Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) and Douglas fir (<Emphasis Type="Italic">Pseudotsuga menziesii</Emphasis>): improving culture initiation and growth with MES pH buffer,biotin, and folic acid

Loblolly pine (Pinus taeda L.) somatic embryogenesis initiation was improved by supplementing the initiation medium with the pH buffer agent 2(n-morpholino)ethanesulphonic acid (MES) at 250 mg l^–1, folic acid at 0.5 mg l¹, and biotin at 0.05 mg l^–1. MES and vitamins increased the percentage of explants with extruded tissue that continued the initiation process to form embryogenic tissue. The increase in initiation was about 12%. Initiation of 12 open-pollinated families averaged 38.5%, which is 16% higher than initiation on medium without these additives. When tested with 18 control-pollinated families, initiation averaged 26.3%. Basal medium contained a combination of modified 1/2 P6 salts, activated carbon (AC) at 50 mg ^–1, Cu and Zn adjusted to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg l^–1 casamino acids, 450 mg l^–1 glutamine, 2 mg l^–1 NAA, 0.63 mg l^–1 BAP, 0.61 mg l^–1 kinetin, 3.4 mg l^–1 silver nitrate, 10 M 8-Br-cGMP, 0.1 M brassinolide, and 2 g l^–1 Gelrite. Early-stage embryo growth and initiation in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were also improved in the presence of these additives.     

The incidence of mistletoe (<Emphasis Type=Italic>Viscum album</Emphasis> ssp. <Emphasis Type=Italic>abietis</Emphasis>) on silver fir (<Emphasis Type=Italic>Abies alba</Emphasis>) in Croatia

The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers. The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence. The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

The expression of analgesic-antitumor peptide (<Emphasis Type=Italic>AGAP</Emphasis>) from Chinese <Emphasis Type=Italic>Buthus</Emphasis><Emphasis Type=Italic>martensii</Emphasis> Karsch in transgenic tobacco and tomato

The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.     

Multiple host-ant use by the predatory social parasite <Emphasis Type=Italic>Phengaris</Emphasis> (=<Emphasis Type=Italic>Maculinea</Emphasis>) <Emphasis Type=Italic>arion</Emphasis> (Lepidoptera,Lycaenidae)

Phengaris (=Maculinea) arion is an endangered social parasite of Myrmica ants, and for a very long time was considered as specific to Myrmica sabuleti. Previous studies carried out in Poland suggested some discrepancies within this assumption, and therefore a much more intensive survey was undertaken. The host ant use of P. arion was studied at five sites in different types of biotopes in Poland, i.e. xerothermal grasslands where Thymus pulegioides was used as a larval food plant by the butterfly, and more or less sandy biotopes with Thymus serpyllum. Altogether nine Myrmica species were recorded, and considerable variation in species composition and density of nests was recorded. At four localities M. sabuleti proved to be the most common ant. A total of 529 Myrmica nests were examined, and only 20 of them contained larvae and pupae of P. arion. Host ants belonged to five different species, i.e. M. sabuleti, Myrmica scabrinodis, Myrmica schencki, Myrmica lobicornis and Myrmica hellenica. Only at one site (NE Poland) was a significant heterogeneity in parasitation rates among Myrmica species detected. M. lobicornis was the most often infested ant there, which may suggest local specialisation of the butterfly. Overall low parasitism rates may explain the vulnerability of P. arion in Central Europe but further studies are also necessary.     

Hormone Profiling by LC-QToF-MS/MS in Dormant <Emphasis Type="Italic">Macadamia integrifolia</Emphasis>: Correlations with Abnormal Vertical Growth

A method for analyzing multiple plant hormone groups in small samples with a complex matrix was developed to initiate a study of the physiology of abnormal vertical growth (AVG) in Macadamia integrifolia (cv. HAES344). Cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA), and auxins were detected in xylem sap and apical and lateral buds using high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS). The extraction method separated compounds with high sensitivity in positive (CKs) and negative (ABA, auxins, GAs) modes of QToF-MS/MS. CK profiles differed in xylem sap and apical and lateral buds irrespective of AVG symptoms. Trans-zeatin riboside (t-ZR) was dominant in sap of normal and AVG trees (∼4 and 6 pmol g⁻¹ FW, respectively). In apical buds isopentenyl adenine (iP) (∼30 pmol g⁻¹ FW) was the most abundant CK, and in lateral buds trans-zeatin (t-Z) (22–24 pmol g⁻¹ FW) and iP (24–30 pmol g⁻¹ FW) were the most abundant. t-Z levels of AVG trees were higher in apical buds (13.88 vs. 6.6 pmol g⁻¹ FW, p < 0.05) and lower in sap (0.16 vs. 0.51 pmol ml⁻¹, p < 0.005) compared to normal trees. ABA in lateral buds was 1.9 times higher (p < 0.001) in AVG. IAA was below quantification, whereas indole-3-butyric acid (IBA) was consistently present. GA₇ was the dominant GA in apical and lateral buds of all trees (100–150 pmol g⁻¹ FW). GA_{3, 4, & 9} were consistently present at low concentrations (<12 pmol g⁻¹ FW) in buds. GAs_{1, 3, & 9} were detected in xylem sap at low concentrations (<0.5 pmol g⁻¹ FW). Differences in sap amino acids (AA) were also assessed. In sap from AVG trees, asparagine and glutamine increased significantly (p < 0.05) in their contribution to total AA. Potential AVG hormone correlations are discussed.     

High-density AFLP map of nonbrittle rachis 1 (<Emphasis Type=Italic>btr1</Emphasis>) and 2 (<Emphasis Type=Italic>btr2</Emphasis>) genes in barley (<Emphasis Type=Italic>Hordeum vulgare</Emphasis> L.)

Wild relatives of barley disperse their seeds at maturity by means of their brittle rachis. In cultivated barley, brittleness of the rachis was lost during domestication. Nonbrittle rachis of occidental barley lines is controlled by a single gene (btr1) on chromosome 3H. However, nonbrittle rachis of oriental barley lines is controlled by a major gene (btr2) on chromosome 3H and two quantitative trait loci on chromosomes 5HL and 7H. This result suggests multiple mutations of the genes involved in the formation of brittle rachis in oriental lines. The btr1 and btr2 loci did not recombine in the mapping population analyzed. This result agrees with the theory of tight linkage between the two loci. A high-density amplified fragment-length polymorphism (AFLP) map of the btr1/btr2 region was constructed, providing an average density of 0.08 cM/locus. A phylogenetic tree based on the AFLPs showed clear separation of occidental and oriental barley lines. Thus, barley consists of at least two lineages as far as revealed by molecular markers linked to nonbrittle rachis genes.Electronic Supplementary Material Supplementary material is available for this article at An erratum to this article can be found at     

Synthesis of orthogonally protected (<Emphasis Type=Italic>3R</Emphasis>,<Emphasis Type=Italic>4S</Emphasis>)- and (<Emphasis Type=Italic>3S</Emphasis>,<Emphasis Type=Italic>4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Cell-free culture medium of <Emphasis Type=Italic>Burkholderia cepacia</Emphasis> improves seed germination and seedling growth in maize (<Emphasis Type=Italic>Zea mays</Emphasis>) and rice (<Emphasis Type=Italic>Oryza sativa</Emphasis>)

The saprophytic bacterium Burkholderia cepacia has been shown to play an active role as plant growth promoting bacteria (PGPB). In this study, the ability of cell-free culture medium (CFCM) of B. cepacia to improve early developmental stages of plants has been assessed on two agronomically important crops, maize (Zea mays) and rice (Oryza sativa). Treating maize and rice seeds for 45 min before germination significantly improved seed germination and consequent seedling growth. The effect of CFCM was confirmed by the increased biomass of the shoot and, mainly, the root systems of treated seedlings. Chromatographic characterization of the CFCM revealed that the spent culture medium of B. cepacia is a complex mix of different classes of metabolites including, among others, salicylic acid, indole-3-acetic acid (IAA) and several unidentified phenolic compounds. Fractionation of the CFCM components revealed that the impressive development of the root system of CFCM-treated seedlings is due to the synergistic action of several groups of components rather than IAA alone. The data presented here suggest that a CFCM of B. cepacia can be used to improve crop germination.     

Effect of seed extracts of <Emphasis Type=Italic>Withania somnifera,Croton tiglium</Emphasis> and <Emphasis Type=Italic>Hygrophila auriculata</Emphasis> on behavior and physiology of <Emphasis Type=Italic>Odontotermes obesus</Emphasis> (Isoptera,Termitidae)

In addition to antibiotic properties, medicinal plants are important sources of chemicals with potential application as pesticides. The present study deals with antitermitic potential of seed extracts of Withania somnifera (Indian ginseng), Croton tiglium (jamalgoota) and Hygrophila auriculata (talimkhana). The seed extracts caused changes in tunneling behaviour, number of bacterial colonies in hindgut and activities of enzymes in midgut of Odontotermes obesus. C. tiglium showed the lowest LT₅₀ (12.85 and 2.65 h) among the three seed extracts at concentrations of 50% (half dilution of the extract) and 100% (extract without dilution), respectively. There was no tunneling in soil treated with 100% concentration of seed extracts of W. somnifera and C. tiglium. Numbers of bacterial colonies in the gut of termites from soils treated with 50% and 100% concentrations of the three plants did not differ significantly, but they differed from those in termites from untreated soil. At 50% concentrations of seed extracts of the tested plants, the difference in hindgut enzyme activities was not obvious, however, at 100% concentrations the enzyme activities in the termites from soils treated with seed extracts significantly differed from controls and differences were also recorded between the plants.     

Characterization of <Emphasis Type=Italic>SpAPETALA3</Emphasis> and <Emphasis Type=Italic>SpPISTILLATA</Emphasis>, B class floral identity genes in <Emphasis Type=Italic>Spinacia oleracea</Emphasis>, and their relationship to sexual dimorphism

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3 and 5 RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.Electronic Supplementary Material Supplementary material is available for this article at Edited by G. Jürgens     

<Emphasis Type=Italic>In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type=Italic>Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

Oviposition site selection by the bromeliad-dweller harvestman <Emphasis Type=Italic>Bourguyia</Emphasis><Emphasis Type=Italic>hamata</Emphasis> (Arachnida: Opiliones)

Bourguyia hamata females oviposit almost exclusively inside the rosette formed by the curled leaves of the epiphytic bromeliad Aechmea nudicaulis. We investigated whether the architecture of the individual bromeliads influences oviposition site selection by this harvestman species. We collected data on the presence of clutches inside bromeliads, rosette length, rosette slope in relation to tree trunks, and the amount of debris inside the rosette. Additionally, we measured the water volume inside the rosettes as well as the variation in the humidity inside and outside bromeliads with long and short rosettes. Longer rosettes were preferred as oviposition site possibly because they accumulate more water and maintain lower internal humidity variation than the external environment. Although the slope of the rosettes did not influence the occurrence of oviposition, the probability of debris accumulation inside the rosettes increased with their slope, and the frequency of clutches was greater in bromeliads with small amounts of debris. A field experiment showed that bromeliads with water inside the rosette were more frequently used as oviposition sites than bromeliads without water. In conclusion, females oviposit predominantly in bromeliads that accumulate more water and have small amounts of debris inside the rosettes, probably because these characteristics promote a more adequate microhabitat for egg development.     

Food web overlap among native axolotl (<Emphasis Type=Italic>Ambystoma mexicanum</Emphasis>) and two exotic fishes: carp (<Emphasis Type=Italic>Cyprinus carpio</Emphasis>) and tilapia (<Emphasis Type=Italic>Oreochromis niloticus</Emphasis>) in Xochimilco,Mexico City

Two exotic fishes, common carp (Cyprinus carpio) and tilapia (Oreochromis niloticus), were introduced more than 20 years ago into Xochimilco for aquaculture, and now dominate the system in terms of biomass and numbers. Over this same period, wild populations of the microendemic axolotl salamander (Ambystoma mexicanum) have been dramatically reduced, and it currently persists in isolated areas of this aquatic system, which is situated at the edge of Mexico City. In this study, we examine potential trophic interactions and niche overlap among two exotic fishes: carp and tilapia, and the native axolotl. Axolotl had more diverse diets and a higher trophic position compared to the exotics. Stable isotope analysis revealed substantial trophic niche overlap among axolotl and the exotics. The two exotics occupied a larger niche area than the axolotl, suggesting higher levels of omnivory and diet specialization. Current exotic fish removal efforts will further our understanding of interactions between the axolotl and exotic species, and are expected to benefit axolotl recovery efforts.     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Proteomic analysis of seed development in Chinese fir (<Emphasis Type=Italic>Cunninghamia lanceolata</Emphasis>)

Seed development is a complex process governed by highly coordinated changes in the expression of a large protein set. DIGE (Difference Gel Electrophoresis)-based proteomics was used to study developing Chinese fir seeds. 153 spots were obtained by using the analysis of DeCyder software (v. 6.5). Cluster analysis showed that they could be joined into three main groups. Eleven spots, more actively expressed at early cotyledonary stage of developing seeds, were identified by LC/MS/MS (tandem MS). Ten spots were identified by searching NCBInr or EST databases. They included two legumin-like storage proteins, LEA protein, small heat-shock protein, PR10-1.13, a protein similar to eukaryotic translation initiation factor, a protein similar to maternal effect embryo arrest 51, ORF115, a protein similar to monodehydroascorbate reductase, and unknown proteins. The potential function of these proteins during the precotyledonary stage of seed development was discussed.