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Production of fertile hybrids by electrofusion of vacuolated and evacuolated tobacco mesophyll protoplasts
Plant Science, 85 (1992) 197-208
197
Elsevier Scientific Publishers Ireland Ltd.
Production of fertile hybrids by electrofusion of vacuolated and evacuolated tobacco mesophyll protoplasts* Beatrix Naton, Margret Ecke and Riidiger Hampp Lehrstuhl Physiologische ¢~kologie tier Pflanzen, lnstitut J~r Botanik, Universitiit Tiibingen, Auf der Morgenstelle 1, W-7400 Tiibingen
(FRG) (Received March 23rd, 1992; revision received May 15th, 1992; accepted June 4th, 1992)
The aim of our experiments was to produce somatic hybrids without using mutants as fusion partners and an expensive cell sorter. Vacuolated mesophyll protoplasts of Nicotiana tabacum U cv. Xanthi were electrically fused with evacuolated protoplasts of the same species or genus (N. rustica L.) in a mass fusion chamber. Fusion yields of 6-11% were obtained. After density gradient centrifugation the fraction enriched in fusion products (90-100%) was embedded in low melting point agarose lenses fixed to the bottom of plastic petri dishes and cultured in the presence of feeder cells. The development of individual fusion products could be followed for several weeks. Plating efficiencies were estimated to be 10-39% for heterokaryocytes. Hybrid character was tested by chromosome counting, isozyme analysis and comparison of morphological properties. About 86% of the regenerants showed hybrid character and 4% were putative cybrids. This study demonstrates that somatic hybrid plants may be selected after electrofusion of mesophyll protoplasts from cultivates lacking specific markers and without using micro-methods or a cell sorter.
Key words: heterokaryon enrichment; iso-osmotic density gradient centrifugation; Nicotiana; evacuolation; protoplast electrofusion; somatic hybridization
Introduction
Protoplast fusion provides a means to hybridize plant species over wide taxonomic distances such as potato and tomato [1] and has become a powerful technique to overcome crossability problems [2,3]. A common limitation in somatic hybridization is the lack of a convenient selection method for the desired heterospecific fusion products [4], because genetic markers are not available for all varieties. Usually fusion frequencies do not exceed 10% irrespective of the applied fusion procedure. Thus culture of the whole fusion population and identification of hybrids at the plant level is quite Correspondence w: Beatrix Naton, Lehrstuhl Physiologische Okologie der Pflanzen, Institut ffir Botanik, Universit~t Tfibingen, Auf der Morgenstelle i, W-7400 Tiibingen, FRG. *These results were presented in part at the 7th International Congress on Plant Tissue and Cell Culture, Amsterdam, The Netherlands, 24-29 June 1990.
elaborate. Microfusion and microculture of single protoplasts as developed by Koop and Schweiger [5], isolation of labelled fusion products by means of micropipettes (e.g. Ref. 6) or enrichment of double-stained fusion products by flow sorting (e.g. Refs. 7-9) are time-consuming and/or expensive methods and require specialized technical expertise. So-called 'universal hybridizers' were produced in a very limited number of plant species (e.g. for Nicotiana tabacum [10]). But the introduction of a resistance gene in the parental species (e.g. Ref. 11) and the additional in vitro culture period may increase somaclonal variation [12], especially in the important crop plant potato [13]. However, this is a disadvantage if certain breeding lines are required. Therefore there is a need of a universal, convenient and low-priced selection method for somatic hybrids without manipulating the genome. Harms and Potrykus [14] used differences in
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
198 boyant density between different types of protoplasts to enrich heterokaryocytes after isoosmotic density gradient centrifugation. In general natural differences between the fusion partners (e.g. epidermis and mesophyll protoplasts) are small [14-16]. As a result the heterokaryocytes are severely contaminated with parental protoplast [14,16]. Additionally, the yield of epidermis protoplasts is very low and handling is complicated, because these protoplasts are very fragile and not pellettable in salt-free solutions, a prerequisite for electrofusion [16; own unpublished observations]. Evacuolation of protoplasts can be used to generate fusion partners distinguishable from ordinary vacuolate protoplasts. Due to distinct differences in the specific density of the respective protoplast populations, heterokaryocytes can be separated from homofusion products as well as from unfused parental protoplasts by iso-osmotic density gradient centrifugation [17]. Evacuolated protoplasts are less fragile than epidermis protoplasts and the degree of somaclonal variation is not increased over that of suspension culture protoplasts. Evacuolation of mesophyll protoplasts is reported for many species [18]. Differences in specific density can be utilized to achieve an ordered pair formation by differential sedimentation. By application of asymmetric membrane breakdown high rates of heterokaryotic fusion products were obtained [19]. Here we demonstrate that this system can be used to recover fertile hybrids from a highly enriched fraction of fusion products. For that purpose mesophyll protoplasts of Nicotiana tabacum were fused either with evacuolated protoplasts of the same species or the same genus (N. rustica), because somatic hybrids between these two species are known from literature [10,20]. Materials and Methods
Enzyme solutions, media containing Percoll® (Pharmacia, Uppsala, Sweden) and liquid protoplast culture media were sterilized by membrane filtration through 0.22-#m Sartorius cellulose nitrate filters (Sartorius, Grttingen, FRG). Callus, suspension- and shoot-culture media were sterilized by autoclaving (20 min, 117°C). All solutions
used for density gradient centrifugation were adjusted to 560 ± 20 mOsm. The osmolality was determined using a Vescor Vapor Pressure Osmometer (Logan, Utah, USA).
Isolation and evacuolation of tobacco mesophyll protoplasts Axenic shoot cultures of N. tabacum L. (cv. Xanthi and Samsun) and N. rustica L. NRT were initiated from sterilized seeds. Shoot cultures were maintained on hormone free MS medium [21] and cultured in 500 ml Weck® jars at 23°C and a light period of 16 h (3500 Lux). Mesophyll protoplasts from 6-8-week-old plants were isolated and purified on iso-osmotic step gradients as described elsewhere [17]. Evacuolation was performed on a self-generating iso-osmotic Percoll® gradient [22] modified according to Naton et al. [17]. Fusion of protoplasts Electrofusion was carried out as previously described [17] using a Zimmermann Cell Fusion TM generator (GCA Corporation, Chicago, IL) in a self-constructed mass fusion chamber, consisting of a glass slide with combe-shaped chromium electrodes (distance: 100 #m). For PEG-induced fusion protoplasts were suspended in W5 solution and fused as described elsewhere [23]. Selection or fusion products Fused protoplasts were resuspended in 6 nil 0.44 M raffinose-buffer, transferred to a 10-ml centrifuge tube and overlaid with 2 ml 0.5 M mannitol-buffer. After centrifugation (10 min at 300 x g) mesophyll protoplasts and fusion products were collected from the interphase, washed once with 0.5 M mannitol-buffer, resuspended and layered on top of a preformed Percoll gradient (20% (v/v) Percoll, 0.5 M mannitol-buffer; 5000 x g, 2 h, swing out rotor). After a second centrifugation step (20 min, 300 x g), a highly enriched fraction of fusion products was recovered at the bottom of the gradient [17]. The modification of the sorting procedure (introduction of an additional centrifugation step to separate the evacuolated protoplasts from the vacuolated protoplasts and the heterokaryons) prevented
199 overloading of the gradient and aggregation of protoplasts,
Culture of fusion products The fraction enriched in fusion products was washed once in a modified MS-medium [24] with 4.7 rag/1 p-CPA and 1 rag/1 kinetin [25], all supplemented with 3% (w/v) sucrose and 9% (w/v) mannitol and embedded in 0.8% (w/v) low melting point agarose (Sea-Plaque®, FMC Marine Colloids, Rockland, NY, USA [26]). Droplets of the agarose-protoplast suspension were placed in 5-ml plastic Petri dishes (Greiner, Niirtingen, FRG). After hardening, the lenses adhered at the bottom and were surrounded with 4 ml of feeder cell suspension (tobacco mesophyll protoplasts or carrot suspension culture cells; density 50 000 or 100 000 ceUs/ml). The development of individual fusion products could be followed for several weeks. Tobacco mesophyll protoplast feeder cells were diluted every 10 days with 1 ml osmoticum free culture medium. Carrot suspension culture cells were diluted every 4 days (1 ml of suspension was replaced by 1 ml culture medium with decreasing osmolality). After 6 weeks the lenses free from feeder cells were transferred onto agar solidified NT-medium [27] complemented with 3 mg/1 NAA and 1mg/l kinetin. Seven days later mlcrocalli were dispersed on the agar by means of an inoculator. Another 2 weeks later the calli were transferred on shoot regeneration medium (MS complemented with 0.1 mg/1 NAA and 1 mg/1 BAP). Developing shoots were cut off and rooted in MS medium without hormones. Culture of suspension culture cells and preparation of conditioned medium For cross feeder experiments Daucus carota L. ssp. sativus suspension cultures were kindly provided by Prof. H.-U. Seitz, Tiibingen [24] and Panicum maximum Jacq. (guinea grass) cells by Prof. I.K. Vasil, Gainesville, FL, USA [28]. Cells from 4-6-day-old suspensions were washed once with protoplast culture medium and diluted to the desired suspension density. Conditioned medium was prepared from 5-day-old suspensions. Cells were removed from medium by filtration through filter paper. The cell-free supernatant was corn-
plemented with sucrose, vitamins and hormones as described above, osmotically adjusted with mannitol and stored at -25°C.
Analysis of somatic hybrids Ninety-five regenerants were selected randomly at callus stage, and regenerated plants were subjected to isozyme, cytogenetic and morphological analysis. All putative hybrid plants analyzed for isozyme patterns were derived from different calli. The leaf-isozyme patterns of selected enzymes were investigated by non-denaturating polyacrylamide gel electrophoresis using a microgel system [29]. The gels (10% separating gel + 3% stacking gel) were prepared as indicated in the Sigma Technical Bulletin No. MKR-137. For separation of esterases (EC 3.1.1.1) 0.3 g of greenhouse grown leave tissue was homogenized in 1 ml of 0.5 M Tris-HCl buffer (pH 6.8) containing 80 mM DTT (Cleland's reagent), 0.2% Na2SO4, 0.15% Na2S205, 0.2% Polyclar AT and 7.5% sucrose ([6] modified according to Stegemann and Schnick [30]). Esterase isozymes were visualized according to Brewbaker et al. [31]. The extraction medium for separation of acid peroxidases (EC 1.11.1.7) contained 0.26 M Trisphosphoric acid (pH 6.9) and 10 mM DTT. The extracts were dialysed overnight against 1:10 diluted extraction buffer [32]. After electrophoresis the patterns were visualized following a proeedure described by Brewbaker et al. [31] with slight modifications. Five milligrams of o-dianisidine and 3 mg of O-naphthol were dissolved in 2 ml of acetone, 8 ml of 0.1 M acetate buffer (pH 4.5) were added, and shortly before use, 10 #1 of 30% H202. Morphological analysis was performed at the inition of flowering. Leave shape and size, flower size, coloration and shape and growth of at least 4 plants from each line was compared with the traits of parental plants cultivated under the same conditions. Cytological techniques For determination of chromosome numbers root tips from greenhouse plants were excised at 10:00 h and pretreated in 2 mM 8-hydroxychinoline for 2.5 h at 17°C. Root tips were fixed in
200 Carnoy's solution overnight, transferred into 70% ethanol, stored at 4°C until use and stained with acetocarmine. Of each clone several individual plants were investigated.
Determination of fertility Flowers were selfed or back-crossed to N.
tabacum. Seed number was compared to selfpollinated parental plants. Pollen grains were stained with acetocarmine or, in case of in vitro germination, incubated at room temperature in a medium described by Dickinson [33]. Results and Discussion
Fusion conditions and heterokaryon selection The fusion frequencies obtained after PEGinduced fusion were very variable and ranged from 0% to 10%. A large portion of the vacuolated protoplasts stuck at the bottom of the petri dish and lysed. But the main problem was the tendency of the protoplasts to agglutinate in an unspecific way especially after centrifugation. Mesophyll protoplasts from greenhouse plants could be sorted after a post-fusion treatment with osmotically adjusted sea-water (5 h; 4°C). By lowering the amount of DMSO in the fusion solution or the Caconcentration in the elution medium both aggiutination and fusion yield were reduced, but sort purity did not exceed 50% in any case. Fusion frequencies after electrofusion varied between 3% and 5% for protoplasts from greenhouse plants and between 6% and 11% for protoplasts from axenic shoot cultures. This may be due to the greater homogeneity of the latter donor tissue. Upon pulse application about 50% of the protoplasts either lysed or formed multikaryons, which burst during subsequent manipulation. Although the fused protoplasts tended to clump slightly, which was possibly due to an increase in plasmalemma hydrophobicity, the purity of the heterokaryon population ranged between 90% and 100% depending on the time elapsed between prepurification and heterokaryon selection, and sort efficiencies varied from 10% to 39%. As a result, the portion of recovered heterokaryons is comparable to the sort efficiency obtained using a flow sorter [9]. The purity of
heterokaryon populations observed in the present study was generally significantly greater ( > 90%) than that reported by other workers. This finding can be explained in a number of ways. Firstly, the use of purified parental protoplast populations within a distinct range of boyant density was essential for obtaining pure heterokaryon populations [14-16]. This explains the failure of Pfister et al. [34] to recover hybrids without the aid of a previously introduced antibiotic resistance gene. The observation of high sort impurity of PEGobtained fusion products after iso-osmotic density gradient centrifugation is well-known from literature and may be induced by clumping and alterations in specific density. After an overnight culture period Thomas and Rose [26] purified the heterokaryocytes a second time, but nevertheless the population enriched in heterokaryocytes was contaminated by up to 50% with parental protoplasts. These findings agree with the literature [14,22]. Aggregation of cells hinders physical enrichment of heterokaryocytes even by cell sorting (e.g. compare Afonso et al. [7] with Hammatt et al. I91). It is worth noting that protoplasts seem to alter boyant density even when stored on ice in isoosmotic solutions [19]. Therefore it is necessary to keep the time between fractionation of parental protoplasts and selection of heterokaryons as short as possible. Furthermore it is important to fuse the protoplast under iso-osmotic conditions. Only electrofusion fulfills both requirements.
Cultivation and development of fusion products Fusion products were embedded in agarose lenses and cultivated in the presence of feeder cells. For that purpose different cells were tested for the ability to nurse embedded tobacco mesophyll protoplasts at low density (10 3 protoplasts/ml). Best results were obtained with mesophyll protoplasts of the same origin. Colony formation of the protoplasts within the agarose lenses was identical to that of the surrounding feeder protoplasts. The same nutritional and osmotic requirements were a second advantage over all tested suspension culture cells. To exclude a possible contamination of nursed cells by feeder cells, heterologous combinations were tested for their suitability to pro-
201
mote microcallus formation. Best results gave carrot suspension cells (suspension density 105 cells/ml). Plating efficiencies of the nursed cells were reduced only by 5-10% compared to the control protoplasts cultivated under standard conditions in liquid culture or agarose beads. No divisions of embedded tobacco protoplasts were obtained in the presence of Panicum maximum cells or conditioned medium alone, but a delay in degeneration was observed. These results gave further support to the hypothesis that cells release both 'viability' factors, which prevent degeneration, and 'nursing' factors, which support division of cells at very low density (for detailed discussion: Ref. 35). The high enrichment factor (-100%) together with plating at low density (about 50-100 viable cells/droplet) and the physical separation of cultured cells from feeder cells allowed the observation of individual fusion products over a long period of time. Figures 1 and 2 illustrate the
typical time course of fusion product development. In general, the heterokaryotic character persisted up to the first division which had a delay of about 4 days as compared with unfused mesophyll protoplasts. After fusion the chloroplasts (and probably the nucleus) of the vacuolate mesophyll protoplast started to migrate towards the area of the evacuolate protoplasts (Figs. I a-1 f). The first division occurred in this part of the cell and often led to asymmetric development (Fig. 2). Plating efficiencies were estimated to be 10-40% for heterokaryocytes and about 50-60% for the unfused mesophyll protoplasts. These results are in good agreement with plating efficiencies of tobacco heterokaryons obtained after microfusion [5,36], manual selection [6] and flow sorting [9] of mesophyll or suspension cell culture protoplasts. During the first few days many fusion products showed intense budding (Figs. 1 and 3). Budding and cleavage divisions are indicators of a non-rigid pseudo-wall in general, or of localized weak areas
Fig. 1. Development of a fusion product over a period of 2 weeks: migration of the chloroplasts towards the area of the evacuolated protoplast, budding at the face of the formerly evacuolate protoplast. Bar = 50/~m.
202
Fig. 2. Developmentof a fusion product over a period of 2 weeks: First division is highly asymmetrical. Bar = 50 ~m.
in the newly synthesized cell wall [37]. Budding is a characteristic event in the early development of evacuolate protoplasts even when embedded in agarose solidified media (unpublished results). Evacuolation may disorganize the cytoskeleton which plays an important role in an ordered deposition of newly synthesized cellulose fibrils [38,39], although the direct impact is yet unknown. Cell wall regeneration also depends on the integrity status of the plasma membrane [40,41]. Injury of the plasmalemma due to shearing forces during evacuolation and enrichment of heterokaryons (e.g. Ref. 8) may not be excluded. Nevertheless, we failed to detect an increased callose synthesis of evacuolated over vacuolated mesophyll protoplasts after staining with aniline blue. A cell sorter was not available which allows the observation of fusion products of vacuolated or evacuolated protoplasts. Therefore it can not be excluded that the presence of the evacuolated pro-
toplast caused an aberrant development of the fusion products. Miller et al. [42] and Fowke et al. [43], however, gave strong evidence that the multikaryotic nature of the fusion products by itself can cause a disturbed cell wall regeneration: polykaryotic soybean protoplasts synthesized less cell wall material than monokaryotic protoplasts and showed intense budding.
Analysis of hybrid plants The high sorting and plating efficiencies resulted in high yields of putative hybrids. In a representative experiment 2 x 600 000 protoplasts were fused. A fusion rate of 7.2% was obtained. The purified fraction of fusion products contained about 5000 cells. Plating efficiency was determined to be 35%. Twenty percent of the developing calli were randomly selected for shoot morphogenesis. About 90% of the clones possessed morphogenic potential and were able to root.
203
Fig. 3.
Fusion product showing extensive budding, the part of the evacuolated protoplast is still visible (dark). Bar = 25 #m.
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Fig. 4. Esterase isozymes. The patterns from extracts of N. tabacum, eleven hybrids and N. rustica (left to right); parental bands are indicated by arrows.
204
Fig. 5. Habitus of typical somatic hybrids in comparison with the parental species N. tabacum (left) and N. rustica (right).
Fig. 6.
The hybrid character of 95 regenerants was tested by chromosome counting and isozyme analysis (acid peroxidases and esterases). The isozyme pattern suggested that 82 of the analyzed clones were hybrids. Figure 4 shows the esterase profile of eleven hybrids and the two parental species. Unique hybrid bands or the absence of parental bands were frequently observed. The hybrid identity of these plants was supported by their chromosome number deviating from 48 (N. tabacum 2n = 48; N. rustica 2n = 48). A remarkable variation was found between the different clones (2n = 56-96), but not within different plants of the same line (variation ± 6 chromosomes). With the exception of 2 clones, all hybrid lines had less than the expected doubled chromosome set of 96 chromosomes. The majority of the hybrid plants grew vigorously. Apical dominance was weak and the plants produced several shoots originating from auxiliary buds. Hybrid vigour was exhibited by 36% of the lines, 51% clones had a size comparable to N. tabacum, while the remaining lines were retarded in growth (Fig. 5). The variation in leaf shape and size is shown in Fig. 6. Flower mor-
Leaf morphology of some somatic hybrids produced between N. rustica (left) and N. tabacum (fight).
205
phology was intermediate for corolla length, width and pigmentation (Fig. 7). With 2 exceptions hybrid lines were self fertile, although fertility was reduced and pollen germination frequency did not
exceed 10% (60% for the parental species). Seeds germinated and produced fertile plants. Examination of fertility and subsequent backcrossing with N. tabacum gave evidence that 4 of
Fig. 7. Typical flowers of N. rustica, four somatic hybrids and N. tabacum (left to fight).
206
the supposed parental lines (chromosome number 2n =48, parental isozyme pattern and morphology) were male sterile and thus putative cybrids. In other words, all plants which had been identified as hybrids owing to their isozyme pattern also possessed an expected chromosome number deviating from 2n = 48 and showed morphological characteristics intermediate with respect to the parental species. This is the first report of somatic hybrid plants produced by mass fusion of evacuolated protoplasts with ordinary protoplasts in the absence of a selectable marker. A high portion of the regenerated plants showed hybrid character, 86% of the tested regenerants displayed an isozyme pattern differing from the parental species and chromosome numbers deviated from 2n = 48. This percentage is lower than the sort purity, but this result confirms previous reports on hybridization frequencies after the use of non-selective methods for hybrid screening [2,18,36]. Fusion may not necessarily result in nuclear fusion [2,36,42], and the occurrence of the male sterile regenerants resembling parental plants gives evidence for the segregation of nuclei in heterokaryons and cybrid formation. As reported in literature [42] chromosome and nucleus separation is possible due to budding, which usually occurred (Figs. 1 and 3). If a nucleus is located at or near the constriction site, mitosis results in irregular chromosome segregation [42-44]. Although the early development of the fusion products seemed to be abnormal as compared to unfused protoplasts, the regenerated plants did not differ from previously reported somatic hybrids of N. tabacum and N. rustica [10,20]. They showed the same variability in morphology and distribution of chromosome numbers like hybrids obtained after mutant complementation (see above for N. tabacum/N, rustica hybrids) or by fluorescence activated cell sorting (e.g. Ref. 7). We therefore conclude that aneuploidization due to chromosome loss is a problem of somatic hybridization in Nicotiana in general and not a result of the evacuolation treatment. Taken together, the combination of fusion of protoplasts with artificially induced differences in
specific density with the selection of heterokaryons according to density offers a new approach for convenient selection of electrically induced intraspecific hybrids in appreciable quantities.
Acknowledgments We thank Professors H.-U. Seitz, T/ibingen and I.K. Vasil, Gainesville, FL, for carrot and guinea grass suspension cultures and Ellen Hoffmann for helpful discussion. This work was supported in part by a grant from the BMFT, DARA grant No. 01 QV 88 683.
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39 40 41
combinations for optimal division response in Nicotiana tabacum protoplast cultures. Plant Sci. Lett., 14 (1979) 237-244. M.R. Thomas and R.J. Rose, Enrichment of Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops. Planta, 175 (1988) 396-402. T. Nagata and I. Takebe, Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta, 99 (1971) 12-20. P. Ozias-Akins, R.3. Ferl and I.K. Vasil, Somatic hybridization in the Gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) + Panicum maximum Jacq. (Guinea grass). Mol. Gen. G-enet., 203 (1986) 365-370. H.-M. Pcehling and V. Nenhoff, One- and twodimensional electrophoresis in micro-slab gels. Electrophoresis, 1 (1980) 90-102. H. Stegemann and D. Schnick, Atlas europiischer Kartoffelsortvn. Mitteilungen aus der Biologischen Bundvsanstalt fiir Land und Forstwirtschaft in Berlin-Dahlem, Paul Parey Verlag, Berlin and Hamburg, 1982, p. 19. J.L. Brewbaker, M.D. Upadhya, Y. M~kinen and T. Macdonald, Isoenzyme polymorphism in flowering plants. III. Gel vlectrophoretic methods and application. Physiol. Plant., 21 (1968) 930-940. M. M~ider, Y. Meyer and M. Bopp, Lokalisation der Peroxidase-Isoenzyme in Protoplasten und Zellwinden von Nicotiana tabacum L. Planta, 122 (1975) 259-268. D.B. Dickinson, Rapid starch synthesis associated with increased respiration in germinating lily pollen. Plant Physiol., 43 (1968) 1-18. E. Pfister, G. KlSck and U. Zimmermann, Selection of hybrid plants obtained by electrofusion of vacuolated × evacuolated plant protoplasts in hypoosmolar solution. Biochim. Biophys. Acta, 1062 (1991) 13-18. E. Sch~'fler and H.-U. Koop, Single cell nurse culture of tobacco protoplasts: physiological analysis of conditioning factors. J. Plant Physiol., 137 (1990) 95-101. G. Spangenberg, M. Osusky, M.M. Oliviera, E. Freydl, J. Nagel, M.S. Pals and I. Potrykus, Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological, genetic and molecular characterization. Thenr. Appl. Genet., 80 (1990) 577-587. L.C. Fowke, C.W. Bech-Hansen and O.L. Gamborg, Electron microscopic observations of cell regeneration from cultured protoplasts of Ammi visnaga. Protoplasma, 79 (1974) 235-248. J. Derksen, F.H.A. Wilms and E.S. Pierson, The plant cytoskeleton: its significance in plant development. Acta Bot. Neerl., 39 (1990) 1-18. D.H. Simmonds, Microtubules in cultured plant protoplasts. Acta Bot. Need., 40 (1991) 183-195. D.P. Delmer, Cellulose biosynthesis. Annu. Rev. Plant Physiol., 38 (1987) 259-290. R.L. Legge and R.M. Brown, Modification of protoplast
208 cell wall regeneration by membrane perturbation. Protoplasma, 143 (1988) 38-42. 42 R.A. Miller, O.L. Gamborg, W.A. Keller and K.N. Kao, Fusion and division of nuclei in multinucleated soybean protoplasts. Can. J. Genet. Cytol., 13 (1971) 347-353. 43 L.C. Fowke, C.W. Bech-Hansen, O.L. Gamborg and F. Constabel, Electron-microscope observations of mitosis
44
and cytokinesis in multinucleate protoplasts of soybean. J. Cell Sci., 18 (1975) 491-507. E.G.M. Meijer, W.A. Keller and D.H. Simmonds, Cytological abnormalities and aberrant microtubule organization during early divisions in mesophyll protoplast cultures of Medicago sativa and Nicotiana tabacum. Physiol. Plant., 74 (1988) 233-239.
197
Elsevier Scientific Publishers Ireland Ltd.
Production of fertile hybrids by electrofusion of vacuolated and evacuolated tobacco mesophyll protoplasts* Beatrix Naton, Margret Ecke and Riidiger Hampp Lehrstuhl Physiologische ¢~kologie tier Pflanzen, lnstitut J~r Botanik, Universitiit Tiibingen, Auf der Morgenstelle 1, W-7400 Tiibingen
(FRG) (Received March 23rd, 1992; revision received May 15th, 1992; accepted June 4th, 1992)
The aim of our experiments was to produce somatic hybrids without using mutants as fusion partners and an expensive cell sorter. Vacuolated mesophyll protoplasts of Nicotiana tabacum U cv. Xanthi were electrically fused with evacuolated protoplasts of the same species or genus (N. rustica L.) in a mass fusion chamber. Fusion yields of 6-11% were obtained. After density gradient centrifugation the fraction enriched in fusion products (90-100%) was embedded in low melting point agarose lenses fixed to the bottom of plastic petri dishes and cultured in the presence of feeder cells. The development of individual fusion products could be followed for several weeks. Plating efficiencies were estimated to be 10-39% for heterokaryocytes. Hybrid character was tested by chromosome counting, isozyme analysis and comparison of morphological properties. About 86% of the regenerants showed hybrid character and 4% were putative cybrids. This study demonstrates that somatic hybrid plants may be selected after electrofusion of mesophyll protoplasts from cultivates lacking specific markers and without using micro-methods or a cell sorter.
Key words: heterokaryon enrichment; iso-osmotic density gradient centrifugation; Nicotiana; evacuolation; protoplast electrofusion; somatic hybridization
Introduction
Protoplast fusion provides a means to hybridize plant species over wide taxonomic distances such as potato and tomato [1] and has become a powerful technique to overcome crossability problems [2,3]. A common limitation in somatic hybridization is the lack of a convenient selection method for the desired heterospecific fusion products [4], because genetic markers are not available for all varieties. Usually fusion frequencies do not exceed 10% irrespective of the applied fusion procedure. Thus culture of the whole fusion population and identification of hybrids at the plant level is quite Correspondence w: Beatrix Naton, Lehrstuhl Physiologische Okologie der Pflanzen, Institut ffir Botanik, Universit~t Tfibingen, Auf der Morgenstelle i, W-7400 Tiibingen, FRG. *These results were presented in part at the 7th International Congress on Plant Tissue and Cell Culture, Amsterdam, The Netherlands, 24-29 June 1990.
elaborate. Microfusion and microculture of single protoplasts as developed by Koop and Schweiger [5], isolation of labelled fusion products by means of micropipettes (e.g. Ref. 6) or enrichment of double-stained fusion products by flow sorting (e.g. Refs. 7-9) are time-consuming and/or expensive methods and require specialized technical expertise. So-called 'universal hybridizers' were produced in a very limited number of plant species (e.g. for Nicotiana tabacum [10]). But the introduction of a resistance gene in the parental species (e.g. Ref. 11) and the additional in vitro culture period may increase somaclonal variation [12], especially in the important crop plant potato [13]. However, this is a disadvantage if certain breeding lines are required. Therefore there is a need of a universal, convenient and low-priced selection method for somatic hybrids without manipulating the genome. Harms and Potrykus [14] used differences in
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
198 boyant density between different types of protoplasts to enrich heterokaryocytes after isoosmotic density gradient centrifugation. In general natural differences between the fusion partners (e.g. epidermis and mesophyll protoplasts) are small [14-16]. As a result the heterokaryocytes are severely contaminated with parental protoplast [14,16]. Additionally, the yield of epidermis protoplasts is very low and handling is complicated, because these protoplasts are very fragile and not pellettable in salt-free solutions, a prerequisite for electrofusion [16; own unpublished observations]. Evacuolation of protoplasts can be used to generate fusion partners distinguishable from ordinary vacuolate protoplasts. Due to distinct differences in the specific density of the respective protoplast populations, heterokaryocytes can be separated from homofusion products as well as from unfused parental protoplasts by iso-osmotic density gradient centrifugation [17]. Evacuolated protoplasts are less fragile than epidermis protoplasts and the degree of somaclonal variation is not increased over that of suspension culture protoplasts. Evacuolation of mesophyll protoplasts is reported for many species [18]. Differences in specific density can be utilized to achieve an ordered pair formation by differential sedimentation. By application of asymmetric membrane breakdown high rates of heterokaryotic fusion products were obtained [19]. Here we demonstrate that this system can be used to recover fertile hybrids from a highly enriched fraction of fusion products. For that purpose mesophyll protoplasts of Nicotiana tabacum were fused either with evacuolated protoplasts of the same species or the same genus (N. rustica), because somatic hybrids between these two species are known from literature [10,20]. Materials and Methods
Enzyme solutions, media containing Percoll® (Pharmacia, Uppsala, Sweden) and liquid protoplast culture media were sterilized by membrane filtration through 0.22-#m Sartorius cellulose nitrate filters (Sartorius, Grttingen, FRG). Callus, suspension- and shoot-culture media were sterilized by autoclaving (20 min, 117°C). All solutions
used for density gradient centrifugation were adjusted to 560 ± 20 mOsm. The osmolality was determined using a Vescor Vapor Pressure Osmometer (Logan, Utah, USA).
Isolation and evacuolation of tobacco mesophyll protoplasts Axenic shoot cultures of N. tabacum L. (cv. Xanthi and Samsun) and N. rustica L. NRT were initiated from sterilized seeds. Shoot cultures were maintained on hormone free MS medium [21] and cultured in 500 ml Weck® jars at 23°C and a light period of 16 h (3500 Lux). Mesophyll protoplasts from 6-8-week-old plants were isolated and purified on iso-osmotic step gradients as described elsewhere [17]. Evacuolation was performed on a self-generating iso-osmotic Percoll® gradient [22] modified according to Naton et al. [17]. Fusion of protoplasts Electrofusion was carried out as previously described [17] using a Zimmermann Cell Fusion TM generator (GCA Corporation, Chicago, IL) in a self-constructed mass fusion chamber, consisting of a glass slide with combe-shaped chromium electrodes (distance: 100 #m). For PEG-induced fusion protoplasts were suspended in W5 solution and fused as described elsewhere [23]. Selection or fusion products Fused protoplasts were resuspended in 6 nil 0.44 M raffinose-buffer, transferred to a 10-ml centrifuge tube and overlaid with 2 ml 0.5 M mannitol-buffer. After centrifugation (10 min at 300 x g) mesophyll protoplasts and fusion products were collected from the interphase, washed once with 0.5 M mannitol-buffer, resuspended and layered on top of a preformed Percoll gradient (20% (v/v) Percoll, 0.5 M mannitol-buffer; 5000 x g, 2 h, swing out rotor). After a second centrifugation step (20 min, 300 x g), a highly enriched fraction of fusion products was recovered at the bottom of the gradient [17]. The modification of the sorting procedure (introduction of an additional centrifugation step to separate the evacuolated protoplasts from the vacuolated protoplasts and the heterokaryons) prevented
199 overloading of the gradient and aggregation of protoplasts,
Culture of fusion products The fraction enriched in fusion products was washed once in a modified MS-medium [24] with 4.7 rag/1 p-CPA and 1 rag/1 kinetin [25], all supplemented with 3% (w/v) sucrose and 9% (w/v) mannitol and embedded in 0.8% (w/v) low melting point agarose (Sea-Plaque®, FMC Marine Colloids, Rockland, NY, USA [26]). Droplets of the agarose-protoplast suspension were placed in 5-ml plastic Petri dishes (Greiner, Niirtingen, FRG). After hardening, the lenses adhered at the bottom and were surrounded with 4 ml of feeder cell suspension (tobacco mesophyll protoplasts or carrot suspension culture cells; density 50 000 or 100 000 ceUs/ml). The development of individual fusion products could be followed for several weeks. Tobacco mesophyll protoplast feeder cells were diluted every 10 days with 1 ml osmoticum free culture medium. Carrot suspension culture cells were diluted every 4 days (1 ml of suspension was replaced by 1 ml culture medium with decreasing osmolality). After 6 weeks the lenses free from feeder cells were transferred onto agar solidified NT-medium [27] complemented with 3 mg/1 NAA and 1mg/l kinetin. Seven days later mlcrocalli were dispersed on the agar by means of an inoculator. Another 2 weeks later the calli were transferred on shoot regeneration medium (MS complemented with 0.1 mg/1 NAA and 1 mg/1 BAP). Developing shoots were cut off and rooted in MS medium without hormones. Culture of suspension culture cells and preparation of conditioned medium For cross feeder experiments Daucus carota L. ssp. sativus suspension cultures were kindly provided by Prof. H.-U. Seitz, Tiibingen [24] and Panicum maximum Jacq. (guinea grass) cells by Prof. I.K. Vasil, Gainesville, FL, USA [28]. Cells from 4-6-day-old suspensions were washed once with protoplast culture medium and diluted to the desired suspension density. Conditioned medium was prepared from 5-day-old suspensions. Cells were removed from medium by filtration through filter paper. The cell-free supernatant was corn-
plemented with sucrose, vitamins and hormones as described above, osmotically adjusted with mannitol and stored at -25°C.
Analysis of somatic hybrids Ninety-five regenerants were selected randomly at callus stage, and regenerated plants were subjected to isozyme, cytogenetic and morphological analysis. All putative hybrid plants analyzed for isozyme patterns were derived from different calli. The leaf-isozyme patterns of selected enzymes were investigated by non-denaturating polyacrylamide gel electrophoresis using a microgel system [29]. The gels (10% separating gel + 3% stacking gel) were prepared as indicated in the Sigma Technical Bulletin No. MKR-137. For separation of esterases (EC 3.1.1.1) 0.3 g of greenhouse grown leave tissue was homogenized in 1 ml of 0.5 M Tris-HCl buffer (pH 6.8) containing 80 mM DTT (Cleland's reagent), 0.2% Na2SO4, 0.15% Na2S205, 0.2% Polyclar AT and 7.5% sucrose ([6] modified according to Stegemann and Schnick [30]). Esterase isozymes were visualized according to Brewbaker et al. [31]. The extraction medium for separation of acid peroxidases (EC 1.11.1.7) contained 0.26 M Trisphosphoric acid (pH 6.9) and 10 mM DTT. The extracts were dialysed overnight against 1:10 diluted extraction buffer [32]. After electrophoresis the patterns were visualized following a proeedure described by Brewbaker et al. [31] with slight modifications. Five milligrams of o-dianisidine and 3 mg of O-naphthol were dissolved in 2 ml of acetone, 8 ml of 0.1 M acetate buffer (pH 4.5) were added, and shortly before use, 10 #1 of 30% H202. Morphological analysis was performed at the inition of flowering. Leave shape and size, flower size, coloration and shape and growth of at least 4 plants from each line was compared with the traits of parental plants cultivated under the same conditions. Cytological techniques For determination of chromosome numbers root tips from greenhouse plants were excised at 10:00 h and pretreated in 2 mM 8-hydroxychinoline for 2.5 h at 17°C. Root tips were fixed in
200 Carnoy's solution overnight, transferred into 70% ethanol, stored at 4°C until use and stained with acetocarmine. Of each clone several individual plants were investigated.
Determination of fertility Flowers were selfed or back-crossed to N.
tabacum. Seed number was compared to selfpollinated parental plants. Pollen grains were stained with acetocarmine or, in case of in vitro germination, incubated at room temperature in a medium described by Dickinson [33]. Results and Discussion
Fusion conditions and heterokaryon selection The fusion frequencies obtained after PEGinduced fusion were very variable and ranged from 0% to 10%. A large portion of the vacuolated protoplasts stuck at the bottom of the petri dish and lysed. But the main problem was the tendency of the protoplasts to agglutinate in an unspecific way especially after centrifugation. Mesophyll protoplasts from greenhouse plants could be sorted after a post-fusion treatment with osmotically adjusted sea-water (5 h; 4°C). By lowering the amount of DMSO in the fusion solution or the Caconcentration in the elution medium both aggiutination and fusion yield were reduced, but sort purity did not exceed 50% in any case. Fusion frequencies after electrofusion varied between 3% and 5% for protoplasts from greenhouse plants and between 6% and 11% for protoplasts from axenic shoot cultures. This may be due to the greater homogeneity of the latter donor tissue. Upon pulse application about 50% of the protoplasts either lysed or formed multikaryons, which burst during subsequent manipulation. Although the fused protoplasts tended to clump slightly, which was possibly due to an increase in plasmalemma hydrophobicity, the purity of the heterokaryon population ranged between 90% and 100% depending on the time elapsed between prepurification and heterokaryon selection, and sort efficiencies varied from 10% to 39%. As a result, the portion of recovered heterokaryons is comparable to the sort efficiency obtained using a flow sorter [9]. The purity of
heterokaryon populations observed in the present study was generally significantly greater ( > 90%) than that reported by other workers. This finding can be explained in a number of ways. Firstly, the use of purified parental protoplast populations within a distinct range of boyant density was essential for obtaining pure heterokaryon populations [14-16]. This explains the failure of Pfister et al. [34] to recover hybrids without the aid of a previously introduced antibiotic resistance gene. The observation of high sort impurity of PEGobtained fusion products after iso-osmotic density gradient centrifugation is well-known from literature and may be induced by clumping and alterations in specific density. After an overnight culture period Thomas and Rose [26] purified the heterokaryocytes a second time, but nevertheless the population enriched in heterokaryocytes was contaminated by up to 50% with parental protoplasts. These findings agree with the literature [14,22]. Aggregation of cells hinders physical enrichment of heterokaryocytes even by cell sorting (e.g. compare Afonso et al. [7] with Hammatt et al. I91). It is worth noting that protoplasts seem to alter boyant density even when stored on ice in isoosmotic solutions [19]. Therefore it is necessary to keep the time between fractionation of parental protoplasts and selection of heterokaryons as short as possible. Furthermore it is important to fuse the protoplast under iso-osmotic conditions. Only electrofusion fulfills both requirements.
Cultivation and development of fusion products Fusion products were embedded in agarose lenses and cultivated in the presence of feeder cells. For that purpose different cells were tested for the ability to nurse embedded tobacco mesophyll protoplasts at low density (10 3 protoplasts/ml). Best results were obtained with mesophyll protoplasts of the same origin. Colony formation of the protoplasts within the agarose lenses was identical to that of the surrounding feeder protoplasts. The same nutritional and osmotic requirements were a second advantage over all tested suspension culture cells. To exclude a possible contamination of nursed cells by feeder cells, heterologous combinations were tested for their suitability to pro-
201
mote microcallus formation. Best results gave carrot suspension cells (suspension density 105 cells/ml). Plating efficiencies of the nursed cells were reduced only by 5-10% compared to the control protoplasts cultivated under standard conditions in liquid culture or agarose beads. No divisions of embedded tobacco protoplasts were obtained in the presence of Panicum maximum cells or conditioned medium alone, but a delay in degeneration was observed. These results gave further support to the hypothesis that cells release both 'viability' factors, which prevent degeneration, and 'nursing' factors, which support division of cells at very low density (for detailed discussion: Ref. 35). The high enrichment factor (-100%) together with plating at low density (about 50-100 viable cells/droplet) and the physical separation of cultured cells from feeder cells allowed the observation of individual fusion products over a long period of time. Figures 1 and 2 illustrate the
typical time course of fusion product development. In general, the heterokaryotic character persisted up to the first division which had a delay of about 4 days as compared with unfused mesophyll protoplasts. After fusion the chloroplasts (and probably the nucleus) of the vacuolate mesophyll protoplast started to migrate towards the area of the evacuolate protoplasts (Figs. I a-1 f). The first division occurred in this part of the cell and often led to asymmetric development (Fig. 2). Plating efficiencies were estimated to be 10-40% for heterokaryocytes and about 50-60% for the unfused mesophyll protoplasts. These results are in good agreement with plating efficiencies of tobacco heterokaryons obtained after microfusion [5,36], manual selection [6] and flow sorting [9] of mesophyll or suspension cell culture protoplasts. During the first few days many fusion products showed intense budding (Figs. 1 and 3). Budding and cleavage divisions are indicators of a non-rigid pseudo-wall in general, or of localized weak areas
Fig. 1. Development of a fusion product over a period of 2 weeks: migration of the chloroplasts towards the area of the evacuolated protoplast, budding at the face of the formerly evacuolate protoplast. Bar = 50/~m.
202
Fig. 2. Developmentof a fusion product over a period of 2 weeks: First division is highly asymmetrical. Bar = 50 ~m.
in the newly synthesized cell wall [37]. Budding is a characteristic event in the early development of evacuolate protoplasts even when embedded in agarose solidified media (unpublished results). Evacuolation may disorganize the cytoskeleton which plays an important role in an ordered deposition of newly synthesized cellulose fibrils [38,39], although the direct impact is yet unknown. Cell wall regeneration also depends on the integrity status of the plasma membrane [40,41]. Injury of the plasmalemma due to shearing forces during evacuolation and enrichment of heterokaryons (e.g. Ref. 8) may not be excluded. Nevertheless, we failed to detect an increased callose synthesis of evacuolated over vacuolated mesophyll protoplasts after staining with aniline blue. A cell sorter was not available which allows the observation of fusion products of vacuolated or evacuolated protoplasts. Therefore it can not be excluded that the presence of the evacuolated pro-
toplast caused an aberrant development of the fusion products. Miller et al. [42] and Fowke et al. [43], however, gave strong evidence that the multikaryotic nature of the fusion products by itself can cause a disturbed cell wall regeneration: polykaryotic soybean protoplasts synthesized less cell wall material than monokaryotic protoplasts and showed intense budding.
Analysis of hybrid plants The high sorting and plating efficiencies resulted in high yields of putative hybrids. In a representative experiment 2 x 600 000 protoplasts were fused. A fusion rate of 7.2% was obtained. The purified fraction of fusion products contained about 5000 cells. Plating efficiency was determined to be 35%. Twenty percent of the developing calli were randomly selected for shoot morphogenesis. About 90% of the clones possessed morphogenic potential and were able to root.
203
Fig. 3.
Fusion product showing extensive budding, the part of the evacuolated protoplast is still visible (dark). Bar = 25 #m.
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Fig. 4. Esterase isozymes. The patterns from extracts of N. tabacum, eleven hybrids and N. rustica (left to right); parental bands are indicated by arrows.
204
Fig. 5. Habitus of typical somatic hybrids in comparison with the parental species N. tabacum (left) and N. rustica (right).
Fig. 6.
The hybrid character of 95 regenerants was tested by chromosome counting and isozyme analysis (acid peroxidases and esterases). The isozyme pattern suggested that 82 of the analyzed clones were hybrids. Figure 4 shows the esterase profile of eleven hybrids and the two parental species. Unique hybrid bands or the absence of parental bands were frequently observed. The hybrid identity of these plants was supported by their chromosome number deviating from 48 (N. tabacum 2n = 48; N. rustica 2n = 48). A remarkable variation was found between the different clones (2n = 56-96), but not within different plants of the same line (variation ± 6 chromosomes). With the exception of 2 clones, all hybrid lines had less than the expected doubled chromosome set of 96 chromosomes. The majority of the hybrid plants grew vigorously. Apical dominance was weak and the plants produced several shoots originating from auxiliary buds. Hybrid vigour was exhibited by 36% of the lines, 51% clones had a size comparable to N. tabacum, while the remaining lines were retarded in growth (Fig. 5). The variation in leaf shape and size is shown in Fig. 6. Flower mor-
Leaf morphology of some somatic hybrids produced between N. rustica (left) and N. tabacum (fight).
205
phology was intermediate for corolla length, width and pigmentation (Fig. 7). With 2 exceptions hybrid lines were self fertile, although fertility was reduced and pollen germination frequency did not
exceed 10% (60% for the parental species). Seeds germinated and produced fertile plants. Examination of fertility and subsequent backcrossing with N. tabacum gave evidence that 4 of
Fig. 7. Typical flowers of N. rustica, four somatic hybrids and N. tabacum (left to fight).
206
the supposed parental lines (chromosome number 2n =48, parental isozyme pattern and morphology) were male sterile and thus putative cybrids. In other words, all plants which had been identified as hybrids owing to their isozyme pattern also possessed an expected chromosome number deviating from 2n = 48 and showed morphological characteristics intermediate with respect to the parental species. This is the first report of somatic hybrid plants produced by mass fusion of evacuolated protoplasts with ordinary protoplasts in the absence of a selectable marker. A high portion of the regenerated plants showed hybrid character, 86% of the tested regenerants displayed an isozyme pattern differing from the parental species and chromosome numbers deviated from 2n = 48. This percentage is lower than the sort purity, but this result confirms previous reports on hybridization frequencies after the use of non-selective methods for hybrid screening [2,18,36]. Fusion may not necessarily result in nuclear fusion [2,36,42], and the occurrence of the male sterile regenerants resembling parental plants gives evidence for the segregation of nuclei in heterokaryons and cybrid formation. As reported in literature [42] chromosome and nucleus separation is possible due to budding, which usually occurred (Figs. 1 and 3). If a nucleus is located at or near the constriction site, mitosis results in irregular chromosome segregation [42-44]. Although the early development of the fusion products seemed to be abnormal as compared to unfused protoplasts, the regenerated plants did not differ from previously reported somatic hybrids of N. tabacum and N. rustica [10,20]. They showed the same variability in morphology and distribution of chromosome numbers like hybrids obtained after mutant complementation (see above for N. tabacum/N, rustica hybrids) or by fluorescence activated cell sorting (e.g. Ref. 7). We therefore conclude that aneuploidization due to chromosome loss is a problem of somatic hybridization in Nicotiana in general and not a result of the evacuolation treatment. Taken together, the combination of fusion of protoplasts with artificially induced differences in
specific density with the selection of heterokaryons according to density offers a new approach for convenient selection of electrically induced intraspecific hybrids in appreciable quantities.
Acknowledgments We thank Professors H.-U. Seitz, T/ibingen and I.K. Vasil, Gainesville, FL, for carrot and guinea grass suspension cultures and Ellen Hoffmann for helpful discussion. This work was supported in part by a grant from the BMFT, DARA grant No. 01 QV 88 683.
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