Systematic approach to the correct identification of Asplenium dalhousiae (Aspleniaceae) with their medicinal uses.

In present study, multiple microscope techniques were used for the systematics identification of the species Asplenium dalhousiae. The plant was collected from different phytogeographical and its natural habitat of Pakistan, where it shows higher diversity. Morphology, foliar epidermal anatomy, and spore morphological characters of the species were studied in detailed using multiple microscopic techniques through light microscopy (LM) and scanning electron microscopy (SEM). LM and SEM were used for the systematics identification of the species. Traditionally, the species is used in the ailment of many diseases, so the spore morphology, anatomical features, and morphological characters are relevant to describe the species taxonomy. The importance of multiple methods of taxonomic study (e.g., documentation and morphological characteristics) for characterizing herbs are important step in systematic certification to maintain the efficacy of herbal medicines. The aim of the present study is to examine the morphological, anatomical, and spore morphology of the species A. dalhousiae in more detailed for the correct taxonomic identification and their medicinal validation from Pakistan.

Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway.

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

Pollen grains for oral vaccination.

Oral vaccination can offer a painless and convenient method of vaccination. Furthermore, in addition to systemic immunity it has potential to stimulate mucosal immunity through antigen-processing by the gut-associated lymphoid tissues. In this study we propose the concept that pollen grains can be engineered for use as a simple modular system for oral vaccination. We demonstrate feasibility of this concept by using spores of Lycopodium clavatum (clubmoss) (LSs). We show that LSs can be chemically cleaned to remove native proteins to create intact clean hollow LS shells. Empty pollen shells were successfully filled with molecules of different sizes demonstrating their potential to be broadly applicable as a vaccination system. Using ovalbumin (OVA) as a model antigen, LSs formulated with OVA were orally fed to mice. LSs stimulated significantly higher anti-OVA serum IgG and fecal IgA antibodies compared to those induced by use of cholera toxin as a positive-control adjuvant. The antibody response was not affected by pre-neutralization of the stomach acid, and persisted for up to 7 months. Confocal microscopy revealed that LSs can translocate into mouse intestinal wall. Overall, this study lays the foundation of using LSs as a novel approach for oral vaccination.

Tracking taphonomic regimes using chemical and mechanical damage of pollen and spores: an example from the Triassic-Jurassic mass extinction.

The interpretation of biotic changes in the geological past relies on the assumption that samples from different time intervals represent an equivalent suite of natural sampling conditions. As a result, detailed investigations of taphonomic regimes during intervals of major biotic upheaval, such as mass extinctions, are crucial. In this paper, we have used variations in the frequency of chemical and mechanical sporomorph (pollen and spore) damage as a guide to taphonomic regimes across the Triassic-Jurassic mass extinction (Tr-J; ∼201.3 Ma) at a boundary section at Astartekløft, East Greenland. We find that the frequency of sporomorph damage is extremely variable in samples from this locality. This likely reflects a combination of taxon-specific susceptibility to damage and the mixing of sporomorphs from a mosaic of environments and taphonomic regimes. The stratigraphic interval containing evidence of plant extinction and compositional change in the source vegetation at Astartekløft is not marked by a consistent rise or fall in the frequency of sporomorph damage. This indicates that natural taphonomic regimes did not shift radically during this critical interval. We find no evidence of a consistent relationship between the taxonomic richness of sporomorph assemblages and the frequency of damage among sporomorphs at Astartekløft. This indicates that previously reported patterns of sporomorph richness across the Tr-J at this locality are likely to be robust. Taken together, our results suggest that the patterns of vegetation change at Astartekløft represent a real biological response to environmental change at the Tr-J.

A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

[Pattern formation in microcosm: the role of self-assembly in complex biological envelopes development].

The data on the development of pollen/spore walls (of sporoderm) were reconsidered in the light of our hypothesis regarding a considerable role of self-assembling processes in the formation of this complex pattern. The premises that (1) glycocalyx (cell surface coating) is a self-assembling colloidal solution, and that (2) exine, formed on a glycocalyx framework, appears as a result of the self-assembly of the biopolymer (sporopollenin microemulsion), were independently suggested by the authors of this paper (Gabarayeva, 1990, 1993; Hemsley et al., 1992). Afterwards a joint hypothesis has been worked out which interpreted the processes of sporoderm development through regularities of colloidal chemistry. It was shown that all of the successive developmental stages, seen in transmission electron microscope (TEM) in the course of pollen wall development, correspond to successive micelle mesophases of a colloidal solution of surface-active substances which self-assemble when their concentration increases. Such an interpretation implies that all of the microstructures, observed in mature pollen walls (granules; rods-columellae; hexagonally packed layers of rods; bilayers, separated with a gap) are somewhat like "stiff history" of their appearance as a micellar sequence, immortalized by chemically resistant sporopollenin. Since self-assembling processes have nonlinear, spasmodic character, and microstructures of pollen wall, mentioned above, are arranged, as a rule, in successive layers, it has been suggested that these layers of heterogeneous microstructures occur as a result of the abrupt phase transitions typical for self-assembling micellar systems.

[Double fertilization in flowering plants: 1898-2008].

A short review of the results of investigations in the field of plant embryology in vivo and in vitro which are directly connected with the discovery of double fertilization in flowering plants by S.G. Navashin is presented. These results have been obtained by using the methods of electron and fluorescence microscopy, cytophotometry, cultures of isolated ovules, sperms, eggs, and embryo sac central cells. The question on an origin of the female gametophyte of flowering plants, double fertilization, and endosperm are discussed. It is emphasized that the progress in this field is connected mostly with the study of molecular processes which control the development and functioning of a female gametophyte and sporophyte at the early stages of ontogenesis.

Effects of copper on spore germination, growth and ultrastructure of Polypodium cambricum L. gametophytes.

The effects of different concentrations (10(-5), 5x10(-5) and 10(-4)M) of copper bromide on spore germination, growth and ultrastructure were investigated in Polypodium cambricum L. gametophytes. The inhibitory effect of Cu was observed in spores cultured on medium supplemented with 10(-4)M CuBr(2): germination occurred about 40 days after sowing and was only 25%. Concentrations of 5x10(-5) and 10(-4)M CuBr(2) induced changes in gametophyte development, possibly by re-orientation of growth. Gametophytes treated with 10(-5) and 5x10(-5)M CuBr(2) took up and accumulated a large amount of copper and ultrastructural observations showed that cytoplasmic damage was limited to twisted swollen thylakoids. The ultrastructure of gametophytes treated with 10(-4)M CuBr(2) showed absence of a vacuolar compartment. The present observations suggest that P. cambricum gametophytes could be a suitable material for studying physiological and molecular alterations induced by excess copper.

Temporal and spatial distribution of pectin epitopes in differentiating anthers and microspores of fertile and sterile sugar beet.

We studied the possible involvement of several pectin epitopes in anther differentiation and microsporogenesis in fertile and cytoplasmically male sterile sugar beets. The spatial and temporal distribution of five structural motifs were traced with a panel of monoclonal antibodies in six stages: premeiosis, meiotic prophase, young and mature tetrads, young and expanding microspores. The composition of the walls of sporogenous cells and meiocytes differed than that in the tapetum, as evidenced by the presence of alpha-Fuc(1-->2)-beta-Gal and alpha-(1-->5)-L-Ara epitopes binding CCRC-M1 and LM6 antibodies. At meiotic prophase, the meiocyte walls were additionally marked by the appearance of poorly methyl-esterified domains of homogalacturonan and of (1-->4)-beta-Gal residues, detected by JIM5 and LM5. Some constituents of the meiocyte wall which reacted with JIM5 and JIM7 persisted on the surface of the special callose sheath during tetrad development. In newly formed primexine and exine layers of tetrads and microspores, epitopes that were bound by JIM5, JIM7 and LM5 were abundant. No differences in the deposition or relative abundance of pectins were found between fertile and sterile anthers until microspore release from the callose. Later, at the time of abortion, sterile microspores had much larger amounts of epitopes detected by JIM5 than their fertile counterparts.

Zoosporicidal activity of polyflavonoid tannin identified in Lannea coromandelica stem bark against phytopathogenic oomycete Aphanomyces cochlioides.

In a survey of nonhost plant secondary metabolites regulating motility and viability of zoospores of the Aphanomyces cochlioides, we found that stem bark extracts of Lannea coromandelica remarkably inhibited motility of zoospores followed by lysis. Bioassay-guided fractionation and chemical characterization of Lannea extracts by MALDI-TOF-MS revealed that the active constituents were angular type polyflavonoid tannins. Commercial polyflavonoid tannins, Quebracho and Mimosa, also showed identical zoosporicidal activity. Against zoospores, the motility-inhibiting and lytic activities were more pronounced in Lannea extracts (MIC 0.1 microg/mL) than in Quebracho (MIC 0.5 microg/mL) and Mimosa (MIC 0.5 microg/mL). Scanning electron microscopic observation visualized that both Lannea and commercial tannins caused lysis of cell membrane followed by fragmentation of cellular materials. Naturally occurring polyflavonoid tannin merits further study as potential zoospore regulating agent or as lead compound. To the best of our knowledge, this is the first report of zoosporicidal activity of natural polyflavonoid tannins against an oomycete phytopathogen.

A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3.

Adenine phosphoribosyltransferase (APT) catalyzes the conversion of adenine and cytokinin bases to the corresponding nucleotides. An Arabidopsis thaliana mutant lacking the major APT isoform, APT1, is male sterile due to defects soon after meiosis. We have now used electron microscopy to define the effects of APT1 deficiency on pollen development to determine whether the changes might be attributed to adenine or cytokinin metabolism. Changes were observed in mutant anthers in both tapetal and pollen mother cells prior to meiosis with additional defects found at later stages, in both compartments. Principal changes include altered lipid accumulation in the tapetal cells, changes in pollen cell wall development, and a loss of synchrony in the development of the tapetum and microspores. Taken together our results suggest that APT1 deficiency causes a general metabolic decrease in energy metabolism, due to the lack of adenine recycling into adenylate nucleotides, which ultimately leads to pollen abortion. The early onset of meiosis in the mutant may be associated with altered cytokinin metabolism.

[Morphological and histological study on Pteris multifida].

The thesis studies on the pharmacognostical characteristics of Pteris multifida were carried out in this paper. The blade has special vein-like cell. The stomas in dorsal epidermis are regular. The petiol has V-shaped amphicribral bundles. The rhizome has 6-8 meristeles. The triangle-form spore has tumor-shaped or pellet-shaped decorations on its surface.

[Pharmacognostical studies on adiantum plants. V. Classification based on spore morphology and distributional patterns of silicon and calcium in the ultimate pinnules].

Crude drugs derived from Adiantum species are used as febrifuge, antidote, diuretic, tonic, etc. Some commercial samples of these drugs are composed of finely cut ultimate pinnules only, which have false indusia and spores. In this paper, in order to establish a classification method based on the characteristics of the ultimate pinnules and their attachments, the morphological study using stereoscope and scanning electron microscope, and X-ray microanalysis using an electron probe microanalyzer were carried out on the false indusia and spores, and the ultimate pinnules, respectively, of 19 Adiantum species. The results showed that examined all species could be distinguished from each other by the following characteristics: in the false indusium, the shape and the presence or absence of hairs; in the spore, the shape, the ornamentation, the ratio of the laesura, and the equatorial diameter; in the X-ray images of the ultimate pinnule, the distributional patterns of silicon and calcium. The distributional patterns of silicon were due to the presence of spicular cells, hairs and papillae, and calcium was present as crystals of calcium oxalate. The average content of silicon in the ultimate pinnules of Adiantum species was 1.99%.

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift.

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.

Rapid assessment of microspore and pollen development stage in wheat and maize using DAPI and membrane permeabilization.

The use of the DNA-specific fluorochrome DAPI has been extended to stage assessment of fresh pollen in wheat and maize. Membrane permeabilization by Triton X-100 incorporated in the staining solution allows access of the fluorochrome to nuclear DNA. At all stages of gametophytic development, the nuclei can be sharply visualized. Starch does not interfere with the fluorochrome so that it is possible to study the second pollen grain mitosis and sperm differentiation. With its rapidity and reliability, this technique represents an efficient tool for routine staging or investigation of the nuclear status of the pollen grains.

Ultrastructural aspects of sporogenesis in the apogamous fern Dryopteris borreri.

The events that accompany sporogenesis in the apogamous fern Dryopteris borreri parallel those seen in sexually reproducing ferns. Organelles dedifferentiate and redifferentiate, and form a discrete band across the equator of dyads; nuclear vacuoles and lipid spherosomes appear during prophase, and the major part of the ribosome population is removed and subsequently replaced during meiosis. Similar events have been found to occur during sporogenesis in mosses, gymnosperms and angiosperms, and therefore characteristic of the meiotic transition from sporophyte to gametophyte, even in the absence of a transition from diplophase to haplophase. The novel aspects of meiosis in D. borreri are largely those connected with the restitution event that precedes meiosis I and serves to maintain the sporophytic chromosome number throughout the life cycle of this fern. Pre-meiotic cells are regularly found to be cleaved by annular wall ingrowths, which traverse the cytoplasm but not the nuclei. The significance of these ingrowths in relation to theories concerning apogamy and plant cell division are discussed.

The tonoplast, a specific marker of embryogenic microspores of Datura cultured in vitro.

Microspores of Datura metel L. have a thin tonoplast which, upon culture in vitro, develops a more or less uniform coating or deposit (as early as 12 h). This response was specific for embryogenic microspores, thereby making it possible to distinguish them from the nonembryogenic microspores. Cytochemical tests indicated the presence of tannins in this deposit which persists only until the early globular stage of embryogenesis. Thus tannin-coated tonoplasts can be used as a reliable and specific cytological marker for microspores following the embryogenic pathway in vitro.