Accumulation of Cd2+ and Pb2+ in the suspension cultures of Agave amaniensis and Costus speciosus and the determination of the culture's growth and phytosteroid content

Cell suspension cultures of Agave amaniensis and Costus speciosus were grown in media containing Cd^{2 +} up to 25 and 20 mg l^–1, respectively, and Pb²⁺ up to 40 mg l^–1. The cultures hyper-accumulated Cd²⁺ up to 900 and 530 g g^–1 and Pb²⁺ up to 1390 and 1170 g g^–1 dry wt. in their respective biomasses. Increasing Pb²⁺ up to 30 mg l^–1 increased the biomass production and total sitosterol content of Costus speciosus by up to 1.7- and 1.3-fold, respectively.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

Screening yeast cells for absorption of selenium oxyanions

Certain yeast cells on solid nutrient medium produced colonies surrounded by a light zone of selenite absorption. This screening procedure resulted in the selection of 22 strains out of 200 isolates with different Se⁴⁺-absorbing capacity ranging from 16 to 98.8 g Se⁴⁺ g^–1 l^–1 h^–1. The highest rate of Se⁴⁺ elimination from the Na₂SeO₃ solution was observed with an oval shaped, cream pigmented fermentative yeast, tentatively called Candida sp. strain MS4. This strain was isolated from wastewater and found to accumulate selenium oxyanions. Se⁴⁺ uptake involved both inactive and active phenomena. The amounts of selenium (initial concentration 2 mg Se⁴⁺ l^–1) removed from aqueous solution by inactive and active phenomena were 667 g Se⁴⁺ g^–1 l^–1, and 1580 g Se⁴⁺ g^–1 l^–1, respectively. The strain also removed selenate inactively (135 g Se⁶⁺ g^–1 l^–1).     

Calli and suspension cultures for biomass production ofEuphorbia characias L. subsp.characias

Summary Friable calli and cell suspension cultures were obtained from leaf segments ofEuphorbia characias L. subsp.characias, in Murashige and Skoog (MS) basal medium supplemented with 1g.l^–1 casein hydrolyzate (CH), 5mg.l^–1 ascorbic acid, 1.0mg.l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.75mg.l^–1 benzyl adenine (BA). The highest callus specific growth rate (=0.085.day^–1), calculated for 1 year old calli cultures, was obtained with 0.25 mg.l^–1 2,4-D and 0.50mg.l^–1 BA. Suspension cultures started with an inoculum of 8.0×10⁴ cells.ml^–1 in supplemented liquid MS medium, gave a specific growth rate =0.256.day^–1.     

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.     

Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures

Lower concentrations of CuSO₄ (25–75 M) in the MS medium supplemented with 0.1 mg l^–1 IAA+5.0 mg l^–1 Kn+500 mg l^–1 CH+10 mg l^–1 Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO₄ (75 M) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO₄ (100 M), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.     

Sequential kinetic parameter estimation of anaerobic fluidized bed bioreactor using an optimization technique

Mathematical model parameters for the methanogenic degradation of propylene glycol were estimated in a sequential manner by means of an optimization technique. Model parameters determined from an initial experimental data set using one bioreactor were then verified with the results from a second bioreactor. The proposed methodology is a useful tool to obtain model parameters for continuous flow reactors with completely mixed regime. Abbrevations: S – substrate concentration (mg COD l^–1); S _in – influent substrate concentration (mg COD l^–1); D _L – dilution rate (day^–1); – stoichiometric coefficients (ND); nx – number of microbial species (ND); X _S – fixed biomass concentration (mg biomass l^–1); X _L – suspended biomass concentration of (mg biomass l^–1); k _d – decay rate of biomass (day^–1); b _S – specific detachment rate of biofilm (day^–1); – specific growth rate of biomass (day^–1); _m – maximum specific growth rate of biomass (day^–1); K _S – half saturation constant (mg COD l^–1); K _I – inhibition constant (mg COD l^–1).     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10⁶ g fwt^–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10⁴ ml^–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10⁴ ml^–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl^–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l^–1 sucrose, NAA (0.2–0.5 mg l^–1), zeatin riboside (0.5–2.0 mg l^–1) and GA₃ (0.5–1.0 mg l^–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l^–1 agar-solidified B5 medium containing 30g l^–1 sucrose, IBA (0.01 mg l^–1) and BAP (1.0 mg l^–1). Elongated shoots developed roots after transfer to 8.0g l^–1 agar-solidified, hormone-free MS medium with 30 g l^–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA³ gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathway

Excised root cultures of Gloriosa superba reached 7.5 g dry wt l^–1 and accumulated 240±40 g colchicine g^–1 cell dry wt after 4 weeks growth. While all precursors (except trans-cinnamic acid) enhanced colchicine content of root cultures without adversely affecting root growth, treatment with p-coumaric acid + tyramine (each at 20 mg l^–1) increased colchicine content to 1.9 mg g^–1 cell dry wt.     

Simple strategy for the in vitro conservation of Ipsea malabarica an endemic and endangered orchid of the Western Ghats of Kerala,India

A simple protocol was developed for plantlet regeneration from seedling leaf segments of pigeonpea cultivar ICPL 93115 through shoot organogenesis. Ten-day-old seedlings aseptically grown on MS medium were used for furnishing leaf segments. Initial incubation for 5 days in dark at 25±2 °C followed by transfer to 10/14-h light / dark cycle (12.1 mol photons m^–2 s^–1) favoured regeneration. The decisive role of 6-benzyl adenine at different concentrations was established on shoot organogenesis. Murashige and Skoog's (MS) (1962) medium fortified with BA of 5.0 mg l^–1 was optimum for shoot regeneration and MS + BA of 1.0 mg l^–1 for shoot elongation, while MS + IAA (1.0 mg l^–1) + kinetin (0.1 mg l^–1) showed good results for rooting.     

Enhanced biotransformation of terpenes in plant cell suspensions using controlled release polymer

Plant cell cultures of Peganum harmala converted geranyl acetate to geraniol. Although the reaction started immediately after feeding, there was disappearance of both product and substrate. Geranyl acetate at 100 mg l^–1 when fed to 100 ml Peganum harmala suspensions (16% packed cell volume) was completely used within 24 h without accumulation of any product. Similarly, linalyl acetate and its biotransformation products, linalool and -terpineol, disappeared. Controlled-release polymer discs made from poly-2-hydroxyethyl methacrylate and containing concentrations of geranyl acetate or linalyl acetate produced greatly extended concentrations of these substrates and their biotransformation products (from about 1 day to over 12 days). The concentrations of substrates remained at around 5 mg l^–1throughout the experiments, while the concentrations of biotransformation products increased from 10 mg l^–1to 55.5 mg l^–1 for geraniol, from 5 mg l^–1 to 14 mg l^–1 for linalool, and 5 mg l^–1 to 12 mg l^–1 for -terpineol compared to the control value. Also low concentrations (30–200 g/disc) of product were taken up by the polymer over 10 days.     

Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12

A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of the strain was enhanced by the addition of other organic materials such as YE, peptone, glucose, and sucrose. YE was a particularly effective additive in stimulating cell growth as well as PAH degradation. When 1 g YE l^–1, an optimum concentration, was supplemented into the basal salt medium (BSM) with 215 mg Phe l^–1, the specific growth rate (0.30 h^–1) and Phe-degrading rate (29.6 mol l^–1 h^–1) were enhanced approximately ten and three times more than those obtained in the BSM with 215 mg Phe l^–1, respectively. Both cell growth and PAH degradation rates were increased with increasing Phe and Pyr concentrations, and B. cepacia 2A-12 had a tolerance against Phe and Pyr toxicity at the high concentration of 730–760 mg l^–1. Through kinetic analysis, the maximum specific growth rate ( _max) and PAH degrading rate ( _max) for Phe were obtained as 0.39 h^–1 and 300 mol l^–1 h^–1, respectively. Also, _max and _max for Pyr were 0.27 h^–1 and 52 mol l^–1 h^–1, respectively. B. cepacia 2A-12 could simultaneously degrade crude oil as well as PAHs, indicating that this bacterium is very useful for the removal of oils and PAHs contaminants.     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

Ca2+ and Cu2+ supplementation augments vancomycin production by Amycolatopsis orientalis

Supplementation with CaCl₂2H₂O (40 mg l^–1) or CuSO₄5H₂O (10 mg l^–1) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca²⁺ decreased the intracellular concentration of vancomycin 36%, whereas Cu²⁺ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca²⁺ probably works by altering the permeability of cells to vancomycin, whereas Cu²⁺ increases the activity of an enzyme responsible for vancomycin biosynthesis.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Contractile responses in rat extensor digitorum longus muscles at different times of postnatal development

Some contractile properties of small bundles (100–200 m diameter) of muscle fibres isolated from the extensor digitorum longus muscle of rats at different times of development were compared. An increase of resting potential was observed in these muscles from-26.9 mV at 1 day of age to-72.6 mV at 3 months. Twitch tension and duration of postnatal muscles 1–7 days were diminished by reducing [Ca]_o (substituted by Mg²⁺) or adding inorganic cations (Ni²⁺, Cd²⁺, La³⁺), unlike in the oldest animals (14 days–3 months postnatal) where twitch responses were unaffected. In the latter, potentiation of the twitch tension was even recorded in the presence of Ni²⁺ (0.5–1 mmol·l^-1) and Cd²⁺ (0.5–2 mmol·l^-1). Properties of activation and inactivation of the developed tension following elevation of [K]_o to 15–200 mmol·l^-1 were analysed at the same stages of postnatal development. In contrast to the tension-membrane potential curves for activation, which presented an average negative shift of-17.6 mV between 1 day postnatal and 3 months of age, a voltage dependence of inactivation similar to that encountered in adult extensor digitorum longus muscles, was already reached at 7 days of age. These results suggest an asynchronism in the maturation of the potential-dependent characteristics of the depolarization-contraction coupling mechanism. Furthermore, during the first week postnatal, in relation with poorly developed membrane systems and low [Ca]_i-recycling capability, [Ca]_o plays a fundamental role in maintaining contraction by replenishing the intracellular calcium pool.Abbreviations ATPase adenosine triphosphatase - [Ca]_o ([K]_o) extracellular calcium (potassium) concentration - DC depolarization-contraction - EC excitation-contraction - e.d.l. muscle extensor digitorum longus muscle - E _m membrane potential - E _r resting potential - HEPES N-2 hydroxyethylpiperazine-N-2 ethanesulphonic acid - I _fast fast calcium current - sr sarcoplasmic reticulum - T-tubules transverse tubules