Two-stage system for micropropagation of several Genista plants producing large amounts of phytoestrogens

A two-stage method for in vitro propagation of six Genista species from shoot tips was developed. Multiple microshoot cultures were obtained by growing the shoot tip explants on Schenk and Hildebrandt medium supplemented with 9.84 microM 6-(gamma,gamma-dimethylallylamino)-purine and 0.99 microM thidiazuron. The best shoot elongation was achieved on Schenk and Hildebrandt medium containing 4.92 microM indole-3-butyric acid. The rooting of shoots brought best effects (100%) on Schenk and Hildebrandt medium with 2.68 microM 1-naphthaleneacetic acid. HPLC analysis indicated that six-month-old regenerated plants as well as the herb of intact plants produced a rich set of simple flavones (derivatives of luteolin and apigenin) and isoflavones (derivatives of genistein, daidzein, formononetin and biochanin A). Multiple microshoot cultures of all species produced no simple flavones at all. In vitro shoots accumulated selectively a rich group of phytoestrogens in the form of aglucones, glucosides and esters (derivatives of genistein and daidzein). Cultures obtained in vitro synthesized many times more isoflavones than the intact plants. In all shoots which were micropropagated the dominating compound was genistin (e.g. shoots of G. tinctoria--ca 3281.4 mg per 100 g dry weight). Possible influence of tissue differentiation on isoflavone content under in vitro and in vivo conditions is discussed.     

Light and temperature conditions affect bioflavonoid accumulation in callus cultures of Cyclopia subternata Vogel (honeybush)

Callus cultures of the endemic South-African legume Cyclopia subternata were cultivated under varying light and temperature conditions to determine their influence on biomass growth and bioflavonoids accumulation. Experimental modifications of light included complete darkness, light of different spectral quality (white, red, blue and yellow) and ultraviolet C (UVC) irradiation. The calli were also subjected to elevated temperature or cold stress. Among the tested light regimes, cultivation under blue light resulted in the highest levels of hesperidin (H)—118.00 mg 100 g^?1 dry weight (DW) on 28 days of experiment, as well as isoflavones: 7-O-β-glucosides of calycosin (CG), pseudobaptigenin (PG) and formononetin (FG)—28.74, 19.26 and 10.32 mg 100 g^?1 DW, respectively, in 14-days old calli. UVC irradiation applied on 20 days stimulated the accumulation of H (204.14 mg 100 g^?1 DW), CG (31.84 mg 100 g^?1 DW) and PG (18.09 mg 100 g^?1 DW) in 28 days culture by 140, 46 and 165 %, respectively, without negatively influencing callus growth. Low temperature (13 °C) increased CG content by over 1,500 % (235.29 mg 100 g^?1 DW) when applied during the whole 28-days growth cycle, at the same time causing 95 % decrease in culture growth in comparison to reference calli maintained at 24 °C. On the contrary, elevated temperature (29 °C) applied during the second half of the culture period resulted in over 300 and 500 % increase in CG and PG content (61.76 and 58.89 mg 100 g^?1, respectively) while maintaining relatively high biomass yield.     

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale “fill-and-draw” reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.     

Improved production of dibenzocyclooctadiene lignans in the elicited microshoot cultures of Schisandra chinensis (Chinese magnolia vine)

Dibenzocyclooctadiene lignans are a specific group of secondary metabolites that occur solely in Schisandra chinensis. The aim of the presented work was to boost the accumulation of lignans in the agitated microshoot cultures of S. chinensis, using different elicitation schemes. The experiments included testing of various concentrations and supplementation times of cadmium chloride (CdCl₂), chitosan (Ch), yeast extract (YeE), methyl jasmonate (MeJa), and permeabilizing agent—dimethylsulfoxide (DMSO). After 30 days, the microshoots were harvested and evaluated for growth parameters and lignan content by LC-DAD method. The analyses showed enhanced production of lignans in the elicited S. chinensis microshoots, whereas the respective media samples contained only trace amounts of the examined compounds (< 5 mg/l). Elicitation with CdCl₂ caused up to 2-fold increase in the total lignan content (max. ca. 730 mg/100 g DW after the addition of 1000 μM CdCl₂ on the tenth day). Experiments with chitosan resulted in up to 1.35-fold increase in lignan concentration (max. ca. 500 mg/100 g DW) after the supplementation with 50 mg/l on the first day and 200 mg/l on the tenth day. High improvement of lignan production was also recorded after YeE elicitation. After the elicitation with 5000 mg/l of YeE on the first day of the growth period, and with 1000 and 3000 mg/l on the 20th day, the lignan production increased to the same degree—about 1.8-fold. The supplementation with 1000 mg/l YeE on the 20th day of the growth cycle was chosen as the optimal elicitation scheme, for the microshoot cultures maintained in Plantform temporary immersion system—the total content of the estimated lignans was equal to 831.6 mg/100 g DW.

     

Essential oil from herb and rhizome of Peucedanum ostruthium (L. Koch.) ex DC

Essential oil from herb and rhizome of Peucedanum ostruthium (L.Koch.) ex DC underwent qualitative and quantitative analyses. The content of the oil obtained by hydrodistillation was 0.95% in the herb and 1.25% in the rhizome (per dry weight basis). Gas chromatography (GC) with MS detection and flame ionisation detection showed that the oil from the rhizome contains 39 compounds, of which 29 were identified. Gas chromatography with flame ionisation detection in chiral columns against standard compounds showed the presence of enantiomers of some of the components of the oil. Compounds present in largest quantities are: sabinene (35.2%) of which (+) sabinene accounts for (96.54%) and 4-terpineol (26.6%) of which (+) 4-terpineol accounts for (65.8%). 44 components were found in the herb essential oil, of which 39 compounds were identified. Compounds present in largest quantities were beta-caryophyllene (16.1%) and alpha-humulene (15.8%). The content of sabinene in the herb oil was 4.7%. The following compounds were present in the herb oil only as enantiomers: (+) sabinene (4.7%), (-) limonene (4.4%), (-) beta-pinene (0.4%). A coumarin (osthole) was detected in both essential oils (5.5% in herb oil and 5.1% in rhizome oil).     

Bioreactor shoot cultures of <Emphasis Type="Italic">Rhododendron tomentosum</Emphasis> (<Emphasis Type="Italic">Ledum palustre</Emphasis>) for a large-scale production of bioactive volatile compounds

Rhododendron tomentosum Harmaja (Ledum palustre), a peat bog plant from Ericaceae family, has been used in traditional medicine as the anti-arthritis agent. Although modern researches confirm its anti-inflammatory properties, it remains threatened by habitat degradation and possibilities to collect this endangered species from its natural environment for further biological activity studies are limited. Therefore, R. tomentosum liquid in vitro cultures were established as the alternative source of that valuable plant material. Schenk–Hildebrandt medium with 24.60 μM 2-isopentenyladenine and 592.02 μM adenine provides intensive growth and proper morphology of the obtained microshoots. The R. tomentosum biomass was scaled up using the various bioreactors (immersion, temporary immersion and spraying systems) for better growth and improved volatile oil production. The largest biomass accumulation (fresh weight?=?250 g l^?1, growth index?=?280, dry weight?=?20 g l^?1) and essential oil content (0.5% v/m) were achieved with application of commercially available RITA^® bioreactor. GC/MS analysis revealed the high content of p-cymene (6.9%), alloaromadendrene (5.5%), shyobunone (8.2%) and ledene oxide (II) (13.0%) in the volatile fraction obtained from RITA^® system. The biomass growth parameters and production profile in terms of essential oil and selected terpenoid compounds were determined during the 2 month period. The influence of culture conditions and bioreactor construction on the growth and volatile oil production in R. tomentosum biomasses was discussed.     

Formation of UV-honey guides in Rudbeckia hirta

The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy, LC-MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols present in the petals. It was shown for the first time that the typical bull’s eye pattern is already established at the early stages of flower anthesis on the front side of the petal surface, but is hidden to pollinators until the buds are open and the petals are unfolded. The rear side of the petals remains UV-reflecting during the whole flower anthesis. Studies on the local distribution of 19 flavonols across the petals confirmed that the majority are concentrated in the basal part of the ray flower. However, in contrast to the earlier studies, eupatolitin 3-O-glucoside (6,7-dimethoxyquercetin 3-O-glucoside) was present in both the basal and apical parts of the petals, whereas eupatolin (6,7-dimethoxyquercetin 3-O-rhamnoside) was exclusively found in the apical parts. The enzymes involved in the formation of the flavonols in R. hirta were demonstrated for the first time. These include a rare flavonol 6-hydroxylase, which was identified as cytochrome P450-dependent monooxygenase and did not accept any methylated flavonol as substrate. All enzymes were present in the basal and apical parts of the petals, although some of them clearly showed higher activities in the basal part. This indicates that the local accumulation of flavonols in R. hirta is not achieved by a locally restricted presence of the enzymes involved in flavonol formation.     

Alkaloid formation in cell suspension cultures of Tabernaemontana divaricata after feeding of tryptamine and loganin

A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors was converted into indole alkaloids such as strictosidine, vallesamine, O-acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of subculturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways.     

Isoflavone production in Cyclopia subternata Vogel (honeybush) suspension cultures grown in shake flasks and stirred-tank bioreactor

Suspension cultures of the endemic South-African plant Cyclopia subternata were established for the first time and evaluated for the presence of isoflavones. The influence of light, as well as medium supplementation strategies with phenylalanine, casein hydrolysate and coconut water on biomass growth and isoflavone production were examined. The highest levels of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (275.57, 125.37 and 147.28 mg/100 g DW, respectively) were recorded for cultures grown in the absence of light, whereas coconut water substantially promoted biomass growth. Cell suspensions were subsequently grown in the 2-l stirred-tank bioreactor. Maximum productivity of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (0.96, 0.44 and 0.22 mg l^?1 day^?1, respectively) in bioreactor-cultivated cells was obtained for biomass grown in the dark and supplemented with coconut water. The results indicate that C. subternata suspension cultures can be utilised for the production of the specified isoflavone derivatives absent in the intact plant.