Chromosome Number and Genome Size Evolution in Brasolia and Sobralia (Sobralieae, Orchidaceae).

Despite the clear circumscription of tribe Sobralieae (Orchidaceae), its internal relationships are still dubious. The recently delimited genus Brasolia, based on previous Sobralia species, is now assumed to be paraphyletic, with a third genus, Elleanthus, nested in it. The morphology of these three genera is significantly different, indicating the necessity of new data for a better genera delimitation. Though morphology and molecular data are available, cytogenetics data for Sobralieae is restricted to two Sobralia and one Elleanthus species. Aiming to evaluate the potential of cytogenetic data for Brasolia-Elleanthus-Sobralia genera delimitation, we present chromosome number and genome size data for 21 and 20 species, respectively, and used such data to infer the pattern of karyotype evolution in these genera. The analysis allowed us to infer x = 24 as the base chromosome number and genome size of average 1C-value of 5.0 pg for the common ancestor of Brasolia-Elleanthus-Sobralia. The recurrent descending dysploidy in Sobralieae and the punctual genome upsize suggest a recent diversification in Sobralieae but did not allow differing between Brasolia and Sobralia. However, the basal position of tribe Sobralieae in the subfamily Epidendroideae makes this tribe of interest to further studies clarifying the internal delimitation and pattern of karyotype evolution.

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis.

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the ß-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae).

Background and aims: In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods: A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key Results: Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion: These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

MAIN CONCLUSION: The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

Taxonomic placement of Paphiopedilum canhii (Cypripedioideae; Orchidaceae) based on cytological, molecular and micromorphological evidence.

Paphiopedilum canhii was discovered in Northern Vietnam. Since its description in 2010, it has caused a stir among taxonomists due to its interesting mixture of morphological features, i.e. marbled, relatively thick leaves, flowers similar to species classified in the section Barbata, and unique, large staminodial shield. On the basis of these features, it is difficult to classify the species to existing infrageneric units. Using cytological data, phylogenetic analyses based on plastid and nuclear genes and the study of the adaxial epidermis of the leaves and gynostemium structure obtained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM), the taxonomic position of P. canhii was determined. These results suggest that P. canhii forms an independent phylogenetic line within the genus Paphiopedilum deserving subgeneric status, already proposed by Braem and Gruss (2011) as Megastaminodium.

Cutin plays a role in differentiation of endosperm-derived callus of kiwifruit.

Cutin fluorescence, after auramine O treatment, was detected on the surface of organogenic areas (protuberances) of endosperm derived callus induced on Murashige and Skoog medium with thidiazuron (0.5 mg l(-1)) in darkness. Electron micrographs of the protuberances revealed cuticle, visible as a dark-staining layer, and amorphous waxes on the cell wall. In some cases the cells of the epidermis-like layer and shoot buds at early stages of development showed thick and characteristically wavy cutin. This waviness corresponds with the wrinkled appearance of the cell wall as observed by scanning electron microscopy. The role of multivesicular bodies in cutin production and transfer to the plasma membrane is discussed.

Seasonal and Simultaneous Cleistogamy in Rostrate Violets (Viola, subsect. Rostratae, Violaceae).

The special mixed reproductive system, i.e., the ability of an individual plant to develop both open, chasmogamous (CH) flowers adapted to cross-pollination and closed, cleistogamous (CL) flowers with obligate self-pollinating, is a common phenomenon in Viola L. In most sections of Northern Hemisphere violets, cleistogamy is seasonal, and CH and CL flowers develop sequentially in the season. Non-seasonal cleistogamy (simultaneous) is a rare phenomenon in rostrate violets. In the current study, we focused on modification of the CH/CL mating system in V. caspia by environmental conditions, resulting in a gradual switch from temporal cleistogamy, occurring in nature, to simultaneous cleistogamy under greenhouse conditions. V. reichenbachiana with seasonal cleistogamy was the control for V. caspia with the labile seasonal/simultaneous cleistogamy system. In simultaneous cleistogamy, the CH and CL flowers, fruits and seeds developed on an individual plant at the same time on the same branch. The typical difference between CH and CL flowers' pistils is a straight style ending with a head-like stigma in CH and a curved style in CL adapted to self-pollination. This trait persists in the fruit and seed stages, allowing for easy recognition of fruit of CL and CH flowers in simultaneous cleistogamy. Floral meristems of CH flowers of V. reichenbachiana developed on the rhizome at the end of the growing season under short-day conditions and remained dormant until the following season. The CL floral meristems formed under long-day conditions on elongating lateral branches in the upper leaf axils. The daily temperature influenced the variable CH/CL ratio of V. caspia in nature and greenhouse conditions. Regulation of the CL/CH flower ratio by modifying environmental factors is important for basic research on genetic/epigenetic regulation of cleistogamy and for practical use to produce genetically stable lines of economically important species via CL seeds.

Behavioral and physiological effects of Viola spp. cyclotides on Myzus persicae (Sulz.).

Cyclotides are defense peptides produced by several plant families. Viola spp. (Violaceae) produce an array of cyclotides with varying biological activities. The peach potato aphid Myzus persicae (Sulz.) (Hemiptera: Aphididae) is a generalist that feeds on the secondary hosts of over 40 plant families, including Violaceae. The present work aimed to evaluate the activities of cycloviolacins from Viola odorata L. and V. ulignosa Besser (cyO2, cyO3, cyO13, cyO19) against M. persicae. To investigate the peptides' influence on aphid feeding behavior, we used 20% sucrose diets supplemented with cyclotides and measured the effects with electrical penetration graph (EPG) technique. We also applied anti-cyclotide antibodies and immunohistochemistry to track the peptides in the digestive systems of the aphids. Our study shows that cyclotides affect aphid probing and feeding behavior and limit their diet sap uptake. The cycloviolacin cyclotides: cyO13 (100 µM) and cyO19 (50 µM) most strongly impeded aphid ingestion activities when applied in sucrose diet. Sustained ingestion of the diet was blocked by 100 µM cyO13, and no aphid showed ingestion of the diet for longer than 10 min. Cyclotides were detected in the pharynx, in contact with the epipharyngeal gustatory organ, in the stomach (midgut) and upper intestine. The present study shows the deterrent activity of cycloviolacins on M. persicae. This activity may be related to the peptides' effects on epithelial cells and gustatory organs along the aphid digestive system. We demonstrate that cyclotides may play an important role in plant-aphid interactions.

Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae).

Two representatives of section Lepidorhiza, previously sometimes considered conspecific, Bulbophyllum levanae and Bulbophyllum nymphopolitanum, demonstrated both similarities and differences in floral features. There were significant differences in the length of sepals and micromorphological features of the labellum. In both species, osmophores are located on the extended apices of sepals and possibly on petals. An abundance of proteins in tepals is probably associated with the unpleasant scent of the flowers, whereas the thin wax layers on the epidermis are probably involved in the maintenance of the brilliance of floral tepals, which strongly attracts flies. In all tepals of both species, we noted the presence of dihydroxyphenolic globules in the cytoplasm after staining with FeCl3. Comparison with ultrastructure results revealed that they were associated with plastids containing plastoglobuli. The most remarkable feature was the presence of a prominent periplasmic space in the epidermal cells of both investigated species. Furthermore, in the labellum of B. levanae, the cuticle contained microchannels. The combination of periplasmic space and microchannels has not previously been recorded.

How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System.

Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 µM to 25 µM. Inhibition of phytopathogenic bacteria - Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum - is also observed with MIC = 25-100 µM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Studies on floral nectary, tepals' structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae).

The lip of Epipactis palustris consists of two movably joined parts: the basal part (hypochile) with central broad isthmus and epichile with callus. The analysis of flowers provides strong evidence to conclude that the whole surface of lip callus and abaxial side of isthmus are secretory. The exudation at first appears on callus, at early stages, later on isthmus. It could be a strategy to prolong the emission of volatile substances and nectar, and this means to prolong luring pollinators. The results from transmission electron microscopy (TEM) support this conclusion. The plastids noted in callus were without starch, whereas the isthmus' cells contained partly hydrolyzed starch. Some plastids, noted in callus, had polymorphic shapes, which were often related to a starch reduction. During the depletion of starch in callus cells, the number of plastoglobuli within the plastids increased, and also lipid bodies appeared in the cytoplasm whereas, in isthmus cells, proplastids with phytoferritin were noted. The endoplasmic reticulum was in contact with plasmalemma, and the vesicles were fusing with plasmalemma in secretory cells of callus and isthmus, which is a way of granulocrine secretion. The cross-sections of sepals revealed that abaxial epidermis was tomentose, with stomata at the top of substomatal cavities. The pollen grains adhering to the rostellum-viscidium prove previous ecological observations that the rostellum-viscidium is not a barrier preventing self-pollination.

The growth and differentiation of aortal smooth muscle cells after calcitriol treatment are associated with microtubule reorganisation -- an in vitro study.

The smooth muscle cells (SMCs) of the arterial media play a predominant role in functional and structural alterations of the arterial wall. The transition from the "contractile" to the "synthetic" phenotype appears to be an early critical event in the development of atherosclerotic disease. A number of observations suggest that 1,25(OH)(2)D(3) (calcitriol) is of importance in maintaining normal cardiovascular function through its receptors in cardiac myocytes or aortal SMCs. The present study has focused on the microtubular (MT) network reorganisation after exposure to calcitriol. SMCs isolated by enzymatic digestion from the aortal media of neonatal rats were cultured on glass cover slips. 1 microM of 1,25(OH)(2)D(3) was added to the culture medium every second day. The cytoskeletal features of SMCs after calcitriol were visualised by the immunofluorescence staining of alpha-tubulin. The alterations in alpha-tubulin expression and the distribution of microtubules related to the activities of the vascular smooth muscle cells, namely adhesion, migration, multilayer formation and cell division, were observed. A spindle shape, decreased cell adhesion, low expression of alpha-tubulin and a longitudinally arranged microtubular network manifested the high rate of SMC differentiation in the calcitriol-treated culture. A flat stellate morphology, high expression of alpha-tubulin and a radially distributed three-dimensional microtubular network were observed in the SMCs of the control culture. Destructive changes in the microtubular architecture which altered the cellular shape were evident in SMCs undergoing apoptosis. Cells with apoptotic features were more frequent in calcitriol-exposed culture. In contrast to the regular SMC divisions observed in the control culture, some of the mitotic cells exposed to calcitriol contained broader bipolar, multipolar or disordered spindles. These alterations in the SMCs' microtubular cytoskeleton after calcitriol treatment were concomitant with changes in cell growth, differentiation and apoptosis, and may suggest a similarity to atherosclerotic plaque formation.

A scanning electron microscopic study of phenotypic plasticity and surface structural changes of aortal smooth muscle cells in primary culture.

Phenotypic modulation of smooth muscle cells (SMCs) from a contractile to a synthetic state characterised by active proliferation appears to be an early event in the pathogenesis of atherosclerosis. A similar transition occurs when SMCs are established in culture. In this study the phenotypic plasticity and surface structural changes of aortal smooth muscle cells during the transition from the contractile to the synthetic state and during maturation have been structurally assessed by scanning electron microscope (SEM). The experiments were performed on SMCs obtained from aorta of neonatal rats after enzymatic digestion and then cultured on glass coverslips. SEM observations revealed a three-dimensional appearance characteristic for different stages of SMCs. Intensively proliferating cells from monolayer region were large, polygonal in shape with lamellipodia and well spread. Long, uniform in diameter, finger-like microvilli were densely arranged on the surface of these cells. In the thickened region of culture, the cells were rather small, generally spindle-shaped, not well spread, with low density of short, bubble-like microvilli on the surface. Numerous plasma membrane structural alterations in apoptotic cells were observed by SEM: loss of cellular adhesion, smoothing, shrinkage and outpouching of membrane segments have been recognised as markers associated with the cell injury and death. It was concluded that scanning microscopy observations would allow a more complete understanding of SMCs and their changes in culture and atherosclerotic disease.

Rhinanthus serotinus (Schönheit) Oborny (Scrophulariaceae): immunohistochemical and ultrastructural studies of endosperm chalazal haustorium development.

Chalazal endosperm haustorium in Rhinanthus serotinus consists of a single large binucleate cell. It originates from the primary endosperm cell dividing transversely into two unequal cells: a smaller micropylar cell and a larger chalazal cell. The chalazal cell undergoes a single mitotic division, then lengthens significantly during development and functions as a chalazal endosperm haustorium. In this paper, immunofluorescent techniques, rhodamine phalloidin assay, and electron microscopy were used to examine the actin and tubulin cytoskeleton during the development of the chalazal haustorium. During the differentiation stage, numerous longitudinally oriented bundles of microfilaments ran along the axis of transvacuolar strands in haustorium. Microtubules formed intensely fluorescent areas near the nuclear envelope and also formed radial perinuclear microtubule arrays. In the fully differentiated haustorium cell, the actin cytoskeleton formed dense clusters of microfilaments on the chalazal and micropylar poles of the haustorium. Numerous microfilament bundles occurred near wall ingrowths on the chalazal wall. There were numerous clusters of microfilaments and microtubules around the huge lobed polytenic haustorial nuclei. The microfilaments were oriented longitudinally to the long axis of the haustorium cell and surrounded both nuclei. The microtubules formed radial perinuclear systems which were appeared to radiate from the surface of the nuclear envelope. The early stage of degeneration of the chalazal haustorium was accompanied by the degradation of microtubules and disruption of the parallel orientation of microtubules in the chalazal area of the cell. The degree of vacuolization increased, autophagous vacuoles appeared and the number of vesicles decreased.

Genotype-dependent efficiency of endosperm development in culture of selected cereals: histological and ultrastructural studies.

The paper reports studies, including histological and ultrastructural analyses, of in vitro cell proliferation and development of immature endosperm tissue isolated from caryopses of Triticum aestivum, Triticum durum, and Triticosecale plants. Endosperm isolated at 7-10 days post-anthesis developed well on MS medium supplemented with auxins and/or cytokinins. The efficiency of endosperm response was highly genotype-dependent and best in two winter cultivars of hexaploid species. The pathways of development and proliferation were very similar among the selected species and cultivars. Histological and scanning electron microscope (SEM) analysis revealed that only the part of the endosperm not touching the medium surface continued growth and development, resulting in swelling. The central part of swollen regions was composed mainly of cells containing many large starch grains. The peripheric parts of developed endosperm consisted of highly vacuolated cells and small cells with dense cytoplasm. SEM showed that cells from the swollen region were covered partially with a membraneous structure. Transmission electron microscope studies of cells from the outer part of the developing region showed features typical for cell activity connected with lipid metabolism.

New data about the suspensor of succulent angiosperms: Ultrastructure and cytochemical study of the embryo-suspensor of Sempervivum arachnoideum L. and Jovibarba sobolifera (Sims) Opiz.

The development of the suspensor in two species - Sempervivum arachnoideum and Jovibarba sobolifera - was investigated using cytochemical methods, light and electron microscopy. Cytological processes of differentiation in the embryo-suspensor were compared with the development of embryo-proper. The mature differentiated suspensor consists of a large basal cell and three to four chalazal cells. The basal cell produces haustorial branched invading ovular tissues. The walls of the haustorium and the micropylar part of the basal cell form the wall ingrowths typical for a transfer cells. The ingrowths also partially cover the lateral wall and the chalazal wall separating the basal cell from the other embryo cells. The dense cytoplasm filling the basal cell is rich in: numerous polysomes lying free or covering rough endoplasmic reticulum (RER), active dictyosomes, microtubules, bundles of microfilaments, microbodies, mitochondria, plastids and lipid droplets. Cytochemical tests (including proteins, insoluble polysaccharides and lipids are distributed in the suspensor during different stages of embryo development) showed the presence of high amounts of macromolecules in the suspensor cells, particularly during the globular and heart-shaped phases of embryo development. The protein bodies and lipid droplets are the main storage products in the cells of the embryo-proper. The results of Auramine 0 indicate that a cuticular material is present only on the surface walls of the embryo-proper, but is absent from the suspensor cell wall. The ultrastructural features and cytochemical tests indicate that in the two species - S. arachnoideum and J. sobolifera - the embryo-suspensor is mainly involved in the absorption and transport of metabolites from the ovular tissues to the developing embryo-proper.

Embryogenesis in Sedum acre L.: structural and immunocytochemical aspects of suspensor development.

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.

Unusual electron-dense dome associates with compound plasmodesmata in the embryo-suspensor of genus Sedum (Crassulaceae).

Plasmodesmata ensure the continuity of cytoplasm between plant cells and play an important part in the intercellular communication and signal transduction. During the development of the suspensor of both Sedum acre L. and Sedum hispanicum L., changes in the ultrastructure of plasmodesmata and adjoining cytoplasm are observed. Numerous simple plasmodesmata are present in the inner wall of the two-celled embryo separating the basal cell from the apical cell. From the early-globular to the torpedo stage of embryo development, the part of the wall separating the basal cell from the first layer of the chalazal suspensor cells is perforated by unusual, compound plasmodesmata. The role and the sort of transport through these plasmodesmata are discussed.

Extracellular matrix of plant callus tissue visualized by ESEM and SEM.

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.

Larval Contracaecum sp. (Nematoda: Anisakidae) in the Great Cormorant [Phalacrocorax carbo (L., 1758)] from north-eastern Poland: a morphological and morphometric analysis.

Stomachs of the Great Cormorant (Phalacrocorax carbo) from north-eastern Poland were found to contain adult nematode Contracaecum rudolphii [Hartwich, G., 1964. Revision der Vogelparasitischen Nematoden Mitteleuropas II. Die Gattung Contracaecum Railliet & Henry, 1912. Mitt. Zool. Mus. Berlin 40, 15-53.] sensu lato and nematode larvae which, based on their morphology, were identified as the third (L3) and fourth (L4) stage larvae of Contracaecum sp. Morphology and biometry of the L3 isolated from the Great Cormorant were very similar to those of the L3 of C. rudolphii described by Bartlett [Bartlett, C.M., 1996. Morphogenesis of Contracaecum rudolphii (Nematoda: Ascaridoidea), a parasite of fish-eating birds, in its copepod precursor and fish intermediate hosts. Parasite 4, 367-376.]. In our opinion, L3 and L4 to be larval stages of C. rudolphii. The paper contains detailed descriptions of the L3 and L4 stages of C. rudolphii; the L4 morphology is described in detail for the first time ever. The descriptions are supplemented by drawings and SEM images. Morphology and biometry of larvae were compared with the literature data and discussed.