Proc. Nati. Acad. Sci. USAVol. 81, pp. 5449-5453, September 1984Cell Biology

Dimethyl sulfoxide can initiate cell divisions of arrested callusprotoplasts by promoting cortical microtubule assembly

(colchicine/hibiscus/Nicotiana glutinosalplant tubulin)

GUNTHER HAHNE AND FRANZ HOFFMANNDepartment of Developmental and Cell Biology, University of California, Irvine, CA 92717

Communicated by Anton Lang, May 16, 1984

ABSTRACT A serious problem in the technology of plantcell culture is that isolated protoplasts from many species arereluctant to divide. We have succeeded in inducing consecutivedivisions in a "naturally" arrested system-i.e., protoplastsfrom a hibiscus cell line, which do not divide under standardconditions-and in an artificially arrested system-i.e., col-chicine-inhibited callus protoplasts of Nicotiana glutinosa,which do readily divide in the absence of colchicine. In bothcases, the reinstallation of a net of cortical microtubules,which had been affected either by colchicine or by the proto-plast isolation procedure, resulted in continuous divisions ofthe formerly arrested protoplasts. Several compounds knownto support microtubule assembly in vitro were tested for theirability to promote microtubule assembly in vivo. Best resultswere obtained by addition of dimethyl sulfoxide to the culturemedium. Unlimited amounts of callus could be produced withthe dimethyl sulfoxide method from protoplasts which neverdeveloped a single callus in control experiments.

Isolated protoplasts of higher plants, "naked" cells that havebeen enzymatically stripped of their walls, are an increasing-ly utilized tool for studies in such areas as plant physiology,genetics, and cell biology (1). They provide a fairly uniformpopulation of true single cells, allow comparatively easy ac-cess to organelles, and are well suited for genetic engineeringexperiments such as microinjection (2), transformation (3),and somatic hybridization (4).

Protoplasts isolated from a number of species have beenshown to regenerate callus when cultured under appropriateconditions. In several cases, these calli regenerated fertileplants (5). It has been impossible, however, to induce con-secutive divisions in cultures of isolated protoplasts frommany other species or even from certain genotypes or tissuesof a given species. Consequently, no callus nor plants couldbe regenerated. These include species of great economic im-portance [e.g., the cereals (6)], as well as species and tissuesthat could be used as models in fundamental studies [e.g.,the hibiscus cell line used in this investigation (7)]. The limi-tations inherent in protoplast regeneration techniques of ag-ricultural plants represent a serious impediment to the ad-vancement of in vitro methods for crop improvement.The behavior of nonregenerating protoplasts may be at-

tributed to a loss of genetic material or to a physiologicalblock incurred by the enzymatic removal of the cell wall.However, in most species that do not yield dividing proto-plasts (which will be referred to as recalcitrant in this paper),it is comparatively easy to induce and maintain callus cul-tures. These calli may even regenerate plants, indicating toti-potency of at least some of the callus cells (8). The recalci-trance of protoplasts isolated from such a callus or from tis-sue of the recalcitrant plant itself supports the view thatcertain aspects of the protoplast isolation procedure, rather

than a genetic defect, block protoplast division. In this pa-per, we report that such a physiological block actually is thearresting factor in hibiscus callus protoplasts.The problem of nondividing protoplasts is usually attacked

empirically by attempts at optimizing the composition of theculture media [e.g., their hormone content (9)], but these at-tempts have had only limited success. In our study, we ana-lyzed and compared a "naturally" arrested system, namely,protoplasts from a recalcitrant hibiscus callus line, and anartificially arrested system, namely, drug-inhibited callusprotoplasts of an otherwise not recalcitrant Nicotiana spe-cies. In the latter, division is inhibited by the addition of col-chicine, and therefore it is defined as artificially arrestedwhile protoplasts that do not divide under standard condi-tions (recalcitrant) are called "naturally" arrested. In both ofour systems, the block is accompanied by disappearance ofthe cortical microtubular net and, in both cases, the induc-tion of consecutive divisions was accomplished by reinstalla-tion of a net of cortical microtubules.

MATERIALS AND METHODSCell Cultures. Protoplasts were isolated as described (7)

from callus cultures of Hibiscus rosa-sinensis L. and Nicoti-ana glutinosa L. that had been grown on the medium of Mur-ashige and Skoog (10). Protoplasts from N. glutinosa werecultured in the medium of Nagata and Takebe (11) at a celldensity of 105 cells/ml; protoplasts of H. rosa-sinensiswere cultured at 105 cells/ml in the following medium (Q)modified after Binding (12): (per liter) 180 mg of NH4NO3,1515 mg of KNO3, 220 mg of CaCl2 2H2O, 984 mg ofMgSO4-7H2O, 69 mg of NaH2PO4-H2O, 27.85 mg of Fe-SO4-7H20, 37.25 mg of Na2EDTA, 100 mg of inositol, 1 mgof pyridoxine HCl, 1 mg of thiamine 2HCl, 1 mg of nicotinicacid, 0.01 mg of biotin, 1 mg of Ca panthothenate, 20 ml ofcoconut water, 300 mg of casein hydrolysate (acid; Nutri-tional Biochemicals), 134 mg of glutamine, 75 mg of glycine,0.2 mg of 2,4-dichlorophenoxyacetic acid, 0.5 mg of 6-ben-zylaminopurine, 1.0 mg of a-naphthaleneacetic acid, 100 g ofglucose, 250 mg of sucrose, and trace elements as in B5 me-dium (13), pH 5.6. Inhibitors were added immediately afterisolation of the protoplasts unless stated otherwise.

Colchicine and deuterium oxide (99.8%) were obtainedfrom Sigma. Taxol, a low molecular weight microtubule-sta-bilizing factor occurring in the mountain ash (Taxus brevifo-lia), was obtained from the National Cancer Institute (Be-thesda, MD). All other chemicals used were analyticalgrade. For the 2H20 experiments, the culture medium waslyophilized and redissolved at the appropriate 2H20 concen-tration. Control experiments were run in which lyophilizedmedium was redissolved in water to ensure that the lyophili-zation itself did not influence the experiment.

Calcofluor White Staining. Calcofluor white staining wascarried out as described (14).

Abbreviation: Me2SO, dimethyl sulfoxide.

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Immunofluorescence. Immunofluorescence staining of mi-crotubules followed the procedure described by Lloyd et al.(15). Antibodies against /3-tubulin of suspension cultures ofPaul's Scarlet Rose were those isolated and described byMorejohn et al. (16). Antibodies against bovine brain micro-tubules were isolated by the method of Fuller et al. (17). Af-finity-purified fluorescein-labeled goat anti-rabbit IgG anti-bodies (Sigma) were used at a 1:50 dilution. Fluorescenceobservations were made on a Zeiss universal microscope us-ing the standard epifluorescence filters for fluorescein. Pho-tographs were taken on Kodak Tri-X film.

RESULTS"Naturally" Arrested Hibiscus Protoplasts. Protoplasts iso-

lated from our hibiscus callus culture (7) do not divide instandard culture media. When cultured in Q medium, how-ever, the cells remain alive for up to 4 weeks, as evidencedby the cytoplasmic organization in vigorously streamingstrands. Hibiscus protoplasts form cell walls much moreslowly and show less intense fluorescence than the dividingprotoplasts of other species under comparable conditions, asjudged by calcofluor white staining. Even after prolongedculture, hibiscus still shows a majority (60-70%) of sphericalcells (Fig. lb) that display only faint calcofluor white fluo-rescence. The remaining cells of the population ("30%) areoval and show stronger fluorescence, indicating cell wall for-mation. Occasionally (frequency, -5 x 10-3), first divisionscan be observed, but in all cases the two-cell stages diedsoon after completion of cytokinesis. So far callus has neverbeen obtained in these cultures (Fig. la).When dimethyl sulfoxide (Me2SO, 2-7%) was included in

the culture medium, however, cell wall formation was rapidand intensive. Approximately 5-25% of the cells are inducedto undergo divisions (Fig. ic). This resulted in the formationof normal callus cultures (Fig. la) that could be maintainedon Me2SO-free medium. Protoplasts isolated from these re-generated calli behaved as those isolated from the originalcallus. The addition of Me2SO was still required to induceregeneration. In either case, a short pulse of Me2SO was notsufficient to trigger the onset of divisions. Me2SO must bepresent until at least one cell cycle is completed.

In addition to the long-term effect of Me2SO-i.e., thestimulation of cell division-Me2SO had an immediate andtransient effect on cell morphology. When Me2SO was add-ed to the culture, the cytoplasm of the cell contracted imme-diately, resulting in shrunken or strongly indented cells. Thecontracting cytoplasmic strands appear to pull the corticalcytoplasm and the plasmalemma towards the center of thecell. Within a few minutes, however, the cytoplasm relaxes,and the cells appear normal again.Me2SO has been reported to have a direct effect on the

properties of plasma membranes, including lipid composi-tion, fluidity, and permeability to 86Rb' ions (18-20). To testwhether Me2SO increases membrane permeability nonspe-cifically, the exclusion of trypan blue and lucifer yellow wastested. Both Me2SO-treated and untreated cells excludedboth dyes, indicating that Me2SO did not induce nonspecificincreases in the permeability of the cell membrane for organ-ic molecules of intermediate molecular weight.

Artificially Arrested Tobacco Protoplasts. In an attempt toelucidate the mechanism of action of Me2SO in inducing celldivisions, its effect in a different nondividing system, onewith a known block, was investigated. In isolated proto-plasts of N. glutinosa, division can be completely inhibitedby colchicine at a concentration of 1 mM. At this concentra-tion, the protoplasts grow into giant cells that remain viablefor several weeks (Fig. 2a). In contrast to the "naturally"arrested hibiscus protoplasts, however, these cells shownormal calcofluor white fluorescence (Fig. 2b), indicating

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FIG. 1. Protoplasts isolated from a hibiscus callus line culturedin the presence or absence of Me2SO. (a) After 4 weeks of culture,no callus is formed in controls without Me2SO added to the culturemedium (left Petri dish). Cultures containing 2% Me2SO ab initioexhibit vigorous callus development (right Petri dish). (b) After 4days of culture, controls (without Me2SO) exhibit no divisions. (c)After 4 days of culture with 2% Me2SO added to the culture medi-um, divisions are frequent. (Bar = 50 ,m.)

that cell wall synthesis is functioning properly. Only a slight-ly slower appearance of the fluorescence and perhaps a re-duction in intensity was visible in our experiments. Depend-ing on the concentration of Me2SO in the culture medium,the protoplasts could be induced to undergo developmentalong two different pathways. At 2% Me2SO, cell divisionwas predominant; at 4% Me2SO, spherical cells with one ormore tubular extensions ("viking helmets") that showedsigns of xylogenesis (Fig. 2 c and d) predominated. At 3%Me2SO, both types were present in a mixture. Neither divi-sions nor viking helmets were observed in colchicine-inhibit-ed control cultures without Me2SO (Fig. 2 a and b). Colchi-cine inhibition of division could be reversed by 2% Me2SO in10-60% of the viable cells (as compared with 30-80% divi-sions in untreated control cultures). Me2SO could be addedup to at least 96 hr after the addition of colchicine, although agreater effect was observed with simultaneous addition. IfMe2SO (2%) was added to cultures of nonarrested Nicotiana

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FIG. 2. Protoplasts isolated from a N. glutinosa callus line inhib-ited with colchicine and cultured in the presence or absence ofMe2SO. (a) Nondividing protoplast cultured in a medium containing1 mM colchicine and no Me2SO. (b) Calcofluor white fluorescenceof the protoplast shown in a: the deposition of fluorescing materialis uniform throughout the cell surface. (c) Viking helmets are formedat a high frequency in cultures containing 1 mM colchicine togetherwith 4% Me2SO. (d) Calcofluor white fluorescence of the cellsshown in c: the deposition of fluorescing material is not uniformthroughout the cell surface, suggesting the beginning of xylogenesis.(Bars = 100 Am.)

protoplasts (without colchicine), the frequency of divisionwas slightly lowered and viking helmets were formed at afrequency of <5%. Viking helmets were never observed insimilar experiments with hibiscus.

Effect of Glycerol, 2H20, and Taxol. The ability of Me2SOto assemble microtubules in vitro (21, 22) indicates that thereversal of colchicine inhibition by Me2SO may also be dueto an effect on microtubules in vivo. Therefore, other induc-ers of microtubule assembly in vitro (namely, glycerol, deu-terium oxide, and taxol) were tested for their ability to in-duce divisions in hibiscus cultures (Table 1). Below their lev-el of toxicity, glycerol and taxol were moderately effective ininducing the first division. 2H20 showed lethal effects evenat 50%, which is half the concentration used by Schnepf et

al. (25) on sphagnum leaflets. None of these chemicals wereeffective in inducing repeated divisions or subsequent devel-opment into macroscopic calli.Immunofluorescence Staining of Cortical Microtubules.

The degree of cortical tubulin polymerization and reticula-tion can be directly visualized by immunofluorescence stain-ing of microtubules in plasma membrane preparations [mem-brane ghosts (15), Fig. 3]. After 16 hr of culture of hibiscusprotoplasts without Me2SO, only a few short bundles of mi-crotubules were visible (Fig. 3a). The fluorescence imageprovided by the antibody binding was subject to rapid fad-ing. In contrast, protoplasts cultured for 16 hr in the pres-ence of 2% Me2SO showed a dense network of long bundlesof cortical microtubules (Fig. 3b). Often, these arrays werethree-dimensional. The fluorescence was more resistant tofading, probably because of the larger amount ofantigen pre-sent-i.e., /3tubulin. These results indicate that, after cul-ture in Me2SO, bundles of cortical microtubules are not onlylonger and more abundant but are also composed of moremicrotubules.

Protoplasts of N. glutinosa, which divide under normalculture conditions without Me2SO (control), can be arrestedwith colchicine (1 mM). This effect could be reversed by theaddition of Me2SO (2%). Immunofluorescence staining ofthe microtubules in these protoplasts showed an almost iden-tical pattern when compared with the corresponding situa-tion in the hibiscus cultures: (i) no microtubules in the artifi-cially arrested (1 mM colchicine) cultures, (ii) a dense net-work of microtubules in cultures containing both colchicine(1 mM) and Me2SO (2%), and (iii) an equally dense networkin nonarrested control cultures (without colchicine/Me2SO).Judged by the immunofluorescence images, protoplasts ob-tained from N. glutinosa seemed to contain slightly fewerbundles of microtubules than did Me2SO-treated hibiscusprotoplasts. This observation is consistent with the observa-tion that a suspension culture of hibiscus yields unusuallyhigh amounts of purified tubulin per fresh weight as com-pared with cultures of several other species (D. E. Fosket,personal communication).An interesting observation was made by using antibodies

against bovine brain microtubules. These antibodies, ob-tained from an animal system, do not bind to cortical micro-tubules of the plant species investigated.

DISCUSSIONRecent investigations on cell wall resynthesis in hibiscusprotoplasts, using the calcofluor white method (14), led us tothe conclusion that wall formation is impaired in these natu-rally arrested protoplasts. Since cell wall regeneration is be-lieved to be a prerequisite for isolated protoplasts to divide(26), we tested several culture conditions that we thoughtmight improve the ability of the cell to form a wall. One ofthe conditions is connected to the "microtubule-microfibrilsyndrome" (27): because microtubules are postulated to playan important role in the deposition of microfibrils, we testeda variety of compounds known to support microtubule as-sembly in vitro, such as Me2SO, 2H20, glycerol, and taxol.By far the best results were obtained after addition ofMe2SOto the culture medium. This treatment resulted in continuous

Table 1. Effects of compounds known to induce microtubule assembly in vitro on cell division inotherwise nondividing callus-derived protoplasts of hibiscus

Divisions atCompound optimal Concentration used

(ref.) Range tested Optimal Lethal concentration in vitro, mM

Glycerol (23) 100-1000 300 500 First 1000-4000Taxol (24) 0.0001-0.1 0.001-0.01 100 First 0.02Me2SO (22) 12.8-1280 256-384 (2-3%) 890 Continuous 670-840

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FIG. 3. Cortical microtubules in membrane ghosts of hibiscus protoplasts cultured in the presence or absence of Me2SO. (Bar = 50 /Lm.) (a)Control culture (no Me2SO added to the medium) showing only a few, faint, short microtubules. The faint peripheral fluorescence is due tononspecific binding of the second antibody to membrane proteins. Because of the absence of a three-dimensional array of microtubules, thecells collapse during extraction, resulting in an accumulation of membrane proteins on the polylysine-coated surface of the slide. (b) Culturewith 2% Me2SO added to the culture medium ab initio, showing a dense three-dimensional network of cortical microtubules. Because of thecytoskeletal support, these cells do not collapse during extraction.

divisions in both naturally and colchicine-blocked cells. Col-chicine-inhibited cultured plant protoplasts usually developinto giant spherical cells, although cellulose microfibrils areformed (28). The addition of Me2SO to colchicine-inhibitedcultures of N. glutinosa triggers either cell divisions or, athigher Me2SO concentrations, the formation of differentiat-ed nondividing cells of a distinctive nonspherical shape, or-dered deposition of calcofluor white-positive material, andsigns of xylogenesis (Fig. 2 c and d). These events are ac-companied, if not mediated, by the assembly of microtubules(29).A direct effect of Me2SO on the assembly of microtubules

could be due to one or a combination of two events. Me2SOmay lead to an increase in the pool of unpolymerized tubu-lin-i.e., synthesis of tubulin de novo. Alternatively, Me2SOmay act to decrease the critical concentration of unpolymer-ized tubulin, leading to the polymerization of the tubulin al-ready present. We favor the second explanation because thisis the effect of Me2SO observed in vitro (22), and this wouldbe the action expected for an antagonist to colchicine. Sub-stances other than Me2SO that are also known to induce mi-crotubule assembly in vitro were much less effective in ourin vivo system. This may simply be a consequence of theirtoxicity at the concentrations required.

It is puzzling that a callus can undergo rapid cell divisions,yet the same cells, in some genotypes but not in others, areunable to divide after their isolation and cell wall removal. Itseems that these cells lose their ability to undergo mitosiswhen their protoplasts are isolated. No explanation for this

is known, and a few possibilities should be mentioned here.(i) The enzyme preparations used to remove cell walls maycontain impurities and enzymatic activities that may damagethe cells. (ii) Changes in cell shape are known to disorganizearrays of cortical microtubules (28), in some cells possibly tothe point where the cell is unable to repair the disrupted mi-crotubules and to reverse this process. (iii) Enzymatic diges-tion of the cell walls may liberate molecules of low molecularweight (elicitors) that are taken up and trigger the reaction(s)of the protoplast (30)-e.g., a depolymerization of microtu-bules. The degree of the events suggested above (i-iii) couldbe genotype specific.As mentioned briefly in the Introduction, many plant spe-

cies as well as certain genotypes or tissues from otherwisenonrecalcitrant species do not yield dividing protoplasts un-der the culture conditions tested. Because of their great eco-nomic importance as food crops, it would be particularly de-sirable to be able to regenerate calli and plants from proto-plasts of the extremely reluctant cereals. As shown by ourresults, it may be rewarding to examine the protoplasts inquestion carefully at the subcellular level. They might havecellular properties and anomalies similar to those that we de-tected in our hibiscus cell line that lead to their inability todivide when cultured as isolated protoplasts.The outlined diagnosis and therapy of a case of nondivid-

ing protoplasts is not a universal remedy for all such situa-tions. For example, experiments with a variety of mesophyllprotoplasts showed no response to Me2SO treatment. Sincemesophyll cells do not have a cortical microtubule net as

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found in callus cells, the quantities of depolymerized tubulinavailable in isolated mesophyll protoplasts may not be suffi-cient to build a polymer without the synthesis of new tubu-lin. Consequently, if Me2SO does not support tubulin syn-

thesis, it does not have the therapeutic effect. On the otherhand, the conclusion that Me2SO does not support cell divi-sion can only be drawn if the experiments are carried outwith a high-quality protoplast preparation, The nondividingprotoplast should survive in a standard culture medium for atleast several days. Protoplasts that release their vacuoleshortly after isolation or die otherwise within a few days can-

not be expected to be rescued by Me2SO. The first step inprotoplast regeneration is always the preparation of healthyprotoplasts, which is usually dependent on the quality of theplants or cells used for the isolation. The importance of thisstep cannot be stressed enough because it is often over-

looked or ignored.

We thank Drs. T. J. Bradley and D. Knauer for stimulating dis-cussions and for critical reading of the manuscript. We also thankDrs. L. C. Morejohn and D. E. Fosket for providing us with theanti-plant A-tubulin antibodies and taxol. We acknowledge the helpof Dr. D. A. Pepper in the immunofluorescent studies and for pro-viding us with the bovine brain antimicrotubule antibodies.

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