Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO₂) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO₂ supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L⁻¹ d⁻¹). The highest values of biomass concentration (1.98 g L⁻¹), biomass productivity (131.8 mg L⁻¹ d⁻¹), carbon in biomass (47.9% w w⁻¹), CO₂ fixation rate (231.6 mg L⁻¹ d⁻¹), and CO₂ use efficiency (80.5% w w⁻¹) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO₂ supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w⁻¹). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.     

Morphogenesis in callus tissue cultures of someMatricaria andAchillea Species

In the present paper we deal with the possibility of morphogenesis induction in callus tissue cultures of some representatives ofMatricaria andAchillea species. Shoot regeneration from calli ofMatricaria chamomilla andM. inodora has been induced by 0.1 mg l^-1 kinetin or by combination of 0.5 mg l^-1 kinetin and 0.5 mg l^-1 NAA added to Murashige-Skoog culture medium. Rhizogenesis took place without any other addition of auxin. In callus tissue cultures ofAchillea ptarmica cultivated on Murashige-Skoog medium with 1 mg l^-1 2,4-D after a year long cultivation the whole plant has been regenerated without any change of nutrient requirements. In callus tissue ofA. nobilis under the same conditions only roots wore regenerated.     

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l⁻¹ of NaH₂PO₄), nitrate (2527 mg l⁻¹ of KNO₃), carbon source (10 g l⁻¹ of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (t_D equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

The study of morphological and histological changes in tissue cultures ofmatricaria inodora L

This paper deals with some morphological and histological changes observed during regular intervals inMatricaria inodora L. tissue cultures derived from leaf expiants. The expiants were cultivated on Murashige-Skoog’s culture medium supplemented with 1.0 mg 1⁻¹ 2,4-dichlorophenoxyacetic acid. The callus formation started about a week after isolation. During the second week the meristematic centers were differentiated from which root and shoot apices were later formed. During long term cultivation under the same culture conditions the inhibition of development of shoot apices took place. Only roots of unorganized growth have been regenerated. The influence of culture conditions on morphogenetic potential is discussed.     

The effect of ionizing irradiation on the tissue culture ofCoronilla varia

Long-term callus cultures of crownvetch (Coronilla varia L.) grown on the Murashige and Skoog’s medium with 2,4-D (1 mg 1^-1) and cultures of somatic embryos cultivated on the same basic medium but with IAA (1.0 mg I^-1) were exposed to ionizing irradiation. The irradiation caused a growth inhibition excepting the lowest dose of 2.5 Gy. The highest dose of 160 Gy induced browning of the culture but this colour change was not lethal. The amount of “giant cells” present in both cultures was dependent on the dose of irradiation.     

Shoot Differentiation in Callus Cultures of Datura innoxia

Datura innoxia Mill. callus cultures formed shoots in 2–4 weeks on media containing; a) gibberellic acid, b) indoleacetic acid, c) low concentrations of naphthylacetic acid, d) low concentrations of 2,4-dichlorophenoxyacetic acid, e) benzylaminopurine, f) no growth substance. Benzylaminopurine promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, pH 5.5, no glycine) in light at 30°C. Callus grew well on any single one of the growth substances NAA (10^?5M), 2,4-D (10^?6M) or BAP (3 × 10^?6M). Growth was less and more erratic on GA or IAA. The callus cultures did not grow significantly better when BAP was combined with one of the auxins or with GA.     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.     

Somatic embryogenesis and plant regeneration inCoronilla varia L. (crownvetch) long-term tissue cultures

Spontaneous recovery of regeneration abilities was observed in a long-term (about two-year-old) crownvetch (Coronilla varia L.) tissue culture permanently grown on MS medium containing 1 mg 1^-1 IAA. Somatic embryos and later complete plants differentiated from initially regenerating roots. The formation and development of embryos was accompanied by a 10- to 20-fold increase in the content of cardioactive glycosides hyrcanoside and deglucohyrcanoside in the culture biomass. The effect of auxins (IAA, NAA, 2,4-D) and cytokinins (6-BAP) on calogenesis and somatic embryogenesis was examined; further development of somatic embryos was enhanced by light. Primary explants which were newly isolated from sterile R₁ plantlets showed differential, organ-specific ability of somatic embryogenesis which was highest in root cuttings. Regenerated plants were transferred to field culture; two-year-old cultures of regenerated plants showed in most cases phenotypic deviations from the original material, especially dwarfism.     

Hormonal stimulation of tyrosinase activity in human foreskin organ cultures

Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining. Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle⁴, D-phe⁷]-alpha MSH (10⁻⁸ M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E₁ produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D₃ (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation in human skin. This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST) and by a research grant from the Presbyterian Health Foundation.     

Improved alkaloid production in Catharanthus roseus suspension cell cultures by various chemicals

Fourteen chemicals were used to treat Catharanthus roseussuspension cell cultures to improve ajmalicine, catharanthine or serpentine biosynthesis. Ajmalicine production was increased by betaine (to 55 mg l^–1), n-propyl gallate (to 27 mg l^–1), succinic acid (to 31 mg l^–1), malic acid (to 60 mg l^–1) and tetramethyl ammonium bromide (to 64 mg l^–1). Ajmalicine and catharanthine yields were about 5–6 fold higher than the control. A large portion (up to 50–85%) of total indole alkaloids was released into the medium. For maximal catharanthine production, the optimal doses of malic acid and tetramethyl ammonium bromide were 50 mg l^–1and 120 mg l^–1, respectively. The mechanisms which may be responsible for these treatment effects are discussed.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

In vitro propagation of Centaurea spachii from inflorescence stems

Procedures for micropropagation of Centaureaspachii (Compositae), an endangered rosulate plantendemic from the mediterranean Spain area, have beendeveloped using inflorescence nodal segments asexplants for in vitro establishment. Only 15%of explants remained contaminated using this materialto start the in vitro axenic cultures. Higherproliferation of shoots and multiplication coefficientwas obtained on Murashige and Skoog (MS) mineralmedium supplemented with 1.0 mg l^{minus 1}6-benzyladenine. However, shoot elongation decreasedwith the addition of this cytokinin.Rooting of shoots with only one auxin was very lowafter 6 weeks on the majority of rooting media tested.The best rooting result (60%) was obtained on MSmedium with a combination of 2 mg l^{minus 1}indole-3-acetic acid plus 2 mgl^{minus 1} indole-3-butyric acid. Moreover, in thisculture medium 50% of shoots rooted during the thirdweek of culture. High survival, over 80%, wasobtained when the plantlets were transferred togreenhouse conditions. The endangered Centaureaspachii can be successfully micropropagatedbeginning with a single inflorescence stem and withoutsignificant damage to the mother plant.     

The effect of light on growth and sporulation of certain fungi

Summary The response of the fungi investigated to duration of exposure to light varies with the variation in the light intensity employed. Using relatively low intensity light (160 and/or 300 foot candles/inch²), the growth ofMyrothecium verrucaria &Pestalotia gracilis was not affected by the increase in the time of light exposure, while that ofPleurotus ostreatus was checked. Fruiting under the same conditions was hastened on exposure to light. Under higher light intensity (950 foot candles/inch²), growth ofMyrothecium was not affected, while that ofPestalotia andPleurotus decreased as the daily period of exposure to light increased. Pleurotus cultures exposed continuously to light showed practically no growth, and combined addition of malt and yeast extracts had a noticeable growth promoting effect on cultures exposed continuously to light, but not significantly on those kept in the dark. This was explained by assuming the presence in malt and yeast extracts of light sensitive growth promoting substances. The effect of light on growth ofPleurotus was found to be concerned with both cell mechanism and medium: light probably inhibits inside the cell the synthesis of one or more substances essential for growth and at the same time it favors the breakdown in the medium of one or more substances required for growth.     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative Toxicity of Three Organic Acids to Freshwater Organisms and Their Impact on Aquatic Ecosystems

The comparative toxicity of lactic acid, acetic acid, and benzoic acid to tilapia (Oreochromis mossambicus), cladoceran crustacea (Moina micrura), and oligochaete worm (Branchiura sowerbyi) were determined using static bioassay tests. Worms were found most sensitive to all the acids whereas the cladoceran was found most resistant to lactic acid and the fish most resistant to acetic acid and benzoic acid. The 96h LC₅₀ values of lactic acid, acetic acid, and benzoic acid, were, respectively, 257.73, 272.87, and 276.74 mg L^?1 for O. mossambicus; 329.12, 163.72, and 71.65 mg L^?1 for M. micrura and 50.82, 14.90, and 39.47 mg L^?1 for B. sowerbyi. Tilapia lost appetite at sub-lethal concentrations as low as 2.18 mg L^?1 lactic acid, 1.26 mg L^?1 acetic acid, and 13.84 mg L^{? 1} of benzoic acid. Growth and reproduction of the fish were affected following 90-day chronic exposure to sub-lethal concentrations of the acids. Minimum effective concentration of the acids that significantly reduced food conversion efficiency (FCE), percent increase of weight, specific growth rate, yield and fecundity of the fish were 2.18, 1.47, and 3.95 mg · L^?1 of lactic acid, acetic acid, and benzoic acid, respectively. Effects of acetic acid and benzoic acid on FCE, weight increase, and yield were not significantly different from each other whereas lactic acid produced different effects from acetic acid as well as benzoic acid. Mean values of dissolved oxygen, primary productivity, and plankton populations of the test medium significantly reduced from control at 16.94 mg L^?1 lactic acid, 16.79 mg L^?1 acetic acid, and 13.84 mg L^?1 benzoic acid.     

In vitro culture and micropropagation of the Baetic-Moroccan endemic plant Lapiedra martinezii Lag. (Amaryllidaceae)

Lapiedra martinezii Lag. is a potential medicinal and ornamental plant facing conservation challenges. Thus, this study was focused on determining the conditions for culture initiation and propagation using in vitro techniques. The optimal sterilization procedure combined thermotherapy at 54°C for 60 min and immersion in 7% (w/v) Ca(ClO)₂ solution for 20 min. The most suitable medium to initiate bulb scales cultures was Gamborg B5 medium containing 500 mg L⁻¹ casein, 2 mg L⁻¹ adenine, 10 mg L⁻¹ glutathione and 10 g L⁻¹ sucrose. The most productive multiplication medium tested was Murashige and Skoog medium containing 30 g L⁻¹ sucrose, 4.0 mg L⁻¹ 6-benzylaminopurine, and 0.12 mg L⁻¹ 1-naphtaleneacetic acid. Most plants developed in vitro rooted spontaneously in the multiplication phase. The vast majority of the plants (89%) were successfully transferred to ex vitro conditions, and 100% survived over 1 yr of cultivation outdoors. Sucrose at a concentration of 60 g L⁻¹ was the most effective treatment to increase the biomass of bulblets. High auxin/cytokinin ratios produced the highest callus induction efficiency. The vast majority of callus developed in dark conditions, but none regenerated in the combinations of growth regulators previously tested. The plants obtained by micropropagation did not show significant differences in morphometric traits compared with the wild specimens, which supported the stability of the materials produced in vitro. This is the first report on cell cultures and micropropagation of L. martinezii, and the results can be applied to other Amaryllidaceae for industrial or conservation purposes.