Glycerol effects both carbohydrate metabolism and cytoskeletal rearrangements during the induction of somatic embryogenesis in chicory leaf tissues

Glycerol effects both carbohydrate metabolismand cytoskeletal rearrangements during the inductionof somatic embryogenesis in chicory leaf tissues

Anne Bellettrea§, Jean-Paul Couillerota, Anne-Sophie Blervacqa, Serge Aubertb, Elisabeth Goutc, Jean-Louis Hilberta*,Jacques Vasseura

a Laboratoire de physiologie cellulaire et morphogenèse végétales, université des sciences et technologies de Lille,59655 Villeneuve d’Ascq cedex, Franceb Laboratoire de physiologie cellulaire végétale, CNRS, 17, rue des Martyrs, 38054 Grenoble, Francec Laboratoire de résonance magnétique en biologie et médecine, département de biologie moléculaire et structurale, CEN-G,17, rue des Martyrs, 38054 Grenoble cedex 9, France

Received 21 September 2000; accepted 31 January 2001

Abstract – In leaf tissue of the chicory hybrid ‘474’ (Cichorium intybus L. var. sativum × Cichorium endivia L. var. latifolia),induction and expression of somatic embryogenesis can be separated into a two-step procedure by the presence of glycerolduring the induction step. This effect of glycerol is caused by the inhibition of the initiation of the first divisions of theembryogenic cells by a so far unknown mechanism. In an attempt to unravel this mechanism, the effects of glycerol oncarbohydrate metabolism and cytoskeletal rearrangements were studied. When the induction medium was supplemented with[14C]-glycerol for 24 h, about 25 % of [14C]-glycerol were converted to other soluble carbohydrates. Activity of glycerol kinasewas very high during the induction phase of somatic embryogenesis and decreased after transfer. Glycerol supplementation wasof no avail on glycerol 3-phosphate dehydrogenase activity. A nuclear magnetic resonance study showed that glycerol3-phosphate had accumulated in leaf tissue. Microscopic studies usingα- and�-tubulin antibodies revealed that cytoskeletalrearrangements were greatly affected by the presence of glycerol. Immunobloting using antibodies againstα- and �-tubulinsrevealed that this effect of glycerol was not caused by alteration in the quantity of either type of tubulin monomers. We describethe progress made in the analysis of the effects of glycerol on somatic embryogenesis in chicory. New evidences are given onthe occurrence of an active glycerol metabolism during the inhibitory treatment. Moreover, the effect of glycerol was correlatedwith a defect in the pre-mitosis cytoskeleton organization. © 2001 Éditions scientifiques et médicales Elsevier SAS

Cichorium / cytoskeleton / glycerol / sugar metabolism / somatic embryogenesis

CS, cytoskeleton / DAPI, 4’,6-diamidino-2-phenylindole / DHAP, dihydroxy acetone phosphate / EC, embryogenic cell /G3P, glycerol 3-phosphate / G3P, glycerol 3-phosphate dehydrogenase / GK, glycerol kinase / GPE, glycerylphospho-rylethanolamine / Mts, microtubules / NMR, nuclear magnetic resonance / SE, somatic embryogenesis

1. INTRODUCTION

Somatic embryogenesis (SE) is the process bywhich a somatic cell develops into a plantlet through

orderly characteristic morphological stages thatresemble zygotic embryogenesis [11, 33]. This featureis a characteristic of higher plants without counterpartin the animal kingdom. Somatic embryogenesis is apowerful tool used in the study of developmentalprocesses in plants, especially embryo development.

As in many plants, embryogenesis inCichoriuminvolves two processes: induction of the embryogenicfate and expression of the embryogenic programme [8,29, 32]. Cichorium hybrid ‘474’ leaf tissues is anoriginal and attractive model in which SE is direct,

*Correspondence and reprints.E-mail address: [email protected] (J.L. Hilbert).§ Present address: Laboratoire de phyto–biologie cellulaire,université de Bourgogne, bât. des sciences Mirande, 21078Dijon, France

Plant Physiol. Biochem. 39 (2001) 503−511© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0981942801012633/FLA

rapid and abundant with a unicellular origin [17, 33].Several factors have been identified that can influenceor are directly correlated with the embryogenic path-way, including specific proteins [10, 20], polyamines[12] and temperature [13]. What makes this systemattractive is that by the addition of 330 mM glycerol tothe synthetic culture medium containing 60 mM sucrose(induction medium), the first division of embryogeniccells is delayed until the transfer of the leaf fragmentsto the same medium devoid of glycerol (expressionmedium) [6]. Studies on physiological, biochemicaland molecular aspects of somatic embryogenesis arefacilitated by a relative synchronization of the process.

Sugars alcohols (polyols) are the most abundantcompounds in bacteria, fungi, algae, mammals andhigher plants [2, 23, 24]. Different physiological roleshave been proposed for polyols in plant but osmoregu-lation was the most commonly reported. While man-nitol is reported as an osmotic regulator in salttolerance in Apium graveolens [18], sorbitol accumu-lation is mainly associated with osmotic adjustment inMalus sp. leaves [35, 36]. In Cichorium, althoughinitially supplied to the induction medium as anosmoticum, we have previously shown that glycerolmodified carbohydrate metabolism and was trans-ported into the tissues where it can provide carbon andenergy for leaf tissues which undergo embryogenesis[6]. Furthermore, glycerol modified carbohydratemetabolism, particularly during the induction period ofembryogenesis. Here, we describe some experimentsundertaken to describe how glycerol metabolism couldeffect SE induction.

In mammalians, alcohols are assumed to alter thecytoskeleton (CS) and this particularly concerns glyc-erol, which interacts with tubulins [37, 38]. Tubulinsand microtubules (Mts), the major components of theCS, are involved in several of the most basic processesincluding cell division and the determination of cellmorphology. Dijak and Simmonds [14] establishedthat Mts organization was modified during early directembryogenesis in mesophyll protoplasts of Medicagosativa. Such modifications of the CS were also observedin chicory, especially during the reactivation phase ofthe somatic cell and during the first embryogenicdivision where a pre-prophase band was observed[29]. These results confirmed that the first division ofthe reactivated cell was symmetrical, as it was previ-ously proposed [8]. Moreover, Cichorium tubulin iso-form (α and �) quantities varied during the two-stepprocess, especially when caffeine (5 mM), an inhibitorof cell plate formation (cytokinesis), is added duringSE [9].

In this paper, we focus on the putative role ofglycerol during the two-step protocol of chicory SE, asglycerol is used during the first step for delaying thefirst division. We studied glycerol behaviour duringsomatic embryogenesis and its repercussion on theglycolytic pathway using 14C, enzymatic activities andNMR investigations. The effects of the presence of theglycerol on CS rearrangements during the inductionphase of SE were also commented.

2. RESULTS

2.1. Glycerol effect on embryogenic cells

In a previous study, it was established that glycerolprevents the first division of embryogenic cells inchicory until day 4 [6]. Leaf tissues incubated inglycerol supplemented medium (M17S60Gly330)showed only reactivated cells dispersed among the leafblade (figure 1A), whereas leaf tissue explants culturedin medium devoid of glycerol (M17S60) show EC,characterized by trabecular cytoplasm, dense nucleo-lus, a central nucleus and somatic embryos (figure 1B,C). Chicory leaf tissue explants incubated in inductionmedium with glycerol as the only carbon source(60 mM to replace sucrose) presented embryogeniccells (figure 1D).

The replacement of sucrose by glycerol during theinduction phase seems to have no adversary effects onthe progress of SE during the expression phase.

2.2. Glycerol metabolism during SE

2.2.1. [14C]-glycerol labelling

During SE, glycerol concentration decreased in theM17S60Gly330 liquid incubation medium and wastransported into the leaf tissue [6]. These resultssuggest that glycerol was used as a carbon source byleaf explants during SE induction. To further investi-gate this, the induction medium was supplementedwith [14C]-glycerol. [14C]-Glycerol was readily metabo-lized and converted into carbohydrates during leaftissue induction (figure 2). At the end of the 24-hlabelling period, most of the radioactivity was recov-ered as [14C]-glycerol under embryogenic conditionand 37 % was converted into glycerol 3-phosphate(G3P) and carbohydrates. Only 25 % of the [14C]-molecules were recovered as sucrose, glucose andfructose. During SE culture, the most representedsugar was sucrose. Fructose was always significantlyhigher than glucose. After the transfer at day 4 into

504 A. Bellettre et al. / Plant Physiol. Biochem. 39 (2001) 503–511

M17S60 expression medium devoid of [14C]-glycerol,G3P and unknown carbohydrates were the majorlabelled molecules. Glycerol disappeared rapidly dur-ing the development of somatic embryos. Sucrose wasthe only sugar recovered throughout the culture and[14C]-hexoses were no longer detected in leaf tissues atday 8. The radioactivity level decreased during thissecond period of culture. From these results it wasconcluded that glycerol was metabolized in cultureleaf explants, and that its metabolism increased aftertransfer to glycerol-free medium.

2.2.2. Carbohydrate NMR spectra analyses

HPLC analyses did not allow the detection ofphosphorylated molecules. Consequently a methodusing NMR analyses was performed for the identifi-cation of glycerol metabolism products. Analysis of31P-NMR spectra on leaf explants after 3 d of SEinduction showed similar profile for both conditionswithout (figure 3A) and with (figure 3B) glycerol forUDP-glucose (precursor of sucrose with fructose

6-phosphate). Glycerylphosphorylethanolamine (GPE)was two-fold higher in the presence of glycerol during

Figure 1. Glycerol effect on early stages of embryogenesis in leaves of Cichorium hybrid ‘474’ . Embryogenic cells (EC) in transversal semi-thinsection stained with toluidine-blue after 4 d in M17S60Gly330 (A), induction in M17S60 medium, transversal section stained withsafranin/fast-green showing EC and embryos localization of EC on semi-thin section without staining and with Nomarski contrast (B, C), ECobserved on semi-thin section stained with toluidine-blue after 4 d induction in M17Gly390 (D). SE, Somatic embryo; Nu, nucleus. Bars = 50 µm.

Figure 2. Distribution of [14C]-glycerol into soluble carbohydrates inCichorium leaves during somatic embryogenesis. Each value is themean of three observations. Distribution is shown as a percentage oftotal [14C]-soluble molecules (= disintegrations in [14C]-glycerol + [14C]-sucrose + [14C]-glucose + [14C]-fructose + [14C]-unknown sugar + [14C]-G3P + [14C]-other labelling molecules; dpm,disintegration per minute).

A. Bellettre et al. / Plant Physiol. Biochem. 39 (2001) 503–511 505

the induction step (figure 3B) than in glycerol-freecondition (figure 3A). In the presence of glycerolduring SE induction, we observed a marked increaseof a resonance at 4.39 ppm (figure 3B–D) whichrepresented G3P. The presence of glycerol in theinduction medium resulted in the virtual absence offructose 6-phosphate, whereas the relative amount ofglucose 6-phosphate decreased three-fold (figure 3D)compared to M17S60 condition (figure 3C). These

results confirmed the [14C]-glycerol labelling studiespreviously described, and suggest that glycerol isconverted to G3P and hexoses through the dihydroxyacetone phosphate (DHAP) pathway.

2.2.3. Changes in enzymatic activitiesThe specific activity of glycerol kinase (GK; EC

2.7.1.30) during Cichorium leaves SE is shown infigure 4A. The 4-d induction period was accompaniedby a large increase in GK specific activity when leaftissues were incubated in M17S60Gly330 medium.After transfer to M17S60 expression medium, enzymeactivity declined to reach a level of 20 nmol·min–1·mg–1

proteins. When leaves were cultured during 4 d inM17S60 and transferred 8 d in M17S60 new medium,GK activity level remained low and stable (15nmol·min–1·mg–1 proteins).

The experiments shown in figure 4B were carriedout to investigate the effect of glycerol on glycerol3-phosphate dehydrogenase (G3PD; EC 1.1.1.8) activ-ity. During early stages of embryogenesis, enzymaticspecific activity was not significantly different whenleaf fragments were cultured with or without glycerol.After the transfer, G3PD specific activity increasedwith the same profile under both conditions. In thepresence of glycerol, the accumulation of G3P duringthe induction step was associated with high GKactivity while conversion into DHAP was unchanged.

2.3. Glycerol effect on cytoskeleton

2.3.1. Glycerol effect on CSIn view of the findings in animal cells showing the

interaction of the cytoskeleton [38], we have investi-gated the possible effect of glycerol on the change ofCS in EC. ECs were easily distinguishable as theyshowed central nuclei under both conditions as revealedafter DAPI staining (figure 5A, C). Anti-tubulin anti-bodies were used to examine the chicory Mts network.In cells incubated in M17S60 medium, the microtu-bule network was completely re-arranged compared tomesophyll non-induced cells. EC Mts network wasclearly definite and was characterized by a star-shapeconfiguration around the nucleus (figure 5B). Mtsradiating all around look like fine strands. Exposure toglycerol for 4 d in M17S60Gly330 resulted in aggre-gates of fluorescent material dispersed throughout thecells; organized Mts network was not observed and nopolymers were present (figure 5D).

2.3.2. Glycerol effect on tubulinsGlycerol has been involved in the alteration of Mts

function. We tested the effect of glycerol on tubulin

Figure 3. Representative 31P-NMR spectra of Cichorium leavesduring somatic embryogenesis. Leaf fragments were incubated for 3 din M17S60 (A and C) or M17S60Gly330 (B and D). C and D areenlargements of the boxed regions in A and B. Pi, Inorganicphosphate; GPE, glycerylphosphorylethanolamine; GPI, glycer-ylphosphorylinositol; GPC, glycerylphosphorylcholine; UDP-G,UDP-glucose.


proteins. The level of α-tubulins, visualized as a53-kDa band after SDS-PAGE and immunoblotting,were unchanged in M17S60Gly330 treated tissuescompared to M17S60 induced leaves (figure 6, lanes a,b). The α-tubulin contents increased throughout theculture period. Level of �-tubulins, visualized as a55-kDa band, is similar in both glycerol and glycerol-free cultures during the induction step (figure 6, lanesc, d). The �-tubulin accumulation increased fromday 2 until the end of the culture. The level of�-tubulin was higher during the expression step afteran induction in M17S60Gly330 medium. The level oftubulins was unchanged during the induction step inthe presence of glycerol. Glycerol treatment seems toalter the first mitosis of embryogenic cells by a changein Mts configuration and this could delay furthersomatic embryo development.

3. DISCUSSION

In our study, we have shown the effect of glycerolon EC. We found that glycerol was metabolized inchicory tissue during SE. We focused on the activity ofGK, the essential enzyme in glycerol metabolism, inthe carbohydrate pathway. We do not yet know whetherglycerol is directly responsible for the inhibition of thefirst division or if one of its metabolic product is. We

Figure 4. Time course of glycerol kinase activity (A) and glycerol3-phosphate dehydrogenase activity (B) during somatic embryogen-esis. Cichorium leaves were incubated 4 d in M17S60 (● ) orM17S60Gly330 (■ ) and transferred for 8 d in M17S60. Mean of threecultures of three explants for each culture with three replications;vertical bars indicate standard error.

Figure 5. Immunofluorescent patterns of the re-organization of the cytoskeleton in embryogenic cells. After 4 d in M17S60 or M17S60Gly330,EC shows central nucleus with DAPI staining (A and C). In M17S60, EC shows Mts connected to parietal cytoplasm (arrows) (B). InM17S60Gly330, Mts show aggregates of fluorescent material (arrow heads) (D). Nu, Nucleus. Bars = 10 µm.


have to investigate this in more details. The results ofthis study indicate that glycerol exposure of chicoryleaf tissues changes in the cytoskeleton, accompaniedby inhibition of cell division. We report for the firsttime in plant that the dynamic state of microtubulepolymerization is affected by the presence of glycerol.

Gautheret [19] showed that Daucus carota andHelianthus annuus callus could be cultured in glycerol-enriched media. Glycerol seemed to be the only polyolable to be used as the only carbon source in in vitroculture [22, 23]. Incubation of tissues from Cucurbitamaxima in [14C]-glycerol led to the formation ofconsiderable amounts of [14C]-sucrose indicating thatglycerol could be metabolized in the glycolytic path-way [3, 4]. MacDonald and Ap Rees [25] confirmedthis metabolic pathway in suspension cultures ofGlycine max. Glycerol is also the preferred carbonsource to obtain Citrus somatic embryos [7, 34]. Weproved by using [14C]-glycerol that glycerol can bemetabolized into sucrose, fructose and glucose duringSE in Cichorium. In bacteria and mammals, suchmetabolism was initiated by GK which catalysedglycerol to G3P. G3P was converted to dihydroxyacetone phosphate that is a metabolic crossroad link-ing the carbohydrate and lipid pathways [26]. In higherplants, Hippman and Heinz [21] demonstrated for thefirst time the existence of a GK in Pisum sativumleaves. GK activity was identified and the enzyme wascharacterized from extracts of Cucumis sativa [31]. InCichorium leaf fragments, we noticed that GK activitywas very high during SE and depended directly on theglycerol accumulated in the explants. 31P-NMR spec-trum analysis confirmed the presence of GK localizedin the cytoplasm compartment of Acer pseudoplatanus[1]. Perry and Harwood [26] used [3H]-glycerol in

developing seeds of Brassica napus and showed thatthe preferred orientation for glycerol was the lipidmetabolic pathway. We have observed that glyceroland G3P were not available to G3PD activity inchicory. This suggested that in Cichorium, [14C]-glycerol was used for lipid synthesis and then was lessavailable for conversion to DHAP. If so glycerolshould be metabolized by tissues probably to detoxifythe excess of glycerol. In presence of glycerol in themedium, GPE accumulation was associated with animportant amount of G3P and low hexose level inCichorium explants.

The accumulation of glycerol into the leaf tissue isdemonstrated and therefore it could interact with cells,which were undergoing de-differentiation followed byre-differentiation to give rise to an EC. Rambaud et al.[27] have already described in chicory that a completere-organization of the CS in the EC is a prerequisite forachieving mitosis. We have observed that in thepresence of glycerol, it was not possible to obtain acharacteristic Mts network in EC but some diffusematerial were observed instead. Although the α- and�-tubulin accumulation seemed not to be affected, wesuggest that glycerol or the abundant metabolite G3Pcould generate a reversible complex with tubulins, aswas previously established in vitro [10, 38]; thiscomplex would prevent the re-initiation of mitosis andthus disturb the CS re-organization and finally blockembryo development. Such events were also describedin animal cells where glycerol suppresses cell prolif-eration by destabilizing the CS [15]. Moreover, etha-nol and its derivatives are also known to disorganizeCS and particularly mammal Mts [37]. In vitro studyof Mts polymerization in presence of glycerol wouldbe helpful to see if the tubulin dimerization is affected

Figure 6. Expression of α- and �-tubulin proteinsduring somatic embryogenesis in Cichorium ‘474’leaves. In M17S60Gly330 induction condition (aand c) or in M17S60 induction condition (b and d).Proteins were extracted from leaves fragments and15 µg were used for western blot analysis.


or if the presence of glycerol generates anarchical Mtsarchitecture. It appears that in vitro glycerol treatmentmay be used for the further study of regulation of thecell cycle and cytoskeletal functions in chicory duringSE.

4. METHODS

4.1. Material and culture conditions

The plant material was obtained from a clone of theCichorium hybrid ‘474’ (Cichorium intybus L. var.sativum × Cichorium endivia L. var. latifolia) propa-gated by root somatic embryogenesis as previouslydescribed in Dubois et al. [16]. For our experiments,leaf fragments were cultured according to Bellettre etal. [6]. Briefly, SE was induced on liquid mediumcontaining 60 mM sucrose and 330 mM glycerol(M17S60Gly330, induction medium) during 4 d, at35 °C, in darkness and on orbital agitation. After thisstep, explants were transferred to the same mediumdeprived of glycerol (M17S60, expression medium)for another 4 d under the same conditions of culture.

4.2. Extraction and analysis of carbohydratesand glycerol and [14C]-glycerol labelling

Weighed frozen tissues were ground in a Potterblender and the powder was extracted with 95 %ethanol. After centrifugation (30 000 × g, 10 min,20 °C), the pellet was re-extracted with 95 % ethanol.The two supernatants were combined and dried undervacuum. The lyophilized powder was resuspended in0.01 M sorbitol (used as internal standard) and passedthrough a 0.45-µm Millipore filter.

Soluble carbohydrates and glycerol were analysedin 100-µL aliquots of filtered extract by HPLC. Thechromatographic system consisted of a solvent degas-ser Spectra SYSTEM SCM400 (Spectra Physics, SanJose, California), a Spectra SYSTEM P4000 pump,Waters Sugar-PAK I (Waters Millipore, Milfort, Mas-sachusetts) column (300 × 6.5 mm) maintained at 85 °Cand a differential refractometer. Distilled water con-taining 50 mg·L–1 calcium-EDTA was used as thesolvent at a flow rate of 0.5 mL·min–1. The inductionculture medium M17S60 was supplemented with glyc-erol and 0.33 µM (1.85 MBq) of [14C]-glycerol (spe-cific activity 5.59 GBq·mmol–1; Radiochemical Cen-ter, Amersham, UK). The final glycerol concentrationwas 330 mM.

After separation by HPLC [14C]-glycerol and [14C]-carbohydrates were quantified with the Flow Radio-

chromatography Detector System Flow-Onet/BetaModel A 515 (Radiomatic Instruments and ChemicalCompany Inc., Meriden, USA).

4.3. Perchloric extract and NMR analysisFor perchloric acid extraction, leaf tissues (8 g FW)

were quickly frozen in liquid nitrogen and ground to afine powder with a mortar and pestle with 1 mL 70 %(v/v) perchloric acid. The frozen powder was thenplaced at –10 °C and thawed. The thick suspensionthus obtained was centrifuged at 10 000 × g for 10 minto remove particulate matter, and the supernatant wasadjusted with 2 M KHCO3 to about pH 5.5. Thesupernatant was then centrifuged at 10 000 × g for10 min to remove KClO4. The resulting supernatantwas lyophilized and stored in liquid nitrogen. For theNMR measurements, this freeze-dried material wasredissolved in 2.5 mL water containing 10 % D2O(perchloric acid extract) [30]. 31P-NMR spectra ofneutralized perchloric acid extracts were determinedon a Bruker NMR spectrometer (AM 400, narrowbore) equipped with a 10-mm multinuclear probe.

For 31P-NMR spectra (161.93 MHz) acquisition,10-µs pulses (50 °C) at 1.8-s intervals were used. Thedeuterium resonance of D2O was used as a lock signal,and the spectra were recorded over a period of 2 hunder conditions of broad band proton decoupling.The spectra at the perchloric acid extract were refer-enced to methylene diphosphoric acid at 16.38 ppm.

4.4. Enzymes extractions and assayAll operations were carried out at 4 °C. Cichorium

leaves were ground with a mortar and pestle ingrinding medium (0.5 M sucrose, 1 mM EDTA, 1 mMMgCl2, 10 mM KCl, 5 mM dithiothreitol, 0.3 M Tris-HCl (pH 7.5)). After centrifugation at 5 000 × g for10 min to remove cell wall and debris, the homogenatewas centrifuged at 30 000 × g for 30 min, and thesupernatant was used for the determination of enzy-matic activities. Glycerol kinase (EC 2.7.1.30) wasassayed in MgCl2-hydrazine-glycine buffer (pH 9.8)containing 0.02 M ATP, 0.075 M L-α-glycerol phos-phate dehydrogenase (Sigma) and 0.1 M NAD-glycerolphosphate dehydrogenase (EC 1.1.1.8) was assayed bya modified method [5, 28] in 0.05 M triethanolamine-HCl buffer (pH 7.5) containing 0.1 mM NADH and0.2 mM dihydroxyacetone phosphate. Reactions wereassayed spectrophotometrically [28].

4.5. Immunolocalization of the CSand DAPI staining

The method was previously described in Perry andHarwood [26]. Briefly, leaf fragments were fixed, then


they were digested and dilacerated on a poly-L-lysinecoated coverslips. Preparation were incubated withmouse anti α-tubulin antibodies diluted 1:200 (Amer-sham) then incubated with fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse antibodies diluted1:20 (Jackson Immunoresearch Lab.). Nuclei werestained with 4’ ,6-diamidino-2-phenylindole (DAPI)(5 µg·mL–1). Slides were observed on an OlympusBH-2 microscope using UV light for DAPI fluores-cence and blue light (excitation wavelength 490 nm)for FITC.

4.6. Electrophoresis and tubulin immunoblotting

Total proteins were extracted according to Hilbert etal. [20]. One-dimensional electrophoresis in 10 %polyacrylamide gels and immunoblotting were per-formed as described previously [9]. Briefly, gels wereelectroblotted onto nitrocellulose membranes, whichthen were incubated first with anti-α- and/or anti-�-tubulin antibodies diluted both 1:2 000 (Amersham).Goat anti-mouse antibodies conjugated with alkalinephosphatase (Jackson Immunoresearch Lab.) were usedfor detection of the primary antibodies.

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Glycerol effects both carbohydrate metabolism and cytoskeletal rearrangements during the induction of somatic embryogenesis in chicory leaf tissues