Sex Plant Reprod (2002) 15:2129

DOI 10.1007/s00497-002-0131-y

O R I G I N A L A RT I C L E

A. Majewska-Sawka M.C. Fernndez

M. Mrani-Alaoui A. Mnster
M.I. Rodrguez-Garca

Cell wall reformation by pollen tube protoplasts of olive

(Olea europaea L.): structural comparison with the pollen tube wall
Received: 11 November 2001 / Accepted: 11 February 2002 / Published online: 21 March 2002
Springer-Verlag 2002

Abstract Mature pollen grains of olive (Olea europaea

L.) were germinated in vitro in Brewbaker and Kwack
medium, and emerging pollen tubes were then enzymatically digested in the presence of high osmoticum. This
treatment resulted in simultaneous degradation of pollen
tube walls and fragmentation of their cytoplasm, giving
rise to numerous protoplasts of different sizes and different numbers of nuclei. After the protoplasts had been purified, they were cultured in Murashige and Skoog medium supplemented with auxin and cytokinin. The initial
steps of cell wall reformation were studied after 12 h and
24 h of culture with a series of cytochemical techniques
including periodic acid-Schiff reagent and phosphotungstic acid, as well as with electron microscopy and immunocytochemical techniques using monoclonal antibodies
directed against pectins and -(13)-glucan (callose).
Among the components of new wall in the protoplasts,
callose proved to be the earliest and most abundantly secreted polysaccharide, whereas the deposition of pectins
recognized by the antibody JIM7 started several hours
later. Pectins that bind JIM5 antibody were not detected
in this early stage of development. Cell wall components
deposited by protoplasts were compared with those present in growing pollen tubes. Callose secreted by protoplasts formed a relatively thicker layer than that found in
the tubes, and pectins recognized by JIM7 were highly
abundant, mostly within the cytoplasm and in the apical
zone of the tubes.
Keywords Pollen tubes Protoplasts Wall formation
Callose Pectins
A. Majewska-Sawka A. Mnster
Institute of Plant Breeding and Acclimatization,
Powstancow Wielkopolskish 10, 85090 Bydgoszcz, Poland
M.C. Fernndez M. Mrani-Alaoui M.I. Rodrguez-Garca ()
Department of Plant Biochemistry, Cellular and Molecular Biology,
Estacin Experimental del Zaidn, CSIC, Profesor Albareda 1,
18008 Granada, Spain
e-mail: mirbicep@eez.csic.es
Fax: +34-958-129600

Introduction
Since Takebe and co-workers first succeeded in regenerating tobacco mesophyll protoplasts in normal plants
(Takebe et al. 1971), this approach has gained interest
due to its possible application in genetic improvement.
Since the early 1970s, whole plants have been obtained
from protoplasts derived from a number of somatic cell
types in several hundred plant species, and many species
have been genetically modified through either somatic
hybridization or direct gene transfer into the protoplasts
(Puite 1992).
Gametophytic line cells have additional advantages,
since protoplasts of different ploidy and different nuclear
specificity can be obtained depending on the developmental stage of the cells subjected to enzymatic treatment (Tanaka 1994). To date, protoplasts have been
successfully isolated from tetrads (Arnalte et al. 1991),
microspores (Zhou 1989b), megasporocytes and egg
cells (Mouritzen and Holm 1995; Pnya et al. 1999),
binucleate or trinucleate pollen grains (Tanaka et al.
1987; Zhou 1989a, b; Fellner and Havranek 1992), and
also from pollen tubes (Kroh and Knuiman 1988; Rutten
and Derksen 1990, 1992). These protoplasts were further
used in gametic or gametosomatic fusion experiments
(Lee and Power 1988; Ueda et al. 1990; Desprez et al.
1995) leading at least in some cases to the production
of triploid hybrid plants (Pirrie and Power 1986).
In addition to their application in the formation of
cells with a triploid chromosome set, microspore- or pollen-derived protoplasts can also contribute to new transformation techniques that are potentially useful as alternatives to those in which whole microspores or pollen
grains are used (Tanaka 1994). Protoplasts isolated from
sperm cells and egg cells have recently been used in in
vitro fertilization studies (Kranz 1999; Scholten and
Kranz 2001).
The applications summarized above require that
either gametophytic protoplasts or their fusion products
(zygotes) reform the cell wall, an absolutely essential
event that precedes mitotic division and the subsequent

22

formation of a multicellular callus or embryo. Although

studies with aniline blue and Calcofluor white staining
have shown that gametophytic protoplasts synthesize
callose and possibly some other -linked glucans (Rutten
and Derksen 1990; Mouritzen and Holm 1995), no details of the process of new wall organization or of the
composition of the new tissue have been reported so far.
In this paper we describe a simple and reproducible
procedure for isolating sufficient numbers of pollen tube
protoplasts of olive for further study. In addition, we show
that these protoplasts start to deposit the new cell wall
within several hours of culture in vitro. The results of immunocytochemical studies of the presence and subcellular
location of callose and two different pectin epitopes, recognized by JIM5 and JIM7 antibodies, are presented, and
these findings are compared to the distribution of these
wall components in growing pollen tube walls.

Materials and methods

Germination and fixation of pollen tubes
Mature pollen grains of Olea europaea L. (cultivar Picual, Granada,
Spain) were germinated in Brewbaker and Kwack medium (1963)
at 25C in the dark. Pollen grains (30 mg) were placed on the surface of 10 ml of medium previously poured in plastic Petri dishes of
60 mm diameter. After 1 h of incubation without shaking, the pollen
grains started to germinate and the pollen tubes continued growing
during 16 h of culture. Pollen tubes were fixed 7 h after putting the
pollen in the culture medium, when the tubes reached an average
length of 0.5 mm (Mrani Alaoui 2000). The fixation was carried
out in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde
in 0.025 M cacodylate buffer, pH 7.5, for 2 h at room temperature.
The material was then washed with several changes of buffer, dehydrated in a graded series of ethanol and embedded in Unicryl resin
(Electron Microscopy Sciences, Fort Washington, PA.).
Isolation and fixation of pollen tubes-protoplasts
Germinated pollen tubes were gently centrifuged to remove excess
medium. The pollen tubes were then placed in a solution of 1%
macerozyme R-10 and 2% cellulase R-10 (Yakult Honsha, Tokyo,
Japan) supplemented with macro- and microelements according to
Frearson et al. (1973), with 20 mM N-morpholino ethanesulfonic
acid and 1 M mannitol as the osmoticum. The material was incubated for 1.5 h at 25C in the dark with gentle shaking on a rotary
shaker. After the protoplasts had appeared, a salt solution was
carefully added to lower the concentration of mannitol to 0.6 M.
Then the enzyme was removed by three subsequent washings with
a salt solution containing 0.6 M mannitol, followed by centrifugation at 800 rpm for 5 min. Finally, the suspension consisting of
protoplasts, empty pollen grains and ungerminated pollen grains
was layered over 1.2 M sucrose and centrifuged again at 800 rpm
for 10 min. The protoplast fraction, only slightly contaminated
with empty pollen grains, was collected from the interphase. Protoplasts were centrifuged once again, then placed in Murashige
and Skoog medium (1962) supplemented with 5 M naphthaleneacetic acid, 2 M 6-benzyloaminopurine and 0.6 M sucrose,
pH 5.8, and cultured at 25C in the dark.
Freshly isolated protoplasts, and protoplasts cultured for 12 h
and 24 h, were fixed in a mixture of 4% paraformaldehyde and
2.5% glutaraldehyde in 0.05 M Pipes buffer, pH 7.2, for 2 h. The
material was rinsed several times with buffer, dehydrated in increasing concentrations of ethanol and embedded in LR Gold resin. Semithin sections 0.50 m thick were cut on an RM 2155
microtome (Leica Microsystems, Nussloch, Germany) and placed
on glass slides covered with a 2% acetone solution of Biobond

(Polysciences, Warrington, PA.). Ultrathin sections were obtained

with a Reichert Ultracut E ultramicrotome (Reichert, Austria) and
were collected on formvar-coated nickel grids.
Cytochemical characterization of pollen tubes and protoplasts
The presence of callose in pollen tubes germinating in vitro was detected by staining with 0.01% aniline blue in 0.07 M phosphate
buffer, pH 8.5, for 20 min. Observations were performed with a
Jenalumar 250 fluorescent microscope (Carl Zeiss, Jena, Germany)
with a UV filter (excitation 330380 nm, barrier filter 410 nm,
dichroic mirror 410 nm).
Nuclei within freshly isolated protoplasts were stained with
solution of 4,6-diamidino-2-phenylindole (DAPI) in the culture
medium (10 g/ml) for 20 min in the dark, then washed several
times in pure culture medium, and observed with a UV filter in the
microscope.
Semithin sections of pollen tubes and protoplasts were stained
with a mixture of 0.5% toluidine blue and 0.5% Azur B in 1% borax for general morphology, and with periodic acid-Schiff reagent
(PAS) to visualize polysaccharides. The presence of the latter in
ultrathin sections was detected after 2 h incubation with a 5%
solution of phosphotungstic acid (PTA) in water, pH 1.5.
Immunocytochemical detection of wall components in pollen
tubes and protoplasts
Pectins were immunodetected with JIM7 and JIM5 antibodies, both
provided by Dr. K. Roberts, John Innes Center, Norwich, UK.
Callose was traced with anti--(13)-glucan, a commercial antibody obtained from Australia Biosupplies, Parkville, Australia.
Immunolocalization of cell wall antigens was performed on both
semithin and ultrathin sections. In semithin sections, the nonspecific binding sites were blocked with 5% bovine serum albumin
(BSA) in 0.1 M phosphate-buffered saline (PBS), pH 7.3, for 36 h.
The material was then incubated for 12 h with anti--(13)-glucan
primary antibody diluted 1:50 in 0.1 M PBS with 5% BSA, washed
in several changes of buffer, and incubated for 8 h with anti-mouse
antibody conjugated with fluorescein isothiocyanate (FITC) diluted
1:30 in 0.1 M PBS containing 0.1% BSA. The sections were
washed overnight in several changes of buffer and distilled water,
then air-dried and covered with anti-fade solution composed of
0.5% phenylenediamine in 0.1 M PBS, pH 12.0, and glycerine at a
ratio of 1:4. The FITC signal was observed with a blue filter
(450 nm excitation, 510 nm barrier filter and 510 nm dichroic mirror) in the microscope. Color photographs were taken on Kodak
400 ASA film.
Ultrathin sections were blocked for 3 h with 5% BSA and then
incubated for 3.5 h with primary antibodies diluted 1:10 (JIM5
and JIM7) or 1:50 [anti--(13)-glucan], each in 0.1 M PBS with
0.1% BSA. Incubation with the secondary antibodies lasted 3 h.
The secondary antibodies were goat-anti-rat coupled with 15 nm
colloidal gold (for JIM5 and JIM7) and goat-anti-mouse conjugated with 10 nm gold [for anti--(13)-glucan], both diluted 1:50
in buffer with 0.1% BSA. Co-localization of callose and pectins
was performed by double labelling with both antibodies, using
concentrations and times as described above. The grids were then
thoroughly washed in buffer and distilled water, and the sections
were briefly stained in a 5% aqueous solution of uranyl acetate,
then observed with a Zeiss 10C electron microscope (Carl Zeiss,
Jena, Germany) at 60 kV.

Results
Isolation of pollen tube-protoplasts
After pollen tubes were placed in enzyme solution, within 30 min of incubation the tube wall became thinner,

23

Fig. 1AF Light micrographs of pollen tubes and tube-derived

protoplasts. A Fragmentation of the tube cytoplasm (arrowheads)
and formation of spherical zones at the apical end (asterisk), followed by protoplast release (arrows); 540. B Numerous protoplasts (arrows) after 90 min of incubation; 250. C Karyoplasts
(arrows) and cytoplast (double arrow) within the protoplast popu-

lation (DAPI-staining); [4,6-diamidino-2-phenylindole (DAPI)

staining]; 540. D New cell wall material (arrows) at the surface
of the 12-h-old protoplasts (toluidine blue staining). E, F Polysaccharides in the cell wall (arrows) of the pollen tube (E,400), and
regenerating protoplast after 24 h of culture (F, 100) colored
pink-red [periodic acid-Schiff reagent (PAS)]

24

25

and the cytoplasm underwent plasmolysis and fragmented into several parts of unequal size (Fig. 1A). At the
same time, many pollen tubes displayed characteristic
displacements of the cytoplasm that formed spherical
zones at the apical end, which in the next step apparently
gave rise to separate protoplasts floating in enzyme solution (Fig. 1A). The number of protoplasts increased rapidly between 60 and 90 min of incubation, as the pollen
tubes disintegrated. The isolated protoplasts were spherical varied in size, and ranged in diameter from 4 to
23 m (Fig. 1B). Staining with DAPI clearly showed that
enzymatic digestion of the pollen tubes resulted in the
release of cytoplasts, which lack nuclei, and karyoplasts
that contained one, two or three nuclei (Fig. 1C).
Electron microscope observation of the morphology
of freshly isolated protoplasts revealed that they were
surrounded by plasma membrane and lacked remnants of
the cell wall (Fig. 3A). As was found for the pollen tubes
(Fig. 2A), the protoplast cytoplasm was relatively dense,
rich in ribosomes, dictyosomes, mitochondria, plastids,
short cisterns of endoplasmic reticulum, and sometimes
in electron-dense spherical bodies (Fig. 3A).
Wall components of pollen tubes

Seven hours after the pollen grains were placed in the

culture medium, the emerging pollen tubes reached an
average length of 0.5 mm. They displayed a well-differentiated, two-layered wall structure with a translucent
inner layer in contact with the plasmalemma, and an
electron-dense, fibrillar outer layer (Fig. 2A). Aniline
blue staining of pollen tubes growing in vivo and immunodetection of callose on semithin sections revealed
deposits of callose within the tube plugs, as well as a
thin layer visible along the pollen tube surface except at
the tip (Fig. 2B, C). The plugs were deposited at variable
intervals, and after 7 h of growth in vitro they varied in
number between one and four. Immunogold labelling
showed the anti-callose antibody mostly in the parts of
the cell wall adjacent to the plasma membrane (Fig. 2D).
The pectin epitopes detected by the JIM5 antibody were

Fig. 2AG Detection of polysaccharidic components in the pollen

tube-wall. A Two-layered cell wall in the subapical zone (uranyl
acetate staining). Note the dense cytoplasm rich in ribosomes, dictyosomes (d), endoplasmic reticulum (er) and mitochondria (m);
11,500. B Callose within the plugs (large arrows) and along the
pollen tube surface (aniline blue staining in vivo). Small arrows
indicate the tip of the pollen tube with no fluorescence; 250.
C Immunodetection of callose within the plug (arrow) and the
tube wall in the tube section [anti--(13)-glucan antibody and
fluorescein isothiocyanate (FITC)-conjugated secondary antibody]; 1,300. D Callose distribution in the innermost layer of the
pollen tube wall, adjacent to the plasmalemma [anti--(13)-glucan antibody and gold-conjugated secondary antibody]. A few
gold particles in the cytoplasm correspond to background;
13,500. E JIM5-recognized pectins in the outermost part of
the pollen tube wall; 9,000. F, G JIM7-recognized pectins distributed throughout the tube wall and inside cytoplasmic vesicles
(arrows) in subapical (F) and apical (G) zones of the tube; 15,000

located in the outer part of the wall, both in the proximal

and distal pollen tube regions (Fig. 2E), but they were
not present in the wall of the apical region. Pectins recognized by JIM7 were also present in the pollen tube
wall, and in lower quantities within cytoplasmic, Golgiderived vesicles (Fig. 2F). Golgi-derived vesicles were
most conspicuous in the apical part of the tube (Fig. 2G).
The wall in the region of the tube tip showed a very simple structure consisting of only one layer, enriched in
pectin epitopes that bind JIM7 antibody (Fig. 2G).
Wall components of protoplasts
Within 12 h of culture, the protoplasts started to regenerate the new cell wall (Fig. 1D), which after 24 h showed
a fibrillar structure following standard staining with uranyl acetate (Fig. 3B). The polysaccharidic nature of the
new wall material was traced in 24-h-old protoplasts by
both Calcofluor staining (data not shown) and the PAS
reaction (Fig. 1E, F) with light microscopic observations, and also PTA staining with electron microscopic
observations (Fig. 3C). PTA-positive material was also
present in cytoplasmic vesicles of different sizes
(Fig. 3C).
Immunocytochemical studies of the presence of wall
antigens showed that one of the main wall components
synthesized within 12 h of regeneration was -(13)glucan (Fig. 3D, E). This polymer was also present in
growing pollen tubes, in the innermost part of the wall
adjacent to the plasma membrane (Fig. 2D). In the protoplasts, however, the callose seemed to be more abundant than in the pollen tube wall and formed a very thick
layer around the whole protoplast (Fig. 3E).
Tracing the events related to wall deposition after
12 h of culture revealed that pectins started to be synthesized at about this time, and JIM7-responsive molecules
were observed mostly within the cytoplasmic vesicles
(Fig. 3F). Following another 12 h of protoplast development, these pectic epitopes could also be found on the
protoplast surface (Fig. 3G). The double labelling with
anti-callose and JIM7 antibodies shows random distribution of pectins within the callose layer (Fig. 3G). Pectins
recognized by JIM5 could not be detected after either
12 h or 24 h of culture.

Discussion
Pollen tube wall
The structure of the olive pollen tube wall described here
confirms previous reports on the two-layered structure
observable in subapical regions of tube walls in other
species (Taylor and Hepler 1997). At the proximal tube
end (i.e., close to the grain), the mature tube wall of
some species is composed of three layers: internal callosic, intermediate cellulosic, and the outermost layer composed of pectins (Li et al. 1993). This arrangement was

26

27

also observed in olive pollen tubes, but only as of 7 h

after putting the pollen grains in the culture medium
(Mrani Alaoui 2000).
The distribution of pectins and callose within the pollen tube wall has been previously described in more than
20 angiosperm species (Geitmann et al. 1995; Stpka et
al. 2000; Doblin et al. 2001; Lenartowska et al. 2001).
Our observations provide additional evidence that the
structure of pollen tube walls presents a universal pattern, probably reflecting the physiological significance
of this wall in the process of tube growth.
Protoplast isolation and wall reformation

Protoplasts from mature pollen grains are not as widely

used as somatic protoplasts; this situation obviously reflects the severe technical difficulties of isolating protoplasts from mature grains. The main obstacle is the pollen
exine, a wall not digestible by commercially available
enzymes. Chemical dissolution of exine with 4-methylmorpholine N-oxide monohydrate was effective, but
proved to be lethal for the protoplasts (Loewus et al.
1985). Two other approaches have been developed to
solve the problem: the first is based on the application of
relatively high osmotic pressure that induces protoplast
extrusion through the aperture (Tanaka 1994; Fellner
1995), and the second takes advantage of the natural process of grain germination and release of its content into
the growing pollen tube (Kroh and Knuiman 1988;
Rutten and Derksen 1992). Obtaining protoplasts from
whole pollen grains usually results in the appearance of a
homogenous population of protoplasts, each containing
two or three nuclei depending on the species specificity.
In contrast, pollen tube digestion gives rise to a heterogenous population containing both cytoplasts and karyoplasts with variable numbers and combinations of nuclei,
i.e., vegetative, generative, both kinds, or none (Rutten
and Derksen 1992). The latter phenomenon is highly
desirable for asymmetric fusions, when only part of the
genetic information is to be transferred to the recipient
protoplasts (Lrz et al. 1981).

Fig. 3AG Detection of polysaccharidic components within walls

reformed by pollen tube protoplasts. A Freshly isolated protoplasts
with no traces of the wall, and dense cytoplasm rich in ribosomes,
dictyosomes (d), plastids (p), mitochondria (m) and endoplasmic
reticulum (er); 13,000. B, C Fibrillar structure of new cell wall
(CW) material after 24 h of culture: uranyl acetate staining
(B, 14,000); phosphotungstic acid (PTA) staining (C, 14,000)
of the fibrillar layer of the cell wall and in cytoplasmic vesicles
(arrows). D, E Callose (arrows) at the protoplast surface after 12 h
of culture: anti--(13)-glucan antibody and FITC-conjugated
secondary antibody (D, 540); anti--(13)-glucan antibody and
gold-conjugated secondary antibody (E, 8,000). F Immunodetection of pectins in 12-h-old protoplasts with JIM7 and gold-conjugated secondary antibody. Label located within cytoplasm vesicles
(arrows); 21,800. G Double labelling of callose (small gold
grains) and pectins (big gold grains, arrows) reveals the distribution of both components within the wall (CW) of a 24-h-old protoplast; 52,500

Gametophytic protoplasts also provide a suitable model for studies of pollen or pollen tube biology, in particular the molecular characteristics of the plasma membrane
(Fan et al. 2001) and cell wall ontogenesis (Rutten et al.
1991). Available information about the nature of wall
components deposited by regenerating gametophytic protoplasts is compared to somatic protoplasts scarce and
based only on the results of cytochemical staining (MikiHirosige et al. 1988; Zhou 1989a). Detailed information
on the chemical nature and subcellular distribution of
wall components is still lacking.
The results presented in this paper show that callose
is abundantly synthesized at the olive protoplast surface
and that within 12 h of culture it forms a thick continuous layer. This process has been previously described for
somatic protoplasts of several species (Caumont et al.
1997; Mnster 2001), pollen-derived protoplasts of
Lilium longiflorum and Nicotiana tabacum, and megasporocyte protoplasts of Hordeum vulgare (MikiHirosige et al. 1988; Rutten et al. 1991; Mouritzen and
Holm 1995) on the basis of aniline blue staining. The
precise distribution of this polymer in pollen-derived
protoplasts is, to our knowledge, revealed here for the
first time by electron microscopy and immunocytochemistry. The deposition of callose may reflect at least two
phenomena: (1) the translation of mRNA(s) encoding
enzyme(s) involved in callose synthesis, which previously existed in growing pollen tubes and contributed to the
formation of the callose layer during tube growth, and
(2) de novo transcription and translation of mRNA activated by plasma membrane wounding in the course of
enzymatic treatment or mechanical damage to the protoplast. Currently, we have no direct evidence to state
whether callose also appears around the cytoplasts,
which do not possess the nucleus necessary for the transcriptional processes. For this reason, neither of the phenomena proposed above can be ruled out. In some other
species the callose wall is, however, also synthesized by
cytoplasts, a fact that gives support to the first hypothesis (Kroh and Knuiman 1988; Rutten and Derksen 1992).
On the other hand, support for the second hypothesis
comes from studies of somatic protoplasts isolated from
cells which do not synthesize callose under in planta
conditions. In these protoplasts, secretion of -(13)glucan, the event that typifies the initial phases of cell
wall reformation and precedes cellulose deposition was
found in a number of species, such as soybean, carrot
and sunflower (Klein et al. 1981; Mock et al. 1990;
Caumont et al. 1997). Callose secretion is most frequently ascribed to the cell-wounding-response (Shea
et al. 1989). The fibrillar material deposited on the olive
protoplast surface was identified as PTA-positive, and
may reflect cellulose deposits, but confirmation will
require more specific procedures.
Along with the synthesis of -glucans at the surface
of pollen tube protoplasts of olive, pectic epitopes recognized by the JIM7 antibody start to be secreted. The
structure of these epitopes has not yet been fully characterized, but optimal antibody binding is known to occur

28

when polymers of galacturonic acid are methyl-esterified

in a range of about 15 to 80%, regardless of whether
esterification displays a random or blockwise pattern of
distribution. Esterification of less than 15% of the pectins results in significant weakening of binding properties of these carbohydrates (Willats et al. 2000). No reaction was observed, following either 12 or 24 h of culture,
with the anti-pectin antibody JIM5, which more efficiently binds polymers with a degree of esterification between 31% and slightly over 40% (Willats et al. 2000),
so it seems reasonable to assume that we detected pectins with an esterification level exceeding 40% in our
olive protoplasts.
The presence of pectins with a relatively high degree
of esterification characterizes actively growing cells, including germinating pollen tubes, especially in their rapidly expanding apical zone. Esterification favors cell
wall flexibility and elasticity due to the inability of pectins to form the rigid egg-box structure through Ca2+
bridges. It thus permits adaptation to rapid changes in
cell volume. Methyl-esterified pectic polysaccharides
were also reported to form the wall of actively growing
somatic protoplasts and somatic cells in vitro (Moustacas
et al. 1986; David et al. 1995; Mnster 2001).
To summarize, we show that pollen tube protoplasts
of olive are able to resynthesize new cell wall material
that, like the subapical region of actively growing pollen
tubes, is rich in callose polymers and pectin epitopes recognized by the JIM7 antibody. Whether these protoplasts
can progress in development and enter mitotic division
remains to be determined in future studies.
Acknowledgements The study was supported by project no. PGC
BMC20001484 (DIGYCIT) from the Spanish Ministerio de
Ciencia y Tecnologa, Bilateral Action Spain-Poland 2001PL0023,
and Polish Committee for Scientific Research project no. 5 06A
040 17. The authors thank Conchita Martnez-Sierra for technical
assistance and Karen Shashok for correcting the English version
of the manuscript.

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