J. Krištín

Developmental SEM observations on an extracellular matrix in embryogenic calli of Drosera rotundifolia and Zea mays

SummaryPrimary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.

Direct plant regeneration from leaf explants of Drosera rotundifolia cultured in vitro

Shoot regeneration was obtained from isolated leaves of Drosera rotundifolia L. cultured on MS media with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). The best direct shoot organogenesis was obtained on growth regulator-free medium or medium supplemented with 10-8 M NAA. Liquid culture medium significantly increased regeneration capacity of leaf tissue. Histological and scanning electron microscopy investigations verify direct plant regeneration without intermediate callus formation. Leaf epidermal cells showed the highest regeneration potential leading to the regeneration of buds. Young shoots with three to seven leaflets rooted spontaneously on the growth regulator-free medium within 38 days of culture and isolated mature plants produced fertile seeds.

Papaver somniferum regeneration by somatic embryogenesis and shoot organogenesis

Secondary somatic embryogenesis and shoot organogenesis from primary somatic embryos of Papaver somniferum L. are described. The embryos became malformed, the root meristem expressed dividing activity without position-dependent cell differentiation, causing abnormal development or arrested growth of primary somatic embryos. The adventitious shoots regenerated from embryo hypocotyl, but secondary somatic embryos had an epidermal origin close to the root meristem. The regeneration occurred without hormonal treatment, indicating endogenous nature of triggering signals. These signals are probably related to the integrity loss of morphogenetic steps during development of primary somatic embryos, which appeared to induce an activation of cells competent to regeneration.

Studies of organogenesis from the callus culture of the sundew (Drosera spathulata Labill.)

Summary Histological observation of organogenesis and plant regeneration from callus of sundew ( Drosera spathulata Labill. ) has been studied. The process of organogenesis begins by formation of two meristemoid regions, which have been found in the peripheral cell layer and the inner zone of calli. Stalk-like organoids are formed from the peripheral callus zone and roots from the inner zone. Roots have a typical histological structure and tracheids are present. In some cases, compact globular structures arose from the peripheral callus layer and later developed into bipolar embryo-like structures.

In vitro cytotoxicity testing of coladerm membrane

Atpresent, biodegradable and biocompatible membranes based on collagen andglycosaminoglycans play an important role in substitutive medicine. Modernbiomaterials use a chemically modified collagen-based matrix for implants withprogrammable biodegradability as a substitute of buccal mucosa, skin,cartilage,etc. Besides the requirements for biocompatibility and biodegradability, themembranes must be also non-toxic. Therefore, cytotoxicity testing of thesematerials in vitro is an integral part of introducingnewlydeveloped types of membranes into clinical practice. As a biological model forthe tested COLADERM membrane, cell cultures from human embryonic fibroblasts(B-HEF-2) were used for both cytotoxicity testing as well as in tests to assessthe ability of cells to proliferate on this membrane. Along with the ability ofcells to grow on the surface and inside the membrane, immunohistochemicalexamination and scanning electron microscopy (SEM) were performed as well. Theobtained results have shown that the COLADERM membrane is non-toxic withsuitable structural and biological properties for clinical application as asubstitute of buccal mucosa following surgical ablation of malignant tissuesfrom the oral cavity.

Properties of copolymer of 2,2′:5′,2″-terthiophene-5,5″-dicarboxylic acid and polyethylene oxide

Abstract A new type of alternating regular copolymer of 2,2′:5′,2″-terthiophene-5,5″-dicarboxylic acid (TDCA) with polyethylene oxide (PEO) was synthesized. The product was characterized by NMR, UV-Vis and fluorescence spectra, EPR spectroscopy, X-ray diffraction (XRD) and DSC measurements. Copolymer is dissolved in dimethylformamide (DMFA) and dimethylsulfoxide (DMSO). Using TEM, it was ascertained that due to a different chemical structure of polymeric sub-units the separated phases in the solid state are formed on a sub-nanometer level.

Morphology and ultrastructure of isolated gemmae of Drosera pygmaea and their in vitro germination

Gemma morphology, histology and ultrastructure before and after germination in vitro were studied in Drosera pygmaea. The histology of the gemma is similar to that of a seed, being characterized by an embryo-like structure and storage tissue, although no seed coat is formed. One embryo-like structure within the gemma, which gives origin to a new plant, expresses polar organisation with distinct meristematic regions. Storage tissue surrounding the embryo-like structure resembles endosperm and it is built of parenchyma cells possessing plastids with starch grains and dense material within vacuoles. The regeneration from the gemma may provide useful system to study plant morphogenesis under stress conditions including in vitro culture.

Bundle Sheath Cells are Responsible for Direct Root Regeneration from Leaf Explants of Helianthus occidentalis L.

Summary Direct root organogenesis from isolated leaf explants of Helianthus occidentalis L. was induced using MS culture media supplemented with (x-naphthalene acetic acid (NAA) and benzyladenine (BA). The highest numbers of roots were formed de novo from bundle sheath cells located around leaf veins when explants were cultured on induction medium supplemented with 2.7 pmol/L NAA and 0.44 pmol/L BA. The structural events accompanying reactivation and division of competent bundle sheath cells and cells derived from them were investigated employing light, transmission and scanning electron microscopy in time course experiments. Vein parenchyma and bundle sheath cells located around leaf veins started to divide first after 2 days of culture. Vein parenchyma cells, however, ceased their divisions by day 5, while reactivated bundle sheath cells produced cambium-like cells. These later cells gave origin to circular meristematic tissue, and subsequently to root primordia. Root primordia appeared after 7 days and regenerated roots emerged from leaf explants after 15 days of culture. Ultrastructure of reactivated regeneration competent cells is described and is compared with other examples of regeneration in vitro.

Cytological study on wheat (Triticum timopheevi Zhuk.) protoplasts

Protoplasts were obtained from tetraploid wheat (Triticum timopheevi Zhuk.) suspension culture by incubation in solution of 1 % pectinase 500, 1 % driselase and 1 % cellulase and cultivated in Schenk and Hildebrandt medium. Freshly isolated protoplasts contained dense cytoplasm and constricted organellae exhibited negative contrast of their membranes. Together with normal protoplasts huge multinucleate protoplasts were present in the population. 3 h after plating, the cytoplasm showed normal appearance, the negative contrast of membranes was not evident any longer. Cisternae of endoplasmic reticulum and Golgi apparatus were numerous. There were some vesicles and fibres on the protoplast surface. 8 d after plating, many dividing cells were found out and cell clumps arosen in this way were present in the culture. Some of the protoplasts particularly those originally multinucleate ones were upset.

The influence of elicitation on the subcellular localization and content of sanguinarine in callus cells ofPapaver somniferum L.

The fungal elicitor prepared fromBotrytis cinerea affected sanguinarine alkaloid formation and accumulation in callus cells ofPapaver somniferum L. Ultrastructural changes have been observed in association with the accumulation and secretion of sanguinarine in elicitor-treated cells. Alkaloid content in elicited cells was showed as a electrondense material (osmiophilic aggregations), which occurred on the tonoplast and in freely floating bodies in the vacuole. A 30-times increasing of sanguinarine content was observed in elicitor-treated cultures.