<Emphasis Type="Italic">Artemisia lerchiana</Emphasis> as a producer of essential oil

Abstrac  The composition of essential oil of Artemisia lerchiana Web. plants growing in Volgograd oblast was studied. Sampling was performed from plots contrasting in climatic and soil characteristics. Essential oil was obtained by hydrodistillation. The content of essential oil in shoot biomass increased gradually during shoot formation, flower bud formation, and flowering beginning and then decreased. The highest content of essential oil varied from 1.1 to 1.5% of plant dry weight at the stage of flower bud formation. More than thirty compounds were identified by gas chromatography-mass spectrometry. The following major components were found: camphor, borneol, bornylacetate, camphene, and 1,8-cineole. Some of compounds (sesquiterpenes and sesquiterpenoids) were identified for the first time. The time-course of accumulation of essential oil components strongly depended on habitat edaphic factors and climatic conditions during the year of sampling. The results permit a conclusion that A. lerchiana is a valuable producer of essential oils. Original Russian Text ? E.B. Kirichenko, Yu.V. Orlova, D.V. Kurilov, 2008, published in Fiziologiya Rastenii, 2008, Vol. 55, No. 6, pp. 934–941.     

Manifestation of salt tolerance of <Emphasis Type="Italic">Spirulina platensis</Emphasis> and <Emphasis Type="Italic">Spirulina maxima</Emphasis> cyanobacteria of the genus <Emphasis Type="Italic">Arthrospira (Spirulina)</Emphasis>

The salt tolerance of two representatives of genus Spirulina (Arthrospira) Spirulina platensis and Spirulina maxima has been investigated. They both are the wide-spread objects of photobiotechnology and it has been shown that the content of 5–15 % sea-water in medium has not caused the decreasing of biomass yield more than 15–20% as compared with control. The further decreasing of biomass was proportionate to sea-water content in medium. The investigation of reactivity of native (intravital) exometabolites secreted into cultural medium has showed that the sea-water content influence the oxidative activity (OA) of exometabolites and hour’s rhythmics.     

Phototropic H<Subscript>2</Subscript> production by a newly isolated strain of <Emphasis Type="Italic">Rhodopseudomonas</Emphasis><Emphasis Type="Italic">palustris</Emphasis>

Rhodopseudomonas palustris TN1 was isolated from Songkhla Lake, Thailand. It phototrophically generates H₂ from the predominant volatile fatty acids (VFAs) produced from microbial dark-fermentations of palm oil milling effluent; yields from 20 mM butyrate, acetate and propionate were 4.7, 2.5, and 1.7 mol H₂ mol VFA⁻¹ with light efficiencies of 1.8, 1, and 0.2%, respectively. Optimum conditions were pH 7 and 3000 lux, although production was reduced by only 33% at 1000 lux. CO₂ evolution never exceeded 9 mmol mol VFA⁻¹.     

Utilization of dibenzothiophene as sulfur source by <Emphasis Type="Italic">Microbacterium</Emphasis> sp. <Emphasis Type="Italic">NISOC-06</Emphasis>

Oil-polluted soils were sampled from National Iranian South Oil Company (NISOC) for isolation and screening of C–S and not C–C targeted Dibenzothiophene (DBT) degrading microorganisms. Microbacterium sp. NISOC-06, a C–S targeted DBT degrading bacterium, was selected and its desulfurization ability was studied in aqueous phase and water-gasoline biphasic systems. The 16srRNA gene was amplified using universal eubacteria-specific primers, PCR product was sequenced and the sequence of nearly 1,500 bp 16srDNA was studied. Based on Gas Chromatography results Microbacterium sp. NISOC-06 utilized 94.8% of 1 mM DBT during the 2 weeks of incubation. UV Spectrophotometry and biomass production measurements showed that the Microbacterium sp. NISOC-06 was not able to utilize DBT as a carbon source. There was no accumulation of phenolic compounds as Gibb’s assay showed. Biomass production in a biphasic system for which DBT-enriched gasoline was used as the sulfur source indicated the capability of Microbacterium sp. NISOC-06 to desulfurize gasoline.     

Chemical Composition of Hydrodistilled Essential Oil of Artemisia incana (L.) Druce and Antimicrobial Activity against Foodborne Microorganisms

The oil obtained by hydrodistillation from the aerial parts of Artemisia incana (L.) Druce from Turkey was analyzed by GC and GC/MS. Sixty‐three compounds were characterized, representing 97.2% of the total components detected, and camphor (19.0%), borneol (18.9%), 1,8‐cineole (14.5%), bornyl acetate (7.8%), camphene (4.9%), and α‐thujone (4.8%) were identified as predominant components. The essential oil was also tested for its antimicrobial activity against 44 different foodborne microorganisms, including 26 bacteria, 15 fungi, and 3 yeast species. The essential oil of A. incana exhibited considerable inhibitory effects against all bacteria, fungi, and yeast species tested. However, the oil showed lower inhibitory activity against the tested bacteria than the reference antibiotics.     

HMG-CoA reductase limits artemisinin biosynthesis and accumulation in <Emphasis Type="Italic">Artemisia annua</Emphasis> L. plants

In vivo modulation of HMG-CoA reductase (HMGR) activity and its impact on artemisinin biosynthesis as well as accumulation were studied through exogenous supply of labeled HMG-CoA (substrate), labeled MVA (the product), and mevinolin (the competitive inhibitor) using twigs of Artemisia annua L. plants collected at the pre-flowering stage. By increasing the concentration (2–16 μM) of HMG-CoA (3-¹⁴C), incorporation of labeled carbon into artemisinin was enhanced from 7.5 to 17.3 nmol (up to 130%). The incorporation of label (¹⁴C) into MVA and artemisinin was inhibited up to 87.5 and 82.9%, respectively, in the presence of 200 μM mevinolin in incubation medium containing 12 μM HMG-CoA (3-¹⁴C). Interestingly, by increasing the concentration of MVA (2-¹⁴C) from 2 to 18 μM, incorporation of label (¹⁴C) into artemisinin was enhanced from 10.5 to 35 nmol (up to 233%). When HMG-CoA (3-¹⁴C) concentration was increased from 12 to 28 μM in the presence of 150 μM mevinolin, the inhibitions in the incorporation of label (¹⁴C) into MVA and artemisinin were, however, reversed and the labels were found to approach their values in twigs fed with 12 μM HMG-CoA (3-¹⁴C) without mevinolin. In another experiment, 14.2% inhibition in artemisinin accumulation was observed in twigs in the presence of 175 μM fosmidomycin, the competitive inhibitor of 1-deoxy-d-xylulose 5-phosphate reductase (DXR). HMG-CoA reductase activity and artemisinin accumulation were also increased by 18.6 to 24.5% and 30.7 to 38.4%, respectively, after 12 h of treatment, when growth hormones IAA (100 ppm), GA₃ (100 ppm) and IAA + GA₃ (50 + 50 ppm) were sprayed on A. annua plants at the pre-flowering stage. The results obtained in this study, hence, demonstrate that the mevalonate pathway is the major contributor of carbon supply to artemisinin biosynthesis and HMGR limits artemisinin synthesis and its accumulation in A. annua plants.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Seroprevalence of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> antibodies among students of the <Emphasis Type="Italic">Faculty of Medicine</Emphasis> in Košice (Slovakia)

Titers of immunoglobulin IgG against phases I and II of Coxiella burnetii were determined in 241 students of the Faculty of Medicine by ELISA method and the respective risk factors were evaluated, e.g., rural and urban life, consumption of milk, contact with animals and gender, which may be associated with exposure to C. burnetii. Phase I antibodies (Abs) were detected in 59 serum samples (24.4 %) at antibody level of 1: 100–1: 400. Phase II Abs were found in 179 persons (74.2 %). The titers were in the range of 1: 100–1: 1600. The titer ≥1: 800 of IgG was used as a cut-off level, and was detected only in 20 students (8.2 %). No significant difference in the prevalence of Abs was detected either between the students living in rural and urban environment (78.8 and 73.2 %, respectively) or between males and females (74.0 and 74.7 %, respectively). Abs were detected more frequently in raw milk consumers (68.1 %) and in students who kept some animals (73.7 %).     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Expression of Two Self-enhancing Antifreeze Proteins from the Beetle <Emphasis Type="Italic">Dendroides canadensis</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T ₃ DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.     

Biotransformations of cinnamaldehyde,cinnamic acid and acetophenone with <Emphasis Type="Italic">Mucor</Emphasis>

A strain JX23 was isolated from soil and identified as a species of Mucor according to the morphological characteristics and the nuclear ribosomal internal transcribed spacer sequence and designated as Mucor sp. JX23. Biotransformations of cinnamaldehyde (CMD), cinnamic acid (CMA) and acetophenone (ACP) catalyzed by JX23 were investigated. After JX23 was cultured for 48 h, the substrates CMD, CMA and ACP were added to the growth medium respectively and the products were analyzed by GC–MS and HPLC. Mucor sp. JX23 exhibited considerable redox capability and different catalytic specificity to CMD, CMA and ACP. CMD was selectively hydrogenated to cinnamyl alcohol. CMA was biotransformed to ACP with α, β-oxidation like degradation, and ACP could not be reduced further by JX23. When ACP was added as substrate, it could be asymmetrically reduced to (S)-(−)-1-phenylethyl alcohol (S-PEA) with high stereoselectivity (90%). Further, the biotransformations of different binary mixture substrates with JX23 were also studied. The biocatalytic selectivity depended on the relationship between the binary mixtures in above-mentioned reaction.     

High frequency in vitro propagation of <Emphasis Type="Italic">Trichopus zeylanicus</Emphasis> subsp. <Emphasis Type="Italic">travancoricus</Emphasis> using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.     

Water relations advantages for invasive <Emphasis Type="Italic">Rubus</Emphasis> <Emphasis Type="Italic">armeniacus</Emphasis> over two native ruderal congeners

Despite species in the Rubus fruticosus complex (wild blackberry) being among the most invasive plants globally in regions with large annual fluctuations in water availability, little is known about their water relations. We compared water relations of a prominent member of the complex, R. armeniacus (Himalayan blackberry), with species native to the Pacific Northwest of North America (PNW), R. spectabilis (salmonberry) and R. parviflorus (thimbleberry). In eight stands of each species located near Portland, Oregon, USA, we measured mid-day hydraulic resistance (R _plant), and daily time series of stomatal conductance (g _s), leaf water potential (Ψ_lf), and environmental conditions at four time periods spanning the 2007 growing season. Although all species maintained Ψ_lf above −0.5 MPa in spring, R. armeniacus maintained less negative Ψ_lf (≥−1.0 MPa) than the natives in summer, a factor attributable to advantages in both its root and shoot systems. R _plant of R. armeniacus was ≤0.1 MPa mmol⁻¹ m² s for the duration of the study, and approximately 25–50% of R _plant for the native species in summer. R. armeniacus had higher g _s compared to the native species throughout the spring and summer, with approximately twice their rates in summer. Our R _plant and g _s results show that R. armeniacus has access to more water during PNW summers than congeneric natives, allowing it to maintain high water-use, and potentially helping it achieve higher growth and reproductive rates. Water relations may therefore be a critical component of the competitive and invasive success of R. armeniacus and other R. fruticosus species worldwide.     

Essential oil composition of <Emphasis Type="Italic">Taraxacum officinale</Emphasis>

The gas chromatography–mass spectrometry (GC–MS) analysis of essential oil obtained by hydrodistillation from the flower of Taraxacum officinale L. revealed the presence of 25 compounds with 1,3-dimethylbenzene, 1,2-dimethylbenzene, 1-ethyl-3-methylbenzene, heneicosane and tricosane as the main components.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

<Emphasis Type="Italic">Grias angustipetala</Emphasis> and <Emphasis Type="Italic">G. ecuadorica</Emphasis>, two new species of Lecythidaceae from western Ecuador

Grias angustipetala and G. ecuadorica, two new species from the wet forests of western Ecuador are described. Grias angustipetala occurs in the Awá Indígenous Territory and Reserva Ecológica Cotacachi-Cayapas in the Esmeraldas and adjacent Carchi Provinces between 50–1800 m elevation. Grias ecuadorica is found in the Los Ríos and Bolívar Provinces between 70–600 m elevation. The new species are illustrated and their relationships with related species are discussed.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.