The fine structure of neuromuscular junctions and contact zones between body wall muscle cells of Ascaris lumbricoides (var. suum)

Summary Neuromuscular junctions and close membrane apposition between body wall muscle cells of Ascaris lumbricoides (var. suum) have been examined with the light and electron microscopes. It was found that the body wall muscle cells send out elongate processes from their basal, myofibril containing portion to terminate on dorsal and ventral nerves. When observed with the aid of the electron microscope the neuromuscular junctions were seen to consist of several muscle cell processes in apposition to a single axon. The intersynaptic cleft was approximately 350–500 Å wide. Both the axolemma and sarcolemma were triple layered membranes which were 75–80 Å thick. Electron dense patches were observed at intervals on the apposed membranes which were due to increased thickness of the inner membrane leaflets of axolemma and sarcolemma. Muscle cell membranes, at the level of the neuromuscular junction, were in close apposition resulting in an apparently five-layered membrane complex which was 170–210 Å thick. The sarcolemmata in these regions were separated by 10–50 Å. Presynaptic axons contained mitochondria, microtubules which were 180–270 Å in diameter, and two, morphologically distinct types and sizes of synaptic vesicles. One was 200–600 Å in diameter, with a single, triple-layered membrane bounding a center of low electron density. The other was 600–1200 Å in diameter, with a single, triple-layered membrane bounding a central, electron dense granule of 500–800 Å size.The functional significances of the close membrane appositions between body wall muscle cells and of the two types of synaptic vesicles found at the neuromuscular junctions of Ascaris lumbricoides were discussed with respect to their possible role in neuromuscular physiology.Supported by U.S.P.H.S. Grant No. NB-01528 and Research Career Development Award No. 9-K3-NB-15255. — The author wishes to express his grateful appreciation for the excellent technical assistance given by Miss Gabrielle Rouiller during the course of this investigation.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

An ultrastructural study of the polyp and strobila of Atorella japonica (Cnidaria,Coronatae) with respect to muscles and nerves

Atorella japonica were observed by TEM to examine the nerve plexus in the capitulum of the polyp and the cross-striated muscle cells of the strobila. The nerve plexus included a number of neuromuscular junctions and many interneural synapses. Neuromuscular junctions contained two types of synaptic vesicle: clear and small (ca 75 nm diam.), and dense cored and large (ca 120 nm diam.). The first type of vesicle always appeared near the presynaptic membrane and the second type was distributed behind the former. In interneural synapses, two types of vesicle which were similar to neuromuscular synaptic vesicles were recognized. They were distributed in a pattern similar to that of the neuromuscular synaptic vesicles, but these vesicles were found on both sides of the two synaptic membranes.     

Ultrastructural Studies of the Development of Nerves in Hydra

Three types of mature epidermal neurons and several of theirdifferentiating stages aie presented in this ultrastructuralstudy. Each of the three types, neurosensory, neurosecretory,and ganglionic cells, is derived from interstitial cells, (i)Mature neurosensory cells contain elongated nuclei, a well-developedcilium in each cell, and membrane-bounded neurosecretory droplets(700–1300 A in diameter). There may be two or more neuritesin which are numerous microtubules, glycogen particles, ribosomesand many neurosecretory droplets, (ii) Mature neurosecretorycells closely resemble neurosensory cells, except that no ciliumis present. The perikarya contain small, dense nuclei, neurosecretorydroplets (850–1300 A in diameter), mitochondria, glycogenparticles, and microtubules. Active Golgi complexes are presentin both cell types. The nemites are similar to those describedfor neurosensory cells, (iii) Mature ganglionic cells are bipolaror multipolar. The small, dense nuclei are surrounded by a smallamount of cytoplasm. The neurites contain mostly microtubules;a few mitochondria, ribosomes, and glycogen particles are alsopresent, but there are no secretory droplets. To date, only neurosensory and neurosecretory cells have beenobserved in the gastiodermis. They are structurally indistinguishablefrom their epideimal counterparts. A significant finding is that three types of synapses—neuromuscular,neuronematocyte, and interneuronal—are identified in boththe epidermal and gastrodermal neurons.     

COMPARISON OF THE STRUCTURE OF THE PALLIAL TENTACLES OF SEVEN SPECIES OF SOUTH AFRICAN PATELLID LIMPET

The ultrastructure of the pallial tentacles of seven speciesof patellid limpet is described. The tip of most of the tentaclesexamined bears a crown of long cilia, whereas the shaft of thetentacles has small tufts (5–10 µm diameter) ofshorter cilia. Sections through the ciliated tufts show themto be composed of several cells, each bearing cilia. The ciliacontain 5–7 central microtubules and therefore do nothave the conventional 9 + 2 arrangement of microtubules. Nerveprocesses run from the base of each ciliated cell to a nervebundle in the centre of the tentacle, suggesting a sensory function.Estimates of densities of ciliated tufts suggest that the territoriallimpets (Patella cochlear and P. longicosta) have the greaternumber of tufts. Electron dense plate-like structures are foundin the centre of the pallial tentacles of Patella cochlear,P. longicosta, P. granularis, P. barbara and Helcion pruinosus.Each plate is about 0.2 µm wide and is surrounded by adouble membrane. It is suggested that these may play a rolein scattering or reflecting light and thus form part of thedermal light sensing ability of these animals. (Received 26 January 1987;     

Cytoplasmic pH of Root Hair Cells of Sinapis alba Recorded by a pH-sensitive Micro-electrode. Does Fusicoccin Stimulate the Proton Pump by Cytoplasmic Acidification?

Bertl, A. and Felle, H. 1985. Cytoplasmic pH of root hair cellsof Sinapsis alba recorded by a pH-sensitive micro-electrode.Does fusicoccin stimulate the proton pump by cytoplasmic acidification?—J. exp. Bot. 36: 1142–1149. pH-sensitive micro-electrodes, filled with ion-exchanger resinhave been fabricated with a turgorinsensitive tip and have beenapplied to test the intracellular pH and changes thereof inroot hair cells of Sinapis alba. (1) The cytoplasmic pH of Sinapisroot hairs was determined to be 7.3 ±0.2 (at neutralexternal pH). (2) 10 mol m^–3 sodium azide depolarizesthe membrane potential by about 100 mV and acidifies the cytoplasmby 0.8 pH-units. (3) The change from 1.0 mol m^–3 to 10mol m^–3 external potassium causes a depolarization ofabout 45 mV, but no change in internal pH. (4) At an externalpH of 5.0, sodium acetate hyperpolarizes the plasmalemma byabout 60 mV and acidifies the cytoplasmic pH by 0.2 to 0.3 units.(5) 2.0 mmol m^–3 fusicoccin (FC) hyperpolarizes the plasmalemmaby 20–25 mV, acidifies the cytoplasm by 0.1 to 0.2 pH-units,and acidifies the external medium by about 0–3 pH-units.It is concluded that cytoplasmic acidification stimulates theelectrogenic proton pump in Sinapis root hairs, and it is suggestedthat the FC-induced effects, viz. hyperpolarization and externalacidification, can also be interpreted in this way. Key words: —Cytoplasmic pH, pH-sensitive micro-electrode, fusicoccin     

Cellular Development of the American Lobster Heart

The heart of the American lobster, Homarus americanus, representsa relatively simple nerve-muscle system. A nine-celled cardiacganglion functions to initiate the contractions of the transverselystriated myocardium. To describe the development of this nerve-musclesystem, hearts of lobster embryos in different stages of developmenthave been examined with an electron microscope. The heart wallconsists of an outer adventitial layer of fibroblast cells andan inner layer of transversely striated myocardial cells. Adjacentfibroblast cells form fasciae adherent and gap junctions. Intercalateddiscs and membrane regions of close apposition (4 nm) occurbetween adjacent myocardial cells. A cardiac ganglion that containsnine neurons and synaptic neuropil is present. Neuromuscularsynapses occur in the myocardium. Cells that we have termedstorage cells contain dense cytoplasmic inclusions that resembleyolk material and form gap junctions with myocardial cells.The heart at six weeks is about 200 µm long and 160 µmwide; at six months it is 300 and 250 /m respectively. The myocardiumgrows by an increase in the size and in the number of myocardialcells. The myocardium contains pre-myoblast, myoblast and well-differentiatedmyocardial cells. Sarcomere formation occurs first adjacentto the inner surface of the sarcolemma where the myofilamentsalign and anchor. Distinct A and I bands appear first, followedby the Z lines. The myocardium is sparsely innervated when theinitial heartbeats occur; a preliminary quantitative analysishas shown that three days later, the density of innervationhas increased six-fold.     

The small pyramidal neuron of the rat cerebral cortex

Summary The pyramidal neurons in layers II and III of the rat parietal cortex have dendritic spines which form synapses with axon terminals. These synapses have synaptic clefts containing granular material that is concentrated towards the middle of the cleft to form a plaque. Only a small amount of dense material occurs on the cytoplasmic face of the presynaptic membrane, while there is a prominent dense layer, some 300 Å deep, in the dendritic spine. When the synapses formed by the smallest dendritic spines are examined in a frontal or en face plane of section this postsynaptic density has the form of a disc. In the synapses on larger spines, the disc is perforated to form a ring, and in the largest spines a number of perforations may occur. Because of these perforations, in larger synapses sections passing at right angles to the plane of the synaptic junction may show two or more separate postsynaptic densities. The possible significance of these findings is discussed.This work was supported by United States Public Health Service Research Grant No. NB-07016 from the National Institutes of Neurological Diseases and Blindness. The authors wish to express their sincere thanks to Lawrence McCarthy and Charmian Proskauer for their valuable assistance.     

Modulation of the Plasma Membrane Electron Transfer System in Root Cells of Limnobium stoloniferum by External pH

The influence of ferricyanide on transmembrane electron transfer,proton secretion, membrane potential, and cytoplasmic pH ofLimnobium stoloniferum (G.F. Mey) Griseb. root cells was investigatedat different external pH HCF III reduction by the roots was accompanied by membrane depolarization,an increase in proton secretion and by alkalinization of thecytoplasm. Change of membrane potential and cytoplasmic pH aswell as transmembrane e^– transfer was more pronouncedat acid external pH. The rate of proton flux was linearly dependenton the rate of electron transfer. The slope of the relationshipwas around 1, independent of external pH The data are in agreement with the hypothesis that electrontransfer at the plasma membrane is directly coupled to protonsecretion. It is suggested that both e^– and redox-coupledH⁺ transport are activated by acid external pH Key words: Plasmalemma redox system, electron transfer, proton transport, pH, membrane potential, Limnobium stoloniferum     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Pathway of Photosynthate Transfer in the Developing Seed of Vicia faba L: A Structural Assessment of the Role of Transfer Cells in Unloading from the Seed Coat

Photosynthate movement within the coat of the developing seedof Vicia faba occurs radially inward from the restricted vascularsystem and laterally through the non-vascularized region ofthe seed coat prior to exchange to the seed apoplast. Thin-walledparenchyma/transfer cells line the entire inner surface of theseed coat and thus are located at the terminus of the photosynthatetransfer pathway. The principal cellular route of transfer withinthe seed coat and the role of the thin-walled parenchyma/transfercells in membrane exchange to the seed apoplast has been investigated.Sucrose fluxes, computed from estimates of the plasma membranesurface areas of the cell types of the pathway, the plasmodesmatalcross-sectional areas interconnecting contiguous cells and theobserved rate of sucrose delivery to the embryo indicate thatsieve element unloading and subsequent transfer to the thin-walledparenchyma/transfer cells is through the symplast. For the cellsof the ground tissue, plasmodesmatal density is consistentlyhigher on their anticlinal walls. This observation supportsthe reported pattern of lateral transfer through these tissuesin the non-vascular regions of the seed coat. Wall ingrowthsare initiated sequentially in the thin-walled parenchyma cellsto maintain 1–3 rows of thin-walled parenchyma/transfercells. The development of these wall ingrowths results in a58% increase in the plasma membrane surface area of these cellsand provides them with the capacity to act as the principalcellular site for membrane exchange of sucrose to the seed apoplast.This cellular route of symplastic transfer from the sieve elementsto the ground tissues where membrane exchange to the seed apoplastoccurs is consistent with that reported for Phaseolus vulgaris Key words: Cellular pathway, photosynthate transfer, transfer cell, Vicia seed coat     

Comparison of the Effects of Ammonia and Methylamine on Chloride Transport and Intracellular pH in Chara corallina

Ammonium and methylammonium ions greatly increase the rate ofCl^– transport in Chara corallian. This effect is dependenton the pH of the bathing solution. The amine-stimulated Cl^–influx is small at pH 5·5, increases to a maximum atpH 6·5–7·5, and decreases again as the pHis raised to 8·5. Increased Cl^– influx is accompaniedby an increase in cytoplasmic pH, as calculated from the distributionof DMO. When the external pH lies between 5·5 and 7·3,cytoplasmic pH in the absence of amine is 7·65–7·70,with an increase of 0·15–0·25 in the presenceof amine. As external pH is increased above 7·3, cytoplasmicpH also increases, with progessively less effect of amine. Although the relationship between Cl^– influx and cytoplasmicpH is not simple, the results provide evidence in accord withthe hypothesis that Cl^– transport in Chara involves H⁺—Cl^–symport, or the equivalent OH^–—Cl^– antiport.The possible role of cytoplasmic pH as a factor involved inthe regulation of membrane transport in Chara is discussed.     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis

The membrane potential and the ionic activity gradients of K⁺and Cl^– across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K⁺ and Cl^–.Our results indicate that K⁺ is close to electrochemical equilibriumwhereas Cl^– is not. ¹ Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan.     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

The ultrastructure of giant fibre and serial synapses in crayfish

Summary The ultrastructure of synapses between the cord giant fibres (lateral and medial) and the motor giant fibres in crayfish, Astacus pallipes, third abdominal ganglia have been examined. These electrotonic synapses are asymmetrical, they have synaptic vesicles only in the presynaptic fibre, and they have synaptic cleft widths normally of about 100 Å but narrowed to about 50 Å in restricted areas. Localized increases in density of the synaptic cleft and adjacent membranes also occur within a synapse, and synaptic vesicles are most tightly grouped at the membrane in such areas. Tight or gap junctions with 30 Å or narrower widths have not been found, but the junctions probably function in a similar way to gap junctions.Three small nerves are closely associated with the synapses between the giant fibres. One of these small nerves has round synaptic vesicles and is thought to be excitatory on morphological grounds; one has flattened vesicles and is thought to be inhibitory; and one is postsynaptic to the lateral giant and the two small presynaptic nerves. It is proposed that these small nerves modulate activity in the much larger giant fibre synapse.     

Biophysical and Biochemical Regulation of Cytoplasmic pH in Chara corallina during Acid Loads

The contribution of membrane transport to regulation of cytoplasmicpH in Chara corallina has been measured during proton-loadingby uptake of butyric acid. In the short-term (i.e. up to 20min) uptake of butyric acid is not affected by removal of externalK⁺, Na⁺ or Cl^– but over longer periods uptake is decreased(by 20–50% in different experiments) in the absence ofexternal Na⁺ or, sometimes, K⁺. Influxes of both Na⁺ and K⁺increase temporarily after addition of butyrate, Na⁺ immediatelyand K⁺ after a lag. Effects on Cl^– influx are small butCl^– efflux increases enormously after a short lag. Anapproximate comparison of internal butyrate with changes inthe concentration of K⁺, Na⁺, and Cl^– suggests that initially(i.e. for a few min) cytoplasmic pH is determined by bufferingand possibly by some decarboxylation of organic acids (biochemicalpH regulation), and that biophysical pH regulation involvingefflux of H⁺ balanced by influxes of K⁺, Na⁺ and especiallyefflux of Cl^– progressively becomes dominant. When butyric acid is washed out of the cells, cytoplasmic pHis restored completely or partially (depending on the butyrateconcentration used) and this is independent of the presenceor absence of external Cl^–. Where Cl^– is present,its influx is relatively small. It is suggested that cytoplasmicpH is then controlled biochemically, involving the synthesisof an (unidentified) organic acid and the accumulation of acidicanions in place of butyurate lost from the cell. During thesecond application of butyrate, net Cl^– efflux is small:it is suggested that control of cytoplasmic pH then involvesdecarboxylation of the organic acid anions. The questions of the source of Cl^– lost from the cell(cytoplasm or vacuole) and of possible cytoplasmic swellingassociated with the accumulation of butyrate are discussed. Key words: Chara corallina, butyric acid, cytoplasmic pH, membrane transport     

Electrical Impedance Analysis in Plant Tissues8

Electrical impedance spectra (100 Hz–800 kHz) were measuredin leaves of Peperomia obtusifolia L. (a succulent) and Brassicaoleracea L. (cabbage). By measuring impedances at three or moreinter-electrode distances in a single leaf, electrode impedanceand specific tissue impedance were separated. Analysis of impedance data from B. oleracea leaves in relationto an equivalent circuit model showed that leaf developmentwas accompanied by increases in extracellular resistance, cytoplasmicresistance and vacuole interior resistance, together with decreasesin plasma membrane capacitance and tonoplast capacitance. AfterB. oleracea leaves were subjected to a –6 °C freeze-thawstress, extracellular resistance, cytoplasmic resistance andvacuole interior resistance decreased, but plasma membrane capacitanceand tonoplast capacitance did not change. These results indicatethat useful measurements of leaf parameters can be obtainedby this technique. Examination of the electrode impedance spectrum showed thatelectrode insertion produced a damaged collar, 0·4–0·5mm wide, around the electrode. This was confirmed by visualobservation of the damage in P. obtusifolia leaf. Key words: Peperomia obtusifolia L., Brassica oleracea L. (cabbage), electrical impedance, equivalent circuit, electrode polarization     

Direct Somatic Embryogenesis in Roots of Cichorium: Is Callose an Early Marker?

Direct somatic embryogenesis can be obtained from epidermaland cortical cells in roots from in vitro Cichorium plantlets.The first embryogenic cells are seen after six days of culturein darkness, at 35 °C, in a liquid medium supplemented withNAA (1 x 10^–7 M), 6-dimethylallyl-amino-purine (2·5x 10^–6 M), sucrose (0.03 M) and glutamine (1·7x 10^–3 M). Embryogenic cells undergo first a linear andthen a globular segmentation, with increasing cytoplasmic density.These cells and young embryoids show aniline blue fluorescence.SEM allows the same microglobular pattern to be seen on thesurface of young embryoids and on young microspores of Cichoriumused as controls. In this root system, callose deposition seemsto be an early marker in somatic embryogenesis. Somatic embryogenesis, callose, Cichorium