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

M. OVECKA & M. BOBf~K
point of view redefined plant cell wall (Roberts
1989) appears to be a dynamic and active cellular
compartment, mediating cell signalling through the
special wall integral part referred to as an extracellular
matrix (Roberts 1994). It is not surprising that
plant cell and tissue surfaces became very attractive
objects in the study of cell adhesion and separation
during the cell differentiation and tissue morpho-
~-enesis (Roberts 1989, 1994, Knox 1992a, b).
In embryogenic culture of Papaver somniferum L.
in vitro, we evaluated morphological variability of
somatic embryos (Ove~ka et al. 1996), and the secondary
regeneration ability of embryo cells during
subsequent long-term cultivation was described
(Ove~ka et al. 1997/98). The aim of this study is the
description of the fine structure of cell and early
embryo surfaces (including cell wall, and structures
located extracellularly) during long-term somatic
embryogenesis, compared to the structural characteristics
of cell and tissue surface organisation in
early stages of opium poppy shoot regeneration in
vitro.
Materials and Methods
Somatic embryogenesis of Papaver somniferum L.
was initiated and maintained as previously described
(Ove~ka et al. 1996). Briefly, embryogenic
callus culture was induced from unripped seeds on
MS induction medium (Murashige and Skoog
1962), supplemented with various concentrations
of ct-naphtaleneacetic acid and kinetin. Somatic
embryos regenerating on hormone-free medium
followed a long-term proliferation with the capacity
of secondary somatic embryogenesis.
Long-term organogenic culture of Papaver somniferum
L. was induced from unripe seeds on Murashige
and Skoogs (1962) medium, supplemented
with c~-naphtaleneacetic acid and benzylaminopurine,
or indoleneacetic acid and kinetin (Samaj et al.
1990, Ove6ka et al. 1997).
Resin-embedded samples for transmission electron
microscopy (TEM) were fixed in 5 % glutaraldehyde,
buffered with phosphate buffer for 5 h, and
postfixed in 2 % osmium tetroxide for 2 h, buffered
with the same buffer. After dehydration in acetone,
the samples were embedded in Durcupan ACM
(Fluca). Ultrathin sections stained with uranyl acetate
and lead citrate were examined using Tesla BS
500 electron microscope. Semithin resin sections of
1-1.5 pm thickness prepared for light microscopy
were stained with 1% aqueous toluidine blue and 2
% aqueous basic fuchsine. Samples for scanning
electron microscope (SEM) were fixed in 3 % buffered
(phosphate buffer) glutaraldehyde for 48 h and
2 % buffered osmium tetroxide for 1 h. Samples dehydrated
in ethyl alcohol were critical point dried in
CO2, sputter-coated with gold (20 nm) and examined
in scanning electron microscope JXA 840A
(JEOL) at 15 kV. Some critical point dried samples
were immersed in ethyl alcohol, transferred to butyl
alcohol, infiltrated and embedded in Histoplast S
(Serva). Sections of 8-10 pm thickness were dewaxed
and prepared for light microscopy without
additional staining, or stained with the periodic
acid-Shifts reaction (PAS).
Results
Somatic embryogenesis was induced in embryogenic
callus culture by activation and determination
of competent cells with growth regulators, and
competent cells expressed their embryogenic potential
on hormone-free medium. Cetlular organisation
and cell adhesion were different depending
on particular steps of embryonic activation, determination
and expression. Population of competent
cells were arranged in compact embryogenic clusters,
located predominantly in surface and subsurface
cell layers of callus. The cells in clusters undergoing
frequent cell divisions maintained
meristemic-like appearance, however random orientation
of cell division plane determined cell rearrangement
within the clumps after cell division
(Fig. 1 a). On the other hand, differentiated protodermis
of globular, heart-shaped and torpedo somatic
embryos comprising files of tightly arranged,
convex-shaped cells elongated in the shoot-root direction
was the most remarkable aspect of surface
morphology of regenerated somatic embryos since
the late globular stage (Fig. 1 b). Embryogenic clusters
included somatic proembryos arising from
competent cells together with later developmental
stages of somatic embryos (Fig. lb). When secondary
somatic embryogenesis was induced from the
primary somatic embryos, similar clusters of differ-
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