In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse

Juvenile big bellied seahorse Hippocampus abdominalis were exposed acutely and chronically to elevated ammonia and nitrite {24 h exposure: 0·01, 5·0, 10·1, 14·8 and 19·9 mg l⁻¹ total ammonia-nitrogen [TA-N] and <0·001, 74·4, 99·2 and 123·6 mg l⁻¹ [NO₂-N] nitrite-nitrogen and 35 days exposure: 0·11, 0·55, 1·67 and 3·07 mg l⁻¹ TAN and <0·001, 0·92, 4·67 and 9·10 mg NO₂-N l⁻¹}. Significant ( P <0·001) increases in oxygen consumption rate and ventilation frequency occurred at 14·8, 19·9 mg l⁻¹ TA-N and 99·2, 123·6 mg l⁻¹ NO₂-N for acutely exposed fish. Oxygen consumption rate was significantly ( P <0·05) elevated at 1·67 and 3·07 mg l⁻¹ TA-N in chronically treated fish and ventilation frequency increased significantly ( P <0·05) at 0·55, 1·67, 3·07 mg l⁻¹ TA-N and 4·59, 9·10 mg l⁻¹ NO₂-N. There were no significant differences in growth between controls and ammonia exposed fish. Mortalities occurred at 14·8, 19·9 mg l⁻¹ TA-N.     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

Pathogenesis-related protein b1' in plants and in cell suspension cultures of Nicotiana glutinosa Nicotiana debneyi and an amphidiploid cross (N. glutinosa×N. debneyi)

The expression of PR-protein b₁' in plants and cell suspension cultures of Nicotiana glutinosa L., Nicotiana debneyi Domin, and an amphidiploid cross of these two species, a hybrid, has been investigated. An enzyme linked immunosorbent assay has been employed to determine the concentration of PR-protein b₁' in extracts. The PR-Protein b₁' was constitutively produced in intact plants of the hybrid (around 25 μg g⁻¹ leaf tissue), while only trace amounts of the protein (< 50 ng g⁻¹ leaf tissue) were found in plants of the two parents. In suspension culture, the concentrations of PR-protein b₁' were 8, 0.4 and less than 0.1 mg l⁻¹ medium for the hybrid. N. debneyi and N. glutinosa , respectively. Only trace amounts of the protein were found in extracts from cells. Seven days after infection by tobacco mosaic virus (TMV) the concentration of PR-protein b₁' in leaves of N. glutinosa was 22.5 μg g⁻¹ leaf tissue. In N. debneyi and the hybrid a relatively limited induction of PR-protein b₁' by TMV was observed. The influence of various phenoxyacetic acids on the expression of PR-protein b₁' in the 3 cell cultures has been investigated. Cultures of N. glutinosa responded to treatments with 2,4-D and 2,4,5-T while cultures of N. debneyi and the hybrid were essentially unaffected. In the former case a concentration of 5–10 mg l⁻¹ 2,4,5-T was optimal and cells were most responsive to the treatment 4 days after subcultivation. The concentration of PR-protein b₁' in elicited cell cultures of N. glutinosa was 2 to 4 mg l⁻¹ medium.     

Assimilate relations in source and sink leaves during acclimation to a CO2-enriched atmosphere

Evidence from previous studies suggested that adjustments in assimilate formation and partitioning in leaves might occur over time when plants are exposed to enriched atmospheric CO₂. We examined assimilate relations of source (primary unifoliolate) and developing sink (second mainstem trifoliolate) leaves of soybean [ Glycine max (L.) Merr. cv. Lee] plants for 12 days after transfer from a control (350 μl l⁻¹) to a high (700 μ l⁻¹) CO₂ environment. Similar responses were evident in the two leaf types. Net CO₂ exchange rate (CER) immediately increased and remained elevated in high CO₂. Initially, the additional assimilate at high CO₂ levels in the light and was utilized in the subsequent dark period. After approximately 7 days, assimilate export in the light began to increase and by 12 days reached rates 3 to 5 times that of the control. In the developing sink leaf, high rates of export in the light occurred as the leaf approached full expansion. The results indicate that a specific acclimation process occurs in source leaves which increases the capacity for assimilate export in the light phase of the diurnal cycle as plants adjust to enriched CO₂ and a more rapid growth rate.     

Mycorrhiza formation and elevated CO2 both increase the capacity for sucrose synthesis in source leaves of spruce and aspen

The effects of mycorrhiza formation in combination with elevated CO₂ concentrations on carbon metabolism of Norway spruce ( Picea abies ) seedlings and aspen ( Populus tremula × Populus tremuloides ) plantlets were analysed. Plants were inoculated for 6 wk with the ectomycorrhizal fungi Amanita muscaria and Paxillus involutus (aspen only) in an axenic Petri-dish culture at 350 and 700 μl l⁻¹ CO₂ partial pressure. After mycorrhiza formation, a stimulation of net assimilation rate was accompanied by decreased activities of sucrose synthase, an increased activation state of sucrose-phosphate synthase, decreased fructose-2,6-bisphosphate and starch, and slightly elevated glucose-6-phosphate contents in source leaves of both host species, independent of CO₂ concentration. Exposure to elevated CO₂ generally resulted in higher net assimilation rates, increased starch as well as decreased fructose-2,6-bisphosphate (aspen only) content in source leaves of both mycorrhizal and nonmycorrhizal plants. Our data indicate only slightly improved carbon utilization by mycorrhizal plants at elevated CO₂. They demonstrate however, that both factors which modulate the sink-source properties of plants increase the capacity for sucrose synthesis in source leaves mainly by allosteric enzyme regulation.     

Morphological changes in the gills of Lophiosilurus alexandri exposed to un-ionized ammonia

The average lethal concentration of un-ionized ammonia (48-h LC₅₀NH₃) has been determined by the static assay for larvae (0.48 mg l⁻¹) and alevins (0.92 mg l⁻¹) of 'pacamã' Lophiosilurus alexandri. Studies by light and scanning electron microscopes at the greatest concentration of NH₃ (0.99 mg l⁻¹ for larvae and 1.5 mg l⁻¹ for alevins) have shown that the changes in the cells and branchial tissue were more intense in the alevins.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

Photosynthesis, growth and structural characteristics of holm oak resprouts originated from plants grown under elevated CO2

The physiological characteristics of holm oak ( Quercus ilex L.) resprouts originated from plants grown under current CO₂ concentration (350 μl l⁻¹) (A-resprouts) were compared with those of resprouts originated from plants grown under elevated CO₂ (750 μl l⁻¹) (E-resprouts). At their respective CO₂ growth concentration, no differences were observed in photosynthesis and chlorophyll fluorescence parameters between the two kinds of resprout. E-resprouts appeared earlier and showed lower stomatal conductance, higher water-use efficiency and increased growth (higher leaf, stem and root biomass and increased height). Analyses of leaf chemical composition showed the effect of elevated [CO₂] on structural polysaccharide (higher cellulose content), but no accumulation of total non-structural carbohydrate on area or dry weight basis was seen. Four months after appearance, downregulation of photosynthesis and electron transport components was observed in E-resprouts: lower photosynthetic capacity, photosystem II quantum efficiency, photochemical quenching of fluorescence and relative electron transport rate. Reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) activity, deduced from the maximum carboxylation velocity of RuBisCo, accounts for the observed acclimation. Increased susceptibility of photosynthetic apparatus to increasing irradiance was detected in E-resprouts.     

The effects of variable nitrogen and phosphorous concentrations on Eriophorum vaginatum tillers grown in nutrient solutions

Eriophorum vaginatum tillers were collected at Eagle Creek, Alaska and cultivated in aerated solutions under controlled environmental conditions. The nutrient solutions contained traces, 1.05 and 21 mg l⁻¹ N (nitrate) and traces, 0.15 and 3.10 mg l⁻¹ P (phosphate), pH was maintained at 5.5. The high N, 21 mg l⁻¹, and P, 3.18 mg l⁻¹, nutrient solution produced significant biomass increases. Functional leaf areas were significantly enhanced by high N and P doses in the solutions. Root surface areas varied considerably between treatments; however, significant differences were not found. The mean root surface area of a tiller reached 126 cm² (range 35–290 cm²), whereas the functional leaf area was 6.8 cm² (range 3.3–20.3 cm²). Tillers growing in the highest N + P solutions produced twice the number of daughter tillers as tillers growing in solutions with trace amounts of N and P.     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.     

The effect of humic substances on the toxicity of aluminium to adult rainbow trout, Oncorhynchus mykiss (Walbaum)

Some physiological parameters were measured in adult rainbow trout during a 10-day exposure to 180 μg Al_total l⁻¹ in acid water (pH 4.7) with or without humic substances (10 mg l⁻). The fish were acclimatized to pH 5.0 for 7 days prior to the experimental treatments.
Chemical analyses revealed that, in the presence of human substances, 74–80% of the A1 was organic bound, while in the absence of humic substances most of the Al(987percnt;) occurred in the inorganic form.
Al bound to humic substances (13–150 μg l⁻¹) did not alter the plasma NaCl-concentration, nor the haematocrit value, of rainbow trout during an exposure period of 10 days. This contrasts with the high death rate obtained within 2–3 days when most of the A1 (175 μg l⁻¹) was in the inorganic form. The lethality was accompanied by a 25% decrease in the plasmaconcentration of NaCl and a doubling of the haematocrit value. Bulk analysis revealed that when the metal was present in inorganic forms the total Al content of the gills (75 μg A1 g⁻¹ wet weight) was 15 times higher than when it was present as bound to the humic substances. These experiments showed that the accumulation of A1 at the gills was accompanied by physiological disturbances, both being a function of the chemical speciation of Al.     

Stamen abscission and water balance in Metrosideros flowers

Cymules (3-flowered units borne on single pedicels) were cut from inflorescences of Metrosideros collina J.R. & G. Forst. cv. Tahiti and used to test the effects of ethephon and ethylene on stamen abscission in the presence of silver thiosulphate (STS) and 1-methylcyclopropene (1-MCP), and to test the effects of holding solutions on cymule water balance and the progression of floral development. Flower bud and stamen abscission occurred in response to 0.5–5.0 and 0.1 μl l⁻¹ ethylene, respectively. Ethylene effects were partially negated by scrubbing exogenous ethylene, and more completely negated by STS (2.0 m M ). 1-MCP caused greater ethylene production and inhibited stamen abscission for only 1–2 days after treatment. Ethephon (10-10 000 mg l⁻¹) induced stamen wilting rather than abscission, an effect that was not negated by STS. Stamen wilting was negatively correlated with stamen relative water content, and the increase in stamen wilting was generally reduced by treatments that enhanced cymule mass. Stamen wilting was least using a 100 g l⁻¹ sucrose pulse or holding solutions containing 30–40 g l⁻¹ sucrose, with hydroxyquinoline citrate (200 mg l⁻¹) maintained at pH 5. Our results indicate that 1-MCP may be relatively ineffective in blocking the effects of ethylene on the abscission of organs, such as the stamens of M . collina , which are highly sensitive to this hormone.     

Growth, mineral uptake and skeletal calcium deposition in brown trout, Salmo trutta L., yolk-sac fry exposed to aluminium and manganese in soft acid water

In 30-day exposures in artificial soft water medium, survival of brown trout alevins was not affected by low pH (4.5,4.8, 5.4), by low calcium concentration (10.25 μmol l⁻¹) or by manganese (≤20 μmol l⁻¹), but was impaired by aluminium (6–8 μmol l⁻¹) at low calcium concentration (10 μmol l⁻¹) irrespective of pH (4.5 or 5.4). Manganese (6.6, 20 μmol l⁻¹) impaired net calcium uptake and calcium deposition in the skeleton at low calcium concentration (25 μmol l⁻¹) irrespective of pH. Aluminium (2–8 μmol l⁻¹) impaired gross development, net uptake of calcium, potassium and sodium, and calcium deposition in the skeleton, and slightly increased the net loss of magnesium, some of these effects being more severe at calcium concentration 10 μmol l⁻¹ than 50 μmol l⁻¹, and some more severe at pH 5.4 than pH 4.5. Net uptake of calcium and sodium were impaired at low pH (4.5, 4.8), and skeletal calcium deposition was impaired at low calcium concentration (10 μmol l⁻¹), but these effects of low pH and low calcium concentration were slight compared with those of the trace metals. The possible role of trace metals in reports of the deleterious effects on fish of low pH levels is discussed.     

The dissolved oxygen and temperature requirements of Atlantic salmon, Salmo salar L., in the Thames Estuary

An improvement in water quality in the estuary of the River Thames in recent years, coupled with the return of adult Atlantic salmon following artificial stocking of the headwaters with parr and of the lower river with smolts, has provided an opportunity to define the dissolved oxygen requirements of adult fish ascending the estuary to reach fresh water. Between July and September 1984 the fish traversed a length of 30 km where the concentration of dissolved oxygen was at its lowest, the 5-percentile and median values being 1.6–2.6 and 3.5–5.9 mg l⁻¹, respectively, depending upon exact location. Within this zone there was a length of about 20 km in which the minimum at any one time during the period was always less than 5mg l⁻¹ and a shorter length of 15 km in which it was always less than 4.7 mg l⁻¹, and it is likely that some fish experienced even lower values during their upstream passage. Over lengths of 1, 10 and 30 km, for example, the 10-percentiles were 2, 2.2 and 2.8 mg l⁻¹, respectively, the medians were 3.6, 3.8 and 4.3 mg l⁻¹, respectively and the 90-percentiles were 4.8, 4.9 and 5.3 mg l⁻, respectively. The water temperature during August, when most of the fish were caught, was never lower than 19°C and there was a length of estuary of at least 20 km where it exceeded 22°C.     

Does down‐regulation of photosynthetic capacity by elevated CO2 depend on N supply in Dactylis glomerata?

Dactylis glomerata was grown hydroponically in a controlled environment at ambient (360 μl l⁻¹) or elevated (680 μl l⁻¹) CO₂ and four concentrations of nitrogen (0.15, 0.6, 1.5 and 6.0 m M NO₃⁻), to test the hypothesis that reduction of photosynthetic capacity at elevated [CO₂] is dependent on N availability and mediated by a build-up of non-structural carbohydrates. Photosynthetic capacity of the youngest fully expanded leaf (leaf 5, 2 days after full expansion) was reduced in CO₂-enriched plants at low, but not high N supply and so the stimulation of net photosynthesis by CO₂ enhancement was less at low than at high N supply. CO₂ enrichment resulted in a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content on a leaf area basis at 0.6 and 1.5 m M NO₃⁻, but not at 0.15 and 6.0 m M NO₃⁻, and had no effect on the total N content of the leaf on an area basis. However, decreases in Rubisco content could be primarily accounted for by a decrease in total N content of leaves, independent of [CO₂]. A doubling of the Rubisco content by increasing the N supply beyond 0.6 m M had only a marginal effect on the maximum carboxylation velocity in vivo, suggesting that the fraction of inactive Rubisco increased with increasing N supply. Although CO₂-enriched plants accumulated more non-structural carbohydrates in the leaf, the reduction of photosynthetic capacity at low N supply was not mediated simply by a build-up of carbohydrates. In D . glomerata , the photosynthetic capacity was mainly determined by the total N content of the leaf.     

The survival of smolts of salmon Salmo salar L. at low concentrations of dissolved oxygen

The survival of Atlantic salmon smolts on sudden exposure to low constant concentrations of dissolved oxygen (DO) has been measured under laboratory conditions; the 3-d LC50 is about 3 mg l⁻¹ in freshwater and about 2.5 mg l⁻¹ in 30% and 80%'sea water', even for fish acclimated for several days to low concentrations of DO and high salinity; median threshold concentrations of DO are probably close to 3.3 and 2.6 mg l⁻¹ respectively.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .