M. Bobák

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.

Extracellular Matrix in Early Stages of Direct Somatic Embryogenesis in Leaves of Drosera spathulata

Leaves from mature in vitro grown plants of Drosera spathulata Labill. regenerated new plantlets on solid induction medium in light. Especially vascular sheath parenchyma cells located close to basal part of tentacule showed high embryogenic potential. Proembryoids arrising from the tentacule base part were visible by scanning electron microscopy. Their surface cells were linked and covered with thin external, fibrilar network representing an extracellular matrix (ECM). Proembryogenic surface cells were later connected by coarse strands of fibrils. Young protoderm was formed arround globular embryoids and its cells were characterized by “brain-like” surface structure. However, the surface of fully developed protodermal cells was practicaly smooth and cells were stick to each other very tightly in torpedo and cotyledonary shaped embryoids. The presence of ECM was typical only for somatic proembryos and globular embryos. The ECM network was never observed on the surface of heart and torpedo shaped embryos.

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.

Shoots and embryo-like structures regenerated from cultured flax (Linum usitatissimum L.) hypocotyl segments

Summary The effect of growth regulators and culture media on regeneration of flax from hypocotyl explants were tested. Shoot differentiation was strongly influenced by genotype, concentration of TDZ, combination of BAP and NAA. The highest number of shoots was obtained with the fibre cultivar Alex using TDZ (0.25 mg · L -1 ) on MS medium. Embryo-like structures were formed only on 2,4-D (5 mg · L -1 ) pretreated hypocotyl segments of cultivar Szegedi-30 (oil flax). As a consequence of 2,4-D pretreatment, the embryo-like structures had poorly defined or fused cotyledons and no apparent shoot apices. Morphologically not well developed ELS did not convert to plants.

A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum L. in vitro

Summary Cellular origin of shoot buds, cell morphogenesis and differentiation were studied during direct and indirect shoot regeneration of Papaver somniferum L. in vitro . Direct shoot organogenesis was induced in immature somatic embryos, where cell division and protomeristem formation started in sub-epidermal and epidermal cell layers of hypocotyl. Indirect shoot regeneration was initiated from callus culture using auxins and cytokinins, where compact globular meristemoids were produced. The common morphogenetic event of direct and indirect shoot regeneration was an establishment of non-random cell division and restricted cell expansion within the group of competent cells during protomeristem formation. However, in contrast to direct regeneration, where all activated cells became competent, in indirect regeneration, only peripheral cells of meristemoids acquired morphogenetic competence. The second difference occurred in shoot tunica formation: original hypocotyl epidermal cells participated in tunica formation during direct organogenesis, while this layer regenerated de novo in meristemoids. These results indicate that cell morphogenesis during shoot regeneration is independent of the developmental history of the competent cells.

Effect of Trifluralin and Colchicine on the Extracellular Matrix Surface Networks during Early Stages of Direct Somatic Embryogenesis of Drosera rotundifolia L.

Summary Leaves from mature in vitro grown plants of sundew ( Drosera rotundifolia L.) directly regenerate new plants via somatic embryogenesis using semi-solid induction media. We studied the extracellular matrix (ECM) on the cell surface of somatic embryos developing from the basal part of tentacles using scanning electron microscopy. Surface cells of early somatic embryos were covered with thin external ECM layers and extracellular matrix surface networks (ECMSN) interconnecting neighbouring embryogenic cells, while these external structures disappeared during somatic embryo development. ECM layers and ECMSN can be observed by SEM until the protoderm is formed on the surface of well-developed globular somatic embryos. Treatments with microtubular toxins trifluralin and colchicine removed the ECMSN between cells and reduced the ECM layers on cell surfaces. After treatment with both toxins for 4 h, the ECMSN was partially re-established by subsequent treatment with DMSO for 5 h, but it disappeared completely when toxin application was longer then over 6 h. Treatments with proteases revealed that ECMSN of Drosera pro-embryogenic cells contain proteinaceous components.

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.