Biomass production of Cymbopogon citratus (D.C.) stapf., a medicinal plant, in temporary immersion systems

Summary In vitro plants of lemon grass were established, starting from shoot apices derived from plants cultivated under field conditions. The effect of the immersion frequency (two, four, and six immersions per day) on the production of biomass in temporary immersion systems (TIS) of 1 liter capacity was studied. The highest multiplication coefficient (12.3) was obtained when six immersions per day were used. The maximum values of fresh weight (FW; 62.2 and 66.2 g) were obtained with a frequency of four and six immersions per day, respectively. However, the values for dry weight (DW; 6.4g) and height (8.97cm) were greater in the treatment with four immersions per day. The TIS used in this work for the production of lemon grass biomass may offer the possibility of manipulating the culture parameters, which can influence the production of biomass and the accumulation of secondary metabolites. We describe for the first time the in vitro production of Cymbopogon citratus biomass in TIS.     

Increased cardenolides production by elicitation of Digitalis lanata shoots cultured in temporary immersion systems

Digitalis lanata is an important source of cardenolides such as digoxin and lanatoside C, which have been widely applied in the treatment of cardiac insufficiencies. Elicitation is one of the most effective methods to enhance the biosynthesis of several secondary metabolites in medicinal plants. We studied the effect of elicitation with Chitoplant^?, Silioplant^? and methyl jasmonate on biomass and cardenolides accumulation in shoots of D. lanata cultivated in temporary immersion systems. Morphological response of the shoots was influenced by elicitors. A reduction in length and number of shoots was evident with all MJ concentrations. Regarding biomass production, Chitoplant^? (0.1?g?l^?1) was found to impact significantly on fresh and dry weight of the shoots. HPLC analysis revealed a higher content of lanatoside C compared to digoxin in all treatments. The highest accumulation of lanatoside C was achieved with Chitoplant^? (0.1?g?l^?1), which resulted in 316???g?g-DW^?1 and with Silioplant^? (0.01?g?l^?1; 310???g?g-DW^?1), which accounted for a 2.2-fold increase in lanatoside C content compared to non-elicited shoot cultures. Additionally, elicitation of D. lanata shoots in temporary immersion systems resulted in an oxidative stress characterized by hydrogen peroxide and malondialdehyde accumulation. These observations point to a connection between hydrogen peroxide generation, lipid peroxidation and cardenolide accumulation. The optimization of elicitor treatment and culture conditions for cardenolide production as well as the advantages of TIS for this purpose are discussed.     

Regeneration and cardiotonic glycoside production in <Emphasis Type="Italic">Digitalis davisiana</Emphasis> Heywood (Alanya Foxglove)

A high-frequency in vitro regeneration of Digitalis davisiana Heywood (Alanya foxglove) and cardiotonic glycoside production from both in vitro produced materials (regenerated plantlets or germinated seedlings) and leaves of natural populations were obtained. Cardiac glycosides regulate heart rhythm and are effective in cancer chemotherapy, in particular for prostate and breast cancer treatments. Testing six different types of culture media revealed that Linsmaier and Skoog (LS) was the most effective for shoot production. Shoot regeneration efficiency was higher when flamingo-bill or hypocotyl explants were cultured on LS medium containing 0.5 mg/l thidiazuron (TDZ) and 0.25 mg/l indoleacetic acid (IAA). Rooting of all shoots that regenerated was readily achieved, even in the absence of plant growth regulators (PGRs). Production of cardenolides (lanatoside C and digoxin) in the materials grown in vitro seemed to correlate with several parameters, such as nutritional and hormonal compositions of the culture medium as well as the duration of culture on the initial regeneration and/or final growth medium. Higher amounts of digoxin accumulation were obtained when shoots were regenerated on LS or Gamborg’s B5 medium containing 0.5 mg/l TDZ and 0.25 mg/l IAA, producing 12.59 and 11.93 mg/kg dry weight (dw) digoxin, respectively. For natural populations, seasonal variations seemed to affect the production of digoxin in the leaves. The highest amount of digoxin (246.58 mg/kg dw) was in leaf samples collected in July, which coincides with the flowering stage of the plant in the region of collection.     

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine—BA; kinetin—Kin; and thidiazuron—TDZ) and auxins (indole-3-acetic acid—IAA; α-naphthaleneacetic acid—NAA; and 2,4-dichlorophenoxy acetic acid—2,4-D). Maximum multiple shoots (12.7?±?0.6) were produced from nodal explants on MS?+?7.5 μM BA. Shoots were rooted in vitro on MS containing 15 μM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 μM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.     

Improved growth and quality of <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Siraitia grosvenorii plantlets were investigated. The TIS promoted the growth and quality of S. grosvenorii plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤ 0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also decreased callus formation at the base of shoots. Callus diameter was significantly (P ≤ 0.05) lower in the TIS (3.30 mm) than in gelled medium (6.31 mm) and liquid medium (6.77 mm), respectively. FW (50.83 mg) and DW (7.08 mg) of callus in the TIS were also significantly (P ≤ 0.05) lower than those in gelled medium (80.00 and 10.56 mg, respectively) and liquid medium (218.75 and 23.75 mg, respectively). During rhizogenesis, minimal callus was evident at the base of shoots in the TIS, with a well-developed root system. However, the plantlets in gelled medium just produced thick, brown and easily broken roots with obvious callus and fewer secondary roots. The natural-like plantlets of S. grosvenorii obtained in the TIS would probably have positive effects on ex vitro rooting and transplanting in large-scale commercial production.     

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.     

Cycle characteristics in a temporary immersion bioreactor affect regeneration,morphology, water and mineral status of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) somatic embryos

Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1 min immersions every 24, 12 and 4 h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15 min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15 min immersion duration applied every 4 h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K⁺ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1 min immersions every 4 h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).     

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H₂O₂ level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

     

Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system

Here, we evaluated the efficiency of shoot multiplication of Vanilla planifolia Jacks. ex Andrews using solid medium, partial immersion, and a temporary immersion system (TIS) to improve micropropagation in this species. Clusters of shoots were cultivated in vitro using Murashige and Skoog (MS) medium supplemented with 9.55 μM benzyladenine (BA) and 100 mL L^?1 coconut water. For the TIS, a RITA^® system was used and three immersion frequencies were evaluated (every 4, 8, and 12 h) with an immersion time of 2 min. After 30-d culture, the TIS produced the maximum multiplication rate (14.27 shoots per explant) when using an immersion frequency of 2 min every 4 h, followed by the partial immersion system (8.64 shoots per explant), and solid medium (5.80 shoots per explant). Next, the effect of the volume of culture medium per explant was also evaluated for TIS. The most suitable volume of culture medium for shoot formation was 25 mL per explant, which increased the rate of multiplication to 17.54 shoots per explant. Root initiation was 90% successful in TIS using half-strength MS medium supplemented with 0.44 μM naphthaleneacetic acid (NAA) and an immersion frequency of 2 min every 4 h. With this system, the shoot multiplication rate increased threefold compared to that obtained with solid medium. In addition, this system produced good results for the transplantation and acclimation (90% of survival) of in vitro-derived plants. These results offer new options for large-scale micropropagation of vanilla.     

High-performance liquid chromatography–ionspray mass spectrometry for the specific determination of digoxin and some related cardiac glycosides in human plasma

An original method based upon high-performance liquid chromatography coupled to ionspray mass spectrometry (HPLC–ISP-MS) has been developed for the identification and quantification in plasma of several cardiac glycosides, namely digoxin, digitoxin, lanatoside C and acetyldigitoxin. After single-step liquid–liquid extraction by chloroform–2-propanol (95:5, v/v) at pH 9.5 using oleandrin as an internal standard, solutes are separated on a 4 μm NovaPak C₁₈ (Waters) column (150×2.0 mm, I.D.), using a gradient of acetonitrile–2 mM NH₄COOH, pH 3 buffer (flow-rate 200 μl/min, post-column split 1:3). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with an ISP interface. In most instances the major ion observed is not [M+H]⁺ as expected, but [M+NH₄]⁺. The mean retention times (min) are: lanatoside C, 5.74; digoxin, 6.00; digitoxin, 8.08, oleandrin, 8.30, acetyldigitoxin, 8.66 and 9.01 (isomers α and β, respectively). The lower limits of detection in single ion monitoring mode range from 0.15 ng/ml (α- and β-acetyldigitoxin) to 0.60 ng/ml (lanatoside C), making the method less sensitive than radioimmunoassay, whereas it is much more specific.     

High-yielding repetitive somatic embryogenesis and plant recovery in a selected tea clone, ‘TRI-2025’, by temporary immersion

 Methods for improving the efficiency of repetitive somatic embryogenesis and plant recovery from somatic embryos of clonal tea, TRI 2025 [Camellia sinensis (L.) O. Kuntze] were investigated by optimising the immersion frequencies of the explants using a modified temporary immersion system (TIS). The relative efficiencies of three conventional methods for multiplying embryos were compared with the temporary immersion method. The highest rate of multiplication of secondary embryos (24-fold) was achieved using the TIS. By controlling the immersion cycles, we achieved more consistent, synchronised multiplication and embryo development with a high level of plant recovery. A one-step computer-programmed immersion protocol based on a single, simple medium with no growth regulators was developed, enabling multiplication, maturation, germination and plant recovery within 17 weeks. Plantlets recovered through this method were hardy, with 2- to 5-cm-long shoots containing a minimum of 2–4 lush green leaves and a well-formed taproot. Callus formation, hyperhydricity and other developmental abnormalities were not observed at any stage in the process. Plantlets produced using this method were successfully acclimatised to glasshouse conditions. This protocol avoids culture transfers, and thus minimises the risk of contamination and reduces labour costs. This technique could have significant commercial implications in tea propagation as it has the potential for large-scale production with considerably reduced production costs. Received: 28 June 1999 / Accepted: 20 April 2000     

Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; α-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis)

The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.     

Increased production of digoxin by digitoxin biotransformation using cyclodextrin polymer inDigitalis lanata cell cultures

Addition of β-cyclodextrin (β-CD) polymer during the biotransformation of digitoxin into digoxin using cell suspension cultures ofDigitalis lanata enhanced the conversion yield. Digitoxin showed better adsorption to CD polymer compared to digoxin, so that the optimization of addition time was found to be necessary. In the case of adding CD polymer 24 hours after the feeding of substrate digitoxin, the highest digoxin production could be achieved. At this period, digitoxin was almost consumed by cells and productivity was proportionally enhanced according as the amount of substrate was increased. Immobilization of CD polymer did not promote the biotransformation. When 3.33 g/L of CD polymer was added, 90% and 50% of digitoxin and digoxin was adsorbed respectively. Thus selective inclusion complex formation could be expected. Adsorption rate was found to be rapid and saturation was obtained within 10 hours of contact.     

Optimization of plantain (<Emphasis Type="Italic">Musa</Emphasis> AAB) micropropagation by temporary immersion system

The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 M was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.     

Bioreactor type affects the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis (Turcz.) Baill.

Microshoots of the East Asian medicinal plant species Schisandra chinensis (Chinese magnolia vine) were grown in bioreactors characterized by different construction and cultivation mode. The tested systems included two continuous immersion systems—a cone-type bioreactor (CNB) and a cylindric tube bioreactor (CTB), a nutrient sprinkle bioreactor (NSB), and two temporary immersion systems (TIS)—RITA® and Plantform. Microshoots were grown for 30 and 60 days in the MS medium enriched with 1 mg l^?1 NAA and 3 mg l^?1 BA. The accumulation of two groups of phenolic compounds: phenolic acids and flavonoids in the bioreactor-grown S. chinensis biomass, was evaluated for the first time. In the microshoot extracts, seven phenolic acids: chlorogenic, gallic, p–hydroxybenzoic, protocatechuic, syringic, salicylic and vanillic, and three flavonoids: kaempferol, quercitrin and rutoside, were identified. The highest total amount of phenolic acids (46.68 mg 100 g^?1 DW) was recorded in the biomass maintained in the CNB for 30 days. The highest total content of flavonoids (29.02 mg 100 g^?1 DW) was found in the microshoots maintained in the NSB for 30 days. The predominant metabolites in all the tested systems were: gallic acid (up to 10.01 mg 100 g^?1 DW), protocatechuic acid (maximal concentration 16.30 mg 100 g^?1 DW), and quercitrin (highest content 21.00 mg 100 g^?1 DW).

     

Functional antagonism by a monoclonal antibody to digoxin in a test system of cultured rat heart myocytes

A monoclonal antibody with a high affinity for digitoxin (K_A = 0.50 nM) and digoxin (K_A = 0.55 nM) was produced by somatic cell fusion. This antibody, designated 2A3(47), displayed little cross reactivity with other glycosides. In cultured rat heart myocytes, 2A3(47), antagonized the positive chronotropic effect exerted by digitoxin but did not alter that of ouabain. Our results suggest that this monoclonal antibody may prove to be useful in treating digoxin and digitoxin intoxication.