Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentK _m values of 1.1 M and 10 M for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.     

Male reproductive effect of arsenic in mice

Arsenic, a known human carcinogen, was given to mice via drinking water as sodium arsenite at a dose 53.39, 133.47, 266.95 and 533.90 mol l for 35 days. A decrease in the activity of 17 HSD along with increase in LDH, GT activity were observed at 533.90 mol l. The observed sperm count, motility and morphological abnormalities in sperm were similar to control at lower dose levels. However at 533.90 mol l a significant decrease in sperm count and motility along with increase in abnormal sperm were noticed. Significant accumulation of arsenic in testes and accessory sex organs may be attributed to the arsenic binding to the tissues or greater cellular uptake. No effects were observed on indices studied for reproductive effects at 53.39 mol l arsenic close to which human being are exposed through drinking water under the present set of experimental conditions.     

Uptake of 24Mg by excised pine roots: A preliminary study

Uptake of ²⁴Mg by excised roots of Pinus sylvestris L. during up to 4 h long incubations in 99.9 atom % ²⁴Mg (50 M) was measured by ICP-MS. A rapid initial uptake phase (30 min) was followed by a slower uptake. This was interpreted as a shift from a phase dominated by saturable ion exchange (free space uptake), to a non-saturable phase, during which the rate of uptake was 0.077±0.0.012 mol Mg g^–1 (d.wt.) h^–1. The metabolic uncoupler DNP (2,4-dinitrophenol) at 50 M decreased the Mg uptake rate by 35% only, but the effect of DNP was significant (p<0.01). Several problems related to a high variability in the experimental material were encountered, and further refinement of this approach in studies of plant Mg uptake is suggested.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PP_i) in three phototrophic bacteria. Formation of PP_i was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PP_i synthesis in Rps. viridis was 0.15 mol PP_i formed/(min*mol Bacteriochlorophyll) at 23°C. The synthesis of PP_i was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PP_i synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PP_i after short light flashes was also studied. The yield of PP_i after a single 1 ms flash was equivalent to approximately 1 mol PP_i/500 mol Bacteriochlorophyll. In Rps. viridis chromatophores, PP_i was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.Abbreviations BChl Bacteriochlorophyll - F₀F₁-ATPase Membrane bound proton translocating ATP synthase - FCCP Carbonyl cyanide p-trifluoromethoxyphenylhydrazone - H⁺-PPase Membrane bound proton translocating PP_i synthase - TPP⁺ Tetraphenyl phosphonium ion - TPB^- Tetraphenyl boron ion - Transmembrane electrical potential difference     

Plasma glucose kinetics and tissue uptake in brown trout in vivo: effect of an intravascular glucose load

The object of the present study was to elucidate whether a glucose load modifies glucose uptake by tissues in brown trout in vivo. By the use of 2-[1,2-³H]-deoxyglucose, plasma glucose disappearance rate and tissue glucose uptake were measured after an intraaortic glucose load of 500 mg·kg^-1 (glucose load group) and under normoglycemic conditions (control). We also attempted to determine whether fasting modifies the glucose load disposal (fasted glucose load group). The procedure used to calculate 2-deoxyglucose uptake by tissues was evaluated, and the levels of 2-deoxyglucose uptake were compared with those of 2-deoxyglucose phosphorylation. Uptake and phosphorylation rates were similar in all tissues, except in brain and heart. In all the groups glucose uptake rates were highest in spleen, kidney, brain and gills, and lowest in red muscle, heart and white muscle. However, white muscle was the main site of glucose uptake on a whole tissue basis. The glucose load led to strong, long-lasting hyperglycemia, in spite of the increases observed in plasma insulin levels and in glucose uptake rate by the whole body (control: 4.9 mol·min^-1·kg^-1; glucose load group: 6.5 mol·min^-1·kg^-1). This higher rate was due to the higher glucose uptake only in white and red muscles (four- and threefold, respectively). Fasting halved the uptake of glucose by both red and white muscles in the load condition. In consequence the use of exogenous glucose decreased with fasting (fasted glucose load group: 5.1 mol·min^-1·kg^-1), causing still longer hyperglycemia.Abbreviations bw body weight - 2DG 2-[1,2-³H]-deoxyglucose - 2DG-P 2-[1,2-³H]-deoxyglucose phosphate - dpm disintegrations per min - FGL fasted glucose load group - GL glucose load group - G-6-Pase glucose-6-phosphatase - LG L-[1-¹⁴C]-glucose - MS-222 3-aminobenzoic acid ethyl ester methanesulphonate salt     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Osmotic measurements on stomatal cells ofCommelina communis L.

Summary Observations of aperture changes as sucrose is added to the solution bathing epidermal strips ofCommelina communis L. allow calculation of the osmotic changes required to open or close the stomatal pore, for comparison with changes in potassium content. With isolated guard cells, in strips in which all cells other than guard cells have been killed, the internal osmotic changes required are 83 mosmol kg^–1 m^–1 below 10m aperture, 129 mosmol kg^–1 m^–1 in the range 10–15 m, and 180 mosmol kg^–1 m^–1 above 15 m. For opening against subsidiary cell turgor in addition to guard cell turgor, in intact strips with live subsidiary and epidermal cells, these figures should each be increased by about 33 mosmol kg^–1 m^–1. A change in subsidiary cell turgor is magnified in its effects on the water relations of the guard cell by a factor greater than 3.7 for equal changes in the water potential of the two cells, or greater than 4.7 at constant volume of the guard cell.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Effects of Cd and Zn on the development of annual xylem rings of young Norway spruce (Picea abies) plants

Three-year-old spruce (Picea abies) saplings were planted and cultivated for 2 years in pots with 3 1 substrate, consisting of a homogenized mixture of sand, peat and forest soil with a high organic content (volume ratio 11.52). This substrate was amended with 10–180 mol Cd [kg soil dry weight (DW)]^–1, 50–7500 mol Zn (kg soil DW)^–1 (determined with 1 M ammonium acetate extracts) or combinations of both elements. Annual xylem growth rings in stems of plants treated with 50 mol Cd (kg soil DW)^–1 or 7500 mol Zn (kg soil DW)^–1 were significantly narrower than in control plants. Growth reductions were more pronounced in the second year of the experiment. The contents of Cd and Zn in stem wood and needles were positively correlated with the substrate concentrations. The Mg contents of the spruce needles were inversely correlated with soil concentrations of Cd and Zn. Root development was impeded at moderate concentrations of Cd (50 mol kg^–1) or Zn (1000 mol kg^–1) in the substrate. The adverse effects of potentially toxic trace elements, like Cd or Zn, on xylem growth of spruce plants are discussed with regard to possible growth reductions in forest trees under field conditions.     

The nutritional status of the apical meristem of Lactuca sativa as affected by NaCl salinization: An electron-probe microanalytic study

A volume of tissue of lettuce (Lactuca sativa L.) plants extending 2 mm basipetally from the apical meristem and including leaf primordia and young expanding leaves was surveyed using electron-probe microanalysis (EPMA) on both frozen-hydrated and freeze-dried samples. This analysis was carried out either 2 or 5 d following NaCl salinization of the medium from the 10 mol · m^–-3 control level up to 80 mol · m^–-3. The objective was the investigation of possible changes in the nutritional status of the apical meristem that might account for some aspects of salt-induced growth inhibition. Sodium and chloride increased significantly in tissues basal to the apical meristem, while both phosphorus and potassium decreased in the same region. These changes were evident in specimens collected just 2 d after the commencement of salinization (20 h after completion of the salinization) and were not exacerbated by an additional 3 d of treatment; they were present in tissue as close as 100 m to the meristem and extending down to 500 m. The apical 10–50 m were relatively protected from both the increase in sodium and chloride and the decrease in phosphorus and potassium that occurred in more basal regions. Young leaves (up to 1.5 mm in length) appear to control their own mineral nutrient levels when challenged by salinization of the medium, presumably because of altered growth. A decrease in the concentration of total Ca as a result of salinization was significant in cells 500 m basal to the meristem, but was evident as a tendency in the data even within the first 50 m. Using an improved automatic method for the analysis of calcium by EPMA, it was found that total Ca was reduced by salinization, especially in basal regions (500 m below the apex) and also in young leaves (1–1.5 mm in length). We suggest that the nutrition of the shoot apical meristem may be disturbed soon after salinization and that the shoot meristem might be the source of a signal to expanding leaves, as well as exerting its own direct influence over leaf emergence.Abbreviation EPMA electron-probe microanalysis This work was supported by U.S. Department of Agriculture grant 87-CRCR-1-2462.     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.