A comparative structural analysis of direct and indirect shoot ...

M. OVECKA & M. BOB,4K
Fig. 1. Scanning electron microscopy of critical point dried embryogenic tissue culture.
a. Dividing activated ceils and somatic proembryos in clusters on the surface of embryogenic callus culture. Bar = 100 lain
b. Asynchronously developing somatic proembryos (p), globular (g), heart-shaped (h) and torpedo (t) somatic embryos. Bar = 100
lam
c. Clustered secondary somatic embryos (arrows) originating from primary somatic embryo (E). Bar = 500 ~m
d. External interconnecting strip-like and fibrillar "bridges" among individual proembryos within the cluster in early stages of regeneration.
Bar = 10 ~am
e. Surface cell wails were covered with continuous discrete layer, which partly disappeared into fibrillar network in the sites of cell
separation (arrowheads). Bar = 10 lam
f. Distinct mode of cell adhesion and cell separation between embryonic competent cells (A) and protodermal cells of globular somatic
embryo (B). Embryonic cells were self-covered with tubular extension of the cell wall, which were absent on the surface of
the protodermis. Bar = 10 IJm
g. Smooth protodermis of heart-shaped somatic embryo. Bar = 10 lam
Fig. 2. Organogenic culture.
a. Globular meristemoid shape supported by anticlinal (thick arrow) and periclinat (thin arrow) cell divisions of isodiametric cells.
Mucilage layer (asterisk). Bar = 50 lam
b. Transmission electron microscopy of peripheral meristemoid ceils. Note the apparently thicker outer "boundary" cell walls of the
meristemoid with presence of wall-connecting elemental fibrils (arrows). Bar = 5 IJm
c. Initial stages of globular meristemoid formation (arrows). Bar = 100 lam
d. Synchrony in cell death of the senescing meristemoid. Bar = 100 Inn
e. Two meristemoids surrounded by the dense amorphous mucilage. Bar = 50 lam
f. Extent mucilage accumulation in the region of meristemoid senescence. Bar -- 100 lam
g. Giant starch grains as a source of metabolic energy for future organogenesis in promeristemoid cells. Bar = 50 lam
Fig. 3. Scanning electron microscopy of shoot regeneration.
a. Developing shoot primordium (asterisk) between callus cells. Note the presence of fine layer of amorphous material on the cell
surface (arrows). Bar = 100 lain
b. Leaf of regenerated shoot. Bar = 100 lam
c. Smooth surface of leaf epidermal and guard stomatal cells. Bar = I0 lam
Fig. 4. Formation of intercellular spaces.
a. Intercellular reticular structure in developing leaf tissue. Bar = 1 lam
b. Reticular (R) and fibrillar (F) filling of intercellular space within the developed leaf parenchyma. Ch-chloroplast, V-central vacuole.
Bar = 1 lam
c, d. Light microscopy of serial section of critical point dried parenchymatic tissue in the region of vascular strands of primary somatic
embryo during secondary somatic embryogenesis. Extracellular fibrillar and reticular network was present in large intercellular
spaces. The majority of PAS reaction-positive material occurred on the surface of cell walls (arrows). Bar = 50 lam.
entiating somatic embryos were produced (Fig. lc).
Detailed surface view shows fine structures covering
individual proembryos, connecting them to
each other (Fig. ld). Strip-like and fibrillar strands
bridged proembryos over distances among them
continuing around the neighbouring "connected"
proembryos. Strip-like bridges interconnected proembryos
over longer distances and mostly fine fibrillar
network covered tight, short distances (Fig.
ld). Similar fibrillar network was found to interconnect
surface proembryo ceils themselves continuing
as a more or less coherent layer on the cell
surface (Fig. le). The fine network could cover the
proembryo itself probably since the first cell divisions
(not shown), but single cells in embryogenic
cluster were covered by long tubular extensions radiating
from the surface of each globular cell (Fig.
lf). Late globular somatic embryos where the pro-
todermis have been established together with
heart-shaped and torpedo somatic embryos with
differentiated protodermis (Fig. lg) lacked completely
tubular or fibrillar extensions of outer tangential
cell walls (Figs. le, f) including fibrillar and
strip-like connections, which were observed among
the proembryos (Fig. ld).
Formation of globular meristemoids as a consequence
of frequent mitotic division of activated
cells took place during shoot organogenesis. Meristemoid
cells achieved and propagated their globular
appearance through periclinal, anticlinal and mixoriented
cell divisions predominantly in the meristemoid
periphery followed by isotropic cell expansion
(Fig. 2a). Fibrillar network was not observed
on the meristemoid surface (not shown), however
peripheral cells expressed position-dependent
assymetry in cell wall thickness. Thicker outer cell
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