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

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284 Miroslav Ovečka, Milan Bobák, Jozef Šamaj Figure 2. Direct shoot regeneration. a., b. Longitudinal section of the embryo hypocotyl in the stage of cell activation. a. First localised anticlinal cell divisions of sub-epidermal cells (arrows). Bar = 50 µm b. Subsequent anticlinal and periclinal cell divisions changed size and shape of sub-epidermal activated cells. Bar = 50 µm. c. Localised cell divisions in organogenic nodule. Frequent cell divisions of subepidermal cells considerably reduced their size. First activated epidermal cells are detected (arrows). Bar = 50 µm. d. Organogenic nodules visible on the hypocotyl surface. Bar = 50 µm. e. Concentration of cell proliferation in the centre of organogenic nodule. Bar = 50 µm. f. Protomeristem with established tunica-corpus zonation in the stage of protomeristem transformation into shoot apical meristem. Bar = 50 µm. g. Cross-section of the shoot bud primordium in the vicinity of meristematic root pole of somatic embryo. Note the involvement only of the epidermal and several sub-epidermal cell layers in adventitious shoot production. Bar = 50 µm. h. Surface view on the shoot apical meristem with well defined three layered tunica (arrow) and initiation of leaf primordia outgrowth (arrowheads). Bar = 100 µm. i. Developing leaves from the flattened apical meristem of adventitious shoot bud. Bar = 100 µm. mations in the cells possessing numerous mitochondria (Fig. 3 g) indicated active cell-to-cell transport. Division of the peripheral meristemoid cells was not properly regulated. Divisions took place in all directions, resulting in a random arrangement of meristemoid cells (Fig. 3 c). However, the selection of dividing meristemoid periphery was very important in cell determination during shoot primordia formation. The change in morphogenesis was connected with regulation of cell division of the outermost peripheral meristemoid cells. The cells expanded in a radial direction and divided periclinally (Fig. 4 a). Later, peripherally located daughter cells formed tunica cell layer undergoing anticlinal divisions (Fig. 4 b). Organogenesis by protomeristem formation continued, regularly associated with starch depletion in the shoot-forming meristemoids (Figs. 4 c, d). The shoot primordium was established when cell proliferation in the procambium region and cell division in the peripheral zone of the apical meristem were detected (Figs. 4 c, e). Cell division and cell differentiation in procambial region of both directly and indirectly regenerated shoots were important for differentiation of vascular tissue and laticifer system typical for P. somniferum L. Laticifers appeared first as laticifer initials (Fig. 4 f); later they formed an articulated system during cell expansion (Fig. 4 g). Cell wall perforation also contributed to laticifer coupling in the lateral direction to promote anastomosing of laticifer arrays. Early stages of perforation were characterised by thinner and elastic cell wall within the site of future perforation, which allowed impressing and partial movement of cell contents between neighbouring cells (Figs. 4 h, i). After cell wall perforation had been completed, typical multinuclear, articulated, anastomosing laticifer system was observed in both developing shoot procambium (Fig. 4 j) and developing leaves (Fig. 4 k) of adventitious shoots regenerated in vitro. Our histological observations revealed that peripheral meristemoid cells represent the original site of shoot primordia formation. We used morphometric measurements of undifferentiated and dividing cells of regenerated shoot apical meristems in our effort to properly characterise meristemoid cells during the course of shoot primordia formation. All cells in the central and peripheral zones of shoot apical meristems expressed high correlation between the volumes of the cell and nucleus (N/C ratio). When we compared the measured N/C ratio values of central and peripheral cells within the apical meristem of regenerated shoots and peripheral meristemoid cells, we found a high degree of similarity (Fig. 5). Cell size was only one distinct morphometrical parameter. As the cells of peripheral and central zones of apical meristems, peripheral meristemoid cells were larger. As the cells in the rib-zone of shoot apical meristem (Fig. 6 a), central meristemoid cells were larger. Mathematical evaluation of the cell shape (form factor) revealed an additional difference: lower values for peripheral meristemoid cells and higher values for shoot apical meristem cells (Fig. 6 b). This means that periph-
Shoot regeneration in opium poppy 285 Figure 3. Indirect shoot regeneration. 1. Cell determination. a. Production of meristemoids within the callus tissue. Bar = 50 µm. b. Tight cell adhesion and meristematic nature of the meristemoid cells. Note the cytological differences (cytoplasmic density, thickness and stainability of the cell wall) between peripheral dividing cells and cells accumulating starch granules. Bar = 50 µm. c. Numerous globular meristemoids appearing mostly on the callus surface. Cell arrangement in the meristemoids was random, but they had compact, non-friable nature. Bar = 50 µm. d, e, f, g, – Ultrastructure of the central meristemoid cells. d. Transparent cytoplasm and large deposits of starch in the central cells. Bar = 10 µm. e. Insertion of vesicular and matrix material into the cell wall of central meristemoid cells by exocytosis. L-lipid droplet. Bar = 2 µm. f. Lipid droplets which fill up the cells in the transition layer between centre and periphery of the meristemoid. Bar = 10 µm. g. Cell wall ingrowths (asterisk) in the central meristemoid cell. Note the presence of numerous mitochondria. Bar = 2 µm. eral meristemoid cells were more rounded and cells of shoot apical meristem were more oblong. However, unlike these differences in cell size and shape, in both meristemoid periphery (Fig. 7a) and shoot apical meristem (Fig. 7b), cell growth was closely related to the similar negative correlation between cell size and cell shape. Increase in the mean of cell size was found to be followed by general decrease in the mean of form factor (Figs. 7 a, b). In conclusion, most of the cytological and morphometrical observations indicate that only peripheral meristemoid cells can be identified as determined cells during indirect shoot organogenesis. Discussion This study deals with cytological characterisation of de novo shoot regeneration of P. somniferum L. in vitro, focusing from the morphogenetical point of view on the differences between direct and indirect shoot primordia formation. Shoot organogenesis is a suitable experimental system for use in discerning patterns of cell division and expansion in order to compare direct and indirect caulogenetic determination. Three developmental steps have been identified in distinct shoot-forming cultures: (I) initiation of meristematic activity in callus or explant as an expression of the morphogenetic competence; (II) cell determination during formation of meristematic nodules; and (III) differentiation of adventitious shoot buds (Von Arnold and Eriksson 1985, Bobák et al. 1989, Sharma and Bhojwani 1990, Colby et al. 1991, Lakshmanan et al. 1997). In contrast to well established models of shoot regeneration, the structural details of shoot primordia initiation and development are missing in studies of organogenic poppy callus culture. In addition, direct shoot organogenesis of opium poppy has not been published before now. The question here was what aspects of cell division, cell expansion, and cell differentiation are related to cellular origin of shoot primordia during either direct or indirect shoot regeneration.
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