Effect of exogenous steroid glycosides on cultured cells of potato under oxidative stress

Low doses of furostanol glycosides (FG) were shown to elevate the activity of peroxidases (guaiacol-dependent and ascorbate peroxidases) and reduce peroxidation of lipids (POL) below the control level in the cell culture of potato (Solanum tuberosum L.). Under oxidative stress (OS) induced by paraquat, FG protected the cell culture from injury with peroxidase activity being high and POL level lower as compared with the effect of paraquat alone. FG did not affect the activity of superoxide dismutase and catalase. Dynamics of the levels of chlorophyll (a + b) and carotenoids depended not only on the effect of FG and paraquat but on the composition of cell population as well. Greenish tissue contained more pigments and was more resistant to the herbicide action than whitish tissue was. Possible reasons for the elevation of resistance of the cultured cells treated with FG under OS are discussed as well as similarity and differences in the responses of cells to the effect of inducers.     

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

 Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml^–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l^–1 mannitol and 40 g l^–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml^–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium. Received: 2 September 1998 / Revision received: 20 November 1998 / Accepted: 12 December 1998     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

The sulfide tolerance of milkfish and tilapia in relation to fish kills in farms and natural waters in the Philippines

Fish kills of milkfish Chanos chanos and tilapia Oreochromis spp. now occur frequently in brackish, marine, and freshwater farms (ponds, pens, and cages) in the Philippines. Aquafarms with high organic load, limited water exchange and circulation, no aeration, and high stocking and feeding rates can become oxygen-depleted and allow sulfide from the sediments to appear in the water column and poison free-swimming fish. The sulfide tolerance of 2–5 g milkfish and 5–8 g O. mossambicus was determined in 25-liter aquaria with flow-through sea water (100 ml min-1) at 26–30 °C and sulfide stock solutions pumped in at 1ml min-1. Total sulfide concentrations in the aquaria were measured by the methylene blue method and used in the regression against the probits of % survival. Four experiments showed that the two species have similar sulfide tolerance. In sea water of pH 8–8.5, about 163 ± 68 μM or 5.2 ± 2.2 mg l-1 total sulfide (mean ± 2 se) or 10 μM or 313 μg l-1 H2S was lethal to 50% of the fish in 4–8 h, and 61 ± 3 μM total sulfide or 4 μM H2S in 24–96 h (to convert all sulfide concentrations: 1 μM = 32 μg l-1). Earthen pond bottoms had 0–382 μM total dissolved sulfide (mean ± sd = 54 ± 79 μM, n = 76); a tenth of the samples had >200 μM. The water column may have such sulfide levels under hypoxic or anoxic conditions. To simulate some of the conditions during fish kills, 5–12 g milkfish were exposed to an abrupt increase in sulfide, alone or in combination with progressive respiratory hypoxia and decreasing pH. The tests were done in the same flow-through set-up but with sulfide pumped in at 25 ml min-1. The lethal concentration for 50% of the fish was 197 μM total sulfide or 12 μM H2S at 2 h, but 28–53 μM sulfide allowed fish to survive 6–10 h. Milkfish in aquaria with no aeration nor flow-through sea water died of respiratory hypoxia in 5–8 h when oxygen dropped from 6 to 1 mg l-1. Under respiratory hypoxia with 30–115 μM sulfide, the fish died in 2.5–4 h. Tests with low pH were done by pumping a weak sulfuric acid solution at 25 ml min-1 into aquaria with flow-through sea water such that the pH dropped from 8 to 4 in 5 h. Under these conditions, milkfish died in 7–9 h when the pH was 3.5. When 30–93 μM sulfide was pumped in with the acid, the fish died in 2–6 h when the pH was still 4.5–6.3. Thus, sulfide, hypoxia, and low pH are each toxic to milkfish at particular levels and aggravate each other's toxicity. Aquafarms must be well oxygenated to prevent sulfide toxicity and fish kills. This revised version was published online in July 2006 with corrections to the Cover Date.     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Efficient plant regeneration of garlic (Allium Sativum L.) by root-tip culture

Summary Root apices from in vitro cultured garlic (Allium sativum) cloves of cvs. ABEN and GT96-1 were used as axenic explants for organogenic callus production and plant regeneration experiments. Explants cultured in media based on those of Chu and co-workers (N6) or Murashige and Skoog (MS) could induce organogenic callus after 8 wk culture in darkness. Both media were supplemented with 2,4-dichlorophenoxyacetic acid (2.2–4.5 μM), alone or combined with 6-furfurylaminopurine (kinetin, 2.3–4.6 μM). Shoots started to grow 3 wk after culturing in the presence of light and the addition to culture media of 4.4 μM N⁶-benzyladenine. Plants capable of producing microbulbs regenerated 6 wk later. Up to 170 plants g⁻¹ FW callus were obtained when culturing was initiated in MS medium supplemented with 4.6 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid.     

Microtuberization in potato (Solanum tuberosum L.)

Twenty-two genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under six in vitro culture conditions. Cultures maintained under a short photoperiod (10 h of 6–12 μmol m^–2 s^–1) and low temperatures (day 20°±2°C and night 18°±2°C) had both a higher yield (255 mg/plantlet) and a greater number (2/plantlet) of microtubers than those maintained under long days (16 h of 38–50 μmol m^–2 s^–1) combined with high temperatures (day 28°±2°C and night 25°±2°C) (yield 207 mg/plantlet; microtuber number, 0.9/plantlet), over a wide range of genotypes. After the plantlets had been cultured under long days for an initial period of 60 days, continuous darkness advanced microtuberization by 2–3 months in various genotypes. Under short-day and low-temperature conditions the addition of 6-benzylaminopurine increased microtuber yield from 255 mg/plantlet to 645 mg/plantlet and average microtuber weight from 115 mg to 364 mg. A similar pattern was observed under conditions of long days and high temperature, and continuous darkness and low-temperature. Microtubers produced under light had a greater number of eyes (maximum average: 5.96/microtuber) than those produced in the dark (maximum average: 3.50/plantlet). The genotype × cultural conditions interactions were significant indicating the importance of developing genotype-specific protocols to maximize microtuberization. Received: 17 September 1997 / Revision received: 12 December 1997 / Accepted: 1 January 1998     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Sensitivity of antioxidant status of plant cells to furostanol glycosides

The present paper reports that in vitro plant objects (test tube plants and cell cultures), when subjected to furostanol glycosides (FG), underwent nonspecific reactions related to antioxidant status—decrease in peroxidation of lipids (POL) and increase in guaiacol-dependent peroxidase activity. The level of superoxide increased as early as after 5 min from contact with yam (Dioscorea deltoidea Wall) cells with FG. In this case, changes in POL processes and in activities of peroxidase and aldehyde-disposing emzymes were also observed. Upon a short-term cell exposure to FG, the levels of the primary POL products (conjugated dienes) increased, and that of the secondary POL products decreased compared to the control. These events were preceded by a rise in SOD activity and in an antioxidant activity of peroxidase along with a concurrent decrease in its oxidase (prooxidant) activity. The elevated activities of aldehyde-disposing enzymes aldehyde dehydrogenase and aldehyde reductase favored the reduction in the content of the secondary products of POL. Upon a long contact of FG with cells, the effect of FG was seen only at the initial and final phases of the culture growth cycle. Namely, FG diminished the POL level at the exponential growth phase and at the end of the cell degradation phase but had no effect at the stationary phase and the onset of the degradation phase. Therefore, the treatment with FG retarded the cell culture degradation and made the fall in cell viability not so dramatic by the end of the growth cycle. Actually, by the end of the degradation phase, the viability diminished down to 40% in the control but remained at 70% in the FG-treated counterpart.     

Anthraquinones from in vitro root culture of <Emphasis Type="Italic">Morinda royoc</Emphasis> L.

Morinda royoc L. (Rubiaceae) root cultures were established for the production of anthraquinones (AQs). Three independent experiments were performed to evaluate the effects of different levels of indole-3-acetic acid (0–22.8 μM), culture duration (15–75 days) and subculture number (0–4). The following indicators were recorded: root fresh weight per Erlenmeyer and intracellular and extracellular AQ production. The experiments performed in this study allowed an increase of intracellular AQ content up to a maximum of 4.5 mg g⁻¹ of fresh mass, after 30 days of culture in a medium 5.7 μM of IAA. In addition, isolation and identification of seven AQs from M. royoc L. roots is described, one of them being reported for the first time for this species. The structures of isolated compounds were determined from ¹H-NMR data. To the best of our knowledge, this is the first report on AQ production from root culture of this plant.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

Tracheids in white spruce seedling's long lateral roots in response to nitrogen availability

This study examined how the availability of inorganic nitrogen (N) modified the anatomical characteristics of white spruce (Picea glauca (Moench) Voss) roots related to their hydraulic properties. Seedlings were grown for one growing season in 4 L capacity pots filled with sand under one of three N levels: low (10 ppm), medium (50 ppm) and high (125 ppm). First order lateral roots with intact tips were sampled from dormant seedlings in October. Root segments were collected from 4, 10, and 14 cm distances above the root tip for fixation and sectioning and for maceration. Additional specimens were collected from the 4 and 14 cm distances for maceration and scanning electron microscopy of xylem pits. Root diameter and surface area occupied by the xylem in root cross sections increased basipetally in all treatments but exceptions were found. Higher N-levels significantly increased root diameter and surface area occupied by the xylem. In the two higher N treatments secondary root development was more advanced near the root tip than in the low N treatment. There was a strong positive correlation between root diameter and cross-sectional root area occupied by the xylem (30–50% of the root cross section) but not in portions with little secondary development. Non-conducting space within the xylem occupied 10–13% of its cross-sectional surface. Tracheids of the primary xylem were larger, had larger lumens but thinner cell walls than those of the secondary xylem. Low N treatment seedling tracheids had smaller total cross-sectional area, less lumen, and less cell wall surface area than the two other N treatments. Tracheid diameter means were between 19–20 μm in the high and medium N treatments, and 15.2 μm in the low N treatment. The range was 4.5–51.3 μm. Tracheid length was not significantly affected by N. The average tracheid was about 1000 μm long, and the range was 110–3530 μm. Pit-border diameters ranged between 4.1–20.6 μm (average 10–11 μm) and were not affected by the N treatment. Pit aperture diameters were within 0.62–10.2 μm range (average between 3–4 μm) and were also not significantly affected by the N treatment, although tracheids from the medium N-treatment roots tended to have larger apertures. The pit border diameter equals that of the margo while the aperture size should be similar to that of the torus of the pit membrane. If the capacity for axial water transport in spruce roots is affected by N, it would be by its impact on conduit diameter and, possibly on the pit-membrane pore sizes but not by changes to conduit length and to the size of the pit membrane surface area. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency in vitro propagation of <Emphasis Type="Italic">Trichopus zeylanicus</Emphasis> subsp. <Emphasis Type="Italic">travancoricus</Emphasis> using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.     

Effect of dissolved oxygen and carbon–nitrogen loads on denitrification by an aerobic consortium

Four samples of natural ecosystems and one sample from an activated sludge treatment plant were mixed together and progressively adapted to alternating aerobic/anoxic phases in the presence of nitrate in order to enrich the microflora in aerobic denitrifiers. Aerobic denitrifying performances of this mixed ecosystem at various dissolved oxygen concentrations and various carbon–nitrogen loads were evaluated and compared to those obtained with the aerobic denitrifier Microvirgula aerodenitrificans. The consortium and the pure strain exhibited an aerobic denitrifying activity at air saturation conditions (7 mg dissolved oxygen l^–1), i.e. there was co-respiration of the two electron acceptors with significant specific nitrate reduction rates. Dissolved oxygen concentrations had no influence on denitrifying performances above a defined threshold: 0.35 mg l^–1 for the consortium and 4.5 mg l^–1 for M. aerodenitrificans respectively. Under these thresholds, decreasing the dissolved oxygen concentrations enhanced the denitrifying activity of each culture. The higher the carbon and nitrogen loads, the higher the performance of the aerobic denitrifying ecosystem. However, for M. aerodenitrificans, the nitrate reduction percentage was affected more by variations in nitrogen load than in carbon load. Received: 6 December 1999 / Received revision: 8 March 2000 / Accepted: 10 March 2000     

Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

The ecological niche of the consortium “Pelochromatium roseum”

A dense accumulation of the phototrophic consortium “Pelochromatium roseum” in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per “P. roseum” a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 μmol quanta m^–2 s^–1) and low sulfide concentrations (0–100 μM). In dialysis cultures of “P. roseum”, bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 μmol quanta m^–2 s^–1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m^–3, which was much higher than that of ambient chemocline water (995.8 kg m^–3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves. Received: 27 May 1997 / Accepted: 16 September 1997     

The Method of ABA Application Affects Salt Stress Responses in Resistant and Sensitive Potato Lines

The phytohormone abscisic acid (ABA) has been proposed to act as a mediator in plant responses to a range of stresses, including salt stress. Most studies of ABA response apply ABA as a single dose. This may not resemble the prolonged increasing endogenous ABA levels that can occur in association with slowly increasing salinity stresses in nature or field situations. Salt stress response based on method of ABA application was examined in four potato genotypes of varying salt stress resistance: the sensitive ABA-deficient mutant and its normal sibling, a resistant genotype line 9506, and commercial cultivar ‘Norland’ of moderate resistance. ABA was applied by root drench at 0, 50, 75, or 100 μM concentrations through a single dose, or by slowly increasing multiple ABA doses in a sand-based growing system under greenhouse conditions. Salt tolerance was then evaluated after 2 weeks of exposure to 150–180 mM NaCl stress. The method of ABA application had a marked effect on the responses to salt stress. Plant responses to the method of ABA application were differentiated according to (1) growth rate, (2) root water content, and (3) apparent shoot growth response. Under a single dose, growth rate increased in all genotypes under salt stress, whereas slowly increasing multiple ABA applications generally maintained stable growth rates except in the ABA-deficient mutant where there was an upward growth trend. Percent root water content was elevated only under slowly increasing multiple ABA doses in two genotypes, whereas none of the single-dose treatments induced any change. The single ABA dose enhanced vertical growth, whereas the slowly increasing multiple ABA dose applications enhanced lateral shoot growth. Because exogenous application is still an artificial system, endogenous ABA was supplied through grafting of ABA-deficient mutant scions onto rootstocks with known elevated ABA levels. Multiple exogenous ABA applications as low as 50 μM elicited similar shoot water content responses as grafting treatments without ABA application in the mutant genotype but had no effect on the ABA normal sibling. Shoot dry weight was significantly increased through grafting over all exogenous ABA treatments. Our study further indicates that the method of ABA application regime in itself can alter plant responses under salt stress and that certain application regimes may reflect responses to elevated endogenous levels of ABA.     

Use of a μ-antisense oligodeoxynucleotide as a μ opioid receptor noncompetitive antagonist in vivo

We examined whether μ-antisense (AS) oligodeoxynucleotide (oligo) treatment can be used in a manner similar to the μ-selective irreversible antagonist β-funaltrexamine (β-FNA) for in vivo pharmacology. Rats were injected intracerebroventricularly (icv) with a μ-AS or a missense (MS) oligo on days 1, 3, 5, 7, and 9 and were tested for the antinociceptive effect of sc injection of morphine on days 2, 4, 6, 8, and 10 in the cold water tail-flick (CWT) test. In another set of experiments, rats were also tested for the antinociceptive action of morphine twenty-four hours after icv injection of β-FNA. Both β-FNA and μ-AS produced rightward shifts in the dose-effect curves of morphine. In addition, pretreatment with 2.5 μg or more of β-FNA or the μ-AS oligo for 5–9 days (but not for 1–3 days) reduced the maximal analgesic effect of morphine. The approximate fraction of functional receptor remaining for morphine was determined with the method of Furchgott to be 49.5% following 2.5 μg of β-FNA; that after 5 days of the μ-AS oligo treatment was 50.8%. The results suggest that the μ-AS oligo can be used in the same manner as highly selective, irreversible μ opioid receptor ligands. Thus, properly designed AS oligos against receptors are of particular benefit when irreversible antagonists are not available. AS oligos represent a new class of selective and powerful pharmacological antagonists. Special issue dedicated to Dr. Eric J. Simon.     

Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.