Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 μM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 μM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 μM but decreased at 50 μM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O₂ ^•−) accumulation, and H₂O₂ content at 5 and 10 μM Cu-treated roots were not increased but strongly increased at 50 μM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 μM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 μM Cu, but both G-POD and CAT activities were inhibited at 50 μM Cu. Glutathione metabolism enzymes such as γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 μM Cu but were strongly inhibited at 50 μM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 μM Cu was associated to the strong induction of γ-glutamyltranspeptidase (γ-GGT) activity. These results indicate that plant could grow under Cu stress (5–25 μM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.     

Analysis of tolerance to copper and zinc in Aechmea blanchetiana grown in vitro

The aim of this study was to evaluate the growth and development of Aechmea blanchetiana Baker L.B. Sm. in vitro on medium with 0.0, 0.145, 1.45 and 14.5 μM Cu and 0.0, 2.75, 27.5 and 275 μM Zn. Significant accumulation of Cu and Zn occurred at 14.5 μM Cu and 27.5 and 275 μM Zn, respectively, and there were no significant changes in contents of the other macro- and micronutrients. Superoxide dismutase (SOD) activity significantly changed in the presence of both metals. Spermine content increased as Zn concentration increased and decreased with increasing concentrations of Cu. There was an accumulation of H₂O₂ in the leaf tissue of plants grown in 1.45 and 14.5 μM Cu and 27.5 and 275 μM Zn. A. blanchetiana was found tolerant to the Cu and Zn in concentrations used in this study and displays the capacity to accumulate these metals.     

Effect of copper excess in environment on soybean root viability and morphology

The effects of increase copper concentrations in medium (10–150 μM CuSO₄) on growth and viability of the roots of two-week-old soybean seedlings (Glycine max L., cv. Dorintsa) were studied. Copper excess suppressed biomass accumulation and linear plant growth; copper affected root growth much stronger than shoot growth. The presence of 10 μM CuSO₄ in medium suppressed accumulation of plant biomass by 40% and the root length by 70%; in the presence of 25 μM CuSO₄, these indices were equal to 80 and 90%, respectively. In the presence of 50 μM CuSO₄, roots ceased to grow but biomass and shoot length still increased slightly. 150 μM CuSO₄ was lethal for plants. The earliest sign of excessive copper toxicity was the accumulation of MDA, indicating activation of membrane lipid peroxidation. A significant increase in MDA content was observed at plant incubation in medium with 10 μM CuSO₄ for 1 h; in this case, the content of copper in the roots increased from 36 ±1.8 (in control) to 48 ± 2.4 μg/g dry wt. The number of dead cells (permeable for the dye Evans Blue) was doubled in the presence of 200 μg/g dry wt within the root; this occurred in 72 h of growth in medium with 10 μM CuSO₄, in 6 h at 25 μM CuSO₄, in 3 h at 50 μM CuSO₄, and 1 h at 150 μM CuSO₄. Toxicity of copper excess was manifested stronger in dividing and elongation cells of the root apex (root meristem and the zone of elongation) than in more basal root regions. Copper excess resulted in the formation of breaks in the surface cell layers of the root tips and affect root morphology. When plant grew in medium with 10 μM CuSO₄, a distance of lateral root formation zone from the root tip decreased markedly, and spherical swellings were formed on the tips of lateral roots. The higher copper concentrations (50 and 150 μM) suppressed completely the development of lateral roots.     

Interactions between environmental variables on growth rate and carrageenan content of Solieria chordalis (Solieriaceae,Rhodophyceae) in culture

Using vegetative propagules (ramuli) of the iota carrageenan producing red seaweed Solieria chordalis, a maximum growth rate of 6.8% d⁻¹ was achieved when cultured at 20 °C and 100 μmol photon m⁻² s⁻¹ in seawater supplemented with 20 μM NO₃-N or 10 μM NO₃-N plus 10 μM NH₄-N. Ramuli grew less well when nitrogen was supplied solely as NH ₄ ⁺ . Maximum carrageenan content was observed at the same temperature, irradiance and salinity as growth, but at lower nitrogen concentrations. These findings are discussed in relation to results obtained from studies on other iota-carrageenan producing carrageenophytes.     

Physiological behavior of Scenedesmus sp. during exposure to elevated levels of Cu and Zn and after withdrawal of metal stress

Summary. A 48 h exposure of Scenedesmus sp. to sublethal concentrations of Cu (2.5 and 10 μM) and Zn (5 and 25 μM) caused a concentration-dependent inhibition of growth, photosynthesis, respiration, NO₃ ⁻ uptake, and nitrate reductase (EC 1.6.6.1) activity, and a reduction in protein, carbohydrate, and photosynthetic-pigment levels with a concomitant increase in intracellular levels of the test metals. After exposure, algal cells were transferred to the basal medium without the excess level of test metals, to study the recovery of various processes. The growth of the test algae had not recovered up to 12 h after transfer to the basal medium, but some physiological parameters such as photosynthesis and respiration recovered within 6 h. The quicker recovery of photosynthesis and respiration might be used as acclimatory responses as they prepare a background for the recovery of other parameters, including growth, of the test alga by generating energy, forming photosynthate, and establishing the usual catabolism to attain normal conditions. Most of the processes recovered completely or almost completely after being stressed with 2.5 μM Cu or 5 μM Zn. However, the maintenance of a relatively high level of Cu and Zn in the cells previously exposed to 10 μM Cu and 25 μM Zn slowed down the recovery of different processes, which did not fully recover even at the end of the experiment after 96 h. The present study demonstrates that a chain of metabolic events, beginning with respiration and photosynthesis and continuing with assimilation and uptake of nutrients and subsequent restoration of other metabolic processes, is involved in the recovery of the algae from Cu and Zn stress. Each studied parameter seems to play an important role in balancing the cellular homeostasis during recovery from metal stress. Correspondence and reprints: Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali 304 022, Rajasthan, India.     

Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

Ectoenzymatic Activity and Uptake of Monomers in Marine Bacterioplankton Described by a Biphasic Kinetic Model

Abstract The kinetics of bacterial hydrolytic ectoenzymatic activity and the uptake of monomeric compounds were investigated in the Northwestern Mediterranean Sea. Aminopeptidase and α- and β-glucosidase activities were analyzed by using fluorogenic substrates at 15–22 concentrations ranging from 1 nM to 500 μM. Radiolabeled glucose and a mixture of amino acids were chosen as representatives of monomeric compounds, and the bacterial uptake rates (assimilation plus respiration) were determined over a wide range of substrate concentrations (from 0.2 nM to 3 μM). We found biphasic kinetics both for hydrolytic enzymes and uptake systems: high affinity enzymes at low concentrations of substrates (K _m values ranged from 48 nM to 2.7 μM for ectoenzymes and from 1.4 nM to 42 nM for uptake systems), and low affinity enzymes at high concentrations of substrates (K _m values ranged from 18 μM to 142 μM for ectoenzymes and from 0.1 μM to 1.3 μM for uptake systems). Transition between high and low affinity enzymes was observed at 10 μM for aminopeptidase and from 1 μM to 25 μM for glucosidases, and it was more variable and less pronounced for the uptake of glucose (40 nM–0.28 μM) and amino acids (10 nM–0.16 μM). Results showed that the potential rates of hydrolysis and uptake are tightly coupled only if the high affinity hydrolytic ectoenzymes and the low affinity uptake systems are operating simultaneously. Received: 5 March 1998; Accepted: 31 July 1998     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of three Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. Of six ZIP’s studied, MtZIP1, MtZIP5 and MtZIP6 were the only members from this family determined to transport Zn and were further characterized. MtZIP1 has a low affinity for Zn with a K_m of 1 μM as compared to MtZIP5 and MtZIP6 that have a higher affinity for Zn with K_m of 0.4 μM and 0.3 μM, respectively. Zn transport by MtZIP1 was more sensitive to inhibition by copper (Cu) concentrations than MtZIP5 and MtZIP6, because 3 μM Cu inhibited Zn transport by 80% in MtZIP1 while 5 μM Cu was required to achieve the same inhibition of Zn transport in MtZIP5 and MtZIP6. Cadmium (Cd) had a greater effect on the ability of MtZIP1 to transport Zn than MtZIP5 and MtZIP6, because at a concentration of 3 μM Cd, the Zn transport by MtZIP1 was inhibited 55% and the transport of Zn by MtZIP5 and MtZIP6 was inhibited by 20–30%. However, only MtZIP6 transported Cd at higher rates than those observed in the control plasmid pFL61, demonstrating a low affinity for Cd based on a K_m of 57 μM. These results suggest that Medicago truncatula has both high and low affinity Zn transporters to maintain Zn homeostasis and that these transporters may function in different compartments within the plant.     

Extending the alkene substrate range of vinyl chloride utilizing Nocardioides sp. strain JS614 with ethene oxide

Nocardioides sp. strain JS614 grows on the C₂ alkenes ethene (Eth), vinyl chloride, and vinyl fluoride as sole carbon sources. The presence of 400–800 μM ethene oxide (EtO) extended the growth substrate range to propene (C₃) and butene (C₄). Propene-dependent growth of JS614 was CO₂ dependent and was prevented by the carboxylase/reductase inhibitor 2-bromoethanesulfonic acid, sodium salt (BES), while growth on Eth was not CO₂ dependent or BES sensitive. Although unable to promote growth, both propene and propene oxide (PrO)-induced expression of the genes encoding the alpha subunit of alkene monooxygenase (etnC) and epoxyethane CoM transferase (etnE) to similar levels as did Eth and EtO. Propene was transformed by Eth-grown and propene-grown/EtO-induced JS614 to PrO at a rate 4.2 times faster than PrO was consumed. As a result PrO accumulated in growth medium to 900 μM during EtO-induced growth on propene. PrO (50–100 μM) exerted inhibitory effects on growth of JS614 on both acetate and Eth, and on EtO-induced growth on Eth. However, higher EtO concentrations (300–400 μM) overcame the negative effects of PrO on Eth-dependent growth.     

Inhibitors of animal phospholipase A2 enzymes are selective inhibitors of auxin-dependent growth. Implications for auxin-induced signal transduction

Auxin and elicitors reportedly activate phospolipase A. A number of inhibitors known to inhibit animal phospholipase A₂ were tested for their ability to inhibit hormone and fusicoccin-induced growth. To this end, growth induced by indolyl-3-acetic acid and 2,4-dichlorophenoxyacetic acid in hypocotyl segments of etiolated zucchini (Cucurbita pepo L.) seedlings was determined in the presence of the inhibitors nordihydroguajaretic acid (NDGA), aristolochic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), PB_x (a prostaglandin derivative), and oleylethyl phosphocholine. Each chemical proved inhibitory to auxin-induced growth, oleylethyl phosphocholine being the least effective. The effects of the first three inhibitors were investigated in more detail. Growth induced by 10 μM 2,4-dichlorophenoxyacetic acid or 1 μM indolyl-3-acetic acid was inhibited 50% by about 30–50 μM NDGA, by about 25 μM aristolochic acid, and by about 10–20 μM EYTA. Growth inhibition was reversible and became apparent 0.5–1 h after inhibitor addition. Growth induced by 0.5 or 1 μM fusicoccin was much less inhibited by NDGA and by ETYA, whereas aristolochic acid was only slightly less effective on fusicoccin-induced than on auxin-induced growth. These three inhibitors were also tested for their effects on gibberellin-induced growth in light-grown peas (Pisum sativum L.) and on cytokinin-induced expansion growth in excised cotyledons from radish (Raphanus sativum L.) seedlings. In both tests, aristolochic acid had toxic side-effects although gibberellin-induced growth was still apparent. In the gibberellin test, neither NDGA at up to 100 μM nor ETYA at 80 μM was inhibitory to hormone-induced growth. Moreover, 40 μM ETYA was not inhibitory to kinetin-induced growth. We hypothesize that the selectivity of phospholipase A₂ inhibitors for auxin-induced growth implies a different signal transduction pathway for each of the different signal substances tested, and that auxins might use fatty acid(s) and/or lysophospholipid(s) or their derivatives as the preferred second messengers. Received: 24 September 1996 / Accepted: 18 January 1997     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

Response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots to lipo-chitooligosaccharide from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and other chitin-like compounds

Lipo-chitooligosaccharides (LCOs) are bacteria-to-plant signals required for the establishment of rhizobia–legume nitrogen fixing symbioses. The ability of LCO [Nod Bj V (C_18:1, MeFuc)] isolated from B. japonicum (strain 532C), and of oligomers of chitosan (tetramer, pentamer) and chitin (pentamer) to affect the developmental morphology of roots in Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) was assessed using an interactive scanner-based image analysis system. LCOs have been shown to play a role in plant organogenesis at nanomolar concentrations. LCO and the chitin pentamer promoted root growth and development in Arabidopsis at concentrations of 10 nM and 100 μM, respectively. The LCO treated Arabidopsis plants had about 35% longer roots than untreated control plants. Similarly, treatment with 100 μM chitin pentamer (CHIT5) resulted in 26% longer roots than the untreated plants; however, chitosan oligomer (CH4 or CH5) treated plants did not differ from the control plants at either concentration (100 or 1 μM). Both LCOs and the chitin pentamer at higher concentrations increased root surface area, mean root diameter and number of root tips. However, leaf area increase was observed only in plants treated with LCO at 10 nM.     

Effect of lead (Pb) on the systemic movement of RNA viruses in tobacco (Nicotiana tabacum var. Turkish)

Effect of various lead (Pb) concentrations on the systemic movement of RNA viruses was examined in tobacco plants. Prior to inoculation, plants were grown hydroponically for 6 days in Hoagland’s solution supplemented with five concentrations of lead nitrate [Pb(NO₃)₂]: 0.0 (control), 10, 15, 50, and 100 μM. Four different RNA viruses with different cell-to-cell movement mechanisms were used. Two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for the presence of viral coat protein. In plants inoculated with Tobacco mosaic virus, Potato virus X, and Tobacco etch virus, TEM images and western blot assays confirmed the presence of viral coat proteins in the upper leaves of all lead treatments. However, in plants inoculated with Turnip vein-clearing virus (TVCV), no signs of viral particles were detected in the upper leaves of plants treated with 10 μM or 15 μM lead nitrate. In contrast, plants treated with high concentrations of lead nitrate (50 μM or 100 μM) showed viral particles in their upper leaves. In plants treated with 10 μM or 15 μM lead nitrate, callose accumulation was the same as in control plants. This suggests that non-toxic concentrations of lead nitrate may trigger the production of putative cellular factors in addition to callose that interfere with the TVCV systemic movement. In contrast, plants treated with 100 μM lead nitrate showed less callose as compared to control plants, facilitating the systemic movement of TVCV.     

DNA damage in mouse lymphocytes exposed to curcumin and copper

Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate thein vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 μM curcumin and various concentrations of copper (10 μM, 100 μM and 200 μM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 μM curcumin in the presence of 100–200 μM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 μM H₂O₂ in mouse lymphocytes. Moreover, 50 μM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 μM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.     

Induction of metal binding compounds and antioxidative defence in callus cultures of two black poplar (<Emphasis Type="Italic">P. nigra</Emphasis>) clones with different tolerance to cadmium

Callus cultures of two parental clones of Populus nigra L., Poli and 58-861, originating from contrasting environments, were exposed to different cadmium concentrations (0, 150 and 250 μM CdSO₄). Clones showed different growth responses to cadmium, evaluated by the tolerance index (Ti), with Poli being more tolerant to the metal at both concentrations. The cadmium concentration at the end of the treatment was very similar between clones at 150 μM CdSO₄, while a higher value in 58-861 compared to Poli was detected at 250 μM CdSO₄. The bioconcentration factor evidenced the lowest value in Poli at 250 μM CdSO₄. Unlike 58-861, cadmium provoked a strong induction of thiols and phytochelatins in clone Poli. In both clones, organic acid concentration differed notably in untreated calli and cadmium treatment induced a general lowering of these compounds. A notably higher antioxidant enzyme activity (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX) was measured in control calli of clone Poli compared to 58-861. Cadmium induced a remarkable enhancement of APX and CAT, but not GPX, activity at 150 μM CdSO₄ in Poli. Conversely, in 58-861 at 150 μM CdSO₄, and in both clones at 250 μM CdSO₄, a decrease in the antioxidant activity occurred. This investigation provided evidence that these two contrasting genotypes of P. nigra are characterised by a different response to cadmium in callus cultures. In particular, in Poli, the higher tolerance to cadmium is associated with a higher activity of antioxidative enzymes and the ability to strongly increase thiol and PC concentration in response to metal exposure.     

Distribution and characterization of Aegilops and Triticum species from the Bulgarian Black Sea coast

A total of 158 Aegilops-Triticum samples representing six Aegilops species (one diploid, four tetraploid and one hexaploid) and one diploid Triticum were collected along the Bulgarian Black Sea coast, and their distribution on the 350 km long coastal line was reported. The region south of Kamchia river, accepted as the middle point of the coast, was characterized by the greatest diversity of these wild relatives of wheat. The most widely distributed species in this area was Ae. geniculata. Ae. cylindrica was distributed only in north (Durankulak), while Ae. biuncialis and Ae. triuncialis were collected both north and south of Kamchia river. All samples of Ae. neglecta were hexaploid. Natural hybrids of goatgrass and wheat were found in Ae. cylindrica populations. Triticum monococcum ssp. aegilopoides had limited distribution in the south region. Aegilops uniaristata was recorded as a new species for the Bulgarian flora. Most of the samples expressed resistance to powdery mildew in seedling and adult stage, but all of them were polymorphic regarding the resistance to leaf rust (cultures 73760 and 43763). The study revealed additional data for the distribution of Aegilops and Triticum species in Bulgaria and their potential value as genetic resources in wheat improvement.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.