Plant Science 174 (2008) 597–605

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Early markers of in vitro microspore reprogramming to embryogenesis

in olive (Olea europaea L.)
Marı́a-Teresa Solı́s a, Beatriz Pintos b, Marı́a-Jesús Prado b,1, Marı́a-Ángeles Bueno b, Ivan Raska c,
Marı́a-Carmen Risueño a, Pilar S. Testillano a,*
a
Plant Development and Nuclear Architecture, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
b
Forest Biotechnology Lab. INIA-CIFOR, A-VI, km 7.5, 28040 Madrid, Spain
c
Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague and Department of Cell Biology, Institute of Physiology,
Academy of Sciences of the Czech Republic, v. v. i. Albertov 4, 128 00 Prague, Czech Republic

A R T I C L E I N F O A B S T R A C T

Article history: Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an
Received 23 January 2008 efficient method of producing homozygous lines for crop breeding. In trees, the process is of special
Received in revised form 12 March 2008 interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a
Accepted 14 March 2008
convenient method has been developed for microspore embryogenesis induction by stress in olive
Available online 30 March 2008
isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental
pathway and the formation of microspore-derived multicellular proembryos have been achieved and a
Keywords: cytochemical and immunocytochemical analysis was performed in the early stages. The young
Olive
microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in
Pollen embryogenesis
vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the
Pectins
Cell wall occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal
Cellular markers population, and changes in cellulosic and pectic cell wall components which constituted early markers of
Microspore culture the embryogenic microspore process. They provided new insights on the molecular and cellular events
associated with the microspore reprogramming of woody plants, and specifically in olive, providing
interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of
high economic interest.
ß 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction reprogramming of the immature pollen, the microspore, towards

Microspore embryogenesis is a widely used method to generate
genetic variability by obtaining microspore-derived embryos and
double-haploid plants which have applications for plant breeding
[1]. Efficient protocols for microspore embryogenesis induction in
culture have been developed in recent years for many crops (review
in [2]), and to a less extent for some woody plants [3–6]. In trees,
with a long reproductive cycle, a high heterozygosity level, tendency
for allogamy, and sometimes self-incompatibility, methods for
obtaining homozygous plants are of high interest, but their
production through conventional methods are practically impos-
sible to perform in woody plants. Microspore embryogenesis by
isolated microspore culture, allows the development of complete
homozygous lines from heterozygous parents. It involves the
* Corresponding author. Tel.: +34 918373112; fax: +34 915360432.
E-mail address: testillano@cib.csic.es (P.S. Testillano).
1
Present address: Department of Plant Biology, University of Vigo, Spain.
a different developmental pathway and the onset of proliferation
and differentiation events which finally leads to embryo formation
and haploid and double-haploid plant regeneration [7].
Olive (Olea europaea L.) from the Mediterranean basin supplies
more than 95% of the world production of olive oil; being a crop of
high social and economic interest in the Mediterranean area.
Conventional breeding methods are time-consuming and have the
limitations of the long juvenile period of the species. In this
context, microspore embryogenesis allows the single-step devel-
opment of complete homozygous lines from heterozygous parents
and constitutes a good method to exploit in olive genetic studies
and breeding. Recently, a convenient method for the induction by
stress of early stage embryogenesis from isolated microspore
culture has been developed in different varieties of olive ([8], for a
more detailed protocol description also see: [9]). In this system, the
switch to the embryogenesis pathway could be induced by a
combination of cold and starvation stress treatments, as it has been
shown in other isolated microspore systems such as tobacco,
wheat or rice [10–12]. This system constitutes a crucial step to

0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.03.014
598 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605
further attempt haploid-plant production, via pollen embryogen-
esis-derived structures.
Basic studies on the process of microspore embryogenesis have
mainly been pursued in model species like rapeseed, tobacco and
barley [13–15,11,1,16,17] but reports are very limited on other
economically interesting crops and trees [18–20]. The search of
molecular and cellular markers during early stages of microspore
embryogenesis constitutes an important goal to monitor the
metabolic processes involved in the induction, and also to identify
cells committed to the induced developmental programme.
Changes in various cell activities and structural organization of
subcellular compartments have been reported to accompany the
process in some model and herbaceous species [21,7,22–24], but
much less is known about the cellular mechanisms leading to the
process in woody plants due to the inherent difficulties of the
system [25,19,26], e.g. high asynchrony during pollen develop-
ment, high influence of climatic conditions, low proportions of
viable pollen, etc.
The aim of the present work was to identify in olive microspore
cultures the cellular changes in responsive microspores switched to
the embryogenic pathway, and pollen-derived multicellular struc-
tures. The study was focused to analyse cellular features related to
proliferation and differentiation events, like starch accumulation,
occurrence of symmetrical divisions, formation of new cell walls,
ribosomal population and cell wall components. The results showed
defined cellular changes, some of them being characteristic of cell
proliferation, which can be considered as markers of the microspore
committed to the embryogenesis process.
2. Material and methods
2.1. Plant material and microspore culture
Floral buds were collected in April–May from trees of the cultivar
Cornicabra (Madrid). Microspore cultures were established as
previously described by us ([8], for a more detailed protocol
description see: [9]). Flower buds containing anthers with micro-
spores at the most responsive developmental stage, the vacuolated
microspore, were selected by using the correlation between
sequential flower bud lengths and pollen developmental stages
which had been previously established [8]. Flower buds were
sterilized in fungicide Benomyl (C14H18N4O3, Aragro) solution
(200 mg/l) for 1 min, in 70% ethanol for 1 min and in 2% sodium
hypochlorite solution, with a few drops of Tween-20 for 30 min.
After rinsing in distilled water, anthers were carefully excised from
the flower buds of the olive trees and analyzed as a system of
gametophytic development in vivo, for comparison with the in vitro
development. Microspores were isolated from anthers using the
following protocol: a total of 200 anthers were placed in 2 ml B
medium [27], a starvation medium lacking sugar and nitrogen.
Anthers were agitated on a magnetic stirrer for 1 min at 250 rpm.
The released microspore population was filtered through a 30 mm
sieve which retained the anthers. This extraction process was
repeated a total of four times. The pooled filtrate containing
microspores was centrifuged for 5 min at 220  g at room
temperature. The microspore pellet obtained was resuspended in
5 ml B medium. This process was repeated a total of four times.
Culture density was adjusted to 1–5  105 microspores/ml by
adding B medium to the pellet obtained after the final centrifugation
step. A volume of 1.5 ml was dispensed into each 35  10 mm sterile
Petri dishes, leaving about 500 ml to determine microspores viability
and concentration. These plates were placed in cold stress conditions
(2–6 8C for 96 h). After stress treatment, the microspore population
in B medium was centrifuged for 5 min at 220  g at room
temperature. The microspore pellet was resuspended in induction
medium AT3 I [28], in four-well plates with the final density of the
microspores being determined using a haemocytometer (Neubauer
chamber) and adjusted to 4–8  104 or 1–2  105 microspores/ml.
The microspores were incubated in a dark culture chamber at 25 8C.
2.2. Fixation and processing for microscopic analysis
Anthers from different bud sizes and in vitro cultures
containing microspores and microspore-derived structures at
specific times (0, 3 and 6 months) were fixed in 4% paraformal-
dehyde in phosphate buffered saline (PBS), overnight, at 4 8C.
Microspore cultures were embedded in gelatine and processed as
the anthers, as described before [29]. Samples were dehydrated in
acetone series and embedded in Technovit 8100 resin (Kulzer,
Germany) at 4 8C.
2.3. Cytochemical methods and confocal analysis
Technovit semithin sections (1 mm) were stained with toluidine
blue and observed under bright field for structural analysis. Several
cytochemical methods were performed to preferentially stain
different cell components.
2.3.1. DAPI staining for DNA
40 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) stain-
ing was applied in squashed preparations of fixed anthers and
microspore in vitro cultures [30]. 1 mg/ml DAPI solution in PBS
plus 1% Triton X-100 was applied for 30–60 min (culture
samples) and overnight (anthers) in darkness. After rinsing,
samples were squashed, mounted in Mowiol and observed in a
confocal laser scanning microscope Leica CLSM TCS SP2 under
UV irradiation, DAPI fluorescence optical sections and DIC
(Differential Interference Contrast) images of the same struc-
tures were captured. DAPI staining (1 mg/ml DAPI in PBS) was
also applied to semithin sections, which after rinsing and drying
were mounted in Eukitt and observed under UV irradiation in a
Zeiss Axioplan epifluorescence microscope equipped with a CCD
camera.
2.3.2. Toluidine blue staining and I2KI staining for starch
Staining solutions (0.075% toluidine blue in water, and 2 g of KI
and 0.2 g of I in 100 ml of water) were applied on Technovit
sections for 10–15 min. After rinsing and drying, preparations
were mounted in Eukitt and observed under bright field in a Leitz
Laborlux 12 microscope equipped with a DP10 Olympus digital
camera.
2.3.3. Calcofluor white for cellulosic wall components
Calcofluor stock solution (3.5 mg/ml in water, pH 10–11,
adjusted with NaOH) was diluted 100 times in Tris–HCl 0.1 M,
pH 8.5 and applied on the sections in darkness for 20 min. After
rinsing and drying, sections were mounted in Eukitt and observed
under UV irradiation in an Axioplan epifluorescence microscope
equipped with a CCD camera.
2.4. Antibodies
For total RNA detection an anti-RNA mouse monoclonal
antibody, D44 [31] was used. For localization of pectins, JIM5
and JIM7 rat monoclonal antibodies which respectively recognize
low and high-methyl-esterified pectins were used [32].
2.5. Immunocytochemistry
Immunocytochemistry was performed on Technovit semithin
sections and revealed either by fluorochromes (for anti-RNA) or
 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605 599

by colloidal gold followed by silver enhancement (for JIM5 and 3. Results

JIM7). Sections were blocked with 5% bovine serum albumin
(BSA) in PBS (5 min), and incubated with anti-RNA, JIM5 or
JIM7 monoclonal antibodies for 1 h, undiluted. After washing in
PBS, they were exposed to the corresponding secondary
antibodies following the procedure previously described by us
[23,29].
3.1. Monitoring the in vivo gametophytic development versus the in
vitro development of the embryogenesis induced-microspore
The in vivo gametophytic development of the microspore was
monitored by microscopical analysis of selected anthers. The

Fig. 1. Microspore gametophytic development. Semithin sections of consecutive developmental stages after different cytochemical and immunocytochemical stainings. (a, d, g, j)
Vacuolated microspore stage; (b, e, h, k) young pollen stage; (c, f, i, l) mature pollen stage; (a–c) toluidine blue staining for general structural visualization; (g–i) DAPI staining for
DNA revealing the nuclei; (d–f) Anti-RNA immunofluorescence on the same sections than in the DAPI images; (j–l) iodide-based cytochemistry for starch revealing the starch
deposits as dark precipitates in young and mature pollen grains. Cytoplasmic vacuole, V; vegetative nucleus, VN, generative nucleus, G, nucleolus, arrows; Bars: 10 mm.
600 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605
Fig. 2. Nuclei divisions in microspore in vitro cultures and formation of microspore-derived multicellular structures. Confocal microscopy of the same microspore observed
under UV excitation for nuclei visualization by DAPI (a–d) and DIC (Differential Interference Contrast, Nomarsky) images showing the exine of the microspore (e–h). Two,
three and four nuclei with similar size and chromatin condensation can be seen in individual microspores. Bars: 10 mm.
correlation between sequential flower bud lengths and pollen
developmental stages has previously been established [8] con-
stituting a useful criterion for handling and selection of specific
stages for the microspore culture. After the release from the tetrad,
microspores developed a large vacuole which pushed the
elongated nucleus to a peripheral location; the cytoplasm
appeared as a thin layer (Fig. 1a, d, g). Anti-RNA immunofluor-
escence provided a high signal in the cytoplasm of vacuolate
microspores (Fig. 1d) when compared with younger microspore
developmental stages (data not shown), indicating an abundant
ribosome population. After the first pollen mitosis, the small
generative cell was localized attached to the pollen wall (exine) with
a more condensed chromatin, as revealed by the DAPI staining
(Fig. 1b, h). In contrast, the larger and rounded vegetative nucleus
was in the centre and displayed a more decondensed chromatin
showing less fluorescence intensity by DAPI (Fig. 1b, h). The
vegetative cytoplasm showed small vacuoles and some clear
inclusions (Fig. 1b), as well as an intense anti-RNA immunofluor-
escence (Fig. 1b, e), both nuclei being free of labelling except for the
nucleoli. Controls avoiding the first antibody did not show
significant labelling (data not shown). Later on, at mature pollen
stages, the generative cell migrated to the centre of the pollen grain
(Fig. 1c, i) whose cytoplasm showed abundant ribosomal population
by anti-RNA immunofluorescence (Fig. 1c, f) and numerous small
rounded clear inclusions, in toluidine blue-stained sections (Fig. 1c).
The iodide-based cytochemistry for starch revealed the
dynamics of starch accumulation during the gametophytic
development. No starch-staining was detected in the vacuolated
microspore (Fig. 1j), whereas at later stages an increasing number
of stained inclusions were found in the vegetative cytoplasm,
indicating a progressive accumulation of starch deposits during
pollen maturation (Fig. 1k, l).
For the in the in vitro induced-microspore embryogenesis, the
microspores at the vacuolated stage were selected as the most
responsive developmental stage for the culture, as previously
reported [8]. Microspores were isolated from anthers and put in
liquid medium. After 3 and 6 months, samples from the culture were
DAPI stained and squashed for checking the number of nuclei in the
pollen grains and pollen-derived structures. Confocal analysis
revealed structures with two, three and four nuclei (Fig. 2) still
surrounded by the exine, as revealed by the corresponding DIC
image of the same structures (Fig. 2e–h). The two nuclei of these
microspores (Fig. 2b) were similar in size, shape and chromatin
condensation suggesting that they were formed by a symmetrical
division, in contrast with the bicellular pollen formed during the
gametophytic development (Fig. 1h, i). The microspores with three
nuclei also exhibited nuclei with similar size, shape and organization
(Fig. 2c), which could indicate that the second division occurred
asynchronically in the two previous nuclei, one of them dividing
first. This fact also supported the idea that these two-nuclei
microspores had wall-separated cytoplasms. The multicellular
pollen-derived structures were found at different time points, from
3 to 6 months, during the progression of the culture, indicating a high
asynchrony in the embryogenic response of the microspores.
Samples of the in vitro culture were fixed and processed for
further microscopical analysis which revealed that the cultures
contained pollen-derived structures with differential features. In
toluidine blue-stained sections, some dead (empty) microspores
with irregular shapes were observed together with others showing
larger sizes and a more contrasted cell wall (Fig. 3a). Some of them
exhibited inner walls separating cells (Fig. 3b) as a sign of previous
cell divisions occurring during the culture. Anti-RNA immunofluor-
escence showed a high cytoplasmic signal in these multicellular
structures (Fig. 3c), indicating a high ribosomal population as typical
feature of high metabolic activity. The iodide-based cytochemistry
showed a high content of starch in some of the microspores of the
culture (Fig. 3d) which exhibited numerous amyloplasts revealed as
dark cytoplasmic spots by the iodine staining (Fig. 3d, e). Other
population of the microspores in the culture did not show starch
accumulations. In the microspore-derived structures with inner
walls, no starch deposits were observed.
3.2. Cell wall changes in microspore-derived structures
The modifications in components of the cell wall and in pectin
residues have been reported as responsible for initiating cell
responses in relation to cell fate and development [33]. The
differential composition of the cell walls in the microspore-derived
proembryos in comparison with the pollen grains were studied.
Cytochemistry with calcofluor white for cellulosic components and
immunocytochemistry using JIM5 and JIM7 monoclonal antibo-
dies recognising non-esterified and esterified pectins respectively
 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605 601

Fig. 3. General structure, anti-RNA immunofluorescence and starch deposits of the microspores in the culture. Semithin sections of the microspore cultures after toluidine
blue staining (a, b), anti-RNA immunofluorescence (c) and iodide-based cytochemistry for starch (d, e). (a) Panoramic view with microspores exhibiting different structures.
(b) Detail of a induced-microspore showing walled cytoplasms. (c) Anti-RNA immunofluorescence showing bright signal in the cytoplasms of a two-celled microspore. (d)
Panoramic view showing some microspores with starch deposits, together with others which do not contain them. (e) Detail of microspores containing numerous starch
deposits. Bars: in (a), (d): 50 mm, in (b), (c), (e): 10 mm.

were performed on semithin sections of both in vivo developing
microspores in anthers and microspore cultures.
During the gametophytic development in vivo, at the stage of
vacuolated microspore, calcofluor white staining provided specific
fluorescence to cell walls of the somatic tissue of the anther
whereas no reaction was observed in the microspore wall (Fig. 4a,
d). After the first pollen mitosis, the newly formed wall of the
generative cell appeared specifically stained by calcofluor as a lens-
shaped fluorescent line (Fig. 4b, e). In the mature pollen, no
staining with calcofluor was observed in the pollen wall except for
the apertures which showed specific fluorescence at their inner
region (Fig. 4c, f), where a thick layer of polysaccharides was
secreted to form the aperture of the pollen grain. No positive
staining with calcofluor was observed in the inner wall of the
developing microspore, the intine, at any stage during in vivo
development (Fig. 4).
In contrast, in the induced-microspore in vitro cultures, the wall
of defined microspores and microspore-derived structures showed
specific fluorescence by calcofluor white staining (Fig. 5), the rest
of the structures appearing negative to this cytochemical
technique. The calcofluor-positive microspores exhibited an
intense fluorescence in the thick peripheral wall localized below
the exine (Fig. 5a, b). Some of them also showed dividing walls
intensively stained at their interior (Fig. 5b–f). These dividing walls
occasionally appeared incomplete (Fig. 5c). The results indicated
that the multicellular microspore-derived proembryos showed a
differential staining reaction with calcofluor in their walls in
comparison with the rest of the culture.
Immunocytochemistry with JIM5 and JIM7 antibodies, recogniz-
ing non-esterified and esterified pectins [32] was performed to
analyze cell wall components of the multicellular proembryos.
Results showed a higher and positive immunoreaction with JIM5
antibodies in the peripheral wall below the exine and in the dividing
walls at their interior (Fig. 6a–c), indicating that cell walls of
microspore proembryos were rich in de-esterified pectins. With
JIM7 antibodies recognizing esterified pectins, no immunoreaction
was found in any cell wall of the microspore-derived proembryos
(Fig. 6d), whereas positive controls for JIM7 immunolocalization
602 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605

Fig. 4. Calcofluor fluorescent staining of cell walls during the microspore gametophytic development. Semithin sections of consecutive developmental stages. (a, d)
Vacuolated microspore; (b, e) young pollen; (c, f): mature pollen. Same semithin sections observed under phase contrast for general structural visualization (a–c) and under
UV excitation for calcofluor specific fluorescence of cellulosic cell wall components (d–f). No fluorescence is observed in the vacuolated microspore wall, only walls of the
somatic cells (S) of the anther exhibit calcofluor-positive reaction (a, d). Specific fluorescence by calcofluor is found in the thin wall of the newly-formed generative cell
(arrow) of the young pollen (b, e) and in the developing pollen apertural region (arrows) of the wall (c, f). Bars: 10 mm.

Fig. 5. Calcofluor fluorescent staining of cell walls in microspores in the culture. (a, b) Panoramic views of the microspore cultures showing many non-stained microspores
together with other microspores showing high fluorescence in their cell walls, some of them also exhibit inner separating walls specifically stained. (c–f) Details of
microspore-derived proembryos whose cell walls are differentially stained by calcofluor. Incomplete walls showing calcofluor fluorescence can be occasionally seen at the
interior of microspore proembryos (c). Bars: in (a), (b) 50 mm, in (c)–(f) 10 mm.
 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605 603

Fig. 6. Non-esterified and esterified pectin immunolocalization by JIM5 and JIM7 antibodies in microspores in the culture. Semithin sections after immunogold labelling followed
by silver enhancement observed under bright field. (a) Immunolocalization of non-esterified pectins by JIM5 antibodies exhibits positive reaction, as dark precipitates, on the walls
of microspores and microspore-derived proembryos with inner separating walls. (b, c) Details of the JIM5 immunoreaction on microspore-derived proembryos. (d)
Immunolocalization of esterified pectins by JIM7 antibodies shows no reaction with the microspores in the culture. (e) Positive control of the JIM7 immunoreaction on somatic
cells of the anther. (f) Negative control avoiding the first antibody shows no reaction on any structure. Bars: in (a), (d) 50 mm, in (b), (c), (f), (e) 10 mm.

performed on anther somatic tissues provided a high immunoreac-
tion on their cell walls (Fig. 6e). Negative controls avoiding the first
antibody did not provide significant labelling in any case (Fig. 6f).
4. Discussion
also a gametophytic-like pathway was suggested for a set of the non-
responsive microspores [29]. In olive microspore cultures, the
presence of starch deposits constituted an early marker of the non-
responsive microspores which can follow, at least in part, a
gametophytic-like pathway.

The comparative analysis performed between specific cellular
changes accompanying the in vivo gametophytic development of
the microspore and those observed in the in vitro cultures of stress-
induced microspore embryogenesis has permitted the identifica-
tion of defined features in relation to proliferation and differentia-
tion events, such as the accumulation of starch inclusions, the
occurrence of a first symmetrical division and subsequent
cytokinesis with formation of new cell walls, new cell divisions,
abundant ribosomes, and changes in pectin esterification and
cellulosic cell wall components. These differential features were
not found either in microspores developed in vivo or in non-
responsive microspores in vitro.
4.1. Starch deposits as an early marker of the non-responsive
stress-treated microspores of in vitro cultures
The results illustrated that a characteristic feature of the in vivo
gametophytic development was a high accumulation of starch found
at stages of maturing pollen. The development of plastids and starch
accumulation constitutes a differential process during pollen
formation in many species [34,35], as well as in certain stages of
zygotic embryogenesis [13,14]. In the olive stress-induced micro-
spore cultures, the multicellular structures or proembryos did not
show starch deposits whereas other non-responsive microspores
exhibited a high starch content in the cytoplasms. Non-embryogenic
pathways have been reported in other microspore in vitro systems,
such as Brassica napus, to occur after the inductive treatment, and
4.2. Nuclei with equal structural organization and walled cells
characterize young microspore proembryos
It is widely accepted that the first morphological evidence of the
embryogenic pathway is the symmetric division of the microspore,
as opposed to the asymmetric one of gametophytic pathway
[36,37], even though a few reports in tobacco showed that
symmetrically divided microspores could germinate in vitro [38]
and express the vegetative cell-specific promoter lat52 [39], but
they could not correctly differentiate the generative cell and to
form the mature pollen grain [39]. The analysis reported here
showed that in olive microspores, not only a symmetric mitosis
took place but also the subsequent divisions of the two-celled
embryogenic microspores which produced multicellular proem-
bryos. This fact illustrates that the reprogramming of the olive
microspore was started by the reactivation of the proliferative
activity, the initial event of the switch to embryogenesis. The
observation of a high ribosomal population in the cytoplasms of
the proembryo cells, as revealed by anti-RNA immunofluores-
cence, constituted additional data of the high metabolic activity of
these cells corresponding to those of the active proliferating cells.
The observation, after DAPI staining, of two or more nuclei in
microspore cultures constitutes the first sign that the embryogen-
esis induction has been initiated, but not in many systems has the
presence of cell walls been reported [40,18,19,7]. The fact that the
olive microspore-derived multicellular structures showed the
formation of inner separating cell walls illustrates that a further
604 M.-T. Solı´s et al. / Plant Science 174 (2008) 597–605
event (not only mitosis but also cytokinesis) has been reached in
this system.
4.3. Changes of the cell wall components as markers of
induced-microspores and microspore proembryos
During developmental processes, the structure and compo-
nents of the cell walls change [41,33,42]; some molecular markers
of somatic embryogenesis and organogenesis have been found in
cell walls [43,44]. Differences in the presence and abundance of
various cell wall components have been reported during micro-
spore differentiation and embryogenesis in some herbaceous and
woody species [7,18,19]. Our results showed that a modification in
the microspore wall and also in inner separating cell walls
occurred in the olive microspore multicellular proembryos; this
change was detectable as a differential reactivity by calcofluor
white staining, a cytochemical method preferential for cellulosic
components [45], suggesting that the structure, arrangement and/
or amount of the cellulosic components of these cell walls were
modified after the embryogenic induction.
In B. napus microspore cultures, calcofluor white has been used
to stain whole cells for flow cytometric analysis, the method being
proposed for the sorting of samples enriched in embryogenic
microspores [46]. This was based on a different fluorescence
intensity of microspore cultures in embryogenic and non-
embryogenic conditions, even though it has also found intrinsic
limitations since the UV light for excitation was absorbed by the
exine pollen wall and only the fluorescence emitted by the surface
regions free of exine (apertures, broken areas, . . .) could be
detected [46]. This fact could explain the low fluorescence
intensity of calcofluor on induced-microspore cultures reported
in this paper. Moreover, a problem of permeability cannot be
excluded for whole cell staining. Our results in semithin sections
present a suitable and reliable calcofluor-based technique to label
stress-induced microspores and microspore proembryos, as an
early marker of the microspore reprogramming process, providing
the thick wall underneath the exine and inner walls with specific
fluorescence. Further investigations are needed to determine the
components and distribution pattern of this especially thick wall
under the exine in induced-microspores and young proembryos.
The development of those thick walls in particular stages of
microspore embryogenesis, somatic embryogenesis and organo-
genesis has been proposed as a molecular marker of these
processes [40,43,18,19,7].
In proliferative systems, such as root meristems, and also in very
young microspore proembryos of herbaceous species including
pepper, barley, maize, and tobacco, a high presence of esterified
pectins in cell walls have been reported and has been suggested as a
characteristic feature associated with proliferative activity [18,19].
In contrast, reports on other trees (Quercus suber, Citrus clementina),
have revealed a different pattern of pectin esterification in young
microspore embryo cell walls [18,19], similar to those found in olive
microspore proembryos cell walls which did not show labelling for
esterified pectins. Further investigations will be needed to ascertain
whether the absence of esterified pectins in cell walls at early stages
of microspore embryogenesis could be associated with a lower rate
of proliferating activity occurring in woody species.
Finally, the results reported here provide new insights on the
molecular and cellular events associated with the microspore
switching to the embryogenic pathway in woody plants, in which
there is very little information. This knowledge, in a tree of
agronomic interest like the olive, could guide future strategies to
induce differentiation and organogenesis, addressed to improving
the yield of the process and obtaining regeneration of microspore-
derived embryos and plants.
Acknowledgements
Work supported by projects granted by the Spanish Ministry of
Education and Science (MEC) BFU2005-01094 and AGL2005-
05104, and it was performed in the frame of the CSIC-INIA Joint
Collaboration Program (Convenio CC03-023). MTS is recipient of a
predoctoral fellowship (FPI, BES-2006-14117) granted by the
Spanish MEC. MJP was recipient of a contract granted by the
project CAM 07G/0009/2003 1 funded by the Comunidad de
Madrid. Partly supported by Spanish-Czech Joint project
2006CZ0006 granted by Spanish National Research Council (CSIC)
and Czech Academy of Sciences and Czech grants
MSM0021620806, LC535 and AV0Z50110509.
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