Selection ofMycosphaerella fijiensis-resistant cell lines from micro-cross sections of banana and plantain

Anin vitro selection system using microcross sections of banana and plantain cultivars belonging to AAA and AAB genomic groups were used to produce plants resistant against the Black Sigatoka disease. The fungus resistant plantlets were obtained in a double selection system. This involved in a first step the use of a fungal crude filtrate and in the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted from the fungusMycosphaerella fijiensis (M. fijiensis), the causal agent of Black Sigatoka disease. Resistant plantlets obtained from the double selection system were inoculated with conidia ofM. fijiensis in a growth chamber to reproduce Black Sigatoka symptoms. Compared to non-treated control plantlets, which were highly susceptible to the fungus, 10.7–19.3% toxin-resistant plantlets which arose from tissues that went through the double selection system were resistant againstM. fijiensis. This technique of using micro-cross sections for selection on fungal toxins seems to be amenable to differentMusa genotypes for the production of fungus-resistant plants.F. A. Schulz died 11. 3. 1995     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

Natural nodulation of <Emphasis Type="Italic">Acacia mangium</Emphasis>–<Emphasis Type="Italic">Acacia auriculiformis</Emphasis> hybrids: distribution of the indigenous strains in the nodules

Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) of the InterGenic Spacer (IGS) between rDNA 16S and 23S was used to identify indigenous strains nodulating four clones of Acacia mangium–Acacia auriculiformis hybrids cultivated in non-sterilized sandy soil from Sangalkam (Senegal) under greenhouse conditions. The experiment was for 4 months. The analysis of restriction fragment length polymorphism obtained with MspI and HaeIII restriction enzymes allowed the identification of 15 different IGS Groups with a distribution which significantly differed according to the clone of the hybrid (strains of one clone can belong to three and five different IGS Groups). Three large multi-lobed nodules were obtained on the root system of clone 3.26 within 5 months. Also, the nature of the rhizobia contained in each lobe was determined. The results showed that the lobes of large nodules can be occupied by one or two strains and the nodules analysed were mainly occupied by those belonging to IGS Group 12.     

<Emphasis Type="Italic">In vitro</Emphasis>–<Emphasis Type="Italic">ex vitro</Emphasis> salt (NaCl) tolerance of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) plants

Three cassava clones (SOM-1, “05”, and “50”) were cultured in vitro on MS medium plus sucrose (30 g L⁻¹) and myo-inositol (100 mg L⁻¹) without plant growth regulators and with additions of 0 (control), 0.5, 1, 1.5, 2, 2.5, and 3 g L⁻¹ NaCl to test their salt tolerance. The same cassava clones were cultivated in greenhouse conditions on a sandy soil substratum and irrigated with 20% strength Hoagland solution, and additions of 0, 4, and 8 g L⁻¹ of NaCl. Salinity negatively affected the survival, development, leaf water content, and mineral composition (mainly by accumulation of Cl and Na) of both in vitro and ex vitro plants, but with different intensity in each clone. In both conditions of culture (in vitro and ex vitro) clone SOM-1, from a desert arid saline zone of Somalia, was the most tolerant and clone “05”, from a rainy region of Ivory Coast, the most sensitive. Clone “50” tolerance to in vitro salt treatments, although lower, was not significantly different from that of SOM-1 but the ex vitro response was similar to “05”. In general, there was a correlation between in vitro and ex vitro behavior of the cassava plant regarding salt tolerance, which would allow the in vitro culture method to be used for selection of salt-tolerant plants of this crop.     

Spectral reflectance from a broccoli crop with vegetation or soil as background: influence on immigration byBrevicoryne brassicae andMyzus persicae

Light reflectance in five wavebands of the spectrum was measured from broccoli (Brassicae oleracea var.botrytis [L].) interplanted with leguminous cover crops (cover crop background) or broccoli grown as monoculture (bare soil background), and fertilized with compost or synthetic fertilizer. AlateBrevicoryne brassicae (L.) andMyzus persicae (Sulzer) (Homoptera: Aphididae) were monitored in yellow pan water traps and on broccoli leaves. Reflectance intensity was higher with a background of bare soil at all wavebands except blue (400–455 nm) in the early-season. Intensity decreased as broccoli canopy cover increased at all wavebands except blue and green (515–550 nm), declining-most dramatically in the yellow (550–590 nm). Highest late-season intensities were in plots with bare soil background and fertilized with compost (those stressed for nitrogen). Few differences in spectral composition, expressed for each waveband as a percentage of total intensity, were recorded. Numbers of alatae were lowest in cover crop background plots in the early season, reached equivalency with bare soil background by mid-season, and showed highest positive correlations with intensity in the yellow (550–590 nm). Results correspond to laboratory findings that aphids are attracted to higher intensity light, especially in the yellow waveband, and support a phototactic explanation for aphid orientation in the field.     

Expression ofMycobacterium tuberculosis genes inEscherichia coli

TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.     

Enhancement of marennine production by blue light in the diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis>

The marine diatom Haslea ostrearia Simonsen produces a blue pigment, marennine, which is used for greening oysters. This microalga is cultured industrially indoors with artificial light. The influence of light quality on marennine production by cultures of H. ostrearia was investigated in the laboratory and at a semi-pilot scale (300 L tanks). In the first series of experiments in the laboratory, a clone of H. ostrearia was cultured under light of different colors (white, blue, green, yellow, and red) and at two irradiances (‘low’ and ‘high’, 20 and 100 μmol photons m⁻² s⁻¹, respectively). Compared to the white light controls, growth was increased in blue light at the ‘low’, but not at the ‘high’ irradiance, and marennine production at the end of the exponential phase was the highest in cells grown under blue light, regardless of the light quality or intensity during growth. Increased marennine production during growth was also observed, whichever color of light (blue or white) was used during the acclimation phase. In a second series of experiments, intraclonal differences were studied by comparing marennine production in seven clones differing with regard to their mean cell size. The total marennine expressed either per cell or per culture volume, was higher in blue light for all the clones. Complementary experiments carried out under semi-industrial conditions confirmed this effect of blue light, which could be relevant for the industrial, indoor production of marennine.     

Evidence against the involvement of phytochrome in UVB-induced inhibition of stem growth in green tomato plants

The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white light (150 μmol m⁻² s⁻¹ PAR)/12 h dark cycles, were first transferred to 200 μmol m⁻² s⁻¹ monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 μmol m⁻² s⁻¹ UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after 4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB irradiation substantially reduced elongation growth of all genotypes (4.5 μmol m⁻² s⁻¹ being more effective than 0.1 μmol m⁻² s⁻¹). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited, while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB–induced inhibition of growth, while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of stem growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ribulose-1, 5-bisphosphate carboxylase activity,chlorophyll and protein concentrations in differentPopulus clones

Ribulose-1,5-bisphosphate carboxylase activity (RuBPC), chlorophyll (chl) and protein (prot) concentrations and chlorophyll/protein (chl/prot) ratios were determined in five differentPopulus clones together with their maximal net CO₂ uptake rates (P_max). A classic reference clone (Populus ×euramericana “Robusta” (Dode) Guinier) was compared with four recently selected euramerican and interamerican crossings. Chl/prot ratio and RuBPC activity varied among the different clones, while chl a/chlb ratio showed only a very low coefficient of variation (1.7%) for the five clones. Poplar clone “Robusta” could be distinguished from the recent faster growing clones based on the different biochemical characteristics. A significant correlation was found between both total chl concentration and chl/prot ratio with P_max for the five clones.     

Sexual dimorphism in <Emphasis Type="Italic">Sebastes owstoni</Emphasis> (Scorpaenidae) from the Sea of Japan

Morphological and genetic differences between red and yellow morphotypes of Sebastes owstoni were investigated, utilizing 277 males [84.0–194.3 mm in standard length (SL)] and 542 females (92.3–251.5 mm SL) from the Sea of Japan. All males smaller than 120 mm SL were characterized by red body color. The frequency of specimens with yellow body color thereafter increased gradually with SL, all specimens larger than 170 mm SL being yellow. The specimens with yellow body color were observed throughout the year. All females smaller than 170 mm SL were characterized by red body color, the frequency of specimens with yellow body color tending to slightly increase with SL. However, most females had red body color, except for 16 specimens (177.7–241.5 mm SL) that were yellow, growth-related color change from red to yellow being uncommon. Morphological analysis of 49 males (107.6–193.3 mm SL) and 68 females (108.7–241.5 mm SL) showed the head length, orbit diameter, lower jaw length, and predorsal length to be relatively greater, but the distance between the pelvic and anal fins less, in males. A discriminant analysis using Mahalanobis distances resulted in 100% correct assignment of specimens to sex, regardless of SL and body color. In addition, no genetic differences were apparent between red and yellow individuals in mitochondrial DNA sequence analyses from the threonine tRNA to the first half of the control region (498 bp). Accordingly, the differences in body color, maximum size, and the five morphometric characters listed above were considered to represent sexual dimorphism. That evidenced by body color was considered to appear after that shown by morphometric characters, some exceptions in the former occurring in females. This is the first report of permanent sexual dimorphism in body color in Sebastes.     

Development and identification of wheat-Ag. pulcherrimum addition line and substitution line with BYDV resistance

Breeding materials derived from CPI113500, amphidiploid ofT. turgidum × Ag.pulcherrimum with barley yellow dwarf virus (BYDV) resistance, were evaluated by using BYDV resistance test, morphology observation, cytogenetics analysis, aneuploid analysis, isozyme electrophoresis,in situ hybridization. Two new germplasms resistant to BYDV were obtained. They were T.aestivum-Ag. pulcherrimum disomic addition line 96S16-11, andT. aestivum-Ag. pulcherrimum disomic substitution line 96W14-9. Project supported by the “863” Program and the National Science and Technology Committee.     

Organization and chromosomal localization ofβ-tubulin genes inLeishmania donovani

The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.     

Mechanoreceptive function of pectines in the Brazilian yellow scorpion Tityus serrulatus: perception of substrate-borne vibrations and prey detection

Environment perception by scorpions is an issue of great interest, and there is controversy about the use of pectines in this function. This paper investigates the mechanoreceptive function of pectines of the yellow scorpion Tityus serrulatus. The study was conducted in laboratory conditions from July 2003 to July 2004. Ninety females were maintained in six terraria (N = 15). Three of them were named “control” group (intact pectines) and the others “treatment” group (pectines covered with paraffin). Three experimental tests (an ethogram, a test of prey detection and a test of perception of substrate-borne vibrations) were performed to compare skills of animals with and without blocked pectines. The ethogram showed that treated individuals significantly reduced their exploratory activities, spent more time resting, and aggregated more than the control ones. Prey detection tests showed that the blocking of pectines made the yellow scorpions inefficient to detect and capture prey. Vibration tests showed that animals with blocked pectines also did not respond to the vibrations produced in the substrate, whereas the opposite occurred with the control group. These results suggest a mechanoreceptive function for T. serrulatus pectines, and show that the detection of ground vibrations by these structures is important for prey perception and orientation in the Brazilian yellow scorpion.     

Genetic transformation of Rhamnus fallax and hairy roots as a source of anthraquinones

Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth, reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g⁻¹(d.m.)] and clone 7 [14.21 mg g⁻¹(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue.     

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS ₄ locus (38 of Mut⁺ clone) or into theAOX1 locus (45 of Mut^s clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of the recombinantS. cerevisiae.     

Bioaccumulation of mercury in yellow perch (Perca flavescens) and common loons (Gavia immer) in relation to lake chemistry in Atlantic Canada

Mercury biomagnifies in aquatic foodwebs in freshwater lakes, and common loons (Gavia immer) breeding in eastern Canada can be exposed to reproductively toxic concentrations of mercury in their fish prey. We assessed the bioaccumulation and biomagnification of mercury in juvenile and adult common loons, and their preferred prey: yellow perch (Perca flavescens) in Kejimkujik National Park (KNP), Nova Scotia by measuring mercury levels and stable isotope ratios in tissues. Total mercury levels and stable-carbon (δ¹³C) and nitrogen isotope ratios (δ¹⁵N) were determined in composite whole-fish samples from lakes in KNP and blood samples from juvenile and adult loons captured on lakes in KNP and southern New Brunswick. Geometric mean mercury concentrations were 0.15 and 0.38 μg/g (wet wt.) in small (9-cm fork length) and large (17-cm fork length) yellow perch, and were 0.43 and 2.7 μg/g (wet wt.) in blood of juvenile and adult common loons, respectively. Mercury concentrations in perch and loons were positively associated with body mass and δ¹⁵N values. Juvenile loons and large yellow perch had similar mercury levels and δ¹⁵N values, indicating similar trophic status despite their 22-fold difference in body mass. Mercury concentrations were higher in yellow perch and common loons in acidic lakes. Our findings highlight the importance of both chemical and ecological factors in understanding mercury biomagnification in lakes and associated risks to fish-eating wildlife. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Microsatellite markers for the fungal banana pathogen Mycosphaerella musicola

Yellow Sigatoka caused by the ascomycete Mycosphaerella musicola Leach, is one of the most severe banana diseases worldwide, which spread in most banana growing areas, until Black Sigatoka, a more aggressive disease caused by Mycosphaerella fijiensis, appeared. Because of the highly devastating nature of the latter pathogen, recent research almost exclusively focused on M. fijiensis. To close the gap of knowledge and to study the population structure of M. musicola in Yellow Sigatoka‐infested areas, we cloned and characterized a versatile set of 26 polymorphic locus‐specific microsatellite markers.