Competition between <Emphasis Type="Italic">Eucalyptus victrix</Emphasis> seedlings and grass species

Competition in a natural system may be interspecific or intraspecific. In semiarid ecosystems, competition for resources between established neighboring grass species and newly recruited seedlings is very high. To examine the effects of grass species density, growing space and time of establishment on Eucalyptus victrix seedlings (interspecific competition), and the effect of density and growing space within E.victrix (intraspecific competition) we conducted an experiment under controlled conditions. We tested four hypotheses (i) E.victrix seedling growth is not affected by grass density; (ii) there is no difference in E.victrix survival and growth between early and later grass establishment; (iii) interspecific competition is not more intense than intraspecific competition in E.victrix; and (iv) growth of E.victrix seedlings is not dependent on available growing space. In a monoculture of E.victrix, seedling mortality was higher (10%) in large pots. In mixed culture pots, where E.victrix seedlings and grass seedlings were planted on the same day, E.victrix seedlings survived for up to 4weeks, but started to die after week five in the smallest pots. However, mortalities occurred in pots of all sizes when grass was established before E.victrix seedlings. Results also indicated that the resources necessary for the growth of individual E.victrix seedlings were more limiting under conditions of increased density of neighboring grass species rather than intraspecific competition. In particular, photosynthetic area of E.victrix seedlings was drastically reduced in mixed cultures. Although density, pot size and time of planting had impacts on E.victrix seedlings, the patterns of these impacts were variable.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

A Motility Revertant of the <Emphasis Type="Italic">ndvB</Emphasis> Mutant of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

A motility revertant of a Bradyrhizobium japonicum ndvB mutant was isolated and characterized. The ndvB mutants of B. japonicum have been reported to be osmotically sensitive, as well as defective in motility, periplasmic cyclic -(13), (16)-D-glucan synthesis, and symbiosis with soybean. The motility revertant was restored for osmotic tolerance but not for cyclic -glucan production or effective symbiosis. These results support our hypothesis that cyclic -glucans have an important role in symbiosis—the suppression of a plant defense response—in addition to their role in periplasmic osmoprotection.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Genetic Distance in Housekeeping Genes Between <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium reichenowi</Emphasis> and Within <Emphasis Type="Italic">P. falciparum</Emphasis>

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672±0.0088 and 0.0011±0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30±5.40) × 10⁴ and (11.62±7.56) × 10⁴ years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.This article contains an online supplementary table.Reviewing Editor: Dr. Martin Kreitman     

Rapid micropropagation of <Emphasis Type="Italic">Curcuma longa</Emphasis> using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Multiple shoots of Curcuma longa were induced by culture of bud explants for 1week in Murashige and Skoog (MS) liquid medium supplemented with 72.64M thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8weeks. The regeneration rate was up to 11.4±1.7shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.     

Tooth development in vitro in two teleost fish,the cichlid <Emphasis Type="Italic">Hemichromis bimaculatus</Emphasis> and the cyprinid <Emphasis Type="Italic">Danio rerio</Emphasis>

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.The in vitro infrastructure at the Ghent laboratory was financed through a grant of the Bijzonder Onderzoeksfonds of Ghent University (BOF: 01102995) and a Krediet aan navorsers (no. 31513695) of the Fonds voor Wetenschappelijk onderzoek (FWO-Vlaanderen). This study also benefitted from an exchange program between the Centre National de Recherche Scientifique (CNRS) and the Ministerie van de Vlaamse Gemeenschap. Research performed by C. Van der heyden was partly financed through a specialization grant of the Flemish Institute for the Advancement of Scientific-Technological Research in Industry (IWT).     

Characterization of Na,K-ATPase genes in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) and comparative genomic organization with rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

A combination of molecular and in silico approaches was employed to assemble a survey of Na, K-ATPase genes contained in the ancestrally tetraploid genome of the Atlantic salmon (Salmo salar). Molecular characterization of genomic clones coding for the subunit revealed two single genes (1a and 2) and two pairs of presumably homeologous genes (1b/i-ii and 1c/i-ii). Each of the six genes showed high sequence similarity to isoforms previously isolated from rainbow trout and extensive structural differences relative to putative orthologs in the human genome. In silico analysis of expressed sequence tag (EST) collections indicated that at least five (1a, 1b, 1c, 2, and 3) and four (1a, 1b, 2, and 3b) subunit isoforms are expressed in Atlantic salmon. Meiotic linkage analysis further showed that Na, K-ATPase genes are dispersed throughout the salmon genome, with the exception of two multigene clusters on linkage groups AS-22 and AS-28. Duplicate gene copies for the isoform 1b were assigned to linkage groups with multiple homeologous anchors (AS-22 and AS-23), while 2 duplicates suggested a new homeologous affinity between AS-05 and AS-28. In addition, the comparison of linkage arrangements with rainbow trout also showed that the genomic organization of Na, K-ATPase genes is consistent with the evolutionary conservation of syntenic chromosome regions between these species.Electronic Supplementary Material Supplementary material is available for this article at .     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

Larvae of <Emphasis Type="Italic">Arnoglossus elongatus</Emphasis> (Pleuronectiformes: Bothidae) from northwestern Australia

Eleven postflexion larvae (9.1–23.9mm standard length: SL) of a bothid, Arnoglossus elongatus, from northwestern Australian waters were described. These specimens were characterized by vertebral numbers of 11–12+32–34=43–45, slender body, remarkably elongated second dorsal fin ray, and presence of melanophores on and slightly above midlateral line of body, plus on dorsal wall of abdominal cavity and air bladder (specimens >12.6mm SL), proximally on caudal fin rays (>16.5mm SL), and on pterygiophore zones of dorsal fin and anal fin (>23.8mm SL).     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Perilla frutescens</Emphasis> from hypocotyl and cotyledon explants

Organogenetic buds were induced from hypocotyl and cotyledon explants of oil crop Perilla frutescens in Murashige and Skoog (MS) medium supplemented with 5.7 M indole-3-acetic acid (IAA) and 8.9 – 13.3 M 6-benzylaminopurine (BA). Shoots were rooted on MS medium with 2.9 M IAA and 1.4 M gibberellic acid (GA3) and the regenerated plants flowered and set seeds normally.     

Transformation of artemisinin by<Emphasis Type="Italic"> Cunninghamella elegans</Emphasis>

Semi-synthetic derivatives of the anti-malarial drug artemisinin hold great promise in the search for an effective and economical treatment of chloroquine-resistant forms of malaria. Unfortunately, synthetic functionalization of the artemisinin skeleton is often tedious and/or impractical. We seek to utilize 7-hydroxyartemisinin, obtained from microbial transformation, as a semi-synthetic precursor for the synthesis of novel 7-substituted artemisinin anti-malarial agents. Here we employ liquid cultures of Cunninghamella elegans as a means for the rational and economical bioconversion of artemisinin to 7-hydroxyartemisinin in 78.6% yield. In addition, there were three other bioconversion products: 7-hydroxy-9-artemisinin (6.0%), 4-hydroxy-1-deoxoartemisinin (5.4%), and 6-hydroxyartemisinin (6.5%).     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

Expression of <Emphasis Type="Italic">Treponema denticola</Emphasis> Oligopeptidase B in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases. This bacterium possesses a particular arginine peptidase activity (previously called BANA-peptidase or trypsin-like enzyme) that is common to the three cultivable bacterial species most highly associated with severe periodontal disease. We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T. denticola through DNA sequencing and mutagenesis studies. In the present study, we report expression of T. denticola OpdB peptidase in Escherichia coli. The opdB PCR product was cloned into pET30b and then transformed into the E. coli BL21 (DE3)/pLysS expression strain. Assays of enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola. Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).