Ultrastructural Changes in Coconut Calli Associated with the by ktc20729


									Annals of Botany 88: 9±18, 2001
doi:10.1006/anbo.2001.1408, available online at http://www.idealibrary.com on

Ultrastructural Changes in Coconut Calli Associated with the Acquisition of Embryogenic
        J . L . V E R D E I L *{, V. H O C H E R {, C . H U E T {, F. GRO S D E M A N G E {, J. E S CO U T E {,
                                        N . F E R R I E R E { and M . N ICO L E {
{GENETROP, IRD, 911 av. Agropolis, BP 5045 34032, Montpellier cedex, France and {BIOTROP, CIRAD-AMIS,
            2477 avenue du Val de Montferrand, BP 5035 34032, Montpellier cedex 1, France

                 Received: 6 November 2000         Returned for revision: 9 January 2001 Accepted: 23 February 2001

             Ultrastructural studies of 2,4-D (2,4 dichlorophenoxyacetic acid) induced coconut calli and of untreated controls
             enabled us to characterize early events in cellular reorganization leading to embryogenic cell individualization and
             subsequent development into proembryos. Embryogenic cells were characterized by special features that chie¯y
             a€ected the nucleus, cytoplasm and cell wall: deep invaginations of the nuclear envelope, proliferation of
             dictyosomes, with emission of Golgi vesicles, directly related to an increase in cell wall thickness. Modi®cation of the
             cell wall structure was studied and particular attention was paid to the cytolocalization of b-1,4-glucans, and of
             callose and pectin epitopes, using gold-conjugated probes. The ®rst changes (detected 7±14 d after 2,4-D increase)
             involved the closure of plasmodesmata, breaking of symplastic continuity, and callose deposition. The acquisition of
             embryogenic competence was linked to the appearance of an outer layer of ®brillar material containing pectin epitope
             (mainly un-methyl-esteri®ed), fully coating the embryogenic cells (21 d after the induction treatment). Some of the
             ultrastructural changes observed during the reprogramming of somatic cells towards embryogenesis can be likened to
             those accompanying the maturation of female gamete cells in many plant species. The possible signi®cance of these
             observations is discussed.                                                        # 2001 Annals of Botany Company

             Key words: Callose, cell wall structure, Cocos nucifera L., cytological events, embryogenic cells, pectin, somatic

                     I N T RO D U C T I O N                               least very similar (Van Engelen and De Vries, 1992).
                                                                          Relatively few studies have reported the initial stage of
Somatic embryogenesis is the process by which somatic cells
                                                                          development, in particular the ultrastructural events and
develop into plants through characteristic morphological
                                                                          their possible relation with the initiation of an embryogenic
embryo stages generally encountered in zygotic embryo-
                                                                          pathway. Intermediate ultrastructural features required to
genesis. Somatic embryogenesis is a valid vegetative
                                                                          acquire embryogenic competence are transient and dis-
propagation procedure in terms of the multiplication rate
                                                                          played in heterogeneous somatic tissues. It is therefore
and resulting rejuvenation. It is the only large-scale method
                                                                          dicult at an early stage to identify the original cells actually
for producing tropical plants from cross-fertilizing species
                                                                          involved in the somatic embryogenesis development process.
and/or species with no natural vegetative propagation
                                                                             In coconut, somatic embryogenesis is a slow process
system (e.g. oil palm or coconut) (Dublin et al., 1991).
                                                                          (10 to 12 months to obtain embryos from somatic cells vs.
   Using histological, biochemical and molecular
                                                                          12 months for zygotic embryogenesis). From this view-
approaches, comparisons were made between somatic and
                                                                          point, this species is an attractive one for studying ultra-
zygotic embryogenesis from the globular stage onwards, for
                                                                          structural changes associated with the acquisition of
which parallel development occurs in most plant species
                                                                          embryogenic competence. The use of homogeneous coco-
(Ammirato, 1983). However, whilst the zygote is intrinsi-
                                                                          nut callus strains (Dussert et al., 1995a, b) with high
cally embryogenic, somatic cells are not naturally embryo-
                                                                          embryogenic potential enabled us to characterize early
genic (Dodeman et al., 1997). The transition of somatic
                                                                          events in cellular reorganization leading to individualiza-
cells into cells referred to as embryogenic i.e. that are
capable of forming an embryo, requires the induction of                   tion of embryogenic cells and their subsequent development
embryogenic competence. This transition is the ®rst and                   into proembryos. This study compared 2,4-D (2,4 dichloro-
crucial step in somatic embryogenesis, but it is also the least           phenoxyacetic acid) induced coconut calli and untreated
understood. Increasing our understanding of the switch                    controls cultured on a multiplication medium.
from somatic cells to embryogenic cells is one of the greatest               In coconut, as in other species, individualized embryo-
challenges in plant cell biology (Dodeman et al., 1997).                  genic cells were found to be surrounded by a thickened
   Given the precision of the zygotic embryo pattern                      outer wall. As the nature of the wall surrounding a cell is
formation programme, it was assumed that the ®rst stages                  thought to in¯uence its subsequent development (Knox,
of both zygotic and somatic embryos could be identical or at              1992), our attention was focused on changes occurring in
                                                                          the cell wall. We used an exoglucanase-gold probe
   * For correspondence. Fax 33 (0)4 67 41 61 81, e-mail verdeil@         (cytolocalization of b-1,4-glucans), gold labelled antibodies
cirad.fr                                                                  directed against b-1,3-glucans and pectin epitopes to study
0305-7364/01/070009+10 $35.00/00                                                                     # 2001 Annals of Botany Company
10                           Verdeil et al.ÐUltrastructural Changes in Coconut Calli
structural changes in the cell wall during the acquisition of    ethanol was followed by in®ltration and embedding in
embryogenic competence.                                          Spurr's low viscosity epoxy resin (Spurr, 1969). Semi-thin
   Some of the ultrastructural alterations temporarily seen      sections (1±2 mm thick) were cut using a Reichert Ultracut
during the transition phase could be likened to those            S microtome (Leica, Milton Keynes, UK), collected onto
accompanying the maturation of female gamete cells in            microscope slides on a drop of water and heat-®xed to the
many plants. Their possible signi®cance is discussed.            slides for 30 min at 40 8C. Representative sections were
                                                                 stained with 0.5 % (w/v) toluidine blue in water for 30 s and
                                                                 rinsed in water. Thin sections (50 to 80 nm) were cut using a
          M AT E R I A L S A N D M E T H O D S                   Reichert-Ultracut S microtome with a diamond knife
Plant material                                                   (Diatome, Bienne, Switzerland), and taken up on uncoated
                                                                 300-mesh nickel grids. After speci®c labelling with gold-
The study was conducted using a homogeneous coconut              conjugated probes, sections were stained with 2 % uranyl
callus strain (L82) obtained from immature ¯oral meristems       acetate for 30 min, followed by lead citrate (30 min in the
taken from an adult palm (20 years old) of the Malayan           dark). The specimens were examined with a Jeol 100 EX
Yellow Dwarf x West African Tall hybrid (PB 121 hybrid           (Tokyo, Japan) transmission electron microscope.
obtained by CIRAD and CNRA Ivory Coast). Primary
calli were obtained using the callogenesis protocol
described by Verdeil et al. (1994). The strain was established
by multiplying the primary calli on a medium containing a        Cytochemistry
double MS formulation supplemented with 3 % sucrose,                Cytolocalization of b-1,4-glucans. b-1,4-glucans were
2 % activated charcoal (neutralized from Sigma) and 2,4-D        localized using a puri®ed exoglucanase conjugated to
(2.5 10 À4 M). The calli were kept in the dark at 27 + 1 8C      colloidal gold at pH 9 (Nicole and Benhamou, 1991).
and subcultured every 6 weeks. At T0 , randomly chosen           Sections were labelled for 30 min at 25 8C on a drop of the
callus specimens were transferred to the same basic medium       gold probe diluted 1 : 10 ( pH 6.5) before rinsing and
with a greater concentration of 2,4-D (4.5 10 À4 M) to induce    staining. Labelling speci®city was assessed by incubating
somatic embryogenesis (Dussert et al., 1995b). Some of the       the sections with the gold-conjugated protein, saturated
calli from the initial batch were subcultured on a fresh         with an excess of b-1,4-glucans from barley.
multiplication medium to serve as a control.
   Structural and ultrastructural observations were carried         Immunocytolocalization of b-1,3-glucans and pectin epi-
out on three di€erent callus samples taken at random at 7 d      topes. Rabbit polyclonal antibodies raised against b-1-3
intervals from day 0 to day 60 after their subculture on the     glucans (CRB, Northwich, UK) were used for immunolo-
somatic embryogenesis induction medium.                          calization of callose according to the protocol described by
                                                                 Boher et al. (1995). Sections were incubated for 30 min at
Tissue processing for light microscopy                           25 8C on a drop of primary antibodies (diluted 1 : 2000 in
                                                                 PBS 0.1 M pH 7.2, 1 % BSA, 0.05 % Tween) prior to
   Tissues were ®xed for 48 h using 10 % paraformaldehyde        incubation on gold-labelled goat anti-rabbit antibodies
in 0.2 M phosphate bu€er ( pH 7.2). Fixed samples were           (diluted 1 : 20) (GAR-15, Biocell, Cardi€, UK) for EM
treated according to histological techniques described           observations.
previously (Bu€ard-Morel et al., 1992). After dehydration           Localization of pectin epitopes was carried out using two
through a graded alcohol series and impregnation in methyl       rat monoclonal antibodies (JIM5 and JIM7) as character-
methacrylate, each sample was embedded in polymethy-             ized by Knox et al. (1989), to locate low methyl-esteri®ed
lacrylate LKB Historesin (Leica, Rueil-Malmaison,                and highly methyl-esteri®ed pectic polysaccharides respect-
France). Polymerization was carried out for 2 h at room          ively (Knox et al., 1990). Immunogold localization of pectin
temperature. Sections (3 mm thick) were obtained using a         was performed as previously described by Knox et al.
microtome (Historange, LKB) with steel blades and                (1990). Brie¯y, sections were incubated on a drop of
mounted on glass slides. The sections were double-stained        primary antibodies for 2 h at 37 8C and then on a drop of
with Periodic Acid-Schi€ (PAS) reagent, combined with            gold-labelled goat anti-rat antibodies (GAT15), (Biocell)
protein-speci®c naphthol blue-black (NBB) (Fisher, 1968).        for 30 min at 37 8C. Labelling speci®city was determined
PAS stains starch reserves and cell walls pink and NBB           on the basis of the following controls: (1) incubation of
speci®cally stains soluble or reserve proteins dark blue.        grids with primary antibodies pre-adsorbed with the
                                                                 corresponding antigen (galacturonic acid (Sigma), b-1,4-
                                                                 glucans, b-1, 3-glucans), (2) incubation of grids with the
Tissue processing for electron microscopy (EM)
                                                                 secondary antibodies only and (3) grid treatment with a
   Pieces of both types of callus (1±2 mm3) were ®xed in         pre-immune serum of the primary antibodies.
2.5 % (v : v) glutaraldehyde in 0.1 M cacodylate bu€er
( pH 7.2) for 2 h at room temperature. After transfer to            Labelling assessment. Whatever the probes used, gold
fresh ®xative and overnight incubation at 4 8C, the samples      labelling was assessed qualitatively. It was considered
were washed three times (10 min each) in cacodylate bu€er        intense when labelling density was higher than 50 gold
and post®xed for 1 h in 1 % OsO4 in a bu€er at 4 8C.             particles per mm2 and sparse when labelling density was less
Sample dehydration through 30, 50, 70, 80, 90 and 100 %          than ten gold particles per mm2.
                                 Verdeil et al.ÐUltrastructural Changes in Coconut Calli                                                    11
                           R E S U LT S                                  two to four non-organized layers of homogeneous cells
                                                                         (Fig. 1B). This structure was maintained for the entire
Cytohistological events
                                                                         duration of the subculture on the multiplication medium.
Microscopic analysis showed that under multiplication                    After transfer to embryogenic conditions, with increased
conditions (Fig. 1A) (2.5 Â 10 À4 M of 2,4-D), callus growth             2,4-D (4.5 Â 10 À4 M), acquisition of embryogenic compe-
was ensured by a peripheral meristematic zone comprising                 tence (Fig. 1C) was observed, with the individualization of

F I G . 1. A, Macroscopic aspect of the coconut callus strain on the multiplication medium. Bar ˆ 0.25 cm. B, Callus multiplication ensured by a
peripheral meristematic zone (MZ). Bar ˆ 61 mm. C, Embryogenic structures (ES), 56 d after transfer of the callus to embryogenesis induction
medium. Bar ˆ 0.25 cm. D, Embryogenic cells 28 d after callus transfer to embryogenic conditions. S, Starch; N, nucleus; n, nucleolus; TW,
thickened outer wall. Bar ˆ 30 mm. E and F, Proembryo formed by segmenting division in a single embryogenic cell. TW, thickened outer wall.
                                                     Bar ˆ 12.5 mm for E, and 68 mm for F.
12                           Verdeil et al.ÐUltrastructural Changes in Coconut Calli
embryogenic cells in the peripheral meristematic zone,            plasmalemma-cell wall interface, the site of cell wall
which became discontinuous and partially disorganized.            assembly in plants (Roland and Vian, 1979)]. The number
  Twenty-eight days after transfer, these densely cyto-           of plasmodesmata between adjacent cells was very low
plasmic cells (Fig. 1D) displayed numerous cytological            compared to the number previously observed under
characters consistent with the description of embryogenic         multiplication conditions.
cells provided by other investigators (Halperin and                  Following incubation with antibody raised against b-1,3-
Wetherell, 1964; Schwendiman et al., 1990). They had a            glucans (used for callose immunolocalization), gold
high nucleus : cytoplasm ratio, a central enlarged nucleus        particles were found associated with the remaining plas-
with a single prominent, densely stained nucleolus and            modesmata (Fig. 3E).
small vacuoles. Single or groups of embryogenic cells were           Fourteen days after the ®rst callus subculture under
surrounded by a thickened outer wall, clearly separating          embryogenic conditions, substantial modi®cations occurred
them from the surrounding degenerative tissues. After 35 d        in the cell wall. Localized cell wall thickening could be seen
on the somatic embryogenesis initiation medium, embryo-           (Fig. 3F) concomitant with an increase in intercellular
genic cells started to divide (Fig. 1E), giving rise by           space at the cell wall junctions (Fig. 3G). Locally intense
segmentation to proembryos (Fig. 1F), which emerged at            labelling with the antibodies raised against b-1,3-glucans
the callus surface between days 42 and 56 (Fig. 1C). This         suggested callose deposition associated with wall thickening
shows that proembryos arising from the mitotic division of        (Fig. 3F). The use of JIM5 showed the existence of
embryogenic cells have a unicellular origin.                      polygalacturonic acid at cell wall junctions (Fig. 3G). The
                                                                  middle lamella was also labelled. Highly-esteri®ed homo-
                                                                  galacturonans recognized by JIM7 were localized in the
Ultrastructural events
                                                                  expanded periplasm, in areas similar to those labelled with
   Zero, 7, 14, 21 and 28 d after transfer to a medium with       JIM5, but of lower intensity (Fig. 3H).
increased 2,4-D, calli revealed ultrastructural changes              Modi®cations seen 7 and 14 d after callus transfer to
leading to embryogenic cells.                                     embryogenic conditions foreshadowed further ultra-
                                                                  structural features of embryogenic cell and enabled us
   From meristematic to embryogenic cells. Under callus           to determine the essential events leading to their di€eren-
multiplication conditions (time zero), meristematic cells         tiation.
located at the callus periphery had a dense ribosome-rich            Embryogenic cells exhibiting the cytological features
cytoplasm, a reduced vacuole and a voluminous central             described above were observed in calli 21 d after their
nucleus with one or two nucleoli (diameter between 1.25 and       transfer to a medium with increased 2,4-D (Fig. 1D). On an
1.5 mm) (Fig. 2A). The cytoplasm contained a large amount         ultrastructural scale, the cells displayed nuclear and cell wall
of rough endoplasmic reticulum. A few amyloplasts, usually        modi®cations. The nucleus became irregularly shaped,
with one starch granule, could be seen randomly distributed       resulting from deep invaginations of the nuclear envelope,
in the cytoplasm. Meristematic cells subcultured on the           leading to several nuclear lobes (Fig. 4A). Some of these
multiplication medium were surrounded by a cell wall of           deep and narrow invaginations (diameter around 1±1.5 mm)
uniform thickness (0.4 mm on average) crossed by numerous         delineated nuclear channels containing plastids and mito-
plasmodesmata ensuring symplastic continuity (Fig. 2B).           chondria. The nucleus contained one enlarged granular
   Labelling of b-1,4-glucans using a puri®ed exoglucanase        nucleolus (diameter between 2.5 and 3.5 mm) with one
conjugated to colloidal gold was dense and regular over the       nucleolar vacuole. The nucleolus gave o€ micronucleoli that
wall of meristematic cells (Fig. 2C). Using the monoclonal        reached the nucleus periphery, then the cytoplasm (Fig. 4B).
antibody JIM5 raised against low-esteri®ed epitopes of               Embryogenic cells contained several amyloplasts, with
pectin, gold particles were widely distributed over the middle    poorly developed internal membranes and numerous starch
lamella between adjacent meristematic cells (Fig. 2D). In         granules ( ®ve to nine per plastid) surrounding the nucleus,
contrast, cell walls were sparsely labelled by the monoclonal     close to the nuclear membrane (Fig. 4A). Embryogenic cells
antibody JIM7 and labelling was unevenly distributed,             were surrounded by a thickened modi®ed outer wall that
indicating that high methyl-esteri®ed pectic polysaccharides      isolated them from the surrounding senescent tissues. No
were not abundant (Fig. 2E). No labelling was found in the        plasmodesmata were observed in the modi®ed cell wall,
meristematic cell walls after incubation with polyclonal          illustrating the absence of cytoplasmic continuity between
antibodies raised against b-1,3-glucans (not shown).              neighbouring cells. The thickened cell wall was hetero-
   Seven days after callus transfer to embryogenic con-           geneous with the existence of an outermost ®brillar matrix
ditions, the nucleus lost its regular spherical shape (Fig. 3A)   coating the embryogenic cell (Fig. 4C). This matrix was
and the number of amyloplasts increased, each of them             separated from the amorphous and homogeneous primary
containing several starch granules (Fig. 3B). Localized           cell wall by a thin layer with a higher electron-opacity and a
thickening of the cell wall could be seen. These modi®-           compact ®brillar structure (Fig. 4C). Labelling for b-1,4-
cations coincided with an increase in Golgi apparatus             glucans with puri®ed b-1,4-exoglucanase-gold complex
activity. Dictyosomes released secretory vesicles. At the         revealed that particles were predominantly associated with
plasmalemma, exocytosis was observed, giving the plasma-          the innermost part of the primary cell wall, adjacent to the
lemma a crenelated appearance (Fig. 3C and D). This               plasma membrane (Fig. 4D). Incubation of ultra-thin
activity seemed to be linked to cell wall thickening [Golgi       sections with the antibody raised against b-1,3-glucans
vesicles were mainly located at the cell periphery and at the     resulted in strong labelling over the inner part of the cell
                                  Verdeil et al.ÐUltrastructural Changes in Coconut Calli                                                          13

F I G . 2. Ultrastructural organization of callus meristematic cells cultured on the callus multiplication medium. A, Meristematic cells located at the
periphery of the callus. Bar ˆ 4 mm. B, Detail of a meristematic cell showing the voluminous spherical nucleus (N) with two nucleoli (n) a cell wall
of uniform thickness and numerous plasmodesmata (Pl). Bar ˆ 2 mm. C, Cytolocalization of b-1,4-glucans in the cell wall using a puri®ed
exoglucanase conjugated to colloidal gold. Bar ˆ 250 nm. D, Immunogold labelling of low esteri®ed pectins in the cell wall of meristematic cells,
detected with JIM5 antibody. Gold particles are only located over the middle lamella. Bar ˆ 125 nm. E, Immunogold labelling (arrows) of highly
                     methyl esteri®ed pectins in the cell wall of meristematic cells, detected with JIM7 antibody. Bar ˆ 166 nm.

wall (Fig. 4F). This indicated the existence of callose in the               From embryogenic cells to proembryos
modi®ed cell wall surrounding the embryogenic cells.
Labelling with JIM5 antibody was mostly located over                            Twenty-eight days after callus transfer to embryogenic
the external ®brillar matrix and the intermediate thin layer                 conditions, single embryogenic cells and proembryos
with a higher electron opacity (Fig. 4E). No di€erences                      composed of two to ten cells could be observed on the
were seen in the patterning of labelling with the JIM7                       same callus histological section (Fig. 5A and B). Proem-
antibody, which recognizes a highly methyl-esteri®ed pectin                  bryos resulted from the division of a single embryogenic cell
epitope, but staining was less widespread.                                   isolated by a thickened outer wall. Following successive
14   Verdeil et al.ÐUltrastructural Changes in Coconut Calli
                                 Verdeil et al.ÐUltrastructural Changes in Coconut Calli                                                       15
mitotic divisions, the cells gradually recovered features                  ¯ow of molecules in and out of the cell and might cause
generally encountered in meristematic cells (Fig. 5B). The                 metabolic changes triggering the process of somatic
nucleus contour became regular in shape, resulting in the                  embryogenesis. It is interesting to note that callose is
disappearance of the nuclear channels and nuclear lobes.                   typically synthesized in meiocyte cell walls of angiosperms
Starch granules were less numerous while remaining                         that produce sexual megagametophytes, and is degraded
amyloplasts were dispersed inside the cytoplasm.                           rapidly following meiosis (Rodkiewicz, 1970; Leblanc et al.,
   The internal cell walls of the proembryos were generally                1995). Heslop-Harrison and Mackenzie (1967) assumed
thin (0.5±0.6 mm on average). They had a ®brous electron-                  that callose deposition during sporogenesis acted as a
dense structure, and were crossed by numerous plasmo-                      molecular barrier for proteins and RNAs, preventing their
desmata revealing the recovery of the cytoplasmic con-                     incorporation into meiocytes and insulating the di€erentiat-
tinuum from one cell to another inside the same proembryo                  ing meiocytes from the maternal tissues. Callose deposition
unit (Fig. 5C). These features were present from the ®rst                  was thought to be a prerequisite for somatic embryogenesis
division of the initial embryogenic cell. In contrast, the                 in Cichorium intybus (Dubois et al., 1990, 1991). Our results
external cell wall of the proembryo unit remained thick                    show that callose was no longer detected after the ®rst
without any plasmodesmata; thus proembryos were struc-                     division of embryogenic cells leading to globular pro-
turally isolated from neighbouring cells.                                  embryos. Recently, HelleboõÈ d et al. (1998) suggested that
   Immunolocalization of b-1,4-exoglucan and pectin epi-                   callose degradation was due to the induction of extra-
topes (Fig. 5C) showed a similar pattern to those described                cellular b-1,3-glucanases that may participate in partial
in the initial embryogenic cell. The only di€erence observed               break-down of 1,3-glucans present in the cell walls.
was for b-1,3-glucan epitopes, which were not detected after                  Induction of embryogenic competence is also linked to
the ®rst division (around day 35).                                         the appearance of a layer of ®brillar material, fully coating
                                                                           embryogenic cells 21 d after the induction treatment, just
                                                                           before their ®rst division. This extracellular matrix has been
                         DISCUSSION                                        described in other species but only from the proembryo
                                                                           stage (Sondahl et al., 1979; Dubois et al., 1991, 1992;
To our knowledge, this is the ®rst detailed report of callus
                                                                           Ovecka et al., 1998). Its molecular composition was, until
meristematic cell remodelling leading to the individualiza-
tion of single embryogenic cells surrounded by a thickened                 now, largely undetermined (Chapman et al., 2000a, b). Our
outer wall. This was possible because, in coconut, gradual                 study has revealed that this outer ®brillar coat contains
individualization of single embryogenic cells requires 3                   pectin epitopes which are mainly weakly-methyl-esteri®ed.
weeks, compared to other species for which intermediate                    Recently, using immuno-¯uorescence microscopy with
ultrastructural changes necessary for the acquisition of                   speci®c monoclonal antibody (JIM4; Knox, 1997), Samaj
embryogenic competence are transient and displayed in                      et al. (1999) reported that the extracellular matrix surface
heterogeneous somatic tissues. Homogeneous coconut                         network which covers embryogenic units of friable maize
callus lines are therefore an attractive model system for                  callus contained arabinogalactan-proteins. Since weakly-
investigating early events in somatic embryogenesis.                       methylated-pectins and arabinogalactan-proteins are
   The molecular cytology approach using gold-labelled                     thought to play an important role in cell±cell adhesion
probes, raised against the main cell wall components,                      and plant morphogenesis respectively, we propose that the
enabled us to characterize ultrastructural changes associ-                 extracellular matrix might be involved in the recognition of
ated with cell wall modi®cations during the acquisition of                 embryogenic cells and regulation of early embryogenic
embryogenic competence.                                                    stages. The existence of arabinogalactan protein epitopes at
   The ®rst detected changes occurred early, 7±14 d after                  the surface of sexual cells in the anthers and ovules of
the induction treatment. The main changes observed                         oilseed rape, as well as at the zygote surface, was also
were the closure of plasmodemata, breaking of the sym-                     demonstrated (Pennell et al., 1991), supporting the idea that
plasmic continuum, callose deposition and local cell wall                  common regulatory mechanisms are probably involved in
thickening. These modi®cations back the hypothesis that                    the ®rst stages of somatic and zygotic embryogenesis.
physical and physiological isolation of embryogenic cells is                  Ultrastructural changes occurring during the induction of
a prerequisite for further morphogenesis (Kohlenbach and                   somatic embryogenesis by 2,4-D also a€ected the symplasm.
Wernicke, 1978; Sterk et al., 1991). The isolation of a cell               The outcome was a nucleus with deep invaginations of the
from those which surround it would seem to restrict the                    nuclear envelope forming nuclear channels containing

F I G . 3. From meristematic to embryogenic cells. A, Irregular shape of the cell nucleus 7 d after callus transfer to embryogenic conditions. n,
Nucleolus; vn, nucleolar vacuole. Bar ˆ 3.3 mm. B, Detail of the cytoplasm containing amyloplast with numerous starch grains. G, Golgi
apparatus; V, vacuoles; N, nucleus. Bar ˆ 1 mm. C, Fusion of Golgi vesicle ( fvg) with the plasmalemma (Pl). G, Golgi apparatus; Gv, Golgi
vesicle. Bar ˆ 200 nm. D, Crenellated aspect of the plasmalemma resulting from fusion with Golgi vesicles. Bar ˆ 640 nm. E,
Immunolocalization of callose using antibody directed against b-1,3-glucans. Gold particles are mainly associated with the remaining
plasmodesmata (Pl). Bar ˆ 192 nm. F, Immunolocalization of b-1,3-glucans. Local cell wall thickening with callose deposition detected with
antibody directed against b-1,3-glucans. Bar ˆ 770 nm. G, Immunolocalization of pectin epitope. Increase of the intercellular area at the cell wall
junctions with low esteri®ed pectins, detected with JIM5 antibody. Bar ˆ 500 nm. H, Immunolocalization of pectin epitope. Sparse JIM7
               labelling (arrowheads) indicates few highly methyl esteri®ed pectins in the enlarged intercellular area. Bar ˆ 400 nm.
16                                Verdeil et al.ÐUltrastructural Changes in Coconut Calli

F I G . 4. Ultrastructural organization of embryogenic cells (21 d after transfer to the medium with increased 2,4-D). A, Irregularly-shaped nucleus
with nuclear channels (NC). n, Nucleolus; S, starch; NL, nuclear lobe. Bar ˆ 1.6 mm. B, Micronuclei (mn) localized at the nucleus periphery. n,
Nucleolus. Bar ˆ 0.96 mm. C, Thickened embryogenic cell wall showing an outermost ®brillar matrix (Fm) separated from the primary cell wall
(Pw) by a compact electron dense ®brillar thin layer (Ctl). Bar ˆ 1.1 mm. D, Cytolocalization of b-1,4-glucans. Labelling of the thickened cell wall
with the b-1,4-exoglucanase-gold complex shows that gold particles are associated with the primary cell wall. Labelling is weak over the ®brillar
matrix (Fm) and the cytoplasm. Bar ˆ 385 nm. E, Immunolocalization of low methyl-esteri®ed pectin epitope (arrows) in the external coating
®brillar matrix and in the intermediary electron dense thin layer. Detection with the JIM5 antibody. Bar ˆ 154 nm. F, Immunolocalization of b-
1,3-glucans using a polyclonal antibody. Labelling of the cell wall indicates the presence of callose in the inner part of the thickened cell wall; no
                                             labelling occurs over the ®brillar matrix (Fm). Bar ˆ 1 mm.

organelles. This particular nuclear shape increased the                     displayed a granular structure with only one nucleolar
interface between the nucleus and the cytoplasm, probably                   vacuole. Micronucleolus generation was also seen at the
increasing exchanges between these two cellular compart-                    nuclear periphery. This nucleolar form was observed by
ments. Simultaneously with nuclear changes, changes in                      Vasil (1973) in pea calli subcultured with high 2,4-D con-
nucleolus structure were observed. The enlarged nucleolus                   centrations. According to Tourte (1975) and Deltour and
                                  Verdeil et al.ÐUltrastructural Changes in Coconut Calli                                             17
                                                                               Ultrastructural changes of the cytoplasm associated with
                                                                           the individualization of embryogenic cells include the
                                                                           proliferation and activation of dictyosomes, with the
                                                                           formation of Golgi vesicles directly related to cell wall
                                                                               Nuclear events and cell wall modi®cations (callose
                                                                           deposition) occurring during the individualization of
                                                                           embryogenic cells were comparable to the cytological
                                                                           reorganization events a€ecting female gametic cell lineage
                                                                           that usually occur during the female gametophytic phase in
                                                                           sexual plant reproduction. In this respect, it appears that in
                                                                           addition to sharing a common pattern of development
                                                                           ( from globular embryo to the formation of a complete
                                                                           embryo), somatic and zygotic embryos also share another
                                                                           important feature: the origin from a single cell which has a
                                                                           speci®c cell wall structure and some structural character-
                                                                           istics similar to those observed in the individualization of
                                                                           megaspore mother cells. Nuti-Ronchi et al. (1990, 1992),
                                                                           considered that meiotic-like reductional events and DNA
                                                                           reducing mechanisms were a prerequisite for the acquisition
                                                                           of embryogenic competence. This idea was supported by a
                                                                           cytological study of some carrot embryogenic lines which
                                                                           displayed a high percentage of haploid cells, as the result of
                                                                           aberrant mechanisms of cell division, namely somatic
                                                                           meiosis and prophase reduction (Nuti-Ronchi et al.,
                                                                           1992). It is interesting to note that in microdensitometric
                                                                           DNA measurements, all the embryogenic cells present in
                                                                           the calli studied here were found to have a 2C DNA
                                                                           content. However, during early events (days 7 and 14) some
                                                                           degenerative cells were found to have a 1C DNA content;
                                                                           nevertheless, their early degeneration did not allow
                                                                           subsequent divisions to form proembryos and somatic
                                                                           embryos (Huet et al., unpubl. res.).
                                                                               The mechanisms that trigger the commitment of a single
                                                                           somatic plant cell to develop as an embryo remain poorly
                                                                           understood and o€er exciting prospects for further inves-
                                                                           tigations. On the basis of our observations, it is reasonable
                                                                           to say that gametophytic-like conditions (in terms of
                                                                           environment) are required to commit somatic cells to
                                                                           embryogenesis. The necessary environment can be achieved
                                                                           by increasing the 2,4-D level, which may act as a stress
                                                                           signal inducing an embryogenic reproductive programme.
                                                                           Our results suggest that under these conditions totipotency
                                                                           leading to somatic embryogenesis would be acquired by
                                                                           cells able to undergo major genome reprogramming and
F I G . 5. From embryogenic cells to proembryos. A, Cells generated by     cytological changes similar to those occurring in vivo during
the division of an embryogenic cell. N, Nucleus; n, nucleolus; v,          macrogametogenesis in plants.
vacuole; icw, internal cell wall; Tw, thickened outer wall. Bar ˆ 10 mm.
B, Multicellular proembryo surrounded by a thickened outer wall (Tw).
Bar ˆ 11.6 mm. C, Immunolocalization of low methyl-esteri®ed pectin
epitope. Thickened outer cell wall and inner thin cell wall of a
proembryo, showing immunogold labelling of low esteri®ed pectins in                      AC K N OW L E D G E M E N T S
the outer and inner cell wall with JIM5 antibody. Pl, Plasmodesmata.
                             Bar ˆ 0.96 mm.                                This work was carried out under a joint research pro-
                                                                           gramme between IRD (Institut de Recherche pour le
                                                                           Developpement) and CIRAD (Centre de Cooperation   Â
de Barsy (1985), this should lead to an increase in the                    Internationale en Recherche Agronomique pour le
number of ribosomes in the cytoplasm. Micronucleolus                         Â
                                                                           Developpement). This study was partly supported by the
formation can therefore be considered as a preparatory                     Commission of the European Communities (contract
phase preceding the increase in protein synthesis already                  number: ERBTS3*CT940298). We thank P. Knox and
observed early in coconut embryogenesis (Dussert et al.,                   K. Roberts for providing monoclonal antibodies JIM5 and
1995a, b).                                                                 JIM7.
18                                Verdeil et al.ÐUltrastructural Changes in Coconut Calli
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