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

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CELL SURFACE STRUCTURE AND MORPHOGENESIS IN VITRO walls (Fig. 2b) serve as a mechanical meristemoid separation apart from the surrounding tissue and conditions localised outside. Globular proliferation of the meristemoids was clearly visible indicating division synchrony since early stages of development (Fig. 2c). Globular shape (or shape deformation depending on the inter-meristemoid pressure) thicker outer periclinal wall of peripheral cells and cell synchronisation persisted throughout the meristemoid development until the meristemoid life terminated in the case of unefficient organogenesis without formation of the shoot primordium (Fig. 2d). Meristemoid surfaces and relating callus cells were covered by mucilaginous layer extently stained with basic fuchsine (Fig. 2e). This extracellularly located mucilage was present around the meristemoids during their differentiation (Fig. 2a) where the first indications of mucilage accumulation on the meristemoid surface appeared in the stage of frequent cell division of peripheral cells (Fig. 2b). Mucilage accumulation was supported by initiation of starch utilisation in the meristemoids and the amount of external mucilage increased during the long-term culture cultivation presumably in the regions of meristemoid senescence (Fig. 2f). Development of the meristemoids was closely related to sugar metabolism and restricted to shootforming tissue. Extent local starch accumulation preceded formation of meristemoids, when the surrounding mucilage was absent (Fig. 2c, g). Formation of shoot primordia from meristemoids required utilisation of starch reserves (not shown) and decreasing starch amount in some meristemoids (Fig. 2e) or complete starch depletion in collapsed meristemoids (Fig. 2d, f) correlated with increasing amount of mucilage on their surface (Figs. 2d, e, f). Developing shoot primordium was free of fibrillar matrix on the surface (Fig. 3a), however underlying shoot-forming meristemoids were overlaid by fine layer of amorphous material (Fig. 3a). When shoots were regenerated the outgrowing leaves contained discrete epidermis (Fig. 3b) with developed stomata (Fig. 3c) but lacking both fibrillar and amorphous surface structures (Fig. 3c). Other most conspicuous extracellular material appeared in intercellular spaces of organised parenchyma tissues of developed leaves in the organogenic culture and in the tissues of primary somatic embryos. Intercellular spaces of leaf chlorenchyma tissue possessed more or less dense fibrilIar or reticular network (Figs. 4a, b). Internal histological tissue organization of primary somatic embryos became malformed during secondary somatic embryogenesis forming large intercellular "caves". These intercellular spaces were filled with a dense reticular network radiating from the surface of the parenchyma cells (Figs. 4c, d). The type of extracellular matrix interconnecting cells of re-differentiated tissues and organs expressed different structural characteristics compared to external cell and tissue surfaces in early stages of plant regeneration. Discussion Histological and morphological study of opium poppy culture in vitro revealed distinct modes of cell adhesion, cell-to-cell communication and cell surface structures in different stages of plant regeneration. In somatic embryogenesis, the relationship between cell shape, cell connection and presence or absence of cell wall external fibrillar network in embryonic tissue and differentiating somatic embryos was clearly demonstrated. Competent embryonic cells were localized in dividing regions on the surface of the long-term cultivated embryogenic callus tissue. Small cell size and mostly isodiametric cell shape show their meristematic character. Their cell walls expressed tubular extensions on the surface of the embryogenic clusters. Somatic proembryos formed in the embryogenic clusters were composed of similar isodiametric cells but fibrillar network interconnected all peripheral proembryo cells until the protodermis was formed. The presence of this interconnecting network strongly correlated with tighter adherence of proembryo cells. The surface network became a common structure for peripheral proembryo cells being a supramolecular communication continuum. On the other hand, embryo-forming cells in the programmed state when they must be separated showed limited adherence and distinct contacts via an external wall structures which were rather selfcovering (Fig. lf). The presence of interconnecting fibrillar network provided an early detection of somatic proembryos within the embryogenic clusters. Single-cell origin of secondary somatic embryos from hypocotyl of primary somatic embryos were demonstrated in repetitive regenerations (Ove~ka 123
M. OVECKA & M. BOBAK et al. 1997/98). Surface pattern of clustered secondary somatic proembryos and globular embryos originating from activated hypocotyl cells (Ove~ka et al. 1997/98) expressed similar developmentally regulated cell connection and surface patterning of proembryos and globular embryos described here. The protoderm of somatic embryos from the globular stage with the special cell shape and tight cell adhesion lacked such external wall structures found common for all regenerated organs covered by epidermis (shoots and leaves in organogenic culture). Our observations of early stages during somatic embryogenesis are in agreement with spatial and temporal cell-to-cell communication responsible for a co-ordinated embryonic pattern expression before globular stage previously published. Cells in the embryogenic tissue during development of the globular embryos were linked with the fibrillar structure before acquisition of tight adhering cell contacts, but somatic embryos from the globular stage were free of such structure in Coffea arabica (Sondahl et al. 1979), in direct somatic embryogenesis of Cichorium from root (Dubois et al. 1990, 1992) and leaf tissue (Dubois etal. 1991) and in indirect somatic embryogenesis of Drosera and Zea (Samaj et al. 1995). In Cichorium, the extracellular matrix of tubular structure (Verdus et al. 1993) were shown of a glycoprotein nature functionally interconnecting the cytoskeleton (Dubois et al. 1992). The continuum crossing the cell wall and the plasma membrane and especially extracellular matrix within this continuum were precisely characterised in polarity fixing during Fucus embryogenesis (see Goodner and Qatrano 1993, Brownlee and Berger 1995). Expression and co-ordination of embryonic development especially establishment and maintenance of cell division pattern could be (or must be) regulated by embryo-intrinsic mechanisms requiring effective signalling machinery in the stage of somatic proembryo when cells are still undifferentiated. Cell signalling must be able to block some signals during cell determination but widely spread other signals during subsequent development. Stagedependent regulation ofJ4e epitope expression recognised by JIM4 antibody was shown in somatic embryogenesis of carrot (Stacey et aI. 1990). The epitope expressed in single cultured cells was re- expressed by cells at the periphery of developing cell clusters in embryogenesis-promoting conditions and subsequently the expression in the protoderm cells of the globular somatic embryo was detected (Stacey etal. 1990). Developmental and spatial cell regulation through cell surface epitopes is documented also by emerging data on involvement of some AGPs in cell-cell signalling and cellmatrix interactions during tissue development and cell proliferation (Duet al. 1996 for a review). In embryogenic clumps of carrot callus, cell position and cell type-specific distribution of AGP epitope at the plasma membrane of the superficial cells of the clump were shown (Knox 1990). In Cichorium two embryogenesis-specific proteins absent in non-embryogenic line were detected and their presence was correlated with formed external glycoprotein fibrillar network (Hilbert et al. 1992). From this point of view any structural components of extracellular matrix (Sondahl et al. 1979, Goodner and Qatrano 1993, Brownlee and Berger 1995, Dubois et al. 1991, 1992, Samaj et al. 1995, and structures reported here) could be evaluated as an integral part of the signalling cascade through extracellular matrix-plasmomembrane-cytoskeleton continuum (Reuzeau and Point-Lezica 1995) allowing peripheral proembryo cells to be involved in co-ordinated development. Temporal occurrence of external matrix in developmentally restricted stage indicates its important role in fixing of cell position and in morphogenesis before the protodermis formation. The differentiated protodermis was free of fibrillar and tubular network (Fig. I g, Sondahl et al. 1979, Dubois et al. 1992, Samaj et aL 1995) while other protoderm-specific marker expressions within the somatic embryo could be detected such as EP2 expression in carrot encoding a lipid transfer protein (Sterk et al. 1991). Arrangement of embryogenic cell clusters indicate association of individual proembryos rather than globular appearance of meristemoids in the organogenic culture where cell layer specialisation precedes the shoot regeneration (Ovetka et al. 1997). Plane of cell division randomly oriented within the meristemoids was associated with initiated morphogenetic program of cells activated for regeneration, still undifferentiated, but keeping a tight cell adhesion within the meristemoid lacking any inter- 124
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