Differences in cytokinin control on cellular dynamics of zucchini cotyledons cultivated in two experimental systems

The effect of endogenous cytokinins on the pattern of palisade cell division post-germination does not depend on the conditions of cotyledon development -in planta (attached to seedlings) or in vitro (isolated from dry zucchini seeds and cultured on water). In cotyledons originating from 4-day-old seedlings (experimental system 1), exogenous cytokinin temporarily (in the first 2 day of cultivation) enhanced post-mitotic cell enlargement of palisade cells, mainly due to enhanced water uptake and use of cell storage compounds, all of which lead to cotyledon senescence. Cytokinin is not able to resume the completed palisade cell division on day 5. As a result, the number of cells and the final areas of treated and control cotyledons are quite similar. By contrast, the effects of cytokinin on cotyledons isolated from dry seeds (experimental system 2) are better expressed, promoting an increase in number of palisade cells accompanied by additional cotyledon area enlargement. However, the prolonged post-mitotic cell expansion in control cotyledons compensates for the reduced speed of cell growth and division activity and decreases differences in final cotyledon area between treatments. The results define cell division as the primary target of cytokinin stimulation in cotyledon tissues competent for division, and determine the temporal patterns of palisade cell cycling related to cotyledon age. This knowledge permits a better choice of experimental system to study effects on cell proliferation and cell growth, as well as cell enlargement and senescence-related events using physiologically homogeneous material.     

Cell division and cell enlargement in isolated Cucurbita cotyledons grown in darkness and in light

The spatial and temporal patterns of post-embryonal cell growth and cell division were characterised in excised cotyledons of vegetable marrow (Cucurbita pepo L. var. giromontia Alef.) incubated in water. The concurrent roles of these two processes in cotyledon growth were determined using paradermal sections of the first palisade layer of developing cotyledons. Tissue specificity was observed in the pattern of cell division. The daughter cells derived from an initial cell, which had already differentiated before imbibition of the seeds, were tightly packed in a cluster, which enabled us to monitor cell division during early cotyledon development. Heterogeneity of cell size was recognised during the process of cell proliferation in the cluster, suggesting that cell division is uncoupled from control of cell size. There was significantly more cell division in the marginal part of the cotyledons than in other parts, suggesting high activity of the marginal meristem. Light enhanced cell and cotyledon enlargement, but had no effect on the number of divisions. This study elucidated the cellular basis of post-germinative Cucurbita cotyledon morphogenesis and development. Electronic Publication     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.     

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Differential effects of methyl jasmonate on growth and division of etiolated zucchini cotyledons

The jasmonates are well studied in the context of plant defence but increasingly are also recognised as playing roles in development. In many systems, jasmonates antagonise the effects of cytokinins. The aim of the present work was to elucidate interactions between methyl jasmonate and cytokinin (benzyladenine) in regulating growth of zucchini (Cucurbita pepo L., cv. Cocozelle, var. Tripolis) cotyledons, taking advantage of the ability to simultaneously quantify cell enlargement and division from paradermal sections of the first palisade layer. Growth regulators were applied to cotyledons, excised from dry seeds and grown in darkness. Cytokinin stimulated expansion and division whereas, surprisingly, jasmonate stimulated expansion but inhibited division. Jasmonate antagonised the stimulating effect of cytokinin on division but worked cooperatively with cytokinin in increasing expansion. However, expansion with jasmonate was more isotropic than with cytokinin. Jasmonate also stimulated the loss of cellular inclusions and soluble protein. Soluble proteins revealed a partial antagonism between jasmonate and cytokinin. These results illustrate the complex interplay between jasmonates and cytokinin in the regulatory network of cotyledon development following germination.     

Effects of Benzylaminopurine and Abscisic Acid on Distribution of rRNA in the Palisade Cells of Excised Cucurbita Pepo Cotyledons

rRNA was labelled with RNase-gold complexes applied to ultrathin Lowicryl sections of excised Cucurbita pepo L. (zucchini) cotyledons grown in darkness. Benzylaminopurine-caused stimulation of cotyledon growth decreases the density of ribosomes in palisade cells despite stimulating rRNA synthesis. Abscisic acid inhibits RNA synthesis and transport, but does not visibly affect the number of pre-existing ribosomes in retarded growth cells. The amount of rRNA in the cells of 24 h treated cotyledons is rather consequence of the growth rate than its leading factor.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Cell division patterns in the apices of subterranean axis and aerial shoot of Psilotum nudum (Psilotaceae): morphological and phylogenetic implications for the subterranean axis

The cell division pattern in the apical meristem of Psilotum nudum was examined using epi-illumination microscopy and a paraffin method. In the subterranean axis, about half of the derivative cells of the apical cell produce tetrahedral daughter apical cells by the first three or more oblique divisions. Roughly half of these apical cells give rise to the apical meristems of axes, whereas the other half do not. Various relative activities of the mother and daughter apical cells give rise to disordered branching patterns. In the ill-organized apical meristem as well as the leafless and capless structure, the Psilotum subterranean axis differs from the basic organs of vascular plants such as stem and root and seems to be an independent organ. The cell division pattern characteristic of the subterranean axis persists in the young unbranched aerial shoots, although fewer daughter apical cells are produced. Dichotomous branching of the aerial shoots, as in a variety of organs of pteridophytes, involves loss of the mother apical cell followed by appearance of two daughter apical cells.     

Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana

Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis. Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes. But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions. In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana. The live imaging technique has led to the development of a spatial and temporal map of cell division patterns. We have integrated cell behavior over time to visualize growth. Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells. Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms. Cell division rates vary across the SAM surface. Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ). Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells. We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer. Primordium development is a sequential process linked to distinct cellular behavior. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth. The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud. Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM. We discuss these results in the context of SAM morphogenesis.     

Differential Responses of Buckwheat Leaf Cells to Growth Substances Stimulating Cell Division

Isolated buckwheat cotyledons form calli, roots or buds whencultured in an appropriate medium. A medium containing high2,4-D (5 mg 1^–1) and low KN (01 mg I^–1), which inducescallus formation, was found to stimulate cell division in thelayer between palisade and spongy parenchyma tissue after 72h. Low 2,4-D and low KN (01 mg I^–1 each), which stimulatesroot formation in buckwheat cotyledons, induces divisions primarilyin spongy parenchyma cells. In a high benzylaminopurine (10^–5M) and a low IAA (10^–6 M) medium, which favours bud induction,cell divisions were localized to the palisade layer. The differentialresponsiveness of leaf cells to various hormone treatments isdiscussed.     

Sélection,par les chromosomes en écouvillon,de lignées vectrices de mutations chez l'amphibien urodèle Pleurodeles poireti

Division of the guard mother cell (GMC) in Allium cotyledons has been examined in epidermal slices viewed with Nomarski optics and electron microscopy. Special attention has been directed towards elucidating the process by which the dividing cell determines its plane of division. In normal development, the cell plate formed during GMC division ultimately lies along the longitudinal axis of the cotyledon, in contrast to the transverse planes formed in other epidermal divisions. Our observations reveal that the final plane of division is not determined by the orientation of the spindle at metaphase but instead is established during late anaphase-telophase as a result of directed reorientation movements of the spindle-phragmoplast and associated daughter nuclei. The metaphase plate may lie at an oblique angle, even as great as 90°, from the final plane of the plate. Thus, daughter chromosomes separate into opposite corners of the cell. During late anaphase-telophase, movement of the spindle is activated; the daughter nuclei move along the sides of the cell while the interzone rotates. Movement continues until daughter nuclei reach positions opposite each other along the sides of the cell and the midzone or cell plate is positioned in the longitudinal orientation. Movement requires 15–20 minutes for completion, is highly directional, and does not overshoot the correct alignment. Following movement cytokinesis proceeds to completion forming two young guard cells. Possible mechanisms for reorientation are discussed, including one that suggests that interzone microtubules may interact with a cortical site on the plasmalemma adjacent to the end and paradermal walls. Such a site may be related to and governed by the same properties which controlled the prior formation of the preprophase band of microtubules in these cells.     

Evaluation of parameters affecting the yield, viability and cell division of Pinus pinaster protoplasts

Various factors affecting the yield and viability of Pinus pinaster Ait. cotyledon protoplasts and the mitotic activity of or regenerated cells are described. A study of the effect of sterilization procedures of the plant material showed that whereas the organs collected from disinfested seedlings allow for good yield and viability of isolated protoplasts, germination under non-sterile conditions favours a greater germinating capacity and stronger mitotic activity. Numerous clusters of from 10 to 15 cells were formed after 20 days of culture when a 5% aqueous solution of calcium hypochlorite was used as a sterilizing agent.
The effects of an additional purification of the enzymes showed that although yield and viability of the protoplasts are only slightly improved, the more highly purified enzymes on the other hand enhanced the mitotic activity markedly. Between the two total enzyme concentrations used (0.2 and 0.4%, and in which the relative ratio of each element was unchanged), only the lowest level supplied a debris-free protoplast suspension; mitotic activity occurred only in that case.
Comparison of the populations of cotyledon protoplasts collected from seedlings at two different growth stages (not fully-developed or fully-expanded cotyledons) did not reveal any appreciable difference in their size distribution. Neither was the extent of cellular viability affected by the degree of cell differentiation at the time of collecting. On the other hand, the yield of protoplasts and the mitotic activity of the regenerated cells were greater when partially-developed organs were used. Moreover a pretreatment of the elongating cotyledons with a mineral (half-strength MS macronutrients and full-strength micronutrients) and hormonal (15 μ M BAP, 0.5 μ M NAA) solution improved cell division frequency.     

Developmental process of sun and shade leaves in Chenopodium album L.

The authors’ previous study of Chenopodium album L. revealed that the light signal for anatomical differentiation of sun and shade leaves is sensed by mature leaves, not by developing leaves. They suggested that the two‐cell‐layered palisade tissue of the sun leaves would be formed without a change in the total palisade tissue cell number. To verify that suggestion, a detailed study was made of the developmental processes of the sun and shade leaves of C. album with respect to the division of palisade tissue cells (PCs) and the data was expressed against developmental time (leaf plastochron index, LPI). The total number of PCs per leaf did not differ between the sun and shade leaves throughout leaf development (from LPI ?1 to 10). In both sun and shade leaves, anticlinal cell division of PCs occurred most frequently from LPI ?1 to 2. In sun leaves, periclinal division of PCs occurred synchronously with anticlinal division. The constancy of the total number of PCs indicates that periclinal divisions occur at the expense of anticlinal divisions. These results support the above suggestion that two‐cell‐layered palisade tissue is formed by a change of cell division direction without a change in the total number of PCs. PCs would be able to recognize the polarity or axis that is perpendicular to the leaf plane and thereby change the direction of their cell divisions in response to the light signal from mature leaves.     

Teilungsverhalten der Chromatophoren in bezug auf die Mitose während des Lebenszyklus vonDiatoma hiemale var.mesodon

The vegetative life cycle ofDiatoma hiemale var.mesodon (Ehr.)Grun. living in a spring has been studied under natural conditions. In the beginning the cells have a constant number of 8 chromatophores which are divided into 16 during cell growth. Chloroplast division is finished before nuclear division starts. The young daughter cells have again 8 chromatophores. In the course of cell division a plastic remodelling of the chromatophores and a simplifying of their shape occurs. Besides single cells also populations have been studied to follow the temporal progress of chromatophore division, mitosis and cell growth. The results are evaluated by indices and demonstrated by a diagram. The maximum of chromatophore divisions preceds the maximum of mitoses by several hours, while the cell growth is in correlation with the chromatophore division. Minima of the other parameters were found before mitosis is starting and after it is finished. Our results are discussed with regard to the semiautonomy of the plastids. From the morphological point of view this concept is supported by the mode of division and by the anticipation of the chromatophore division. The number of chromatophores at the beginning (8) and at the end (16) of the life cycle is constant. The life cycle is classified into stages of cell growth, chromatophore division, stagnation, mitosis and differentiation of the daughter cells.     

Cell divisions in cotyledons after germination: localization, time course and utilization for a mutagenesis assay

Post-germinative proliferation of cells was studied in cotyledons of Nicotiana tabacum L., Petunia hybrida Vilm. and Arabidopsis thaliana (L.) Heynh. Patterns of cell divisions after germination were characterized by clonal analysis in cotyledons of N. tabacum. The fate of initial cells, which are formed by the end of embryogenesis, was quite variable: cells could undergo between one to seven, and most often, between three to five anticlinal divisions after germination. Sector shape suggested that there were more divisions in length than in width, particularly at the periphery of the cotyledon. The boundaries of clones generated by irradiation of mature seeds did not intersect the midvein, and in most cases, did not intersect lateral veins. The time course of cell divisions during post-germinative development was analyzed cytologically in cotyledons of N. tabacum and P. hybrida. No divisions were detected up to the second day after sowing (DAS), when the radicle emerged. Cotyledon cells started to divide at a rapid rate between 2 and 3 DAS, reaching a mitotic index of about 2% at 3–4 DAS. A rapid decline followed the peak, and no divisions were detected 6–7 DAS. Similarities between leaf and cotyledon development are discussed. In addition, we show that divisions in cotyledons of N. tabacum and A. thaliana chlorophyll mutants can be exploited for a quick and sensitive bioassay from which the effects of various mutagens and DNA repair genes can be assessed.     

The activity of total histone H1 kinases is related to growth and commitment points while the p13suc1-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda

Under optimal growing conditions, synchronous cultures of the alga Scenedesmus quadricauda underwent three DNA replications and three mitoses during one cell cycle. This resulted in eight daughter cells. By different illumination regimes and temporal addition of cycloheximide, cell divisions resulting in two, four or eight daughter cells per cycle were obtained. The selected cell cycle patterns differed in timing of commitment points, the number of nuclear divisions, and their positioning in the cell cycle. These distinct cell cycle patterns allowed to assess the correlation of histone H1 kinase activity with commitment points and mitoses. The activity of the histone H1 kinases was assayed in cellular protein extracts and after affinity purification using the p13^suc1 protein. The main peaks of kinase activity in the cellular extract were found to correlate with the commitment points. Small histone H1 kinase activity peaks were also found which preceded the nuclear division. Contrary to the histone H1 kinase activity of cellular extracts, the p13^suc1-bound kinase activity preceded the nuclear division, whilst its activity was negligible at the commitment points. Being able to manipulate the timing of commitment points and cell division by manipulating experimental conditions, we could precisely match the commitment points to an as yet unidentified histone H1 kinase activity and mitosis to p13^suc1-bound CDK activity during a particular cell division pattern with overlapping cycles. This provides molecular evidence, that local activation of CDKs regulates distinct events of the cell cycle.     

Maternal genotype influences pea seed size by controlling both mitotic activity during early embryogenesis and final endoreduplication level/cotyledon cell size in mature seed

When reciprocal crosses are made between different pea genotypes, there is a strong maternal influence on mature seed size of the reciprocal hybrids, i.e. their dry weights are similar to that of seeds obtained from their maternal parents. Reciprocal crosses between pea varieties having very different mature seed sizes were used to investigate how the maternal genotype controls seed development and mature seed size. The differences in dry seed weight between genotypes and reciprocal hybrids reflected differences in both cotyledon cell number and mean cell volume, and the maternal control on the establishment of these two traits was investigated. Using flow cytometry, data relative to endoreduplication kinetics in cotyledons during the transition between the cell division phase and maturation were obtained. The appearance of nuclei having an 8C DNA content indicates the initiation of the endoreduplication phenomenon and thus the end of the cell division phase. It was shown that the duration of the cell division phase was the same in the reciprocal hybrids, its value being intermediate between those recorded for their maternal parents. This result indicates that the timing of development of the embryo is not under maternal control, but depends on its own genotype. Consequently, maternal genotype must influence the mitotic rate during the cell division phase to achieve differences in cell number found in the cotyledons of mature F1-reciprocal hybrids. The final level of endoreduplication in cotyledons of mature seeds was also investigated. This study showed that there is a close relationship (r2 = 0.919) between the endoreduplication level in mature cotyledons and seed dry weight or mean volume of cotyledon cells, suggesting that both maternal and non-maternal factors could control the number of endoreduplicating cycles in the cotyledons and, hypothetically, the cotyledon cell size.     

Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

Explants of mature cotyledons of Phaseolus vulgaris form callusrapidly when cultured in vitro with their adaxial surfaces embeddedin a solidified nutrient medium containing coconut milk, kinetinand 2,4-D. Proliferation is confined to the highly polyploidstorage cells and commences near the adaxial epidermis, whichis soon ruptured by the callus developing internally. Callusformation progresses to the abaxial tissue and within 3–4weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls,loss of food reserves, hypertrophy, development of various new-wallsand nuclear activation leading to division. The induction ofnuclear and cell divisions within this mature storage tissuecontrasts with normal germination in which these cells undergorapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainlyamitotic. It is preceded by the development of multiple nucleoli.The nuclear envelope also becomes more complex and deeply lobed;leading to formation of a nuclear isthmus and final separationinto two nuclei. No chromosomes are visible during nuclear fragmentation.Amitosis is accompanied by freely-forming walls, which may developadjacent to a nuclear isthmus and perhaps participate in nuclearfragmentation. Large labyrinthine wall bodies frequently occuron these walls. Mitoses are only observed in already dividedstorage cells. A cell plate forms between the two daughter nuclei,and microtubules are present at its margins in contrast to freely-formingwalls where none are evident. Phaseolus vulgaris L., bean, in-vitro culture, cotyledon, ultrastructure     

The induced sector Arabidopsis apical embryonic fate map

Creation of an embryonic fate map may provide insight into the patterns of cell division and specification contributing to the apical region of the early Arabidopsis embryo. A fate map has been constructed by inducing genetic chimerism during the two-apical-cell stage of embryogenesis to determine if the orientation of the first anticlinal cell division correlates with later developmental axes. Chimeras were also used to map the relative locations of precursors of the cotyledon and leaf primordia. Genetic chimeras were induced in embryos doubly heterozygous for a heat shock regulated Cre recombinase and a constitutively expressed beta-glucuronidase (GUS) gene flanked by the loxP binding sites for Cre. Individual cells in the two-apical-cell stage embryo responding to heat shock produce GUS-negative daughter cells. Mature plants grown from seed derived from treated embryos were scored for GUS-negative sector extent in the cotyledons and leaves. The GUS-negative daughters of apical cells had a strong tendency to contribute primarily to one cotyledon or the other and to physically adjacent true leaf margins. This result indicated that patterns of early cell division correlate with later axes of symmetry in the embryo and that these patterns partially limit the fates available for adoption by daughter cells. However, GUS-negative sectors were shared between all regions of the mature plant, suggesting that there is no strict fate restriction imposed on the daughters of the first apical cells.     

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.