Alžbeta Blehová

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.

The histological analysis of indirect somatic embryogenesis on Drosera spathulata Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 µM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.

Drosera Species (Sundew): In Vitro Culture and the Production of Plumbagin and Other Secondary Metabolites

Carnivorous plants have attracted considerable attention from many biologists for several hundred years, not only because of their special nutritional requirements and ecological adaptations, but also because of their value as medicinal herbs (Lecoufle 1990). Among them, Charles Darwin studied insectivorous plants in detail and used them in his evolutionary studies (Darwin 1875). The genus Drosera, which consists of about 125 species (Culham and Gornall 1994), represents a really good example of plant evolution and functional adaptation. Importantly, extracts from numerous species of Drosera have been traditionally used for various medicinal purposes, especially as efficient agents against respiratory diseases (Table 1). These therapeutic effects are thought to correlate with the content of secondary metabolites, namely naphthoquinones which are synthesized and accumulated in various Drosera species (Zenk et al. 1969; Culham and Gornall 1994). Among naphthoquinones, the most intensively studied has been plumbagin, because of its broad medicinal and other effects (see Finnie and van Staden 1993). However, many Drosera species also contain the second of the two most abundant naphthoquinones of Droseraceae, called 7-methyljuglone. Besides this, Drosera plants synthesize several minor, but unique, naphthoquinones as well as a broad spectrum of flavonoids with practical or potential medicinal use. Plumbagin production in vivo and in vitro has been well described, especially for the two South-African species D. capensis and D. natalensis, by Crouch et al. (1990). Therefore, in this chapter attention is focused on new data on the production of 7-methyljuglone, plumbagin and minor naphthoquinones as well as flavonoids in some new Drosera species, e.g. D. spathulata and D. rotundifolia.

Cuscuta europaea plastid apparatus in various developmental stages: Localization of THF1 protein

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

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.

Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton

Abstract Dodder (Cuscuta) species cause severe agricultural damage in many countries throughout the world. To establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. For these efforts genetic modification would represent a powerful tool. Here we report on Agrobacteriummediated transformation of dodder using green fluorescent protein (GFP) fused to actin-binding protein as a vital marker. Since the shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. The transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. Transient expression of GFP appears to be a suitable method for studying Cuscuta development through cytoskeleton organisation that is presently largely unexplored.