Control of in vitro rooting and plant development in Corymbia maculata by silver nitrate, silver thiosulfate and thiosulfate ion

Plant regeneration and transformation in vitro is often improved by adding silver ion (Ag⁺) to the culture media as AgNO₃ or silver thiosulfate (STS). Ag⁺ reacts with substances to form insoluble precipitates, while thiosulfate (S₂O₃ ²⁻) interferes with these reactions. We studied the implications of silver precipitation and S₂O₃ ²⁻ in the medium for culture development by (1) examining formation of Ag⁺ precipitates from AgNO₃ versus STS in agar gels and their possible dependence on agar type; (2) comparing Corymbia maculata culture responses to AgNO₃ and STS and determining which better suits control of culture development; (3) clarifying whether STS-dependent alterations in culture development are due to Ag⁺ alone or also to a separate influence of S₂O₃ ²⁻. Silver precipitates appeared in aqueous gels of four agar brands supplemented with AgNO₃, but not in Phytagel^™, which remained transparent. No precipitation was observed in gels with STS. Indole-3-butyric acid (IBA)-mediated adventitious root induction and shoot growth were higher in C. maculata shoot tips cultured on gels with STS versus AgNO₃ (6–25 μM Ag⁺). IBA-treated shoot tips exhibited enhanced adventitious root regeneration, accelerated root elongation, increased frequency of lateral root formation, and stimulated shoot growth mediated by 100–250 μM sodium thiosulfate (Na₂S₂O₃) in medium without Ag⁺. The potency of S₂O₃ ²⁻ in facilitating culture development has never been recognized. It is inferred that superiority of STS in stimulating multiple responses of C. maculata culture results from sustained biological activity of Ag⁺ through prevention of its precipitation, and from impact of S₂O₃ ²⁻ on cell differentiation and growth.     

The uptake of silver ions by Escherichia coli K12: toxic effects and interaction with copper ions

Summary Growth of Escherichia coli in chloridefree medium in batch culture is inhibited completely at concentrations of AgNO₃ greater than 2.5x10^-6 M. Incubation of non-growing cells in HEPES buffer (pH 7.4) at increasing levels of Ag⁺ results in the progressive saturation of two types of binding site. At one site, the Ag⁺ is not released by washing with 0.1 M nitric acid, and is probably intracellular. Silver bound to the second site is released by acid-washing, but not by buffer washing, and is assumed to be surface-bound. The amounts of Ag⁺ taken up from solution at the two sites is 1.6x10^-7 and 4.6x10^-7 mol (mg dry weight)^-1, respectively. Total accumulation of silver is 67 mg (g dry weight)^-1, similar to literature values found for silver-resistant bacteria. Binding of Ag⁺ at intracellular sites (observed at low [Ag⁺]) appears to be independent of pH. Addition of AgNO₃ to growing cells in mid-exponential phase of growth in concentrations that will inhibit growth results in substantially decreased accumulation of silver. Growth yield in chemostat culture is diminished in the presence of added Ag⁺, but this effect is moderated by added Cu²⁺, which may protect copper sites from Ag⁺ or compete with Ag⁺ for other sites at which Ag⁺ exerts toxic effects. Very small amounts of Cu²⁺ are found in cell samples from the chemostat compared to the substantial amounts of Ag⁺ taken up, but uptake of Cu²⁺ is decreased at higher [Ag⁺]/[Cu²⁺]ratios.     

Effect of inhibitors of ethylene biosynthesis and action, as well as calcium and magnesium on rose shoot rooting, shoot-tip necrosis and leaf senescence in vitro

Microcuttings of easy-to-root dwarf rose cv. Starina, showing early symptoms of leaf senescence and shoot-tip necrosis in rooting stage, were chosen for the study. The effects of inhibitors of ethylene biosynthesis (AOA, AIB) and action (AgNO₃), and Ca²⁺ and Mg²⁺ were studied in relation to rooting, leaf senescence and shoot-tip necrosis. The effects of these substances were examined with respect to IAA presence in a medium, which stimulated leaf yellowing and shoot-tip necrosis. AOA strongly inhibited rooting of microcuttings, but did not affect ethylene biosynthesis. AIB at 250 mg·l⁻¹ and AgNO₃ 2.5 mg·l⁻¹ in the presence of IAA did not affect rooting but effectively prevented leaf senescence. Ca²⁺ alone or combined with Mg²⁺ at raised concentration, or an ethylene action inhibitor Ag⁺, reduced shoot-tip necrosis in microcuttings treated with IAA. Addition of Ag⁺ to IAA medium drastically increased ethylene production by the shoots. Interaction between endogenous levels of auxin, ethylene and calcium in relation to rooting, shoot-tip necrosis and leaf senescence was discussed. Ethylene could enhance tissue sensitivity to auxin. Moreover, the tissue of rose shoots is very sensitive in the in vitro condition on standard medium because of the calcium deficiency. Thus, the raised Ca/Mg level counteracted shoot-tip necrosis through enhancing cell membrane and wall resistance to ethylene and IAA.     

Stimulation and oxidative catalytic inactivation of thermolysin by copper•Cys-Gly-His-Lys

[Cu²⁺•Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 μM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 μM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 μM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu²⁺–amino-terminal copper and nickel binding motif (λ _max ∼ 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu²⁺ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (ΔE = 92 mV) for the Cu^3+/2+ redox couple obtained for [Cu²⁺•Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu²⁺•Lys-Gly-His-Lys]⁺ (0.84 V, ΔE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu²⁺•Lys-Gly-His-Lys]⁺ homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O₂) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k _obs = 7.7 × 10⁻² min⁻¹, yielding a second-order rate constant of (7.7 ± 0.9) × 10⁴ M⁻¹ min⁻¹. Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.     

Improvement of echinacoside and acteoside production by two-stage elicitation in cell suspension culture of <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

At appropriate concentrations, polyamines promoted the callus growth and echinacoside content of Cistanche deserticola while Ag⁺ increased the content of echinacoside and acteoside. In a 20-day culture period, when putrescine (25 μM) and Ag⁺ (10 μM) were added on day 8 and day 16, respectively, the echinacoside production (1.7 g l⁻¹) and acteoside production (0.4 g l⁻¹) reached the maximum, which were 1.4-fold and 1.5-fold of those in single putrescine treatment, 1.6-fold and 1.4-fold of those in single Ag⁺ treatment, respectively. Exogenous putrescine enhanced cell viability and antioxidant enzyme activity markedly, so increased the final biomass. Ag⁺ addition increased H₂O₂ content and phenylalanine ammonia-lyase activity significantly which led to higher echinacoside and acteoside contents.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

Detoxification of environmental sulfide to sulfane sulfur in the intertidal sipunculid Phascolosoma arcuatum

The capability of Phascolosoma arcuatum to detoxify sulfide in anaerobic conditions was examined. Sulfane sulfur, which underwent cold cyanolysis, was the major excretory end product of sulfide detoxification during anoxia. Thiosulfate was not excreted into the external medium. Instead, it was absorbed by P. arcuatum and its absorption was stimulated by the presence of sodium sulfide (Na₂S) in the incubation medium. The effective formation and excretion of sulfane sulfur by P.␣arcuatum required the presence of both Na₂S and sodium thiosulfate (Na₂S₂O₃). Results obtained indicate that rhodanese might be involved in sulfide detoxification in this sipunculid. Rhodanese could act as a catalyst in the transfer of sulfur atoms from thiosulfate to HS⁻. The body wall and the introvert were the main sites of sulfide detoxification. However, it is unlikely that epibiotic bacteria associated with the outside surface of the worm were involved in the detoxification process. A time-course study on the contents of thiosulfate and sulfane sulfur in the body wall of P. arcuatum incubated anaerobically in the presence of Na₂S + Na₂S₂O₃ verified that thiosulfate absorbed was utilized to detoxify sulfide to sulfane sulfur. Accepted: 24 October 1996     

Evaluation of a closed system,diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO₃ and silver thiosulphate – Ag₂S₂O₃ as inhibitors of ethylene activity, cobalt chloride – CoCl₂ as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm³). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO₃, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO₃ (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO₂ levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content. In the light period, CO₂ depletion occurred in the head-space of the sealed vessels (ca. 40 μl l^-1), the CO₂ concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Silver tolerance and accumulation in yeasts

Summary Debaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO₃. Although Ag⁺ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag⁺ to Ag⁰ resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO₃-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag⁰ although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag⁺ reduction to Ag⁰.     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Accumulation of silver by Chromatium vinosum from solutions containing silver thiosulfate

The photosynthetic sulfur bacterium, Chromatium vinosum, was cultured in inorganic photographic processing solutions containing silver thiosulfate complex salt (AgNa₃(S₂O₃)₂) under light. It was found that Chromatium was resistant to Ag and accumulated granular silver in the membrane during growth. The amount of Ag accumulated in the cells depended on the initial concentrations of the Ag salt in the culture solution. When the concentration of Ag was 300 mg/l, the bacteria accumulated Ag as high as 30% of the dry cell weight. The size of the granules was 0.1 to 0.3 m. Results from X-ray microanalysis indicated that these granules consisted mostly of Ag^o with small fractions of Ag₂S and AgCl.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

Effects of Anions on the Capacity and Affinity of Copper Adsorption in Two Variable Charge Soils

Effects of nitrate,(NO₃⁻) chloride (Cl⁻), sulfate (SO₄^2-, and acetate (Ac⁻) on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were evaluated in two Fe and Al enriched variable charge soils from Southern China. The maximum adsorption of Cu²⁺ (M, a parameter from the Langmuir isotherm model) in the presence of different anions decreased in the order Cl⁻ > Ac⁻ > NO₃⁻ > SO₄^2- for both soils. The clayey loamy soil (mixed siliceous thermic Typic Dystrochrept, TTD), developed on the Arenaceous rock, adsorbed less Cu²⁺ than the clayey soil (kaolinitic thermic Plinthudults, KTP), derived from the Quaternary red earths, regardless of anion type present in the medium. The affinity of adsorbed Cu²⁺ to both soils could be characterized by the Kd (distribution coefficient) values and successive extraction of the adsorbed Cu²⁺ with 1-mol NH₄Ac L⁻¹. The log₁₀Kd value was smaller for the TTD soil than for the KTP soil and decreased in the order of Cl⁻ > NO₃⁻ > SO₄^2- > Ac⁻ at low initial Cu²⁺ concentrations (≤40 mg Cu²⁺L⁻¹), whereas at 80 mg Cu²⁺L⁻¹, the log₁₀Kd value was similar for NO₃⁻, SO₄^2-, and Ac⁻, but was slightly higher for Cl⁻. Complete extraction of Cu²⁺ adsorbed in the presence of Ac⁻ was achieved. Influence of NO₃⁻ and SO₄^2- on the affinity of adsorbed Cu²⁺ was similar, but the effects of Cl⁻ depended on the initial Cu²⁺ concentrations. The extracted percentage of the adsorbed Cu²⁺ in the presence of NO₃⁻ or SO₄^2- increased with increasing Cu²⁺ adsorption saturation. The presence of Cl⁻, NO₃⁻, or SO₄^2- markedly decreased the equilibrium solution pH for both soils with increasing initial Cu²⁺ concentrations, and the delta pH values at the highest Cu²⁺ level were 0.5, 0.63, and 0.55 U for the TTD soil and 0.79, 0.84, and 0.93 U for the KTP soil, respectively for the three anions. The presence of Ac⁻ had a minimal influence on the equilibrium solution pH because of the buffering nature of the NaAc/HAc medium which buffered the released protons. The effects of anions on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were dependent on anion types and were apparently related to the altered surface properties caused by anion adsorption and/or the formation of anion– Cu²⁺ complexes.     

Effects of low nitrogen medium on endogenous changes in ethylene, auxins, and cytokinins in in vitro shoot formation from rhizomes of Cymbidium kanran

Summary Shoot formation from rhizome explants of Cymbidium kanran was promoted on Murashige and Skoog (MS) medium: (1) with 1 mgl⁻¹ (4.4μM) 6-benzyladenine (BA) and 0.1 mgl⁻¹ (0.54μM) α-naphthaleneacetic acid (NAA); (2) with ethylene inhibitor (silver nitrate, AgNO₃); or (3) by reducing ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) to 25 and 50%, respectively, of their original concentrations. Shoot formation by BA and NAA was strongly inhibited with the application of ethephon, an ethylene releaser. The ethylene production from the rhizome explants was reduced 30–55% on low nitrogen medium after 1–3 mo. of culture and 52% on BA and NAA medium after 1 mo. of culture compared with explants on standard MS medium. No difference in endogenous auxin (indole-3-acetic acid, IAA) and cytokinin (isopentenyl adenosine, iPA) contents in the rhizomes was found between the treatments. Low ethylene levels were correlated with higher frequency of shoot formation from the rhizomes.     

An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)

Direct comparisons between photosynthetic O₂ evolution rate and electron transport rate (ETR) were made in situ over 24 h using the benthic macroalga Ulva lactuca (Chlorophyta), growing and measured at a depth of 1.8 m, where the midday irradiance rose to 400–600 μmol photons m⁻² s⁻¹. O₂ exchange was measured with a 5-chamber data-logging apparatus and ETR with a submersible pulse amplitude modulated (PAM) fluorometer (Diving-PAM). Steady-state quantum yield ((F_m′−F_t)/F_m′) decreased from 0.7 during the morning to 0.45 at midday, followed by some recovery in the late afternoon. At low to medium irradiances (0–300 μmol photons m⁻² s⁻¹), there was a significant correlation between O₂ evolution and ETR, but at higher irradiances, ETR continued to increase steadily, while O₂ evolution tended towards an asymptote. However at high irradiance levels (600–1200 μmol photons m⁻² s⁻¹) ETR was significantly lowered. Two methods of measuring ETR, based on either diel ambient light levels and fluorescence yields or rapid light curves, gave similar results at low to moderate irradiance levels. Nutrient enrichment (increases in [NO₃ ⁻], [NH₄ ⁺] and [HPO₄ ^2-] of 5- to 15-fold over ambient concentrations) resulted in an increase, within hours, in photosynthetic rates measured by both ETR and O₂ evolution techniques. At low irradiances, approximately 6.5 to 8.2 electrons passed through PS II during the evolution of one molecule of O₂, i.e., up to twice the theoretical minimum number of four. However, in nutrient-enriched treatments this ratio dropped to 5.1. The results indicate that PAM fluorescence can be used as a good indication of the photosynthetic rate only at low to medium irradiances. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency plant regeneration from the cotyledonary node of common bean

An efficient regeneration system for Phaseolus vulgaris was developed from mature seeds germinated on Murashige and Skoog (MS) medium supplemented with thidiazuron or N⁶-benzylaminopurine (BA) for 6 d. Using cotyledonary nodes, multiple buds were induced on the MS medium supplemented with 5.0 mg dm⁻³ BA with the induction frequency 71.9 % after 4-week culture. The buds were then transferred onto shoot formation medium containing 1.0 mg dm⁻³ BA, 0.1 mg dm⁻³ gibberellic acid and 2.0 mg dm⁻³ silver nitrate. The addition of AgNO₃ enhanced the frequency of the shoot formation from 61.3 to 87.6 %. Root induction medium was half-strength MS medium with 0.75 mg dm⁻³ indolebutyric acid and 0.02 mg dm⁻³ BA. The average root frequency was 84.3 %. The regenerated plantlets with healthy roots grew successfully when transferred to soil. Using this system we obtained over 10 regenerated plantlets from one explant.     

Oxygen consumption and sulfide detoxification in the lugworm Arenicola marina (L.) at different ambient oxygen partial pressures and sulfide concentrations

The lugworm Arenicola marina is a typical inhabitant of intertidal flats. In its L-shaped burrow the animal is exposed to varying concentrations of O₂ and toxic sulfide depending on the tides. The lugworm is able to detoxify sulfide through its oxidation to thiosulfate. When exposed to declining O₂ tensions Arenicola marina reacted as an oxyconformer. In the presence of 25 μmol · l⁻¹ sulfide the respiration was not affected. In contrast, the lugworm consumed significantly less O₂ at any Po₂ in the presence of 200 μmol · l⁻¹ sulfide. Without sulfide anaerobic metabolism started at a Po₂ of approximatedly 10 kPa. Even at high O₂ tensions animals exposed to sulfide produced significantly more anaerobic metabolites compared with the controls. Accordingly the critical value Pc_M, the ambient Po₂ below which anaerobic metabolism starts, was shifted towards normoxia. Since O₂ supply was sufficient for aerobic metabolism, anaerobiosis was induced by sulfide. An influx of sulfide was observed at 25 as well as at 200 μmol · l⁻¹ sulfide. The main product of sulfide detoxification in the lugworm was thiosulfate. Its synthesis increased with ambient Po₂ and depended on the sulfide concentration. Sulfide and thiosulfate were detected in the coelomic fluid, the blood, and the body wall of Arenicola marina. Only about 2% of the ambient O₂ was used for sulfide detoxification at 25 μmol · l⁻¹ sulfide and about 50% at 200 μmol · l⁻¹ sulfide, respectively. Even at the low sulfide concentration Arenicola marina's capacity to detoxify sulfide was too low to maintain a complete aerobic metabolism. Accepted: 19 February 1997     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.