduction
A common limitation to producing somatic hybrids is the absence of selection schemes for the species under study, as genetically and biochemically characterised mutants are not always available. Furthermore, it may be undesirable to work with mutants when specific genotypes are to be used. To overcome the problem of using mutants as fusion partners to identify somatic hybrids, a procedure used in recent years has been the visual selection and manual isolation of heterokaryons. The heterokaryons are produced by fusion of a mesophyll protoplast with a cell-suspension protoplast and after isolation are cultured in a microdrop (Kao 1977; Gleba 1978; Bates and Hasenkampf 1985; Flick et al. 1985). Single protoplasts have been cultured in 10-25 nl of unconditioned medium (Koop et al. 1983), by achieving the same population density as in mass culture. Similar systems can be utilised in somatic-hybridisation studies (Koop and Schweiger 1985). Growth of heterokaryons in microdrops is frequently achieved with "conditioned" medium (Gleba and Sytnik 1984, pp. 17-19; Bates and Hasenkampf 1985). Gleba et al. (1984) have emphasized in their studies that fusion and manual isolation of heterokaryons overcomes the shortcomings of hybrid-screening methods that result in an automatic rejection of the theoretically possible recombinant forms. The culture of single cells or small cell numbers in microdrops requires specialised culture conditions to prevent evaporation from the microdrop,
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
as well as correct media composition, concentration, volume and in some cases "conditioning" to stimulate the heterokaryons to divide. One way to overcome the difficulties associated with the microdrop method has been to culture the manually isolated heterokaryons in a nurse culture that is genetically different, for example chlorophyll-deficient mutants (Menczel et al. 1978; Hamill et al. 1984) or auxotrophic mutants (Hein et al. 1983). In the present study we have utilised polyethylene glycol (PEG)-induced protoplast fusion, heterokaryon-enrichment procedures, and a nurse-culture system for the growth of Nicotiana tabacum L. x Nicotiana suaveotens Lehm. hybrid calli. These procedures are easily carried out and do not require mutant genotypes. The advantages of manual isolation and microdrop culture, particularly in being able to achieve the widest range of variation from somatic hybridisation, are largely maintained. Also, these procedures are suitable for studying the early development of somatic hybrids in an analogous way to our studies with N. tabacum (Thomas and Rose 1983).
Materials and methods Enzyme solutions, wash solutions, fusion solutions, and liquid culture media were sterilised by membrane filtration through 0.22-~tm Nalgene filters (Nalge Co., Rochester, N.Y., USA). The sterile Percoll (Pharmacia, Uppsala, Sweden) solution was used as supplied and should not be autoclaved in the procedures described. Callus and cell-suspension media were sterilised by autoclaving.
Isolation and purifieation oJmesophyll protoplasts of N. tabacum. Seedlings of N. tabacum L. cv. Xanthi (from seed maintained by us) were grown as described by Thomas and Rose (1983). Plants were grown for five to six weeks before transfer to a growth cabinet for 4-5 d with a daylength of 16 h, day and night temperatures of 27 ~ C and 25 ~ C, respectively, and relative humidity of 70 80%. Photon fluence rate provided by VHO cool-white fluorescent tubes (GTE Sylvania Canada, Drummondville, P.Q., Canada) and incandescent lamps was 85-100 ~tmol.s 1 -m-2 of photosynthetically active radiation (PAR) as measured by a Li-Cor quantum sensor (Li-Cor, Lincoln, Neb., USA). Leaves that were almost fully expanded, No. 7 or 8 from the apex, were selected for protoplast isolation. Leaf tissue from the central region of leaf 7 or 8 was surfacesterilised for 2 rain in 70% ethanol followed by 7 min in a calcium-hypochlorite solution (2% minimum available chlorine). After rinsing twice in sterile, glass-distilled water the leaf pieces (0.5-0.6 g FW) were cut into l-ram 2 sections and vacuum-infiltrated (400 mm Hg) for 1 min with 8 ml of enzyme solution. The enzyme solution contained 0.5% (w/v) Cellulase RI0 and 0.5% (w/v) Macerozyme R10 (enzymes from Yakult Honsha Co. Tokyo, Japan), dissolved in complete modified Kao (1977) medium for protoptast culture (PKM; Table 1) at pH 5.8, with 0.4 M mannitol in place of 0.38 M glucose. Incubation was in 100-ml Erlenmeyer flasks, in the dark at 27 ~ C for 12 16 h. After enzyme incubation the mesophyll protoplasts were collected by filtration through 70-gin stainless-steel mesh
397
(Hunter Wire Products, Warners Bay, N.S.W., Australia) into 15-ml glass centrifuge tubes. Percoll was then added to 20% (v/v) and mixed with the protoplasts, and 1 ml of PKM medium gently layered on top. Viable protoplasts that band at the PercolI/PKM interface after 5 min centrifugation at 50-g were col= lected.
Isolation and purification of cell-suspension protoplasts ojN. suaveolens. Cell suspensions were initiated from callus derived from leaf tissue. Callus was initiated from leaf sections of shoot cultures by incubation on basal medium (BM) as described in Table 1 +0.5 gM 2,4-dichlorophenoxyacetic acid (2,4-D)+ 5 gM l-naphthaleneacetic acid (NAA) in 0.8% agar. Callus cultures were grown in the dark at 27 ~ C and subcultured every three weeks. Cell suspensions were initiated by transferring calli to liquid BM medium and shaking in 250-ml Erlenmeyer flasks on a B. Braun HT orbital shaker (B. Braun, Melsungen, FRG) at 120 rpm in the dark at 27 ~ C. For protoplast isolation, cells were collected by centrifugation 4 d after subculture and 1.5-2 ml of packed cells were suspended in 8 ml of enzyme solution. The enzyme solution contained 0.5% (w/v) Cellulase RS (Yakult Honsha) 0.5% w/v Macerozyme R10 and 0.02% w/v Pectolyase Y23 (Seishu Pharmaceutical industry Co., Tokyo, Japan), dissolved in complete PKM medium (Table 1) at pH 5.5 with 0.4 M mannitol in place of 0.38 M glucose. Incubation on an orbital shaker (60 rpm) was in 100-ml Erlenmeyer flasks, in the dark at 27~ for 12-16 h. Protoplasts were collected by filtration through 44-gm stainless-steel mesh into 15-ml glass centrifuge tubes. Purification of viable protoplasts was the same as that described for N. tabacum mesophyll protoplasts except an inorganic salt solution which we designated MW5 was used (180 mM CaC12 2H20, 18 m M NaC1, 5 mM KC1, 5 m M glucose, 3 m M 2-(Nmorpholino)ethane sulfonic acid (Mes) buffer, pH 5.5) in place of the PKM medium as an overlay solution. The MW5 inorganic salt solution was modified from Medgyesy et al. (1980) and its use was necessary since few cell-suspension protoplasts form a pellet when centrifuged at 50 .g in P K M medium. Protoplast fusion. The purified protoplasts of the two species were resuspended in MW5 solution in separate 15-ml glass centrifuge tubes and pelleted by 5 min centrifugation at 50.g. The pellets were resuspended in twice their packed cell volume with MW5 solution. The two protoplast populations were then mixed at a volume:volume ratio of 1:2, N. tabacum mesophyll protoplasts to N. sauveolens cell-suspension protoplasts, to give a final volume of 0.4 ml. This gave approx. 4-105 N. tabacum protoplasts and 2.106 N. suaveolens protoplasts. A 1.2-ml volume of a polyethylene-glycol (PEG) fusion solution was then added along the side of the centrifuge tube and the protoplasts were gently resuspended with a siliconised Pasteur pipette. The PEG fusion solution consisted of 50% (w/v) PEG 1540 (Polysciences, Northhampton, UK), 10.5 mM Ca(NO3)z-4H20, 0.1 M glucose, 0.7 mM KHzPO4 and 2% (v/ v) dimethyl sulfoxide (DMSO) at pH 5.5. The protoplasts were incubated in the PEG fusion solution for 5 min. During this incubation the protoplasts were pelleted (2 min, 50 "g). An equal volume (1.6 ml) of a high-pH-calcium-DMSO elution solution was then added and the protoplast pellet resuspended to break up large clumps. This solution consisted of a solution of 0.4 M glucose, 55.5 m M Ca(NO3)2-4H20 and 8.88% (v/v) DMSO and a solution of 0.3 M glycine adjusted with K O H to pH 10.5 ; mixed just before use at 9:1. After 10 min in the elution solution, PKM medium (but buffered with 50 mM Mes buffer; Menczel and Wolfe 1984) was added over 20 min to give a total volume of 8 ml.
398
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
Table 1. Composition of the basal medium (BM) and the modified Kao (1977) medium (PKM), in mg/l. The BM medium is modified from Murashige and Skoog (1962), vitamins are from Gamborg et al. (1968). Coconut water is present in P K M at 10 ml/1 and is obtained from mature nuts then heated to 60 ~ C for 30 min and filtered. Both BM and P K M have iron concentration and chelation according to Dalton et al. (1983). Adjustment of ~H is made with 1 M K O H to 5.8 for BM and 5.5 for PKM. Casamino acids are vitamin-free from Difco (Difco Labs., Detroit Mich., USA). Compound
Compound
Medium BM
PKM
1650 1900 440 370 170 -
600 1900 600 300 170 300
FeSO4" 7H20 Na2EDTA'2H20 a
9.3 37.3
9.3 37.3
H3BO 3
6.2 16.9 8.6 0.83
3 10 2 0.75
MnSO4" H 2 0 ZnSOa "7H20 KI Na2MoO4" 2H20 CuSO,~" 5H20 CoC12 - 6H20
0.25 0.025 0.025
0.25 0.025 0.025
Organic acids Sodium pyruvate Citric acid Malic acid Fumaric acid
---
5 10 10 10
PKM
Sucrose Glucose Fructose Ribose Xylose Mannose Rhamnose Cellobiose Sorbitol Mannitol
30000 -
125 68400 125 125 125 125 125 125 125 125
Vitamins myo-Inositol Nicotinamide Nicotinic acid Pyridoxine. HC1 Thiamine. HC1 D-Calcium panthothenate Biotin Folic acid p-Aminobenzoic acid Choline chloride
100 1 1 i0 -
100 1 1 10 0.5 0.005 0.2 0.01 0.5
250
195 125
Other
Hormones NAA 2,4-D BAP
BM
Sugars
Mineral salts NH4NO3 KNO3 CaC12 2 H 2 0 MgSOr 7H20 KHzPO4 KC1
Medium
--
0.93 0.11 0.56
Mes buffer Casamino acids
a Disodium-ethylenediaminetetraacetic acid
Enrichment for heterokaryons after protoplast fusion. Percoll was added to 20% (v/v) to the post-fusion preparation and the preparation was mixed and overlayed with 1 ml of P K M medium. Centrifugation at 50 .g for 5 rain allowed viable protoplasts rich in heterokaryons to be collected at the interface of the two solutions. The protoplast population enriched for heterokaryons was washed once with P K M medium, diluted to 4 ml, and plated 0.5 ml/well in 24-well Linbro tissue-culture plates (Flow Laboratories, Sydney, N.S.W., Australia). After overnight culture the protoplast population enriched for heterokaryons was collected into a glass centrifuge tube and pelleted for 5 rain at 50.g. The protoplast pellet was resuspended in 0.6 ml P K M medium containing 20% (v/v) Percolt, transferred to a 1.5-ml siliconised Eppendorf tube, overlayed with P K M medium, and centrifuged for 5 min at 50.g to obtain viable protoplasts. Viable protoplasts were collected from the interface and washed once in 1 ml of P K M medium in an Eppendorf tube before final resuspension in 0.1 ml of P K M medium. Aliquots (0.01 ml) of the protoplasts enriched in heterokaryons were distributed to wells of 24-well Linbro tissue-culture plates and mixed with 0.01 ml of 0.7% molten (35 ~ C) Seaplaque agarose (FMC Marine Colloids Division, Rockland, N.Y., USA) in P K M medium. After the agarose microdrops
had solidified a liquid nurse culture (0.5 ml) was then added to each well containing the heterokaryons embedded in the 0.02-ml agarose microdrops.
Culture of heterokaryons. The liquid nurse cultures were either N. tabacum- or N. suaveolens-protoplast cultures that came from protoplasts not used in the fusion treatment. The N. suaveolens-protoplast nurse cultures were at a density of 105-2 . 105 protoplasts/ml and the N. tabacum-protoplast nurse cultures were at a density of 10~-2 - 104 protoplasts/ml. Five to seven days after fusion, 0.18 ml of the nurse culture from each well was removed and replaced with 0.18 ml of C K M medium. The C K M medium was as for P K M medium but with glucose at 0.253 M and sucrose at 29 mM. On day 12 of culture an additional 0.18 ml of C K M medium was added to each well. After 2 d the nurse culture was removed from each well with a Pasteur pipette and the wells were washed twice with C K M medium having lower hormone concentrations (0.05 gM NAA, 0.005 gM 2,4-D, 0.25 pM N6-benzylaminopurine (BAP)) before finally covering the microdrop with 0.5 ml of this medium. Three weeks after fusion the agarose microdrops containing the hybrid cell colonies were transferred to agar plates contain-
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana ing shoot-induction medium (BM medium with 0.05 laM N A A + 2.5 g M BAP). Washing in the low-hormone C K M medium prior to transfer ensured no nurse colonies were transferred. Approximately 0.5 ml of the low-hormone C K M medium was added around the transferred microdrops to prevent osmotic stress to the cell colonies. Large cell colonies were present after 6 d and at this time the agarose microdrop was broken up under a stereo-microscope ( x 16) to separate out the colonies to obtain callus formation.
Fluoreseein-isothiocyanate labelling of N. suaveolens cell suspension protoplasts. At the beginning of enzyme incubation, 25 gl of freshly prepared fluorescein isothiocyanate (FITC; Sigma Chemical Co., St. Louis, Mo., USA) in acetone (5 mg/ml) was added to 8 ml of the cell-wall-degrading enzyme solution. The protoplast isolation, purification and fusion were as described previously. Protoplasts labelled with FITC were visualised with a Leitz Orthoplan fluorescence microscope (E. Leitz, Wetzlar, F R G ) under epi-fluorescent illumination using a BG12 excitation filter and a K490 suppression filter. All protoplasts were labelled.
Results
Growing the plants under controlled conditions gave yields of intact N. tabacum protoplasts averaging 106 per I g of leaf tissue. Cell suspension cultures of N. suaveolens gave yields averaging 5" 105 per 2 ml of packed cell volume. The use of Percoll flotation to separate viable from non-viable protoplasts after enzyme treatment proved to be a simple and reliable method for both the mesophyll protoplasts of N. tabacum (Fig. 1) and the cell-suspension protoplasts of N. suaveolens (Fig. 1). This procedure was an improvement over the use of a Percoll cushion that we had used previously (Thomas and Rose 1983). Fusion in a centrifuge tube (Douglas et al. 1981) has a number of advantages. Maximum contact between protoplasts can be achieved during PEG treatment and the heterokaryons produced are readily available for further manipulation. The sticking of heterokaryons to the coverslip makes manipulation of heterokaryons difficult in fusion procedures on coverslips. However, Kao (1986) has recently overcome this by fusion of the protoplasts with PEG at the interface of two solutions. A mixed protoplast suspension of N. tabacum mesophyll protoplasts and N. suaveolens cell-suspension protoplasts before fusion is shown in Fig. 1. There is a higher proportion of the smaller cell-suspension protoplasts (see Materials and methods) to increase the chance of heterokaryon formation. Protoplast populations enriched for heterokaryons after fusion and Percoll purification are shown in Figs. 2 and 3. The mesophyll protoplasts were most sensitive to the fusion treatment and after purification the viable protoplasts were
399
mainly heterokaryons or cell-suspension protoplasts of N. suaveolens (Fig. 2, Table 2). The purified protoplasts were cultured overnight before being purified again to remove dead protoplasts and embedded in agarose microdrops (Fig. 3) for culture in a nurse culture (see Materials and methods). Counts from three separate experiments, carried out on enriched heterokaryon protoplast populations after embedding in agarose microdrops showed that on average 49.7% of the population were heterokaryons, 44.1% cell-suspension protoplasts of N. suaveoIens and 6.2% mesophyll protoplasts of N. tabacum (Table 2). These figures were confirmed in a separate experiment to check our identification of heterokaryons, by using fluorescein-isothiocyanate-labelled cell-suspension protoplasts of N. suaveolens with mesophyll protoplasts of N. tabacum. Counts by fluorescence microscopy after fusion indicated that 92.5% (n = 200) of the protoplasts containing chloroplasts were heterokaryons, and the remainder were N. tabacum mesophyll protoplasts. The presence of the fluorescent stain in the heterokaryons was very specific since a control treatment where fluorescein-isothiocyanate-stained protoplasts of N. suaveolens were fused and mesophyll protoplasts ofN. tabacum added after the high-pH-calcium-dimethylsulfoxide treatment (same contact time as normal fusion) showed no uptake of the stain by protoplasts of N. tabacum because of release by dead protoplasts of N. suaveolens. The protoplasts enriched for heterokaryons and embedded in an agarose microdrop were able to grow in conditions favourable for heterokaryon growth and the agarose provided a physical boundary between the nurse culture and the heterokaryons. Figure 3 shows heterokaryons embedded in an agarose microdrop. Manually isolated heterokaryons can be readily used in this system. Figure 4 shows a cell colony that developed from a heterokaryon isolated (1 d after fusion) with a Gilson Pipetman (Gilson France, Villiers Le Bel, France). Division frequencies of chloroplast-containing cells (heterokaryons and N. tabacum cells; see Table2) averaged 31% after 8 d of culture while the frequency of N. suaveolens cells was 9%. These frequencies are considerably lower than that observed for unfused control protoplast cultures of N. tabacum (80%) and N. suaveolens (67%). Electron microscopy is facilitated by the use of the agarose microdrops which are readily embedded. The heterokaryons contain two types of plastids (data not shown). One type of plastid represents dedifferentiation of chloroplasts from the N. tabacum mesophyll fusion partner (see Thomas
400
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
Fig. 1. Mixed protoplast suspension of iV. tabacum mesophyll protoplasts and N. suaveolens cell-suspension protoplasts before fusion. There is a higher proportion of cell-suspension protoplasts to mesophyll protoplasts. Bar =20 btm; x 460 Fig. 2. Protoplast population enriched for heterokaryons after fusion of N. tabacum mesophyll protoplasts with N. suaveolens cell-suspension protoplasts. Heterokaryons can be recognised (arrowheads) because of the presence of chloroplasts from the mesophyll protoplasts and the cytoplasm of the cell-suspension protoplasts. Bar = 20 gm; x 460 Fig. 3. Protoplast population after fusion and enrichment for heterokaryons embedded in an agarose microdrop. All chloroplastcontaining protoplasts in this field are heterokaryons (arrowheads). Bar=100 gm; x 170 Fig. 4. Division of manually isolated heterokaryon embedded in an agarose microdrop. Out-of-focus cells are nurse cells outside the agarose drop. Bar=20 #m; x 350
Table 2. Proportion of heterokaryons after protoplast fusion, enrichment for heterokaryons, and embedding in agarose microdrops. Number of protoplasts counted in the agarose microdrops for the different experiments were 1462 (Expt. 1), 1000 (Expt. 2), 200 (Expt. 3)
Expt.
Protoplast types (%) N. suaveolens
N. tabaeum
Heterokaryons
1 2 3
48.4 48.2 35.7
0.3 1.6 16.8
51.3 50.2 47.5
Mean
44.1 +_2.4
6.2_+3.1
49.7_+0.7
and Rose 1983) and the other plastid type is representative of the small proplastids observed in the N. suaveolens cell suspension fusion partner. The cell colonies in the agarose microdrops were grown through to calli and some formed shoots on regeneration medium. More cell colonies grew through to calli when N. tabacum mesophyll protoplasts were used as a nurse culture. Esterase profiles of callus protein extracts run on nondenaturing polyacrylamide gels showed that N. tabacum calli had two major low-molecularweight bands close together while N. suaveolens calli had four higher-molecular-weight bands. Hy-
M.R. Thomas and R.J. Rose: tteterokaryon enrichment and growth in microdrops in Nicotiana
brid calli had both N. tabacum and N. suaveolens bands (data not shown). About 45% of the calli appear to be derived from heterokaryons. The N. suaveolens cell line used does not develop past the callus stage. Approximately one-third of the regenerated plants were N. tabacum and two-thirds differed morphologically from N. tabacum. Analyses to date on these latter plants indicate the presence of cybrids rather than hybrids. In sexual hybridisation between N. tabacum and N. suaveolens only N. tabacum lines with N. suaveolens cytoplasm have been obtained (Schweppenhauser and Mann 1968). Discussion
The basic PEG fusion procedure introduced by Kao and Michayluk (1974), though widely and succesfully used in various modified forms to produce somatic hybrids (Gleba and Sytnik 1984) gives relatively low and variable fusion frequencies (Table 3). However Kao (1986) has recently obtained fusion frequencies of up to 30% using a modified coverslip procedure (Table 3). Utilising the protocols outlined in this study with N. tabacure x N. suaveolens allows a preparation to be obTable 3. Heterokaryons produced by different fusion and enrichment protocols. In the reports cited, "division of heterokaryons, b calli from heterokaryons, ~ plants from heterokaryons. Heterokaryons ( % ) -
heterokaryons x 100 total No. of protoplasts
Authors
Basic method
Heterokaryons
Kao and Michayluk (1974)
PEG, coverslip
up to 10 a
Harms and Potrykus (1978)
PEG, rolling tube, iso-osmotic sucrose gradients
up to 31 b
Bates (1985)
Large-scale etectrofusion
20_+ 1- 0c
Tempelaar and Jones (1985)
Large-scale electrofusion
up to 16 c
Kao (1986)
purified PEG, at interface of glucose and sucrose-PEG solution
up to 30 a
Kao and Saleem (1986)
purified PEG, coverslip
17-41 a
Kamata and Nagata (1987)
Electrofusion, PercolI and seawater step gradients
71-83 ~
Present paper
PEG, test tube, Percoll flotation
50 • 0- 7 c
(%)
401
tained with 50% heterokaryons (Tables 2, 3) that are capable of subsequent growth into hybrid calli and plants. In keeping with our own experience with PEG giving variable fusion frequencies (Hodgson and Rose 1984) the absolute quantity of heterokaryons we recover does vary. This is of little consequence however if the base-line is minimised by taking the appropriate precaution with protoplast quality, and particularly given the subsequent nurse system where cell density is not a problem. In a typical fusion experiment, between 3.6" 105 mesophylt protoplasts of N. tabacum and 1.7.106 cell-suspension protoplasts of N. suaveolens, 2750 viable heterokaryons can be embedded in agarose. Recent studies by Kao and Saleem (Table 3) have shown improved fusion with PEG deionized by a mixed-bed ion-exchange resin. We have not tested this procedure but it may increase the heterokaryon recovery rate further. The 50% fusion frequency following purification (Table 2) compares favourably with the recently published electrofusion procedures (Table 3) based on the original technique of Zimmerman and Scheurich (1981). The Percoll procedure that we have used is simpler than the iso-osmotic density gradients of Harms and Potrykus (1978) which yielded up to 31% heterokaryons. The procedure used in our study has two features which facilitate the enrichment; the sensitivity of the mesophyll protoplasts to the PEG and dimethylsulfoxide concentrations used, and a different buoyant density of many of the N. suaveolens protoplasts compared with the heterokaryons. Based on available information these two conditions are likely to be met in other hybrid combinations, giving the technique more general applicability. Mesophyll protoplasts of other species commonly are more sensitive to PEG than protoplasts from cell suspensions (Kao and Michayluk 1974; Gleba and Sytnik 1984, p. 24; Menczel and Wolfe 1984). Mesophyll protoplasts differing in buoyant density from a protoplast fusion partner from a cell-suspension line should commonly occur given that mesophyll cells frequently differ in buoyant density from other cell types that lack or contain few chloroplasts (Harms and Potrykus 1978; Gleba and Sytnik 1984, p. 50, Kamata and Nagata 1987). The nurse-culture system used in this study is similar in principle to other feeder-system techniques (Menczel et al. 1978; Hein et al. 1983; Hamill et al. 1984; Shneyour et at. 1984; Lawrence and Davies 1985). The main value of our system is that it is a robust technique which retains many of the features of the microdrop techniques while
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M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
avoiding the very small liquid volumes required in some highly refined protocols (Koop and Schweiger 1985) and the possible difficulties with media composition (Caboche et al. 1984). The use of an agarose microdrop surrounded by nurse cells for somatic hybridisation has general application where low numbers of protoplasts or cells need to be cultured. Immobilization of heterokaryons in agarose microdrops also allows heterokaryons to be followed and manipulated during culture. The procedures we have utilised overcome the need for selection methods based on appropriate mutants or genetically modified parents and allow exploiting the total variation possible from a heterokaryon population. Hybrid and cybrid forms can be obtained in the same way they are obtained by manual isolation and subsequent growth in conditioned media (Gleba et al. 1984). We wish to thank Mr. Gary Weber of the University of Newcastle Electron Microscope Unit, the University of Newcastle Medical Communications Unit and Mr. John Fitter. This research was supported by the Australian Research Grants Scheme.
References Bates, G.W. (1985) Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana. Planta 165, 217-224 Bates, G.W., Hasenkampf, C. (1985) Culture of plant somatic hybrids following electrical fusion. Theor. Appl. Genet. 70, 227-233 Caboche, M., Aranda, G., Poll, A.M., Huet, J.-C., Leguay, J.-J. (1984) Auxin conjugation by tobacco mesophyll protoplasts. Correlations between auxin cytotoxicity under low density growth conditions and induction of conjugation processes at high density. Plant Physiol. 75, 54-59 Dalton, C.C., Iqbal, K., Turner, D.A. (1983) Iron phosphate precipitation in Murashige and Skoog media. Physiol. Plant. 57, 472-476 Douglas, G.C., Keller, W.A., Setterfield, G. (1981) Somatic hybridisation between Nicotiana rustica and N. tabacum. II. Protoplast fusion and selection and regeneration of hybrid plants. Can. J. Bot. 59, 22~227 Flick, C.E., Kut, S.A., Bravo, J.E., Gleba, Y.Y., Evans, D.A. (1985) Segregation of organelle traits following protoplast fusion in Nicotiana. Bio/Technology 3, 559-560 Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Nutrient requirements of a suspension culture of soybean root cells. Exp. Cell Res. 50, 151-158 Gleba, Y.Y. (1978) Microdroplet culture. Tobacco plants from mesophyll protoplasts. Naturwissenschaften. 65, 158-159 Gleba, Y.Y., Sytnik, K.M. (1984) Protoplast fusion, genetic engineering in higher plants. Springer Verlag, Berlin Gleba, Y.Y., Kolesnik, N.N., Meshkene, I.V., Cherep, N.N., Parokonny, A.S. (1984) Transmission genetics of the somatic hybridisation in Nicotiana. I. Hybrids and cybrids among the regenerates from cloned protoplast fusion products. Theor. Appl. Genet. 69, 121-128 Hamill, J.D., Patnaik, G., Pental, D., Cocking, E.C. (1984) The culture of manually isolated heterokaryons of Nicotiana tabacum and Nicotiana rustica. Proc. Indian Acad. Sci. (Plant Sci.) 93, 317-328
Harms, C.T., Potrykus, I. (1978) Enrichment for heterokaryocytes by the use of iso-osmotic density gradients after plant protoplast fusion. Theor. Appl. Genet. 53, 49-55 Hein, T., Przewozny, T., Schieder, O. (1983) Culture and selection of somatic hybrids using an auxotrophic cell line. Theor. Appl. Genet. 64, 119-122 Hodgson, R.A.J., Rose, R.J. (1984) Fusion of spinach mesophyll protoplasts with carrot root parenchyma protoplasts and the effect on spinach chloroplasts. J. Plant Physiol. 115, 69-78 Kamata, Y., Nagata, T. (1987) Enrichment of heterokaryocytes between mesophyll and epidermis protoplasts by density gradient centrifugation after electric fusion. Theor. Appl. Genet. 75, 26-29 Kao, K.N. (1977) Chromosomal behaviour in somatic hybrids of soybean-Nicotiana glauca. Mol. Gen. Genet. 150, 225 230 Kao, K.N. (1986) Fusion of plant protoplasts at the interface of a glucose and a sucrose-polyethylene glycol solution. J. Plant Physiol. 126, 55-58 Kao, K.N., Michayluk, M.R. (1974) A method for high-frequency intergeneric fusion of plant protoplasts. Planta 115, 355-367 Kao, K.N., Saleem, M. (1986) Improved fusion of mesophyll and cotyledon protoplasts with PEG and high pH-Ca 2+ solutions. J. Plant Physiol. 122, 217-225 Koop, H.U., Schweiger, H.G. (1985) Regeneration of plants after electrofusion of selected pairs of protoplasts. Eur. J. Cell Biol. 39, 46-49 Koop, H.U., Weber, G., Schweiger, H.G. (1983) Individual culture of selected single cells and protoplasts of higher plants in microdroplets of defined media. Z. Pflanzenphysiol. 112, 21 34 Lawrence, W.A., Davies, D.R. (1985) A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep. 4, 33-35 Medgyesy, P., Menczel, L., Maliga, P. (1980) The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris and isolation of their somatic hybrids. Mol. Gen. Genet. 179, 693 698 Menczel, L., Lazar, G., Maliga, P. (1978) Isolation of somatic hybrids by cloning Nicotiana heterokaryons in nurse culture. Planta 143, 29 32 Menczel, L., Wolfe, K. (1984) 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. 57, 473-497 Schweppenhauser, M.A., Mann, T.J. (1968) Restoration of staminal fertility in Nicotiana by introgression. Can. J. Genet. Cytol. i0, 401-411 Shneyour, Y., Zelcer, A., Izhar, S., Beckman, J.S. (1984) A simple feeder-layer technique for the plating of plant cells and protoplasts at low density. Plant Sci. Lett. 33, 293 302 Tempelaar, M.J., Jones, M.G.K. (1985) Directed electrofusion between protoplasts with different responses in a mass fusion system. Plant Cell Rep. 4, 92-95 Thomas, M.R., Rose, R.J. (1983) Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration. Planta 158, 329-338 Zimmermann, U., Scheurich, P. (1981) High frequency fusion of plant protoplasts by electric fields. Planta 151, 26-32
Received I February; accepted 11 April 1988
Planta (1988) 175:396-402
9 Springer-Verlag 1988
Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops M.R. Thomas* and R.J. Rose** Department of Biological Sciences, University of Newcastle, N.S.W. 2308, Australia
Abstract. Protoplasts isolated from Nicotiana tabacure L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures. Heterokaryons obtained were embedded in 20-~tl drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development. The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed. * Present address: Department of Plant Pathology, Kansas State University, Throckmorton Hall, Manhattan, KS 66506, USA ** To whom correspondence should be addressed Abbreviations: BAP=N6-benzylaminopurine; BM=basal me-
dium; 2,4-D = 2,4-dichlorophenoxyacetic acid; NAA = 1-naphthalene acetic acid; PEG =polyethylene glycol; PKM =modified Kao (1977) medium for protoplast culture
Key words: Heterokaryon enrichment - Nicotiana (protoplasts) - Protoplast fusion.
Introduction
A common limitation to producing somatic hybrids is the absence of selection schemes for the species under study, as genetically and biochemically characterised mutants are not always available. Furthermore, it may be undesirable to work with mutants when specific genotypes are to be used. To overcome the problem of using mutants as fusion partners to identify somatic hybrids, a procedure used in recent years has been the visual selection and manual isolation of heterokaryons. The heterokaryons are produced by fusion of a mesophyll protoplast with a cell-suspension protoplast and after isolation are cultured in a microdrop (Kao 1977; Gleba 1978; Bates and Hasenkampf 1985; Flick et al. 1985). Single protoplasts have been cultured in 10-25 nl of unconditioned medium (Koop et al. 1983), by achieving the same population density as in mass culture. Similar systems can be utilised in somatic-hybridisation studies (Koop and Schweiger 1985). Growth of heterokaryons in microdrops is frequently achieved with "conditioned" medium (Gleba and Sytnik 1984, pp. 17-19; Bates and Hasenkampf 1985). Gleba et al. (1984) have emphasized in their studies that fusion and manual isolation of heterokaryons overcomes the shortcomings of hybrid-screening methods that result in an automatic rejection of the theoretically possible recombinant forms. The culture of single cells or small cell numbers in microdrops requires specialised culture conditions to prevent evaporation from the microdrop,
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
as well as correct media composition, concentration, volume and in some cases "conditioning" to stimulate the heterokaryons to divide. One way to overcome the difficulties associated with the microdrop method has been to culture the manually isolated heterokaryons in a nurse culture that is genetically different, for example chlorophyll-deficient mutants (Menczel et al. 1978; Hamill et al. 1984) or auxotrophic mutants (Hein et al. 1983). In the present study we have utilised polyethylene glycol (PEG)-induced protoplast fusion, heterokaryon-enrichment procedures, and a nurse-culture system for the growth of Nicotiana tabacum L. x Nicotiana suaveotens Lehm. hybrid calli. These procedures are easily carried out and do not require mutant genotypes. The advantages of manual isolation and microdrop culture, particularly in being able to achieve the widest range of variation from somatic hybridisation, are largely maintained. Also, these procedures are suitable for studying the early development of somatic hybrids in an analogous way to our studies with N. tabacum (Thomas and Rose 1983).
Materials and methods Enzyme solutions, wash solutions, fusion solutions, and liquid culture media were sterilised by membrane filtration through 0.22-~tm Nalgene filters (Nalge Co., Rochester, N.Y., USA). The sterile Percoll (Pharmacia, Uppsala, Sweden) solution was used as supplied and should not be autoclaved in the procedures described. Callus and cell-suspension media were sterilised by autoclaving.
Isolation and purifieation oJmesophyll protoplasts of N. tabacum. Seedlings of N. tabacum L. cv. Xanthi (from seed maintained by us) were grown as described by Thomas and Rose (1983). Plants were grown for five to six weeks before transfer to a growth cabinet for 4-5 d with a daylength of 16 h, day and night temperatures of 27 ~ C and 25 ~ C, respectively, and relative humidity of 70 80%. Photon fluence rate provided by VHO cool-white fluorescent tubes (GTE Sylvania Canada, Drummondville, P.Q., Canada) and incandescent lamps was 85-100 ~tmol.s 1 -m-2 of photosynthetically active radiation (PAR) as measured by a Li-Cor quantum sensor (Li-Cor, Lincoln, Neb., USA). Leaves that were almost fully expanded, No. 7 or 8 from the apex, were selected for protoplast isolation. Leaf tissue from the central region of leaf 7 or 8 was surfacesterilised for 2 rain in 70% ethanol followed by 7 min in a calcium-hypochlorite solution (2% minimum available chlorine). After rinsing twice in sterile, glass-distilled water the leaf pieces (0.5-0.6 g FW) were cut into l-ram 2 sections and vacuum-infiltrated (400 mm Hg) for 1 min with 8 ml of enzyme solution. The enzyme solution contained 0.5% (w/v) Cellulase RI0 and 0.5% (w/v) Macerozyme R10 (enzymes from Yakult Honsha Co. Tokyo, Japan), dissolved in complete modified Kao (1977) medium for protoptast culture (PKM; Table 1) at pH 5.8, with 0.4 M mannitol in place of 0.38 M glucose. Incubation was in 100-ml Erlenmeyer flasks, in the dark at 27 ~ C for 12 16 h. After enzyme incubation the mesophyll protoplasts were collected by filtration through 70-gin stainless-steel mesh
397
(Hunter Wire Products, Warners Bay, N.S.W., Australia) into 15-ml glass centrifuge tubes. Percoll was then added to 20% (v/v) and mixed with the protoplasts, and 1 ml of PKM medium gently layered on top. Viable protoplasts that band at the PercolI/PKM interface after 5 min centrifugation at 50-g were col= lected.
Isolation and purification of cell-suspension protoplasts ojN. suaveolens. Cell suspensions were initiated from callus derived from leaf tissue. Callus was initiated from leaf sections of shoot cultures by incubation on basal medium (BM) as described in Table 1 +0.5 gM 2,4-dichlorophenoxyacetic acid (2,4-D)+ 5 gM l-naphthaleneacetic acid (NAA) in 0.8% agar. Callus cultures were grown in the dark at 27 ~ C and subcultured every three weeks. Cell suspensions were initiated by transferring calli to liquid BM medium and shaking in 250-ml Erlenmeyer flasks on a B. Braun HT orbital shaker (B. Braun, Melsungen, FRG) at 120 rpm in the dark at 27 ~ C. For protoplast isolation, cells were collected by centrifugation 4 d after subculture and 1.5-2 ml of packed cells were suspended in 8 ml of enzyme solution. The enzyme solution contained 0.5% (w/v) Cellulase RS (Yakult Honsha) 0.5% w/v Macerozyme R10 and 0.02% w/v Pectolyase Y23 (Seishu Pharmaceutical industry Co., Tokyo, Japan), dissolved in complete PKM medium (Table 1) at pH 5.5 with 0.4 M mannitol in place of 0.38 M glucose. Incubation on an orbital shaker (60 rpm) was in 100-ml Erlenmeyer flasks, in the dark at 27~ for 12-16 h. Protoplasts were collected by filtration through 44-gm stainless-steel mesh into 15-ml glass centrifuge tubes. Purification of viable protoplasts was the same as that described for N. tabacum mesophyll protoplasts except an inorganic salt solution which we designated MW5 was used (180 mM CaC12 2H20, 18 m M NaC1, 5 mM KC1, 5 m M glucose, 3 m M 2-(Nmorpholino)ethane sulfonic acid (Mes) buffer, pH 5.5) in place of the PKM medium as an overlay solution. The MW5 inorganic salt solution was modified from Medgyesy et al. (1980) and its use was necessary since few cell-suspension protoplasts form a pellet when centrifuged at 50 .g in P K M medium. Protoplast fusion. The purified protoplasts of the two species were resuspended in MW5 solution in separate 15-ml glass centrifuge tubes and pelleted by 5 min centrifugation at 50.g. The pellets were resuspended in twice their packed cell volume with MW5 solution. The two protoplast populations were then mixed at a volume:volume ratio of 1:2, N. tabacum mesophyll protoplasts to N. sauveolens cell-suspension protoplasts, to give a final volume of 0.4 ml. This gave approx. 4-105 N. tabacum protoplasts and 2.106 N. suaveolens protoplasts. A 1.2-ml volume of a polyethylene-glycol (PEG) fusion solution was then added along the side of the centrifuge tube and the protoplasts were gently resuspended with a siliconised Pasteur pipette. The PEG fusion solution consisted of 50% (w/v) PEG 1540 (Polysciences, Northhampton, UK), 10.5 mM Ca(NO3)z-4H20, 0.1 M glucose, 0.7 mM KHzPO4 and 2% (v/ v) dimethyl sulfoxide (DMSO) at pH 5.5. The protoplasts were incubated in the PEG fusion solution for 5 min. During this incubation the protoplasts were pelleted (2 min, 50 "g). An equal volume (1.6 ml) of a high-pH-calcium-DMSO elution solution was then added and the protoplast pellet resuspended to break up large clumps. This solution consisted of a solution of 0.4 M glucose, 55.5 m M Ca(NO3)2-4H20 and 8.88% (v/v) DMSO and a solution of 0.3 M glycine adjusted with K O H to pH 10.5 ; mixed just before use at 9:1. After 10 min in the elution solution, PKM medium (but buffered with 50 mM Mes buffer; Menczel and Wolfe 1984) was added over 20 min to give a total volume of 8 ml.
398
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
Table 1. Composition of the basal medium (BM) and the modified Kao (1977) medium (PKM), in mg/l. The BM medium is modified from Murashige and Skoog (1962), vitamins are from Gamborg et al. (1968). Coconut water is present in P K M at 10 ml/1 and is obtained from mature nuts then heated to 60 ~ C for 30 min and filtered. Both BM and P K M have iron concentration and chelation according to Dalton et al. (1983). Adjustment of ~H is made with 1 M K O H to 5.8 for BM and 5.5 for PKM. Casamino acids are vitamin-free from Difco (Difco Labs., Detroit Mich., USA). Compound
Compound
Medium BM
PKM
1650 1900 440 370 170 -
600 1900 600 300 170 300
FeSO4" 7H20 Na2EDTA'2H20 a
9.3 37.3
9.3 37.3
H3BO 3
6.2 16.9 8.6 0.83
3 10 2 0.75
MnSO4" H 2 0 ZnSOa "7H20 KI Na2MoO4" 2H20 CuSO,~" 5H20 CoC12 - 6H20
0.25 0.025 0.025
0.25 0.025 0.025
Organic acids Sodium pyruvate Citric acid Malic acid Fumaric acid
---
5 10 10 10
PKM
Sucrose Glucose Fructose Ribose Xylose Mannose Rhamnose Cellobiose Sorbitol Mannitol
30000 -
125 68400 125 125 125 125 125 125 125 125
Vitamins myo-Inositol Nicotinamide Nicotinic acid Pyridoxine. HC1 Thiamine. HC1 D-Calcium panthothenate Biotin Folic acid p-Aminobenzoic acid Choline chloride
100 1 1 i0 -
100 1 1 10 0.5 0.005 0.2 0.01 0.5
250
195 125
Other
Hormones NAA 2,4-D BAP
BM
Sugars
Mineral salts NH4NO3 KNO3 CaC12 2 H 2 0 MgSOr 7H20 KHzPO4 KC1
Medium
--
0.93 0.11 0.56
Mes buffer Casamino acids
a Disodium-ethylenediaminetetraacetic acid
Enrichment for heterokaryons after protoplast fusion. Percoll was added to 20% (v/v) to the post-fusion preparation and the preparation was mixed and overlayed with 1 ml of P K M medium. Centrifugation at 50 .g for 5 rain allowed viable protoplasts rich in heterokaryons to be collected at the interface of the two solutions. The protoplast population enriched for heterokaryons was washed once with P K M medium, diluted to 4 ml, and plated 0.5 ml/well in 24-well Linbro tissue-culture plates (Flow Laboratories, Sydney, N.S.W., Australia). After overnight culture the protoplast population enriched for heterokaryons was collected into a glass centrifuge tube and pelleted for 5 rain at 50.g. The protoplast pellet was resuspended in 0.6 ml P K M medium containing 20% (v/v) Percolt, transferred to a 1.5-ml siliconised Eppendorf tube, overlayed with P K M medium, and centrifuged for 5 min at 50.g to obtain viable protoplasts. Viable protoplasts were collected from the interface and washed once in 1 ml of P K M medium in an Eppendorf tube before final resuspension in 0.1 ml of P K M medium. Aliquots (0.01 ml) of the protoplasts enriched in heterokaryons were distributed to wells of 24-well Linbro tissue-culture plates and mixed with 0.01 ml of 0.7% molten (35 ~ C) Seaplaque agarose (FMC Marine Colloids Division, Rockland, N.Y., USA) in P K M medium. After the agarose microdrops
had solidified a liquid nurse culture (0.5 ml) was then added to each well containing the heterokaryons embedded in the 0.02-ml agarose microdrops.
Culture of heterokaryons. The liquid nurse cultures were either N. tabacum- or N. suaveolens-protoplast cultures that came from protoplasts not used in the fusion treatment. The N. suaveolens-protoplast nurse cultures were at a density of 105-2 . 105 protoplasts/ml and the N. tabacum-protoplast nurse cultures were at a density of 10~-2 - 104 protoplasts/ml. Five to seven days after fusion, 0.18 ml of the nurse culture from each well was removed and replaced with 0.18 ml of C K M medium. The C K M medium was as for P K M medium but with glucose at 0.253 M and sucrose at 29 mM. On day 12 of culture an additional 0.18 ml of C K M medium was added to each well. After 2 d the nurse culture was removed from each well with a Pasteur pipette and the wells were washed twice with C K M medium having lower hormone concentrations (0.05 gM NAA, 0.005 gM 2,4-D, 0.25 pM N6-benzylaminopurine (BAP)) before finally covering the microdrop with 0.5 ml of this medium. Three weeks after fusion the agarose microdrops containing the hybrid cell colonies were transferred to agar plates contain-
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana ing shoot-induction medium (BM medium with 0.05 laM N A A + 2.5 g M BAP). Washing in the low-hormone C K M medium prior to transfer ensured no nurse colonies were transferred. Approximately 0.5 ml of the low-hormone C K M medium was added around the transferred microdrops to prevent osmotic stress to the cell colonies. Large cell colonies were present after 6 d and at this time the agarose microdrop was broken up under a stereo-microscope ( x 16) to separate out the colonies to obtain callus formation.
Fluoreseein-isothiocyanate labelling of N. suaveolens cell suspension protoplasts. At the beginning of enzyme incubation, 25 gl of freshly prepared fluorescein isothiocyanate (FITC; Sigma Chemical Co., St. Louis, Mo., USA) in acetone (5 mg/ml) was added to 8 ml of the cell-wall-degrading enzyme solution. The protoplast isolation, purification and fusion were as described previously. Protoplasts labelled with FITC were visualised with a Leitz Orthoplan fluorescence microscope (E. Leitz, Wetzlar, F R G ) under epi-fluorescent illumination using a BG12 excitation filter and a K490 suppression filter. All protoplasts were labelled.
Results
Growing the plants under controlled conditions gave yields of intact N. tabacum protoplasts averaging 106 per I g of leaf tissue. Cell suspension cultures of N. suaveolens gave yields averaging 5" 105 per 2 ml of packed cell volume. The use of Percoll flotation to separate viable from non-viable protoplasts after enzyme treatment proved to be a simple and reliable method for both the mesophyll protoplasts of N. tabacum (Fig. 1) and the cell-suspension protoplasts of N. suaveolens (Fig. 1). This procedure was an improvement over the use of a Percoll cushion that we had used previously (Thomas and Rose 1983). Fusion in a centrifuge tube (Douglas et al. 1981) has a number of advantages. Maximum contact between protoplasts can be achieved during PEG treatment and the heterokaryons produced are readily available for further manipulation. The sticking of heterokaryons to the coverslip makes manipulation of heterokaryons difficult in fusion procedures on coverslips. However, Kao (1986) has recently overcome this by fusion of the protoplasts with PEG at the interface of two solutions. A mixed protoplast suspension of N. tabacum mesophyll protoplasts and N. suaveolens cell-suspension protoplasts before fusion is shown in Fig. 1. There is a higher proportion of the smaller cell-suspension protoplasts (see Materials and methods) to increase the chance of heterokaryon formation. Protoplast populations enriched for heterokaryons after fusion and Percoll purification are shown in Figs. 2 and 3. The mesophyll protoplasts were most sensitive to the fusion treatment and after purification the viable protoplasts were
399
mainly heterokaryons or cell-suspension protoplasts of N. suaveolens (Fig. 2, Table 2). The purified protoplasts were cultured overnight before being purified again to remove dead protoplasts and embedded in agarose microdrops (Fig. 3) for culture in a nurse culture (see Materials and methods). Counts from three separate experiments, carried out on enriched heterokaryon protoplast populations after embedding in agarose microdrops showed that on average 49.7% of the population were heterokaryons, 44.1% cell-suspension protoplasts of N. suaveoIens and 6.2% mesophyll protoplasts of N. tabacum (Table 2). These figures were confirmed in a separate experiment to check our identification of heterokaryons, by using fluorescein-isothiocyanate-labelled cell-suspension protoplasts of N. suaveolens with mesophyll protoplasts of N. tabacum. Counts by fluorescence microscopy after fusion indicated that 92.5% (n = 200) of the protoplasts containing chloroplasts were heterokaryons, and the remainder were N. tabacum mesophyll protoplasts. The presence of the fluorescent stain in the heterokaryons was very specific since a control treatment where fluorescein-isothiocyanate-stained protoplasts of N. suaveolens were fused and mesophyll protoplasts ofN. tabacum added after the high-pH-calcium-dimethylsulfoxide treatment (same contact time as normal fusion) showed no uptake of the stain by protoplasts of N. tabacum because of release by dead protoplasts of N. suaveolens. The protoplasts enriched for heterokaryons and embedded in an agarose microdrop were able to grow in conditions favourable for heterokaryon growth and the agarose provided a physical boundary between the nurse culture and the heterokaryons. Figure 3 shows heterokaryons embedded in an agarose microdrop. Manually isolated heterokaryons can be readily used in this system. Figure 4 shows a cell colony that developed from a heterokaryon isolated (1 d after fusion) with a Gilson Pipetman (Gilson France, Villiers Le Bel, France). Division frequencies of chloroplast-containing cells (heterokaryons and N. tabacum cells; see Table2) averaged 31% after 8 d of culture while the frequency of N. suaveolens cells was 9%. These frequencies are considerably lower than that observed for unfused control protoplast cultures of N. tabacum (80%) and N. suaveolens (67%). Electron microscopy is facilitated by the use of the agarose microdrops which are readily embedded. The heterokaryons contain two types of plastids (data not shown). One type of plastid represents dedifferentiation of chloroplasts from the N. tabacum mesophyll fusion partner (see Thomas
400
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
Fig. 1. Mixed protoplast suspension of iV. tabacum mesophyll protoplasts and N. suaveolens cell-suspension protoplasts before fusion. There is a higher proportion of cell-suspension protoplasts to mesophyll protoplasts. Bar =20 btm; x 460 Fig. 2. Protoplast population enriched for heterokaryons after fusion of N. tabacum mesophyll protoplasts with N. suaveolens cell-suspension protoplasts. Heterokaryons can be recognised (arrowheads) because of the presence of chloroplasts from the mesophyll protoplasts and the cytoplasm of the cell-suspension protoplasts. Bar = 20 gm; x 460 Fig. 3. Protoplast population after fusion and enrichment for heterokaryons embedded in an agarose microdrop. All chloroplastcontaining protoplasts in this field are heterokaryons (arrowheads). Bar=100 gm; x 170 Fig. 4. Division of manually isolated heterokaryon embedded in an agarose microdrop. Out-of-focus cells are nurse cells outside the agarose drop. Bar=20 #m; x 350
Table 2. Proportion of heterokaryons after protoplast fusion, enrichment for heterokaryons, and embedding in agarose microdrops. Number of protoplasts counted in the agarose microdrops for the different experiments were 1462 (Expt. 1), 1000 (Expt. 2), 200 (Expt. 3)
Expt.
Protoplast types (%) N. suaveolens
N. tabaeum
Heterokaryons
1 2 3
48.4 48.2 35.7
0.3 1.6 16.8
51.3 50.2 47.5
Mean
44.1 +_2.4
6.2_+3.1
49.7_+0.7
and Rose 1983) and the other plastid type is representative of the small proplastids observed in the N. suaveolens cell suspension fusion partner. The cell colonies in the agarose microdrops were grown through to calli and some formed shoots on regeneration medium. More cell colonies grew through to calli when N. tabacum mesophyll protoplasts were used as a nurse culture. Esterase profiles of callus protein extracts run on nondenaturing polyacrylamide gels showed that N. tabacum calli had two major low-molecularweight bands close together while N. suaveolens calli had four higher-molecular-weight bands. Hy-
M.R. Thomas and R.J. Rose: tteterokaryon enrichment and growth in microdrops in Nicotiana
brid calli had both N. tabacum and N. suaveolens bands (data not shown). About 45% of the calli appear to be derived from heterokaryons. The N. suaveolens cell line used does not develop past the callus stage. Approximately one-third of the regenerated plants were N. tabacum and two-thirds differed morphologically from N. tabacum. Analyses to date on these latter plants indicate the presence of cybrids rather than hybrids. In sexual hybridisation between N. tabacum and N. suaveolens only N. tabacum lines with N. suaveolens cytoplasm have been obtained (Schweppenhauser and Mann 1968). Discussion
The basic PEG fusion procedure introduced by Kao and Michayluk (1974), though widely and succesfully used in various modified forms to produce somatic hybrids (Gleba and Sytnik 1984) gives relatively low and variable fusion frequencies (Table 3). However Kao (1986) has recently obtained fusion frequencies of up to 30% using a modified coverslip procedure (Table 3). Utilising the protocols outlined in this study with N. tabacure x N. suaveolens allows a preparation to be obTable 3. Heterokaryons produced by different fusion and enrichment protocols. In the reports cited, "division of heterokaryons, b calli from heterokaryons, ~ plants from heterokaryons. Heterokaryons ( % ) -
heterokaryons x 100 total No. of protoplasts
Authors
Basic method
Heterokaryons
Kao and Michayluk (1974)
PEG, coverslip
up to 10 a
Harms and Potrykus (1978)
PEG, rolling tube, iso-osmotic sucrose gradients
up to 31 b
Bates (1985)
Large-scale etectrofusion
20_+ 1- 0c
Tempelaar and Jones (1985)
Large-scale electrofusion
up to 16 c
Kao (1986)
purified PEG, at interface of glucose and sucrose-PEG solution
up to 30 a
Kao and Saleem (1986)
purified PEG, coverslip
17-41 a
Kamata and Nagata (1987)
Electrofusion, PercolI and seawater step gradients
71-83 ~
Present paper
PEG, test tube, Percoll flotation
50 • 0- 7 c
(%)
401
tained with 50% heterokaryons (Tables 2, 3) that are capable of subsequent growth into hybrid calli and plants. In keeping with our own experience with PEG giving variable fusion frequencies (Hodgson and Rose 1984) the absolute quantity of heterokaryons we recover does vary. This is of little consequence however if the base-line is minimised by taking the appropriate precaution with protoplast quality, and particularly given the subsequent nurse system where cell density is not a problem. In a typical fusion experiment, between 3.6" 105 mesophylt protoplasts of N. tabacum and 1.7.106 cell-suspension protoplasts of N. suaveolens, 2750 viable heterokaryons can be embedded in agarose. Recent studies by Kao and Saleem (Table 3) have shown improved fusion with PEG deionized by a mixed-bed ion-exchange resin. We have not tested this procedure but it may increase the heterokaryon recovery rate further. The 50% fusion frequency following purification (Table 2) compares favourably with the recently published electrofusion procedures (Table 3) based on the original technique of Zimmerman and Scheurich (1981). The Percoll procedure that we have used is simpler than the iso-osmotic density gradients of Harms and Potrykus (1978) which yielded up to 31% heterokaryons. The procedure used in our study has two features which facilitate the enrichment; the sensitivity of the mesophyll protoplasts to the PEG and dimethylsulfoxide concentrations used, and a different buoyant density of many of the N. suaveolens protoplasts compared with the heterokaryons. Based on available information these two conditions are likely to be met in other hybrid combinations, giving the technique more general applicability. Mesophyll protoplasts of other species commonly are more sensitive to PEG than protoplasts from cell suspensions (Kao and Michayluk 1974; Gleba and Sytnik 1984, p. 24; Menczel and Wolfe 1984). Mesophyll protoplasts differing in buoyant density from a protoplast fusion partner from a cell-suspension line should commonly occur given that mesophyll cells frequently differ in buoyant density from other cell types that lack or contain few chloroplasts (Harms and Potrykus 1978; Gleba and Sytnik 1984, p. 50, Kamata and Nagata 1987). The nurse-culture system used in this study is similar in principle to other feeder-system techniques (Menczel et al. 1978; Hein et al. 1983; Hamill et al. 1984; Shneyour et at. 1984; Lawrence and Davies 1985). The main value of our system is that it is a robust technique which retains many of the features of the microdrop techniques while
402
M.R. Thomas and R.J. Rose: Heterokaryon enrichment and growth in microdrops in Nicotiana
avoiding the very small liquid volumes required in some highly refined protocols (Koop and Schweiger 1985) and the possible difficulties with media composition (Caboche et al. 1984). The use of an agarose microdrop surrounded by nurse cells for somatic hybridisation has general application where low numbers of protoplasts or cells need to be cultured. Immobilization of heterokaryons in agarose microdrops also allows heterokaryons to be followed and manipulated during culture. The procedures we have utilised overcome the need for selection methods based on appropriate mutants or genetically modified parents and allow exploiting the total variation possible from a heterokaryon population. Hybrid and cybrid forms can be obtained in the same way they are obtained by manual isolation and subsequent growth in conditioned media (Gleba et al. 1984). We wish to thank Mr. Gary Weber of the University of Newcastle Electron Microscope Unit, the University of Newcastle Medical Communications Unit and Mr. John Fitter. This research was supported by the Australian Research Grants Scheme.
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Received I February; accepted 11 April 1988
