Sources of measurement error in assays of EROD activity of fish for biological effects monitoring

The paper describes the technical, procedural and biological aspects of 7-ethoxyresorufin O-deethylase (EROD) activity measurements in dab, Limanda limanda, from the North Sea. Several sources of measurement error in assays of EROD activity measurements for biological effects monitoring exercises could be defined, particularly for assay conditions, sample storage and sampling. In addition to variability caused by technical errors, biological features inherent in the surveyed fish may lead to even more severe errors in EROD activity measurements. This is particularly true of the choice of sampling time. Disregarding the species-specific spawning periods may lead to a loss of activity by a factor of as much as 90 in females and 40 in males, thus making it necessary to design sampling surveys appropriately. Received: 14 April 1999 / Received in revised form: 10 July 1999 / Accepted: 15 July 1999     

Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999     

Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): transformation frequency and integration pattern

Two non-linked marker genes (gus and bar) were co-introduced by microprojectile bombardment into wheat cells. Four different DNA structures were compared with respect to ability to integrate into the wheat genome: circular or linear (l) DNA as a single- or double-stranded plasmid (ss and ds, respectively). In eight independent experiments, linearized DNA integrated in the ds or ss form with a high efficiency of up to 14% for l-ssDNA. Molecular analyses by Southern blotting showed that all DNA forms gave a similar complicated integration pattern of the bar gene. Received: 20 July 1998 / Accepted: 30 January 1999     

QTL mapping for the paste viscosity characteristics in rice (Oryza sativa L.)

In order to understand the genetic basis of the paste viscosity characteristics (RVA profile, which is tested on the Rapid Visco Analyser) of the rice grain, we mapped QTLs for RVA profile parameters using a DH population derived from a cross between an indica variety, Zai-Ye-Qing 8 (ZYQ8), and a japonica variety, Jing-Xi 17 (JX17). Evidence of genotype-by-environment interaction was found by comparing the mapped QTLs between two locations, Hainan (HN) and Hangzhou (HZ). A total of 20 QTLs for six parameters of the RVA profiles were identified at least one location. Only the waxy locus (wx) located on chromosome 6 was detected significantly at both environments for five traits, i.e. hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), consistency viscosity (CSV) and setback viscosity (SBV). This locus explained 19.5%–63.7% of the total variations at both environments, suggesting that the RVA profiles were mainly controlled by the wx gene. HPV, CPV, BDV, CSV and SBV were also controlled by other QTLs whose effects on the respective parameter were detected only in one environment, while for the peak viscosity (PKV), only 2 QTLs, 1 at HN,the other at HZ, were identified. These results indicate that RVA profiles are obviously affected by environment. Received: 18 July 1999 / Accepted: 27 August 1999     

Changes in Tissue Cellularity Are Associated with Growth Enhancement in Genetically Modified Arctic Char (Salvelinus alpinus L.) Carrying Recombinant Growth Hormone Gene

Biochemical and histological analyses were used to study the number and size of cells (cellularity) in tissues of fast-growing, genetically modified Arctic char (Salvelinus alpinus L.), overexpressing sockeye salmon (Oncorhynchus nerka) growth hormone gene (OnGH1). DNA contents of muscle, heart, and liver were compared in transformed, sibling (age control) and 1 year older (size control) char. Total white muscle cross-sectional area, white muscle fiber number, and total nuclei number within the muscle tissue were determined from one complete half-section of each fish. The analyzed tissues responded differently to growth hormone overproduction. In muscle tissue of OnGH1-transformed char, the enhanced growth was clearly associated with proliferation of muscle cells (hyperplasia), whereas in heart tissue both cell proliferation and increase in cell size (hypertrophy) were enhanced. The relative DNA concentration in the liver of transformed char was significantly greater than that of control fish, suggesting reduction in size of hepatic cells. Received June 29, 2000; accepted October 25, 2000     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Recovery of Historically Contaminated Watercourse Polluted by the Chemical Wood Industry: EROD Activity in Fish as Biomarker

Despite outstanding process alterations over decades, pulp- and paper-mill-contaminated sediments and continuing exposure by the effluents may still have effects on biota. In this study, ecotoxicological impacts in the boreal watercourse were analyzed by measuring ethoxyresorufin-O-deethylase (EROD) induction from wild fish populations and from experimentally exposed fish. In order to assess the role of sediment-borne chemicals, juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to the surface sediments of Lake Vatianjärvi and Southern Lake Saimaa, both watercourses impacted by the chemical wood industry for approximately a century. Hepatic EROD activity was also measured from roach (Rutilus rutilus) and perch (Perca fluviatilis) caught in Lake Vatianjärvi. Increased EROD activity was not observed in wild fish caught in Lake Vatianjärvi nor in rainbow trout exposed to the sediment of Lake Vatianjärvi, but it was observed in rainbow trout exposed to the sediment of Southern Lake Saimaa, probably explained by critically high concentrations of retene. The results of both the laboratory and field study indicate the absence of EROD-inducing compounds of the previously heavily contaminated Lake Vatianjärvi.     

Metabolic disturbances in fish exposed to sodium pentachlorophenate (NaPCP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126), individually or combined

The Swan River Estuary is the recipient of multiple urban and agricultural contaminants which have the potential to induce liver detoxication enzymes as well as altering the metabolism of aquatic organisms. To test if altered liver metabolism would influence liver detoxication capacities, pink snapper (Pagrus auratus) were i.p. injected with peanut oil (controls), or pentachlorobiphenyl #126 (PCB126), with sodium pentachlorophenate (NaPCP), or PCB126+NaPCP. Relative to controls, ethoxyresorufin-O-deethylase (EROD) activity was induced in the PCB126 and PCB126+NaPCP fish, but not in the NaPCP group. In the liver, cytochrome c oxidase (CCO) activity was enhanced by the treatments while citrate synthase (CS) activity remained unchanged and lactate dehydrogenase (LDH) activity was increased in the NaPCP treatment only. The results suggest that liver CCO activity may be a suitable biomarker of effect following exposure to PCBs or phenolic compounds. In the white muscle, only the PCB126+NaPCP treatment enhanced CCO activity, with all other enzymatic activities remaining unchanged. It appears that the resilience to metabolic perturbations is greater for white muscle than for liver. Low serum sorbitol dehydrogenase (sSDH) activity and histopathology of the liver indicated no significant alteration of cellular structure, albeit the lipid droplet size was increased in the PCB126 and in the PCB126+NaPCP treatments. It is concluded that the hepatic metabolic changes correspond to histopathological observations, but an altered metabolic capacity do not influence the metabolism of xenobiotics by liver enzymes, as measured by EROD activity.     

Microsatellite fingerprinting in lettuce (Lactuca sativa L.) and wild relatives

Southern hybridisation with a single microsatellite probe, (TCT)₁₀, sufficed to discriminate between a representative set of cultivars/accessions of lettuce, Lactuca sativa L., and its wild relatives L. serriola, L. saligna and L. virosa. Variability within cultivars was tested in a relatively modern cultivar (Hector), where no variation was found, and in an older and morphologically more variable cultivar (Madrilene), where heterogeneity was observed in the TCT fingerprint. (TCT)₁₀ fingerprinting should be useful for variety identification and homogeneity testing in lettuce. Received: 25 July 1997 / Revision received: 5 August 1997 / Accepted: 30 August 1997     

Trace element levels in fish from clean and polluted coastal marine sites in the Mediterranean Sea, Red Sea and North Sea

The bioaccumulation of Hg, Cd, Zn, Cu, Mn and Fe was evaluated in the muscle and liver tissue of four fish species (Siganus rivulatus, Diplodus sargus, Lithognatus mormyrus and Plathychtis flesus) from clean and polluted marine coastal sites in the Red Sea, Mediterranean Sea and North Sea within the framework of the MARS 1 program. Representative liver samples were screened for organic contaminants (DDE, PCBs and PAHs) which exhibited very low concentrations. The levels of Cd, Cu, Zn, Fe and Mn found in the muscle tissue in this study were similar among the four species and within the naturally occurring metal ranges. However, differences were found among the sites. In the Red Sea, Cu was higher in the muscle of S. rivulatus at Ardag and Zn at the Observatory (OBS). Cu, Zn and Mn were higher in the Red Sea than in the specimens from the Mediterranean. The differences were attributed to different diets derived from distinctively different natural environments. D. sargus from Haifa Bay (HB) had higher Cd, Cu and Mn values than specimens from Jaffa (JFA), and L. mormyrus higher Cd, Fe and Mn in HB, corresponding to the polluted environmental status of the Bay. No differences in metal levels were found among the North Sea sites, except for Fe that was lower at the Eider station. Hg was low in all the specimens, but the values varied with species and sites. The lowest Hg values were found in S. rivulatus, the herbivorous species, as expected from its trophic level. Hg in P. flesus was higher than in S. rivulatus but still low. Higher Hg values were found in the muscle tissue of L. mormyrus,with the highest values in D. sargus, both carnivorous species from the same family. Hg in D. sargus was higher in HB than in JFA, as expected, but in the larger specimens of L. mormyrus from JFA values were higher, while in the small specimens there were no differences in Hg values. The levels of all metals were higher in the liver than in the muscle, with enrichment factors ranging from 3 to 104, depending on species and sites. The lowest enrichment values were found for Hg. Based on liver values, the specimens of S. rivulatus from the OBS had the highest levels, as well as D. sargus and L. mormyrus from JFA, contrary to the known relative environmental status of the sites. Received: 25 February 1999 / Received in revised form: 5 June 1999 / Accepted: 7 June 1999     

PCB contamination of the common barbel,Barbus barbus (pisces,cyprinidae), in the River Meuse in relation to hepatic monooxygenase activity and ultrastructural liver change

To evaluate the impact of PCBs on a wild population of a regressing fish species, we have measured the levels of these toxicants in common barbel (Barbus barbus) from the Belgian part of the river Meuse. We have expressed these levels in terms of the most suitable composition of commercial PCB mixture (Aroclor 1254 and 1260 20/80; v/v), and related them to hepatic xenobiotic-metabolising enzyme activities and to hepatocyte ultrastructure.PCB concentrations in barbel organs were extremely high, with no statistical difference between the two sexes at equal weight. In liver, muscle, and gonads, PCB concentrations increased significantly with age, reaching 20 g g^–1 DW in 12- to 15-year-old individuals. The activities of the monooxygenases (ethoxyresorufin o-deethylase, EROD, and ethoxycoumarin o-deethylase, ECOD) and the cytochrome P-450 content correlated closely with the PCB concentration in fish liver. Moreover, wild fish presented a markedly altered liver ultrastructure, as compared to controls; the rough endoplasmic reticulum (RER) was particularly abundant and the mitochondrial membranes were altered. PCBs thus alter essential metabolic functions in wild barbels, which constitute a highly sensitive tool for biomonitoring wild fish populations. While the effects of PCBs on metabolic pathways may combine additively or synergistically with effects of other xenobiotics, it has been demonstrated elsewhere that they decrease successful reproduction. Their chronic negative effects have thus played a role in reducing barbel populations in highly polluted areas.     

The role of seed reserves in arbuscular mycorrhizal formation and growth of Leucaena leucocephala (Lam.) de Wit. and Zea mays L.

We show here that seed reserves in Leucaena leucocephala (Lam.) de Wit. and Zea mays L. (maize) are important for mycorrhizal formation and seedling growth. Seed reserve removal reduced mycorrhizal formation markedly in Leucaena but not in maize, except at 15 and 45 days after seed reserve removal. Partial or total removal of seed reserves decreased plant growth and tissue nutrient concentrations in both hosts. Nodule number in Leucaena, which was related positively to plant biomass and mycorrhizal infection levels, was reduced when one or both cotyledons were severed. Leucaena seedlings without or with partial seed reserves had higher nutrient use efficiencies throughout seedling growth. But such an effect was observed only initially in maize. Seed reserve removal increased the specific absorption rates of nutrients in both hosts. Phosphorus absorption rate was significantly and positively related to root infection levels in both Leuceana and maize. Though the growth rates of plants without seed reserves were low initially, these plants had higher growth rates during later stages. We conclude that seed reserves are not only important for seedling growth, but also for mycorrhizal formation and nodulation. Received: 15 July 1999 / Accepted: 6 December 1999     

Heteroblasty in Arabidopsis thaliana (L.) Heynh

 Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific β-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emf1lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position. Received: 9 July 1999 / Accepted: 17 August 1999     

Field Studies Using European Starlings to Establish Causality between PCB Exposure and Reproductive Effects

Accumulation and effects of polychlorinated biphenyls (PCBs) in avian species were evaluated at a Superfund site located at Crab Orchard National Wildlife Refuge, Illinois, and seven criteria were used to assess whether there was a causal relationship between PCB exposure and observed reproductive effects. European starlings (Sturnus vulgaris) were monitored at nest boxes constructed at each of two exposed and two reference sites. During the breeding season, starling productivity (number of chicks produced per nest) and adult nest attentiveness behavior (provisioning behavior) were monitored. At 15 days post-hatch, chicks were collected for contaminant and biomarker analyses. Chicks were necropsied, ethoxyresorufin-O-deethylase (EROD) activity measured in liver tissue, and PCB (Aroclor 1254) and 34 chlorinated biphenyl (CB) congener concentrations measured in carcasses using gas chromatography. PCB and CB concentrations also were measured in eggs that failed to hatch. Mean Aroclor 1254 and quantified CB concentrations were greater (P<0.001) in eggs that failed to hatch and 15-day-old chicks collected from PCB sites compared to those collected from reference sites. EROD activity was greater (P=0.005) in 15-day-old chicks collected from PCB sites and was correlated with carcass PCB concentrations. Reduced adult nest attentiveness behavior and decreased chick survival were observed at PCB sites. Six of the seven causal criteria evaluated provided evidence that observed reproductive effects resulted from exposure to PCBs. Using this weight-of-evidence approach provided a means for establishing the likely cause of effects and thus provided managers with information needed in decision-making processes.     

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis

 This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×10⁶), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×10⁴ cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil. Received: 1 April 1999 / Revision received: 27 July 1999 / Accepted: 23 August 1999     

Localising QTLs for leaf rust resistance and agronomic traits in barley (Hordeum vulgare L.)

The Hordeum vulgare accession ’HOR 1063’ was crossed with the barley cultivar Krona, and 220 doubled haploid lines were produced based on this cross. A molecular map was constructed based on RFLP markers. Field trials were performed over 2 years and at two locations. In field trials, resistance to leaf rust by means of artificial infection, heading date, plant height and Kernel weight were assessed. For leaf rust resistance, 4 QTLs were localised, that explained 96.1% of the genetic variation. One QTL on chromosome 4H confirmed a position found in another genetic background and one mapped to the same position as Rph16 on chromosome 2H. All digenic effects decreased the effects of the respective QTLs. In addition to the denso-locus and the hex-v locus, other QTLs influencing heading date, plant length and kernel weight were found in this cross. Received: 16 July 1999 / Accepted: 7 September 1999     

Is self-compatibility the main breeding system in borage (Borago officinalis L.)?

The polygenic postzygotic self-incompatible system reported in borage (Borago officinalis L.) is refuted. The behaviour of pollen tubes after self- and cross-pollination, analysis of the crossability coefficient, seed set after continuous generations of self-pollination and the effects of inbreeding depression in borage were studied. Evidence of self-compatibility was established. The influence of protandry in a self-compatible system is also discussed. Received: 15 July 1999 / Accepted: 3 November 1999     

Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer

 By using highly regenerative calluses, we developed a stable transformation system in garlic (Allium sativum L.). The temperature and number of days of co-cultivation with Agrobacterium tumefaciens was shown to be an important factor in transient expression of the uid A gene. After a culture period of 5 months in selection medium containing hygromycin, 20 shoots were induced from ca. 1000 calluses, among which 15 plants expressed β-glucuronidase activity upon staining with X-Gluc. Shoots developed into transgenic garlic after 1 month. Integration of the uid A gene was confirmed by Southern blot analysis for genomic DNA of transgenic garlic plants. Received: 25 October 1999 / Revision received: 16 February 2000 / Accepted: 22 February 2000     

Cryopreservation and plant regeneration from somatic embryos of Pinus patula

 Embryogenic tissue of Pinus patula Scheide et Deppe was cryopreserved for 8 weeks using sorbitol and dimethylsulfoxide (DMSO) as cryoprotectants. Results indicate that 0.3 M sorbitol and 5% DMSO had the best cryoprotecting effect. The recovered tissue initially underwent a lag phase but then continued to proliferate normally on MSG3 maintenance medium. Recovered tissue was placed onto MSG5 maturation medium, and embryos were isolated and germinated. Plantlet regeneration from the recovered tissue was achieved. Received: 16 April 1999 / Revision received: 26 July 1999 / Accepted: 17 August 1999