Plant Science 174 (2008) 597–605

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Plant Science

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Early markers of in vitro microspore reprogramming to embryogenesisin olive (Olea europaea L.)

Marıa-Teresa Solıs a, Beatriz Pintos b, Marıa-Jesus Prado b,1, Marıa-Angeles Bueno b, Ivan Raska c,Marıa-Carmen Risueno a, Pilar S. Testillano a,*a Plant Development and Nuclear Architecture, Centro de Investigaciones Biologicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spainb Forest Biotechnology Lab. INIA-CIFOR, A-VI, km 7.5, 28040 Madrid, Spainc 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

Article history:

Received 23 January 2008

Received in revised form 12 March 2008

Accepted 14 March 2008

Available online 30 March 2008

Keywords:

Olive

Pollen embryogenesis

Pectins

Cell wall

Cellular markers

Microspore culture

A B S T R A C T

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an

efficient method of producing homozygous lines for crop breeding. In trees, the process is of special

interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a

convenient method has been developed for microspore embryogenesis induction by stress in olive

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

cytochemical and immunocytochemical analysis was performed in the early stages. The young

microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in

vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the

occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal

population, and changes in cellulosic and pectic cell wall components which constituted early markers of

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.

� 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Microspore embryogenesis is a widely used method to generategenetic variability by obtaining microspore-derived embryos anddouble-haploid plants which have applications for plant breeding[1]. Efficient protocols for microspore embryogenesis induction inculture have been developed in recent years for many crops (reviewin [2]), and to a less extent for some woody plants [3–6]. In trees,with a long reproductive cycle, a high heterozygosity level, tendencyfor allogamy, and sometimes self-incompatibility, methods forobtaining homozygous plants are of high interest, but theirproduction through conventional methods are practically impos-sible to perform in woody plants. Microspore embryogenesis byisolated microspore culture, allows the development of completehomozygous 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.

0168-9452/$ – see front matter � 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.plantsci.2008.03.014

reprogramming of the immature pollen, the microspore, towardsa different developmental pathway and the onset of proliferationand differentiation events which finally leads to embryo formationand haploid and double-haploid plant regeneration [7].

Olive (Olea europaea L.) from the Mediterranean basin suppliesmore than 95% of the world production of olive oil; being a crop ofhigh social and economic interest in the Mediterranean area.Conventional breeding methods are time-consuming and have thelimitations of the long juvenile period of the species. In thiscontext, microspore embryogenesis allows the single-step devel-opment of complete homozygous lines from heterozygous parentsand constitutes a good method to exploit in olive genetic studiesand breeding. Recently, a convenient method for the induction bystress of early stage embryogenesis from isolated microsporeculture has been developed in different varieties of olive ([8], for amore detailed protocol description also see: [9]). In this system, theswitch to the embryogenesis pathway could be induced by acombination of cold and starvation stress treatments, as it has beenshown in other isolated microspore systems such as tobacco,wheat or rice [10–12]. This system constitutes a crucial step to

M.-T. Solıs et al. / Plant Science 174 (2008) 597–605598

further attempt haploid-plant production, via pollen embryogen-esis-derived structures.

Basic studies on the process of microspore embryogenesis havemainly been pursued in model species like rapeseed, tobacco andbarley [13–15,11,1,16,17] but reports are very limited on othereconomically interesting crops and trees [18–20]. The search ofmolecular and cellular markers during early stages of microsporeembryogenesis constitutes an important goal to monitor themetabolic processes involved in the induction, and also to identifycells committed to the induced developmental programme.Changes in various cell activities and structural organization ofsubcellular compartments have been reported to accompany theprocess in some model and herbaceous species [21,7,22–24], butmuch less is known about the cellular mechanisms leading to theprocess in woody plants due to the inherent difficulties of thesystem [25,19,26], e.g. high asynchrony during pollen develop-ment, high influence of climatic conditions, low proportions ofviable pollen, etc.

The aim of the present work was to identify in olive microsporecultures the cellular changes in responsive microspores switched tothe embryogenic pathway, and pollen-derived multicellular struc-tures. The study was focused to analyse cellular features related toproliferation and differentiation events, like starch accumulation,occurrence of symmetrical divisions, formation of new cell walls,ribosomal population and cell wall components. The results showeddefined cellular changes, some of them being characteristic of cellproliferation, which can be considered as markers of the microsporecommitted 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 cultivarCornicabra (Madrid). Microspore cultures were established aspreviously described by us ([8], for a more detailed protocoldescription see: [9]). Flower buds containing anthers with micro-spores at the most responsive developmental stage, the vacuolatedmicrospore, were selected by using the correlation betweensequential flower bud lengths and pollen developmental stageswhich had been previously established [8]. Flower buds weresterilized in fungicide Benomyl (C14H18N4O3, Aragro) solution(200 mg/l) for 1 min, in 70% ethanol for 1 min and in 2% sodiumhypochlorite solution, with a few drops of Tween-20 for 30 min.After rinsing in distilled water, anthers were carefully excised fromthe flower buds of the olive trees and analyzed as a system ofgametophytic development in vivo, for comparison with the in vitro

development. Microspores were isolated from anthers using thefollowing protocol: a total of 200 anthers were placed in 2 ml Bmedium [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 mmsieve which retained the anthers. This extraction process wasrepeated a total of four times. The pooled filtrate containingmicrospores was centrifuged for 5 min at 220 � g at roomtemperature. The microspore pellet obtained was resuspended in5 ml B medium. This process was repeated a total of four times.Culture density was adjusted to 1–5 � 105 microspores/ml byadding B medium to the pellet obtained after the final centrifugationstep. A volume of 1.5 ml was dispensed into each 35� 10 mm sterilePetri dishes, leaving about 500 ml to determine microspores viabilityand concentration. These plates were placed in cold stress conditions(2–6 8C for 96 h). After stress treatment, the microspore populationin B medium was centrifuged for 5 min at 220 � g at roomtemperature. The microspore pellet was resuspended in induction

medium AT3 I [28], in four-well plates with the final density of themicrospores being determined using a haemocytometer (Neubauerchamber) 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 culturescontaining microspores and microspore-derived structures atspecific 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 asthe anthers, as described before [29]. Samples were dehydrated inacetone 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 toluidineblue and observed under bright field for structural analysis. Severalcytochemical methods were performed to preferentially staindifferent 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 andmicrospore in vitro cultures [30]. 1 mg/ml DAPI solution in PBSplus 1% Triton X-100 was applied for 30–60 min (culturesamples) and overnight (anthers) in darkness. After rinsing,samples were squashed, mounted in Mowiol and observed in aconfocal laser scanning microscope Leica CLSM TCS SP2 underUV 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) wasalso applied to semithin sections, which after rinsing and dryingwere mounted in Eukitt and observed under UV irradiation in aZeiss Axioplan epifluorescence microscope equipped with a CCDcamera.

2.3.2. Toluidine blue staining and I2KI staining for starch

Staining solutions (0.075% toluidine blue in water, and 2 g of KIand 0.2 g of I in 100 ml of water) were applied on Technovitsections for 10–15 min. After rinsing and drying, preparationswere mounted in Eukitt and observed under bright field in a LeitzLaborlux 12 microscope equipped with a DP10 Olympus digitalcamera.

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. Afterrinsing and drying, sections were mounted in Eukitt and observedunder UV irradiation in an Axioplan epifluorescence microscopeequipped with a CCD camera.

2.4. Antibodies

For total RNA detection an anti-RNA mouse monoclonalantibody, D44 [31] was used. For localization of pectins, JIM5and JIM7 rat monoclonal antibodies which respectively recognizelow and high-methyl-esterified pectins were used [32].

2.5. Immunocytochemistry

Immunocytochemistry was performed on Technovit semithinsections and revealed either by fluorochromes (for anti-RNA) or

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by colloidal gold followed by silver enhancement (for JIM5 andJIM7). Sections were blocked with 5% bovine serum albumin(BSA) in PBS (5 min), and incubated with anti-RNA, JIM5 orJIM7 monoclonal antibodies for 1 h, undiluted. After washing inPBS, they were exposed to the corresponding secondaryantibodies following the procedure previously described by us[23,29].

Fig. 1. Microspore gametophytic development. Semithin sections of consecutive developm

Vacuolated microspore stage; (b, e, h, k) young pollen stage; (c, f, i, l) mature pollen stage; (

DNA revealing the nuclei; (d–f) Anti-RNA immunofluorescence on the same sections than

deposits as dark precipitates in young and mature pollen grains. Cytoplasmic vacuole, V;

3. Results

3.1. Monitoring the in vivo gametophytic development versus the invitro development of the embryogenesis induced-microspore

The in vivo gametophytic development of the microspore wasmonitored by microscopical analysis of selected anthers. The

ental stages after different cytochemical and immunocytochemical stainings. (a, d, g, j)

a–c) toluidine blue staining for general structural visualization; (g–i) DAPI staining for

in the DAPI images; (j–l) iodide-based cytochemistry for starch revealing the starch

vegetative nucleus, VN, generative nucleus, G, nucleolus, arrows; Bars: 10 mm.

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.

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correlation between sequential flower bud lengths and pollendevelopmental stages has previously been established [8] con-stituting a useful criterion for handling and selection of specificstages for the microspore culture. After the release from the tetrad,microspores developed a large vacuole which pushed theelongated nucleus to a peripheral location; the cytoplasmappeared as a thin layer (Fig. 1a, d, g). Anti-RNA immunofluor-escence provided a high signal in the cytoplasm of vacuolatemicrospores (Fig. 1d) when compared with younger microsporedevelopmental stages (data not shown), indicating an abundantribosome population. After the first pollen mitosis, the smallgenerative cell was localized attached to the pollen wall (exine) witha more condensed chromatin, as revealed by the DAPI staining(Fig. 1b, h). In contrast, the larger and rounded vegetative nucleuswas in the centre and displayed a more decondensed chromatinshowing less fluorescence intensity by DAPI (Fig. 1b, h). Thevegetative cytoplasm showed small vacuoles and some clearinclusions (Fig. 1b), as well as an intense anti-RNA immunofluor-escence (Fig. 1b, e), both nuclei being free of labelling except for thenucleoli. Controls avoiding the first antibody did not showsignificant labelling (data not shown). Later on, at mature pollenstages, the generative cell migrated to the centre of the pollen grain(Fig. 1c, i) whose cytoplasm showed abundant ribosomal populationby anti-RNA immunofluorescence (Fig. 1c, f) and numerous smallrounded clear inclusions, in toluidine blue-stained sections (Fig. 1c).

The iodide-based cytochemistry for starch revealed thedynamics of starch accumulation during the gametophyticdevelopment. No starch-staining was detected in the vacuolatedmicrospore (Fig. 1j), whereas at later stages an increasing numberof stained inclusions were found in the vegetative cytoplasm,indicating a progressive accumulation of starch deposits duringpollen maturation (Fig. 1k, l).

For the in the in vitro induced-microspore embryogenesis, themicrospores at the vacuolated stage were selected as the mostresponsive developmental stage for the culture, as previouslyreported [8]. Microspores were isolated from anthers and put inliquid medium. After 3 and 6 months, samples from the culture wereDAPI stained and squashed for checking the number of nuclei in thepollen grains and pollen-derived structures. Confocal analysisrevealed structures with two, three and four nuclei (Fig. 2) stillsurrounded by the exine, as revealed by the corresponding DIC

image of the same structures (Fig. 2e–h). The two nuclei of thesemicrospores (Fig. 2b) were similar in size, shape and chromatincondensation suggesting that they were formed by a symmetricaldivision, in contrast with the bicellular pollen formed during thegametophytic development (Fig. 1h, i). The microspores with threenuclei also exhibited nuclei with similar size, shape and organization(Fig. 2c), which could indicate that the second division occurredasynchronically in the two previous nuclei, one of them dividingfirst. This fact also supported the idea that these two-nucleimicrospores had wall-separated cytoplasms. The multicellularpollen-derived structures were found at different time points, from3 to 6 months, during the progression of the culture, indicating a highasynchrony in the embryogenic response of the microspores.

Samples of the in vitro culture were fixed and processed forfurther microscopical analysis which revealed that the culturescontained pollen-derived structures with differential features. Intoluidine blue-stained sections, some dead (empty) microsporeswith irregular shapes were observed together with others showinglarger sizes and a more contrasted cell wall (Fig. 3a). Some of themexhibited inner walls separating cells (Fig. 3b) as a sign of previouscell divisions occurring during the culture. Anti-RNA immunofluor-escence showed a high cytoplasmic signal in these multicellularstructures (Fig. 3c), indicating a high ribosomal population as typicalfeature of high metabolic activity. The iodide-based cytochemistryshowed a high content of starch in some of the microspores of theculture (Fig. 3d) which exhibited numerous amyloplasts revealed asdark cytoplasmic spots by the iodine staining (Fig. 3d, e). Otherpopulation of the microspores in the culture did not show starchaccumulations. In the microspore-derived structures with innerwalls, no starch deposits were observed.

3.2. Cell wall changes in microspore-derived structures

The modifications in components of the cell wall and in pectinresidues have been reported as responsible for initiating cellresponses in relation to cell fate and development [33]. Thedifferential composition of the cell walls in the microspore-derivedproembryos in comparison with the pollen grains were studied.Cytochemistry with calcofluor white for cellulosic components andimmunocytochemistry using JIM5 and JIM7 monoclonal antibo-dies recognising non-esterified and esterified pectins respectively

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.

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were performed on semithin sections of both in vivo developingmicrospores in anthers and microspore cultures.

During the gametophytic development in vivo, at the stage ofvacuolated microspore, calcofluor white staining provided specificfluorescence to cell walls of the somatic tissue of the antherwhereas no reaction was observed in the microspore wall (Fig. 4a,d). After the first pollen mitosis, the newly formed wall of thegenerative cell appeared specifically stained by calcofluor as a lens-shaped fluorescent line (Fig. 4b, e). In the mature pollen, nostaining with calcofluor was observed in the pollen wall except forthe apertures which showed specific fluorescence at their innerregion (Fig. 4c, f), where a thick layer of polysaccharides wassecreted to form the aperture of the pollen grain. No positivestaining with calcofluor was observed in the inner wall of thedeveloping 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 showedspecific fluorescence by calcofluor white staining (Fig. 5), the rest

of the structures appearing negative to this cytochemicaltechnique. The calcofluor-positive microspores exhibited anintense fluorescence in the thick peripheral wall localized belowthe exine (Fig. 5a, b). Some of them also showed dividing wallsintensively stained at their interior (Fig. 5b–f). These dividing wallsoccasionally appeared incomplete (Fig. 5c). The results indicatedthat the multicellular microspore-derived proembryos showed adifferential staining reaction with calcofluor in their walls incomparison with the rest of the culture.

Immunocytochemistry with JIM5 and JIM7 antibodies, recogniz-ing non-esterified and esterified pectins [32] was performed toanalyze cell wall components of the multicellular proembryos.Results showed a higher and positive immunoreaction with JIM5antibodies in the peripheral wall below the exine and in the dividingwalls at their interior (Fig. 6a–c), indicating that cell walls ofmicrospore proembryos were rich in de-esterified pectins. WithJIM7 antibodies recognizing esterified pectins, no immunoreactionwas found in any cell wall of the microspore-derived proembryos(Fig. 6d), whereas positive controls for JIM7 immunolocalization

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. Solıs et al. / Plant Science 174 (2008) 597–605602

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.

M.-T. Solıs et al. / Plant Science 174 (2008) 597–605 603

performed on anther somatic tissues provided a high immunoreac-tion on their cell walls (Fig. 6e). Negative controls avoiding the firstantibody did not provide significant labelling in any case (Fig. 6f).

4. Discussion

The comparative analysis performed between specific cellularchanges accompanying the in vivo gametophytic development ofthe 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, theoccurrence of a first symmetrical division and subsequentcytokinesis with formation of new cell walls, new cell divisions,abundant ribosomes, and changes in pectin esterification andcellulosic cell wall components. These differential features werenot 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 foundat stages of maturing pollen. The development of plastids and starchaccumulation constitutes a differential process during pollenformation in many species [34,35], as well as in certain stages ofzygotic embryogenesis [13,14]. In the olive stress-induced micro-spore cultures, the multicellular structures or proembryos did notshow starch deposits whereas other non-responsive microsporesexhibited a high starch content in the cytoplasms. Non-embryogenicpathways have been reported in other microspore in vitro systems,such as Brassica napus, to occur after the inductive treatment, and

also a gametophytic-like pathway was suggested for a set of the non-responsive microspores [29]. In olive microspore cultures, thepresence of starch deposits constituted an early marker of the non-responsive microspores which can follow, at least in part, agametophytic-like pathway.

4.2. Nuclei with equal structural organization and walled cells

characterize young microspore proembryos

It is widely accepted that the first morphological evidence of theembryogenic 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 thatsymmetrically divided microspores could germinate in vitro [38]and express the vegetative cell-specific promoter lat52 [39], butthey could not correctly differentiate the generative cell and toform the mature pollen grain [39]. The analysis reported hereshowed that in olive microspores, not only a symmetric mitosistook place but also the subsequent divisions of the two-celledembryogenic microspores which produced multicellular proem-bryos. This fact illustrates that the reprogramming of the olivemicrospore was started by the reactivation of the proliferativeactivity, the initial event of the switch to embryogenesis. Theobservation of a high ribosomal population in the cytoplasms ofthe proembryo cells, as revealed by anti-RNA immunofluores-cence, constituted additional data of the high metabolic activity ofthese cells corresponding to those of the active proliferating cells.

The observation, after DAPI staining, of two or more nuclei inmicrospore cultures constitutes the first sign that the embryogen-esis induction has been initiated, but not in many systems has thepresence of cell walls been reported [40,18,19,7]. The fact that theolive microspore-derived multicellular structures showed theformation of inner separating cell walls illustrates that a further

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event (not only mitosis but also cytokinesis) has been reached inthis 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 markersof somatic embryogenesis and organogenesis have been found incell walls [43,44]. Differences in the presence and abundance ofvarious cell wall components have been reported during micro-spore differentiation and embryogenesis in some herbaceous andwoody species [7,18,19]. Our results showed that a modification inthe microspore wall and also in inner separating cell wallsoccurred in the olive microspore multicellular proembryos; thischange was detectable as a differential reactivity by calcofluorwhite staining, a cytochemical method preferential for cellulosiccomponents [45], suggesting that the structure, arrangement and/or amount of the cellulosic components of these cell walls weremodified after the embryogenic induction.

In B. napus microspore cultures, calcofluor white has been usedto stain whole cells for flow cytometric analysis, the method beingproposed for the sorting of samples enriched in embryogenicmicrospores [46]. This was based on a different fluorescenceintensity of microspore cultures in embryogenic and non-embryogenic conditions, even though it has also found intrinsiclimitations since the UV light for excitation was absorbed by theexine pollen wall and only the fluorescence emitted by the surfaceregions free of exine (apertures, broken areas, . . .) could bedetected [46]. This fact could explain the low fluorescenceintensity of calcofluor on induced-microspore cultures reportedin this paper. Moreover, a problem of permeability cannot beexcluded for whole cell staining. Our results in semithin sectionspresent a suitable and reliable calcofluor-based technique to labelstress-induced microspores and microspore proembryos, as anearly marker of the microspore reprogramming process, providingthe thick wall underneath the exine and inner walls with specificfluorescence. Further investigations are needed to determine thecomponents and distribution pattern of this especially thick wallunder the exine in induced-microspores and young proembryos.The development of those thick walls in particular stages ofmicrospore embryogenesis, somatic embryogenesis and organo-genesis has been proposed as a molecular marker of theseprocesses [40,43,18,19,7].

In proliferative systems, such as root meristems, and also in veryyoung microspore proembryos of herbaceous species includingpepper, barley, maize, and tobacco, a high presence of esterifiedpectins in cell walls have been reported and has been suggested as acharacteristic 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 youngmicrospore embryo cell walls [18,19], similar to those found in olivemicrospore proembryos cell walls which did not show labelling foresterified pectins. Further investigations will be needed to ascertainwhether the absence of esterified pectins in cell walls at early stagesof microspore embryogenesis could be associated with a lower rateof proliferating activity occurring in woody species.

Finally, the results reported here provide new insights on themolecular and cellular events associated with the microsporeswitching to the embryogenic pathway in woody plants, in whichthere is very little information. This knowledge, in a tree ofagronomic interest like the olive, could guide future strategies toinduce differentiation and organogenesis, addressed to improvingthe yield of the process and obtaining regeneration of microspore-derived embryos and plants.

Acknowledgements

Work supported by projects granted by the Spanish Ministry ofEducation and Science (MEC) BFU2005-01094 and AGL2005-05104, and it was performed in the frame of the CSIC-INIA JointCollaboration Program (Convenio CC03-023). MTS is recipient of apredoctoral fellowship (FPI, BES-2006-14117) granted by theSpanish MEC. MJP was recipient of a contract granted by theproject CAM 07G/0009/2003 1 funded by the Comunidad deMadrid. Partly supported by Spanish-Czech Joint project2006CZ0006 granted by Spanish National Research Council (CSIC)and Czech Academy of Sciences and Czech grantsMSM0021620806, LC535 and AV0Z50110509.

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