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A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum L. in vitro
J. Plant Physiol. 157.281-289 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
• JOURNAL OF • PLANT PHYSIOLOGY
A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum l. in vitro Miroslav Ovecka 1 *, Milan Bobak2, Jozef Sarna?
1
Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, SK-842 23 Bratislava, Slovak Republic
2
Department of Plant Physiology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B-2, SK-842 15, Bratislava, Slovak Republic
3
Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P.O. Box 39 A, SK-950 07 Nitra, Slovak Republic
Received January 11, 2000 . Accepted May 22,2000
Summary Cellular origin of shoot buds, cell morphogenesis and differentiation were studied during direct and indirect shoot regeneration of Papaver somniferum L. in vitro. Direct shoot organogenesis was induced in immature somatic embryos, where cell division and protomeristem formation started in sub-epidermal and epidermal cell layers of hypocotyl. Indirect shoot regeneration was initiated from callus culture using auxins and cytokinins, where compact globular meristemoids were produced. The common morphogenetic event of direct and indirect shoot regeneration was an establishment of non-random cell division and restricted cell expansion within the group of competent cells during protomeristem formation. However, in contrast to direct regeneration, where all activated cells became competent, in indirect regeneration, only peripheral cells of meristemoids acquired morphogenetic competence. The second difference occurred in shoot tunica formation: original hypocotyl epidermal cells partiCipated in tunica formation during direct organogenesis, while this layer regenerated de novo in meristemoids. These results indicate that cell morphogenesis during shoot regeneration is independent of the developmental history of the competent cells.
Key words: morphogenesis - morphometry - Papaver somniferum L. - regeneration - shoot organogenesis
Introduction Adventitious shoot regeneration is an important method of in vitro plant biotechnology. Efficiency and yield of this type of regeneration are closely related to culture initiation in some species, regardless of whether shoot organogenesis can be induced directly without callus production. A very important • E-mail correspondingauthor:[email protected]
factor determining culture response to a given culture conditions is the state of differentiation of the participating cells. In this respect, cell polarity, regulation of cell division, cell expansion, and cell differentiation are important parameters in the effort to understand the process of cell determination during the early stages of shoot organogenesis (Samaj et al. 1997). In P somniferum L., only callus induction from isolated hypocotyls and indirect shoot organogenesis have been achieved. A general rule of the induction was an application 0176-1617/00/157/03-281 $15.00/0
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Miroslav Ovecka, Milan Bobak, Jozef Samaj
of auxins and cytokinins (Nessler and Mahlberg 1979, Kamo et al. 1982, Yoshikawa and Furuya 1985, Griffing et al. 1989), or a transformation of the callus tissue originating from hypocotyl by Agrobacterium rhizogenes (Yoshimatsu and Shimomura 1992). Organogenesis in the callus is connected to the formation of meristematic centres-meristemoids with non-random distribution of starch and lipids (Nessler and Mahlberg 1979, Samaj et al. 1990 a, Ovecka et al. 1997). Both starch and lipids have been shown to be related to shoot regeneration: the amount of lipids in callus cells is 1.6-2.6 %, while meristemoid cells contain 13 % in solid culture and 34.1 % in liquid culture (Yoshikawa and Furuya 1985). Cell specification in meristemoids takes place during transition phase of organogenesis (meristemoid "maturation") when the initial accumulation and subsequent utilisation of starch and lipids change the nature of the cells. As a consequence, the meristemoid cells express structural changes of nuclei (Bobak et al. 1990), plastids (Samaj et al. 1988), and vacuolar system (Samaj et al. 1990 b). However, the precise data on cellular origin of shoot bud primordia are scarce or nonexistent in early works dealing with opium poppy shoot organogenesis. In our previous study, we documented some cytological and morphometrical differences among meristemoid cells during indirect shoot organogenesis of P somniferum L. (Ovecka et al. 1997). Alternatively, bud production was achieved from cultivated somatic embryos, and direct shoot regeneration of opium poppy was documented for the first time (Ovecka et al. 1997/98). The aim of the present work was to study the similarities and differences in morphogenetical steps of shoot primordia formation during direct and indirect shoot regeneration. We characterised in detail: i. the cellular origin of shoot primordia and shoot buds directly regenerated from somatic embryos; and ii. the cell differentiation during shoot primordia formation from meristemoids. In addition to cytological and histological analyses, indirect shoot regeneration was studied by morphometrical analysis.
Material and Methods In vitro cultures Direct shoot regeneration was induced from somatic embryos of P. somniferum L. Embryogenic callus culture was initiated from septa of poppy capsules using a combination of a-naphtaleneacetic acid (0.537-5.37IlmoI/L) and kinetin (0.23-0.46IlmoI/L). Regeneration of somatic embryos took place on the growth regulator-free medium, as described by Ovecka et al. (1996). Arrested torpedo somatic embryos unable to develop functional root, spontaneously produced secondary somatic embryos and adventitious shoots during cultivation on the regeneration medium without growth regulators (Ovecka et al. 1997/98). Organogenia callus culture was induced from unripe seeds on solidified MS media (Murashige and Skoog 1962), supplemented with 0.5371lmo1/L a-naphtaleneacetic acid and 0.46Ilmol/L kinetin. Long-term shoot regeneration continued during culture cultivation on the: (I) induction medium, (II) medium with 0.57Ilmol/L in-
dole-3-acetic acid and 2.22 limol/L 6-benzylaminopurine, and (III) growth regulator-free medium (Ovecka et al. 1997).
Microscopy Morphological observations were performed directly on living material or using scanning electron microscopy (SEM). The samples for SEM were fixed with 3 % glutaraldehyde (48 h, 0.1 mol/L phosphate buffer, pH 7.2) and 2 % OS04 (1 h, the same buffer and pH). After washing in buffer, the samples were dehydrated in ethanol, critical point dried in liquid CO 2 , sputter coated with 20 nm layer of gold-palladium, and observed with a JEOL JXA 840A (JEOL, Japan) microscope. Samples for transmission electron microscopy (TEM) were fixed in 5 % glutaraldehyde (5 h, 0.1 mol/L phosphate buffer, pH 7) and 1 % OS04 (2 h, 0.1 mol/L phosphate buffer, pH 7), dehydrated in acetone and embedded in Durcupan ACM (Fluca, Buchs, Switzerland). Ultrathin sections were stained according to Reynolds (1963) and observed in ATEM 2000FX (JEOL, Japan) and TESLA BS 500 (Tesla, Czech Republic) electron microscopes. Histological and cytological analyses of shoot regeneration were performed at the light microscopy level. Paraffin sections (7-10 lim) were prepared after sample fixation in FAA (formalin 40 %, acetic acid 5 %, ethyl alkohol 50 %), embedded in Histoplast S (Serva, Heidelberg, Germany) and stained with hematoxylin-eosin, PAS reaction or Feuglen reaction. Sections (1-2 lim thick) for the fine cytological observation were prepared from the samples embedded for TEM. After SEM observation, some dried bulk samples were immersed in ethanol and embedded in Histoplast S (Serva, Heidelberg, Germany). The sections (7-10 lim thick) were observed without additional staining under bright-field microscopy (Ovecka and Bobak 1999). All prepared samples were studied using ZEISS Jenalumar (Carl Zeiss, Jena, Germany) dissection light microscope. Pictures digitalised using a Kappa CF 8/1 FMCC camera (Kappa Messtechnik, Germany), and a Leica Q500MC image analysis system (Leica Cambridge Ltd. England, UK) were processed using Corel Photo Paint 8 (Corel Corporation, Canada).
Morphometry All planimetric measurements of meristemoid and shoot primordia cells during indirect shoot regeneration were made using an ASBA (Wild, Heerbrugg, Switzerland) image analyser. Cell size, cell shape, and nucleus size were measured on the basis of cell cross-section area in 1-2 lim thick sections. The mean values were compared among the above-mentioned three culture media with different amounts of growth regulators. The cell shape (form factor) was calculated from the cell area and cell projections (a, b, a+b, a-b). The form factor 5.09 means a circular shape, 6 means a square and 7 a rectangle shape with a side ratio of 2: 1 (Baluska et al. 1990). Morphometrical values of cells in central and peripheral zones of the regenerated shoot apical meristems were measured as standards for the comparative characterisation of meristemoid cells. The computed sizes, shapes of cells, and N/C ratios were used as parameters of meristemoid cell activity (cell division and cell expansion). Linear regression analyses, coefficients of correlation, and N/C ratios were calculated using Sigma Plot (Jandel Scientific, USA) statistical software.
Shoot regeneration in opium poppy
Results Shoot regeneration was initiated from hypocotyls of primary somatic embryos (Fig. 1 a). The emerging shoot primordia appeared on the hypocotyl surface of the somatic embryo hypocotyl, but they did not disrupt the surface tissues (Fig. 1 b). In addition, no general tissue reorganisation was observed (Fig. 1 b). Due to these features, this kind of regeneration was termed direct shoot organogenesis in this study. Indirect shoot regeneration involved tissue dedifferentiation, callus formation, and cell re-differentiation leading to de novo shoot regeneration (Fig . 1c). Tissue organisation and cell morphogenesis of indirectly regenerated shoot primordia were completely different in comparison to underlying callus tissue (Fig . 1d).
Direct shoot regeneration Surface integrity of the embryo hypocotyl suggested that adventitious shoots arose from superficial cell layers (Fig . 1b). Initially, sub-epidermal cells were activated and divided. The plane of the first cell division seemed to be anticlinal (Fig. 2 a), but subsequent cell divisions were observed in both anti- and periclinal planes (Fig. 2 b). Afterwards, dividing subepidermal and epidermal cells created organogenic nodules, the first recognisable structures on the hypocotyl surface (Figs . 2c, d). The two most important morphogenetic features of these cells were dense non-vacuolated cytoplasm (Figs. 2 a , b,c) and reduced cell size (Fig . 2d). The first apparent indication of cell specification was observed in the phase of apical meristem formation . The cells
Figure1. Shoot buds regenerated in the callus culture of P somniferum l. a. Somatic embryo (SE) in the culture where
the shoot (SH) regeneration from the hypocotyl was induced. Bar = 1mm. b. Directly regenerated shoots with leaf primordia, visible as protuberances on the hypocotyl surface of somatic embryo. Bar = 100 11m. c. Shoot bud regenerated in the organogenic callus culture. The white compact tissue represents meristemoids. Bar = 1 mm. d. Shoot primordium on the surface of callus tissue. Bar = 1001lm.
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within nodule apices adopted tunica-corpus zonation (Figs. 2 e, f) . The original epidermal cells participated in differentiation of the tunica layer (Fig . 2 c) . This explains why the tunica and protomeristem maintained morphological and histological continuity between the forthcoming buds and the remaining hypocotyl surface (Figs. 1 b, 2 c , d,g). Cell specialisation continued by both histodifferentiation of the shoot apical meristem (Figs. 2 e, f) and transformation of organogenic nodules into buds (Figs. 2 g ,h). Adventitious buds were visible on the hypocotyl surface (Figs . 2 g h) , and regular activity of the apical meristem was detected, including formation and growth of leaf primordia (Figs. 2 h ,i).
Indirect shoot regeneration Distinct mode of indirect induction of organogenesis was based on callus production. Cell activation triggered a morphogenetic switch in some cells within rarely dividing callus tissue , resulting in the establishment of dividing , meristemlike, and shoot-forming tissue (Fig . 3a). Typical features of meristemoids (representing population of competent cells) were small cell size, cytoplasmic density, minimal vacuolation (Figs . 3 a b), , and cell adhesion after cell division (Figs . 1d, 3 a ,b, c). The first cell differentiation events within the multicellular meristemoids resulted in the formation of both peripheral and central cell layers, with only peripheral cells continuing their divisions (Fig. 3 b). Reserves were stored in central , and lipids (Figs. 3 e ,f). cells in the form of starch (Figs . 3 b d) These cells were non-dividing but their cytological parameters indicated high metabolic activity. Frequent endo- and exocytosis in these cells (Fig . 3e) and cell wall ingrowth for-
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Figure2. 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 flm b. Subsequent anticlinal and periclinal cell divisions changed size and shape of sub-epidermal activated cells. Bar = 50 flm. 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 11m. d. Organogenic nodules visible on the hypocotyl surface. Bar = 50 11m. e. Concentration of cell proliferation in the centre of organogenic nodule. Bar = 50 flm. f. Protomeristem with established tunica-corpus zonation in the stage of protomeristem transformation into shoot apical meristem. Bar = 50 flm. 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 flm. h. Surface view on the shoot apical meristem with well defined three layered tunica (arrow) and initiation of leaf primordia outgrowth (arrowheads). Bar = 100 flm. i. Developing leaves from the flattened apical meristem of adventitious shoot bud. Bar = 100l1m.
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. 4c, 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. 6a), 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 peri ph-
Shoot regeneration in opium poppy
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Figure3. Indirect shoot regeneration . 1. Cell determination.
a. Production of meristemoids with in the callus tissue. Bar = 50 ~m . 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 cell s accumulating starch granules. Bar = 50 ~m . 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~m . d, e, f, g, - Ultrastructure of the central meristemoid cells. d. Transparent cytoplasm and large deposits of starch in the central cells. Bar = 10 ~m . e. Insertion of vesi cular a nd matrix material into the cell wall of central meristemoid cells by exocytos is. L-lipid droplet. Bar = 2f1m. f. Lipid droplets which fill up the cells in the transition layer between centre and periphery of the meristemoid . Bar = 10~m . g. Cell wall ingrowths (asterisk) in the central meristemoid cell . Note the presence of numerous mitochondria. Bar = 21J.m.
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. 7a, 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 or-
ganogenesis 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 d si tinct 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, Bobak 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 cal lus 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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Figure4. Indirect shoot regeneration. 2. Formation of shoot primordia. a. Morphogenesis of peripheral meristemoid cells during shoot primordia formation. Cells expanded in anticlinal direction (arrowhead) and subsequently divided periclinally (arrows). Bar = 50 11m. b. Establishment of the tunica layer on the meristemoid surface by anticlinal cell division. Bar = 50 11m. c. Histodifferentiation of the shoot apical meristem (SAM). Bar = 50 11m. d. Cell proliferation concentrated in the dome-like protomeristem. Cells located away of the centre of cell division are devoid of starch and start to vacuolate. Bar = 50 11m. e. Developed shoot bud with leaf primordia and vascular tissue (VT) in the procambium. Bar = 100 11m. f. Laticifer initials (arrows) in the procambial region of shoot apical meristem. Bar = 50 11m. g. Initial perforation of cross cell wall between two laticifer initials. Bar = 50 11m. h. Formation of articulated, anastomosing laticifer system. Note the elastic properties of longitudinal cell wall in the site of future perforation (arrow). Bar = 50 11m. i. Insertions of cell volume between future anastomosing laticifers in cross-section (arrows). Bar = 50 11m. j. Articulated, anastomosed laticifer system in shoot procambium. Bar = 50 11m. k. Articulated, anastomosed laticifer system in developing leaves of regenerated shoots. Bar = 50 11m.
90
II
~80
Figure 5. Similarity in the correlation curves between cell size and nucleus size of peripheral meristemoid cells (-e-) and cells of peripheral and central zones of the apical meristems in regenerated shoots (-0-). 30-50 cells of each zone were measured on the medium supplemented with 0.537Ilmol/L of a-naphtaleneacetic acid and 0.46Ilmol/L kinetin (I), or with 0.577llmoI/L indole-3-acetic acid and 2.221lmo1/L 6-benzylaminopurine (II), or growth
III
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100
200
300
100
200
300
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Direct shoot formation was initiated in the culture where somatic embryos had failed to develop correctly, but maintained continuously juvenile, undifferentiated state of their cells in the root pole which were able to generate new somatic embryos (Ovecka et al. 1997/98). However, as we show here, adventitious shoots originated from the differentiated, non-dividing tissue of hypocotyl. The competent cells have to be re-activated and they enter cell division. The culture un-
3DD
400
regulator-free medium (III). Cell size values and nucleus size values are presented as planimetric values of cell and nucleus area measured from cross sections.
derwent spontaneous repetitive regeneration including shoot organogenesis and somatic embryogenesis (Ovecka et al. 1997/98). Some internal factors determining cell competence in the course of shoot bud regeneration have been identified, such as reduced .cellulase activity in tobacco callus culture (Truelsen and Ulvskov 1995), or importance of polar auxin transport, because its inhibition by TIBA (2,3,5-triiodobenzoic acid) strongly enhanced shoot regeneration, but not somatic
Shoot regeneration in opium poppy Figure6. Mean of the cell cross section area (a), and mean of the form factor describing cell shape (b) , observed during shoot regeneration in the medium supplemented with 0.5371lmo1/L of a-naphtaleneacetic acid and 0.4611mo1/L kinetin. Peripheral meristemoid cells (A) were compared to cells of central and peripheral zones of regenerated shoot apical meristems (8), and central meristemoid cells (e) were compared to cells in the rib-zone of shoot apical meristems (D). Percentage amount of cells with isodiametric (a side ratio app. 1 1), : and oval shape (side ratio up to 2: 1) is indicated (b). The rest of the cells had a more elongated shape. Results from measurements of app. 300 cells of each selected part of meristemoids and shoot apical meristems ... - significant at P<0.01 according to t-test.
a -
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Figure 7. a. Negative correlation between cell size and cell shape in meristemoids (r = -0.51) . b. Negative correlation between shoot apical meristems of regenerated shoots (r = -0.343). Mean values of cell sizes and cell shapes (form factors) of all measured cells in the present study were compared.
6.4 6.3 100
150
200
100
150
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embryogenesis (Tetu et al. 1990). The study of morphology
1995, Gilissen et al. 1994). This data indicates that a limited
and conversion ability of poppy somatic embryos revealed
number of participating cells, even with a distinct fate before
some structural abnormalities (fused cotyledons, embryo
activation, can form shoot primordium through regulated and
twins, and developmental arrest with continual cell prolifera-
co-ordinated cell morphogenesis.
tion, Ovecka et al. 1996) which resembled abnormalities of
The second morphological aspect of direct shoot regen-
Panax ginseng C. A. Meyer somatic embryos reported after
eration was an apparent participation of the hypocotyl epi-
polar auxin transport inhibition (Choi et al. 1997). From this point of view it seems that discrepancy in endogenous hor-
dermal cells in the formation of shoot apical meristem. Epidermis is formed by polarised cells. However, epidermal
mone (especially auxin) level would be an internal selecting factor supporting activation of competent cells in the embryo
cells were reported to be competent in some models of direct shoot regeneration. Meristematic regions formed by anticlinal
hypocotyl. The two most important aspects of direct poppy shoot re-
and periclinal divisions of epidermal cells, and even sub-epidermal cells, as well as continuity of meristematic domes of
generation were connected with proper regulation of morphogenesis in multicellular organogenic nodule. First, all activated cells were synchronised and became determined and
developing shoot primordia with the remaining epidermis have been observed (Tetu et al. 1990, Bronner et al. 1994, Burritt and Leung 1996, Konieczny 1996). This is a strong in-
thus were involved in shoot primordia formation . In periclinal
dication of a totipotent nature of epidermal cells (Burritt and
chimeras of Nicotiana tabacum L. and Nicotiana g/auca
Leung 1996, and references therein). Activation and periclinal
Grahm . it was clearly shown that shoots can originate in any
cell division of epidermis are effective mostly in organogenic
cell layer of the leaf tissue , and the origin of some shoots can
nodule formation, while each anticlinal cell division maintain-
be multicellular and multihistogenic (Marcotrigiano 1986). In
ing original cell polarity participates in the further tunica forma-
contrast to the cortex layer, in shoot-forming thin cell layers of
tion of the shoot primordium derived from sub-epidermal tissue
Nicotiana tabacum L., the cell expansion in epidermis and
(Fig. 2). These observations document the importance of an
sub-epidermal layers was limited and initial periclinal cell di-
early regulation of the plane of cell division during the shoot
vision indicated a modification of cell polarity (Altamura et al.
apical meristem formation in directly regenerated shoot buds .
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Miroslav Ovecka, Milan Bobak, Jozef Samaj
Shoot regeneration in callus culture requires a combination of auxins and cytokinins (Christianson and Warnick 1983, De Klerk et al. 1997). The main change in cell division and cell expansion was observed during competence acquisition. Meristemoids were produced from rarely dividing callus cells. Cell division was more patterned within meristemoids. Their cells were small, micro-vacuolated, and able to stick together. This indicates that the fate of the callus culture is altered through cell morphogenesis of the activated competent cells as a result of the action of growth regulators. The second switching point in cell morphogenesis of poppy callus culture occurred in the multicellular meristemoids as a defined step of cell determination within the shootforming tissue. A critical event was the localisation of cell division to the meristemoid periphery. The cell determination has been expressed when modified polar periciinal cell division occurred in meristemoids. Apical daughter cells generated dividing peripheral layers with morphogenetic competence while the distal daughter cells became more differentiated and generated storage cells possessing metabolic sources. The previously observed differences in cell size and shape showed that the peripheral cells were smaller, having an oblong shape as a result of frequent cell division, while the central cells became larger due to globular expansion (Ovecka et al. 1997). Starch-containing cells were the largest ones. They had isodiametric shape, and any changes (cell division, plastid division, vacuolation) took place only after starch utilisation had been initiated (not shown). Central meristemoid cells thus can support organogenesis by the energy. It was clearly shown that some metabolic indications of shoot regeneration (protein inclusions, starch, lipids, high enzyme activities) were found only in meristemoids (Ross et al. 1973, Nessler and Mahlberg 1979, Patel and Berlyn 1983). Meristemoids possessed a high starch content, while high activities of starch-degrading enzymes were observed in shoot buds (Thorpe and Murashige 1970, Thorpe and Meier 1974, Patel and Berlyn 1983). A high N/C ratio of peripheral meristemoid cells of opium poppy, comparable with cells in shoot apical meristem (Fig. 5), together with comparable rate of chromatin de-condensation (Ovecka et al. 1997) are also important parameters of cell determination within the meristemoids. Variability in the size of nucleus and the correlation with DNA amount is closely related to shoot regeneration (Flinn et al. 1989, Fournier et al. 1991). A two-phase organogenesis, with meristemoid formation during the first phase and formation of globular structures from the surface during the second phase, was observed in sundew callus tissue (Bobak et al. 1993). This data indicates that "mitotic zonation" established a distinct morphological competence of mer istemoid cells in the process of shoot primordia formation when the site of origin of shoot buds was located at the meristemoid periphery. Concluding our comparative analysis of direct and indirect shoot regeneration in P somniferum L., important changes in cell morphogenesis, characterised by non-random cell divi-
sions, took place during dermal layer and protomeristem formation in shoot primordia. We observed a random orientation of cell divisions in meristemoids, while protomeristem and especially tunica formation were accompanied by regulated periclinal and subsequently anticlinal cell divisions. In directly regenerated shoots, the dermal layer was formed by original epidermal cells through anticlinal cell divisions. This type of division maintains epidermal cell polarity. Peripheral meristemoid cells are polarised with non-homogenous cell surfaces due to the presence of adhered and outer non-adhered cell walls (Ovecka and Bobak 1999). This polarity and cell position are important morphogenetic factors in cell determination. Cell expansion was found to be minimal in cells involved in formative, morphogenetically important cell diviSion. The cells expanded, but not over the cell volume necessary for a subsequent cell division. In addition, cell size and cell shape have to be negatively correlated. This relationship would indicate an independence of post-mitotic cell growth and an extensive cell vacuolation, originally observed in maize root apex (Baluska et al. 1990). A comparative analysis revealed that cell activation in shoot organogenesis of P somniferum L. depends on the stage of cell differentiation before initiation, while cell morphogenesis of the determined cells is similarly regulated both spatially and temporally, during direct and indirect shoot regeneration. Acknowledgements. We gratefully acknowledge D. Jantova for technical assistance, and Dr. M. Ciamporova for the English correction. This work was supported by Grant Agency VEGA, Grant No. 1/6030/99 and Grant No. 1/6107/99 from the Slovak Academy of Sciences. M.O. gratefully acknowledges the Alexander von Humboldt-Stiftung (Bonn, Germany) for donation of the Leica Q500MC image analysis system.
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spathulata L. cultivated in vitro. Biol6gia (Bratislava) 44: 785-792 Bobak M, Blehova A, Samaj J, Ovecka M, Kristin J (1993) Studies of organogenesis from the callus culture of the sundew (Drosera
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Shoot regeneration in opium poppy Burritt OJ, Leung DWM (1996) Organogenesis in cultured petiole explants of Begonia x erythrophylla: the timing and specificity of the inductive stimuli. J Exp Bot 47: 557-567 Choi YE, Kim HS, Soh WY, Yang DC (1997) Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid. Plant Cell Rep 16: 738-744 Christianson ML, Warnick DA (1983) Competence and determination in the process of in vitro shoot organogenesis. Dev Bioi 95: 288293 Colby SM, Juncosa AM, Stamp JA, Meredith CP (1991) Developmental anatomy of direct shoot organogenesis from leaf petioles of Vitis vinifera (Vitaceae). Amer J Bot 78: 260-269 De Klerk G-J, Arnholdt-Schmitt B, Lieberei R, Neumann K-H (1997) Regeneration of roots, shoots and embryos: physiological, biochemical and molecular aspects. Bioi Plant 39: 53-66 Flinn BS, Webb DT, Newcomb W (1989) Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern white pine (Pinus strobus) cotyledons in vitro. Can J Bot 67: 779-789 Fournier 0, Bonnelle Ch, Tourte Y (1991) Ultrastructural features of soybean somatic cells at the beginning of an organogenic process: toward a new concept. Bioi Cell 73: 99-105 Gilissen LJW, Van Staveren MJ, Hakkert JC, Smulders MJM, Verhoeven HA, Creemers-Molenaar J (1994) The competence of cells for cell division and regeneration in tobacco explants depends on cellular location, cell cycle phase and ploidy level. Plant Sci 103: 81-91 Griffing LR, Fowke LC, Constabel F (1989) Radioimmunoassay of morphinan alkaloids in Papaver somniferum hypokotyls, callus and suspension cultures. J Plant Physiol 134, 645-650 Kamo KK, Kimoto W, Hsu AF, Mahlberg PG, Bills DO (1982) Morphine alkaloids in cultured tissues and redifferentiated organs of Papaver somniferum. Phytochemistry 21: 219-222 Konieczny R (1996) Plant regeneration from immature embryo culture of Trifolium michelianum Savi. - histological observations on adventitious shoot induction. Acta Soc Bot Pol 65: 261-266 Lakshmanan P, Ng SK, Loh CS, Goh CJ (1997) Auxin, cytokinin and ethylene differentially regulate specific developmental states associated with shoot bud morphogenesis in leaf tissues of mangosteen (Garcinia mangostana L.) cultured in vitro. Plant Cell Physiol 38: 59-64 Marcotrigiano M (1986) Origin of adventitious shoots regenerated from cultured tobacco leaf tissue. Amer J Bot 73: 1541-1547 Murashige T, Skoog F (1962) A revised medium for rapid grow1h and bioassay with tobacco tissue cultures. Physiol Plant 15: 473-497 Nessler CL, Mahlberg PG (1979) Ultrastructure of laticifers in redifferentiated organs on callus from Papaver somniferum. Can J Bot 57: 675-685 Ovecka M, Bobak M (1999) Structural diversity of Papaver somniferum L. cell surfaces in vitro depending on particular steps of plant regeneration and morphogenetic program. Acta Physiol Plant 21: 117-126
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Morphology and conversion ability of somatic embryos in longterm embryogenic callus culture of Papaver somniferum. Biologia (Bratislava) 51: 417-423 Ovecka M, Bobak M, Samaj J (1997) Development of shoot primordia in tissue culture of Papaver somniferum L. Bioi Plant 39: 499-506 Ovecka M, Bobak M, Blehova A, Kristin J (1997/98) Papaver somniferum regeneration by somatic embryogenesis and shoot organogenesis. Bioi Plant 40: 321-328
Patel KR, Berlyn GP (1983) Cytochemical investigations on multiple bud formation in tissue cultures of Pinus coulteri. Can J Bot 61: 575-585 Reynolds ES (1963) The use of the lead citrate at high pH as an electron opaque in electron microscopy. J Cell Bioi 17: 208-213 Ross MK, Thorpe TA, Costerton JW (1973) Ultrastructural aspects of shoot initiation in tobacco callus cultures. Amer J Bot 60: 788-795 Sharma KK, Bhojwani SS (1990) Histological aspects of in vitro root and shoot differentiation from cotyledon explants of Brassica juncea (L.) Czern. Plant Sci 69: 207-214 Samaj J, Bobak M, Erdelsky K (1990a) Histological-anatomical studies of the structure of the organogenic callus in Papaver somniferum L. Bioi Plant 32: 14-18 Samaj J, Bobak M, Erdelsky K (1990 b) Vacuolar system in the cells of organogenic callus cultures of Papaver somniferum L. in vitro. Acta FRN Univ Comen Physiol Plant XXV: 21-32 Samaj J, Bobak M, Erdelsky K (1988) Plastids in cells of Papaver somniferum L. organogenic calli grown on various media. Biol6gia (Bratislava) 43: 577-588, in Slovak Samaj J, Bobak M, Ovecka M, Blehova A, Pret'ova A (1997) Structural features of plant morphogenesis in vitro. Veda, Bratislava Tetu T, Sangwan RS, Sangwan-Norreel BS (1990) Direct somatic embryogenesis and organogenesis in cultured immature zygotic embryos of Pisum sativum L. J Plant Physiol137: 102-109 Thorpe TA, Murashige T (1970) Some histochemical changes underlying shoot initiation in tobacco callus cultures. Can J Bot 48: 277285 Thorpe TA, Meier DO (1974) Starch metabolism in shoot-forming tobacco callus. J Exp Bot 25: 288-294 Truelsen TA, Ulvskov P (1995) The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus. Planta 196: 727731 Von Arnold S, Eriksson T (1985) Initial stages in the course of adventitious bud formation on embryos of Picea abies. Physiol Plant 64: 41-47 Yoshikawa T, Furuya T (1985) Morphinan alkaloid production by tissue differentiated from cultured cells of Papaver somniferum. Planta Med 44: 110-113 Yoshimatsu K, Shimomura K (1992) Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724. Plant Cell Rep 11: 132-136
• JOURNAL OF • PLANT PHYSIOLOGY
A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum l. in vitro Miroslav Ovecka 1 *, Milan Bobak2, Jozef Sarna?
1
Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, SK-842 23 Bratislava, Slovak Republic
2
Department of Plant Physiology, Faculty of Natural Sciences, Comenius University, Mlynska dolina B-2, SK-842 15, Bratislava, Slovak Republic
3
Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P.O. Box 39 A, SK-950 07 Nitra, Slovak Republic
Received January 11, 2000 . Accepted May 22,2000
Summary Cellular origin of shoot buds, cell morphogenesis and differentiation were studied during direct and indirect shoot regeneration of Papaver somniferum L. in vitro. Direct shoot organogenesis was induced in immature somatic embryos, where cell division and protomeristem formation started in sub-epidermal and epidermal cell layers of hypocotyl. Indirect shoot regeneration was initiated from callus culture using auxins and cytokinins, where compact globular meristemoids were produced. The common morphogenetic event of direct and indirect shoot regeneration was an establishment of non-random cell division and restricted cell expansion within the group of competent cells during protomeristem formation. However, in contrast to direct regeneration, where all activated cells became competent, in indirect regeneration, only peripheral cells of meristemoids acquired morphogenetic competence. The second difference occurred in shoot tunica formation: original hypocotyl epidermal cells partiCipated in tunica formation during direct organogenesis, while this layer regenerated de novo in meristemoids. These results indicate that cell morphogenesis during shoot regeneration is independent of the developmental history of the competent cells.
Key words: morphogenesis - morphometry - Papaver somniferum L. - regeneration - shoot organogenesis
Introduction Adventitious shoot regeneration is an important method of in vitro plant biotechnology. Efficiency and yield of this type of regeneration are closely related to culture initiation in some species, regardless of whether shoot organogenesis can be induced directly without callus production. A very important • E-mail correspondingauthor:[email protected]
factor determining culture response to a given culture conditions is the state of differentiation of the participating cells. In this respect, cell polarity, regulation of cell division, cell expansion, and cell differentiation are important parameters in the effort to understand the process of cell determination during the early stages of shoot organogenesis (Samaj et al. 1997). In P somniferum L., only callus induction from isolated hypocotyls and indirect shoot organogenesis have been achieved. A general rule of the induction was an application 0176-1617/00/157/03-281 $15.00/0
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of auxins and cytokinins (Nessler and Mahlberg 1979, Kamo et al. 1982, Yoshikawa and Furuya 1985, Griffing et al. 1989), or a transformation of the callus tissue originating from hypocotyl by Agrobacterium rhizogenes (Yoshimatsu and Shimomura 1992). Organogenesis in the callus is connected to the formation of meristematic centres-meristemoids with non-random distribution of starch and lipids (Nessler and Mahlberg 1979, Samaj et al. 1990 a, Ovecka et al. 1997). Both starch and lipids have been shown to be related to shoot regeneration: the amount of lipids in callus cells is 1.6-2.6 %, while meristemoid cells contain 13 % in solid culture and 34.1 % in liquid culture (Yoshikawa and Furuya 1985). Cell specification in meristemoids takes place during transition phase of organogenesis (meristemoid "maturation") when the initial accumulation and subsequent utilisation of starch and lipids change the nature of the cells. As a consequence, the meristemoid cells express structural changes of nuclei (Bobak et al. 1990), plastids (Samaj et al. 1988), and vacuolar system (Samaj et al. 1990 b). However, the precise data on cellular origin of shoot bud primordia are scarce or nonexistent in early works dealing with opium poppy shoot organogenesis. In our previous study, we documented some cytological and morphometrical differences among meristemoid cells during indirect shoot organogenesis of P somniferum L. (Ovecka et al. 1997). Alternatively, bud production was achieved from cultivated somatic embryos, and direct shoot regeneration of opium poppy was documented for the first time (Ovecka et al. 1997/98). The aim of the present work was to study the similarities and differences in morphogenetical steps of shoot primordia formation during direct and indirect shoot regeneration. We characterised in detail: i. the cellular origin of shoot primordia and shoot buds directly regenerated from somatic embryos; and ii. the cell differentiation during shoot primordia formation from meristemoids. In addition to cytological and histological analyses, indirect shoot regeneration was studied by morphometrical analysis.
Material and Methods In vitro cultures Direct shoot regeneration was induced from somatic embryos of P. somniferum L. Embryogenic callus culture was initiated from septa of poppy capsules using a combination of a-naphtaleneacetic acid (0.537-5.37IlmoI/L) and kinetin (0.23-0.46IlmoI/L). Regeneration of somatic embryos took place on the growth regulator-free medium, as described by Ovecka et al. (1996). Arrested torpedo somatic embryos unable to develop functional root, spontaneously produced secondary somatic embryos and adventitious shoots during cultivation on the regeneration medium without growth regulators (Ovecka et al. 1997/98). Organogenia callus culture was induced from unripe seeds on solidified MS media (Murashige and Skoog 1962), supplemented with 0.5371lmo1/L a-naphtaleneacetic acid and 0.46Ilmol/L kinetin. Long-term shoot regeneration continued during culture cultivation on the: (I) induction medium, (II) medium with 0.57Ilmol/L in-
dole-3-acetic acid and 2.22 limol/L 6-benzylaminopurine, and (III) growth regulator-free medium (Ovecka et al. 1997).
Microscopy Morphological observations were performed directly on living material or using scanning electron microscopy (SEM). The samples for SEM were fixed with 3 % glutaraldehyde (48 h, 0.1 mol/L phosphate buffer, pH 7.2) and 2 % OS04 (1 h, the same buffer and pH). After washing in buffer, the samples were dehydrated in ethanol, critical point dried in liquid CO 2 , sputter coated with 20 nm layer of gold-palladium, and observed with a JEOL JXA 840A (JEOL, Japan) microscope. Samples for transmission electron microscopy (TEM) were fixed in 5 % glutaraldehyde (5 h, 0.1 mol/L phosphate buffer, pH 7) and 1 % OS04 (2 h, 0.1 mol/L phosphate buffer, pH 7), dehydrated in acetone and embedded in Durcupan ACM (Fluca, Buchs, Switzerland). Ultrathin sections were stained according to Reynolds (1963) and observed in ATEM 2000FX (JEOL, Japan) and TESLA BS 500 (Tesla, Czech Republic) electron microscopes. Histological and cytological analyses of shoot regeneration were performed at the light microscopy level. Paraffin sections (7-10 lim) were prepared after sample fixation in FAA (formalin 40 %, acetic acid 5 %, ethyl alkohol 50 %), embedded in Histoplast S (Serva, Heidelberg, Germany) and stained with hematoxylin-eosin, PAS reaction or Feuglen reaction. Sections (1-2 lim thick) for the fine cytological observation were prepared from the samples embedded for TEM. After SEM observation, some dried bulk samples were immersed in ethanol and embedded in Histoplast S (Serva, Heidelberg, Germany). The sections (7-10 lim thick) were observed without additional staining under bright-field microscopy (Ovecka and Bobak 1999). All prepared samples were studied using ZEISS Jenalumar (Carl Zeiss, Jena, Germany) dissection light microscope. Pictures digitalised using a Kappa CF 8/1 FMCC camera (Kappa Messtechnik, Germany), and a Leica Q500MC image analysis system (Leica Cambridge Ltd. England, UK) were processed using Corel Photo Paint 8 (Corel Corporation, Canada).
Morphometry All planimetric measurements of meristemoid and shoot primordia cells during indirect shoot regeneration were made using an ASBA (Wild, Heerbrugg, Switzerland) image analyser. Cell size, cell shape, and nucleus size were measured on the basis of cell cross-section area in 1-2 lim thick sections. The mean values were compared among the above-mentioned three culture media with different amounts of growth regulators. The cell shape (form factor) was calculated from the cell area and cell projections (a, b, a+b, a-b). The form factor 5.09 means a circular shape, 6 means a square and 7 a rectangle shape with a side ratio of 2: 1 (Baluska et al. 1990). Morphometrical values of cells in central and peripheral zones of the regenerated shoot apical meristems were measured as standards for the comparative characterisation of meristemoid cells. The computed sizes, shapes of cells, and N/C ratios were used as parameters of meristemoid cell activity (cell division and cell expansion). Linear regression analyses, coefficients of correlation, and N/C ratios were calculated using Sigma Plot (Jandel Scientific, USA) statistical software.
Shoot regeneration in opium poppy
Results Shoot regeneration was initiated from hypocotyls of primary somatic embryos (Fig. 1 a). The emerging shoot primordia appeared on the hypocotyl surface of the somatic embryo hypocotyl, but they did not disrupt the surface tissues (Fig. 1 b). In addition, no general tissue reorganisation was observed (Fig. 1 b). Due to these features, this kind of regeneration was termed direct shoot organogenesis in this study. Indirect shoot regeneration involved tissue dedifferentiation, callus formation, and cell re-differentiation leading to de novo shoot regeneration (Fig . 1c). Tissue organisation and cell morphogenesis of indirectly regenerated shoot primordia were completely different in comparison to underlying callus tissue (Fig . 1d).
Direct shoot regeneration Surface integrity of the embryo hypocotyl suggested that adventitious shoots arose from superficial cell layers (Fig . 1b). Initially, sub-epidermal cells were activated and divided. The plane of the first cell division seemed to be anticlinal (Fig. 2 a), but subsequent cell divisions were observed in both anti- and periclinal planes (Fig. 2 b). Afterwards, dividing subepidermal and epidermal cells created organogenic nodules, the first recognisable structures on the hypocotyl surface (Figs . 2c, d). The two most important morphogenetic features of these cells were dense non-vacuolated cytoplasm (Figs. 2 a , b,c) and reduced cell size (Fig . 2d). The first apparent indication of cell specification was observed in the phase of apical meristem formation . The cells
Figure1. Shoot buds regenerated in the callus culture of P somniferum l. a. Somatic embryo (SE) in the culture where
the shoot (SH) regeneration from the hypocotyl was induced. Bar = 1mm. b. Directly regenerated shoots with leaf primordia, visible as protuberances on the hypocotyl surface of somatic embryo. Bar = 100 11m. c. Shoot bud regenerated in the organogenic callus culture. The white compact tissue represents meristemoids. Bar = 1 mm. d. Shoot primordium on the surface of callus tissue. Bar = 1001lm.
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within nodule apices adopted tunica-corpus zonation (Figs. 2 e, f) . The original epidermal cells participated in differentiation of the tunica layer (Fig . 2 c) . This explains why the tunica and protomeristem maintained morphological and histological continuity between the forthcoming buds and the remaining hypocotyl surface (Figs. 1 b, 2 c , d,g). Cell specialisation continued by both histodifferentiation of the shoot apical meristem (Figs. 2 e, f) and transformation of organogenic nodules into buds (Figs. 2 g ,h). Adventitious buds were visible on the hypocotyl surface (Figs . 2 g h) , and regular activity of the apical meristem was detected, including formation and growth of leaf primordia (Figs. 2 h ,i).
Indirect shoot regeneration Distinct mode of indirect induction of organogenesis was based on callus production. Cell activation triggered a morphogenetic switch in some cells within rarely dividing callus tissue , resulting in the establishment of dividing , meristemlike, and shoot-forming tissue (Fig . 3a). Typical features of meristemoids (representing population of competent cells) were small cell size, cytoplasmic density, minimal vacuolation (Figs . 3 a b), , and cell adhesion after cell division (Figs . 1d, 3 a ,b, c). The first cell differentiation events within the multicellular meristemoids resulted in the formation of both peripheral and central cell layers, with only peripheral cells continuing their divisions (Fig. 3 b). Reserves were stored in central , and lipids (Figs. 3 e ,f). cells in the form of starch (Figs . 3 b d) These cells were non-dividing but their cytological parameters indicated high metabolic activity. Frequent endo- and exocytosis in these cells (Fig . 3e) and cell wall ingrowth for-
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Figure2. 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 flm b. Subsequent anticlinal and periclinal cell divisions changed size and shape of sub-epidermal activated cells. Bar = 50 flm. 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 11m. d. Organogenic nodules visible on the hypocotyl surface. Bar = 50 11m. e. Concentration of cell proliferation in the centre of organogenic nodule. Bar = 50 flm. f. Protomeristem with established tunica-corpus zonation in the stage of protomeristem transformation into shoot apical meristem. Bar = 50 flm. 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 flm. h. Surface view on the shoot apical meristem with well defined three layered tunica (arrow) and initiation of leaf primordia outgrowth (arrowheads). Bar = 100 flm. i. Developing leaves from the flattened apical meristem of adventitious shoot bud. Bar = 100l1m.
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. 4c, 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. 6a), 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 peri ph-
Shoot regeneration in opium poppy
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Figure3. Indirect shoot regeneration . 1. Cell determination.
a. Production of meristemoids with in the callus tissue. Bar = 50 ~m . 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 cell s accumulating starch granules. Bar = 50 ~m . 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~m . d, e, f, g, - Ultrastructure of the central meristemoid cells. d. Transparent cytoplasm and large deposits of starch in the central cells. Bar = 10 ~m . e. Insertion of vesi cular a nd matrix material into the cell wall of central meristemoid cells by exocytos is. L-lipid droplet. Bar = 2f1m. f. Lipid droplets which fill up the cells in the transition layer between centre and periphery of the meristemoid . Bar = 10~m . g. Cell wall ingrowths (asterisk) in the central meristemoid cell . Note the presence of numerous mitochondria. Bar = 21J.m.
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. 7a, 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 or-
ganogenesis 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 d si tinct 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, Bobak 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 cal lus 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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Figure4. Indirect shoot regeneration. 2. Formation of shoot primordia. a. Morphogenesis of peripheral meristemoid cells during shoot primordia formation. Cells expanded in anticlinal direction (arrowhead) and subsequently divided periclinally (arrows). Bar = 50 11m. b. Establishment of the tunica layer on the meristemoid surface by anticlinal cell division. Bar = 50 11m. c. Histodifferentiation of the shoot apical meristem (SAM). Bar = 50 11m. d. Cell proliferation concentrated in the dome-like protomeristem. Cells located away of the centre of cell division are devoid of starch and start to vacuolate. Bar = 50 11m. e. Developed shoot bud with leaf primordia and vascular tissue (VT) in the procambium. Bar = 100 11m. f. Laticifer initials (arrows) in the procambial region of shoot apical meristem. Bar = 50 11m. g. Initial perforation of cross cell wall between two laticifer initials. Bar = 50 11m. h. Formation of articulated, anastomosing laticifer system. Note the elastic properties of longitudinal cell wall in the site of future perforation (arrow). Bar = 50 11m. i. Insertions of cell volume between future anastomosing laticifers in cross-section (arrows). Bar = 50 11m. j. Articulated, anastomosed laticifer system in shoot procambium. Bar = 50 11m. k. Articulated, anastomosed laticifer system in developing leaves of regenerated shoots. Bar = 50 11m.
90
II
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Figure 5. Similarity in the correlation curves between cell size and nucleus size of peripheral meristemoid cells (-e-) and cells of peripheral and central zones of the apical meristems in regenerated shoots (-0-). 30-50 cells of each zone were measured on the medium supplemented with 0.537Ilmol/L of a-naphtaleneacetic acid and 0.46Ilmol/L kinetin (I), or with 0.577llmoI/L indole-3-acetic acid and 2.221lmo1/L 6-benzylaminopurine (II), or growth
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cell area (11m2)
Direct shoot formation was initiated in the culture where somatic embryos had failed to develop correctly, but maintained continuously juvenile, undifferentiated state of their cells in the root pole which were able to generate new somatic embryos (Ovecka et al. 1997/98). However, as we show here, adventitious shoots originated from the differentiated, non-dividing tissue of hypocotyl. The competent cells have to be re-activated and they enter cell division. The culture un-
3DD
400
regulator-free medium (III). Cell size values and nucleus size values are presented as planimetric values of cell and nucleus area measured from cross sections.
derwent spontaneous repetitive regeneration including shoot organogenesis and somatic embryogenesis (Ovecka et al. 1997/98). Some internal factors determining cell competence in the course of shoot bud regeneration have been identified, such as reduced .cellulase activity in tobacco callus culture (Truelsen and Ulvskov 1995), or importance of polar auxin transport, because its inhibition by TIBA (2,3,5-triiodobenzoic acid) strongly enhanced shoot regeneration, but not somatic
Shoot regeneration in opium poppy Figure6. Mean of the cell cross section area (a), and mean of the form factor describing cell shape (b) , observed during shoot regeneration in the medium supplemented with 0.5371lmo1/L of a-naphtaleneacetic acid and 0.4611mo1/L kinetin. Peripheral meristemoid cells (A) were compared to cells of central and peripheral zones of regenerated shoot apical meristems (8), and central meristemoid cells (e) were compared to cells in the rib-zone of shoot apical meristems (D). Percentage amount of cells with isodiametric (a side ratio app. 1 1), : and oval shape (side ratio up to 2: 1) is indicated (b). The rest of the cells had a more elongated shape. Results from measurements of app. 300 cells of each selected part of meristemoids and shoot apical meristems ... - significant at P<0.01 according to t-test.
a -
-
it
-=
,.-.it it
7.4
b
r=-it it
it
~
6~
rff%
o 7.0 6.9
.. 6.8 6.7 .. 6.56.6
A B
CD
it it
c- 7.2 88
c-
7.0
c- 6.8
-
A B
287
m
r- 6.6
CD
- 6.2
-
6.4
... 0
u ~
j
6.0
a
b
!
0
t;
J! E
0
Figure 7. a. Negative correlation between cell size and cell shape in meristemoids (r = -0.51) . b. Negative correlation between shoot apical meristems of regenerated shoots (r = -0.343). Mean values of cell sizes and cell shapes (form factors) of all measured cells in the present study were compared.
6.4 6.3 100
150
200
100
150
200
embryogenesis (Tetu et al. 1990). The study of morphology
1995, Gilissen et al. 1994). This data indicates that a limited
and conversion ability of poppy somatic embryos revealed
number of participating cells, even with a distinct fate before
some structural abnormalities (fused cotyledons, embryo
activation, can form shoot primordium through regulated and
twins, and developmental arrest with continual cell prolifera-
co-ordinated cell morphogenesis.
tion, Ovecka et al. 1996) which resembled abnormalities of
The second morphological aspect of direct shoot regen-
Panax ginseng C. A. Meyer somatic embryos reported after
eration was an apparent participation of the hypocotyl epi-
polar auxin transport inhibition (Choi et al. 1997). From this point of view it seems that discrepancy in endogenous hor-
dermal cells in the formation of shoot apical meristem. Epidermis is formed by polarised cells. However, epidermal
mone (especially auxin) level would be an internal selecting factor supporting activation of competent cells in the embryo
cells were reported to be competent in some models of direct shoot regeneration. Meristematic regions formed by anticlinal
hypocotyl. The two most important aspects of direct poppy shoot re-
and periclinal divisions of epidermal cells, and even sub-epidermal cells, as well as continuity of meristematic domes of
generation were connected with proper regulation of morphogenesis in multicellular organogenic nodule. First, all activated cells were synchronised and became determined and
developing shoot primordia with the remaining epidermis have been observed (Tetu et al. 1990, Bronner et al. 1994, Burritt and Leung 1996, Konieczny 1996). This is a strong in-
thus were involved in shoot primordia formation . In periclinal
dication of a totipotent nature of epidermal cells (Burritt and
chimeras of Nicotiana tabacum L. and Nicotiana g/auca
Leung 1996, and references therein). Activation and periclinal
Grahm . it was clearly shown that shoots can originate in any
cell division of epidermis are effective mostly in organogenic
cell layer of the leaf tissue , and the origin of some shoots can
nodule formation, while each anticlinal cell division maintain-
be multicellular and multihistogenic (Marcotrigiano 1986). In
ing original cell polarity participates in the further tunica forma-
contrast to the cortex layer, in shoot-forming thin cell layers of
tion of the shoot primordium derived from sub-epidermal tissue
Nicotiana tabacum L., the cell expansion in epidermis and
(Fig. 2). These observations document the importance of an
sub-epidermal layers was limited and initial periclinal cell di-
early regulation of the plane of cell division during the shoot
vision indicated a modification of cell polarity (Altamura et al.
apical meristem formation in directly regenerated shoot buds .
288
Miroslav Ovecka, Milan Bobak, Jozef Samaj
Shoot regeneration in callus culture requires a combination of auxins and cytokinins (Christianson and Warnick 1983, De Klerk et al. 1997). The main change in cell division and cell expansion was observed during competence acquisition. Meristemoids were produced from rarely dividing callus cells. Cell division was more patterned within meristemoids. Their cells were small, micro-vacuolated, and able to stick together. This indicates that the fate of the callus culture is altered through cell morphogenesis of the activated competent cells as a result of the action of growth regulators. The second switching point in cell morphogenesis of poppy callus culture occurred in the multicellular meristemoids as a defined step of cell determination within the shootforming tissue. A critical event was the localisation of cell division to the meristemoid periphery. The cell determination has been expressed when modified polar periciinal cell division occurred in meristemoids. Apical daughter cells generated dividing peripheral layers with morphogenetic competence while the distal daughter cells became more differentiated and generated storage cells possessing metabolic sources. The previously observed differences in cell size and shape showed that the peripheral cells were smaller, having an oblong shape as a result of frequent cell division, while the central cells became larger due to globular expansion (Ovecka et al. 1997). Starch-containing cells were the largest ones. They had isodiametric shape, and any changes (cell division, plastid division, vacuolation) took place only after starch utilisation had been initiated (not shown). Central meristemoid cells thus can support organogenesis by the energy. It was clearly shown that some metabolic indications of shoot regeneration (protein inclusions, starch, lipids, high enzyme activities) were found only in meristemoids (Ross et al. 1973, Nessler and Mahlberg 1979, Patel and Berlyn 1983). Meristemoids possessed a high starch content, while high activities of starch-degrading enzymes were observed in shoot buds (Thorpe and Murashige 1970, Thorpe and Meier 1974, Patel and Berlyn 1983). A high N/C ratio of peripheral meristemoid cells of opium poppy, comparable with cells in shoot apical meristem (Fig. 5), together with comparable rate of chromatin de-condensation (Ovecka et al. 1997) are also important parameters of cell determination within the meristemoids. Variability in the size of nucleus and the correlation with DNA amount is closely related to shoot regeneration (Flinn et al. 1989, Fournier et al. 1991). A two-phase organogenesis, with meristemoid formation during the first phase and formation of globular structures from the surface during the second phase, was observed in sundew callus tissue (Bobak et al. 1993). This data indicates that "mitotic zonation" established a distinct morphological competence of mer istemoid cells in the process of shoot primordia formation when the site of origin of shoot buds was located at the meristemoid periphery. Concluding our comparative analysis of direct and indirect shoot regeneration in P somniferum L., important changes in cell morphogenesis, characterised by non-random cell divi-
sions, took place during dermal layer and protomeristem formation in shoot primordia. We observed a random orientation of cell divisions in meristemoids, while protomeristem and especially tunica formation were accompanied by regulated periclinal and subsequently anticlinal cell divisions. In directly regenerated shoots, the dermal layer was formed by original epidermal cells through anticlinal cell divisions. This type of division maintains epidermal cell polarity. Peripheral meristemoid cells are polarised with non-homogenous cell surfaces due to the presence of adhered and outer non-adhered cell walls (Ovecka and Bobak 1999). This polarity and cell position are important morphogenetic factors in cell determination. Cell expansion was found to be minimal in cells involved in formative, morphogenetically important cell diviSion. The cells expanded, but not over the cell volume necessary for a subsequent cell division. In addition, cell size and cell shape have to be negatively correlated. This relationship would indicate an independence of post-mitotic cell growth and an extensive cell vacuolation, originally observed in maize root apex (Baluska et al. 1990). A comparative analysis revealed that cell activation in shoot organogenesis of P somniferum L. depends on the stage of cell differentiation before initiation, while cell morphogenesis of the determined cells is similarly regulated both spatially and temporally, during direct and indirect shoot regeneration. Acknowledgements. We gratefully acknowledge D. Jantova for technical assistance, and Dr. M. Ciamporova for the English correction. This work was supported by Grant Agency VEGA, Grant No. 1/6030/99 and Grant No. 1/6107/99 from the Slovak Academy of Sciences. M.O. gratefully acknowledges the Alexander von Humboldt-Stiftung (Bonn, Germany) for donation of the Leica Q500MC image analysis system.
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