Effects of sucrose and plant growth regulators on acetylcholinesterase inhibitory activity of alkaloids accumulated in shoot cultures of Amaryllidaceae

The influence of sucrose (30, 60, 90 and 120 g/L), activated charcoal (5 and 10 g/L), and various levels of several plant growth regulators (6-benzyladenine, naphthalene-1-acetic acid, 2,4-dichlorophenoxyacetic acid, and picloram) on organogenesis (bulb and root development) and the accumulation of alkaloid and galanthamine in shoot cultures of three Amaryllidaceae species (Narcissus pseudonarcissus, Galanthus elwesii, and Leucojum aestivum) was investigated in a full-factorial experiment. Alkaloid extracts were analyzed by gas chromatography–mass spectrometry, leading to the quantification of galanthamine and to the identification of other alkaloids. The different extracts were then subjected to an Ellman test to evaluate the inhibitory activity of acetylcholinesterase. The highest contents of galanthamine [0.02–0.1% dry weight (DW) depending on the plant species] were always accompanied with a high level of acetylcholinesterase inhibition (>30%). However, some samples containing low amounts of galanthamine (0.005% DW) showed high inhibitory activities (40–80%). These findings demonstrate the presence of Amaryllidaceae alkaloids that have not yet been identified as having anti-acetylcholinesterase activity.     

Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures

A callus strain with stable ability for vindoline synthesis was selected from many prepared Catharanthus roseus leaf calli to study the regulation of vindoline biosynthesis as well as other indole alkaloids. It was shown that light and plant growth regulators significantly influenced the biosynthesis of vindoline and other alkaloids as well as acidic and basic peroxidase activities. Light promoted vindoline and serpentine biosynthesis, and stimulated plastid development and peroxidase activity. However, 2,4-D suppressed the biosynthesis of all indole alkaloids and peroxidase activity. Our results suggest that light or plant hormones regulate vindoline, serpentine and other alkaloid biosynthesis and accumulation by influencing peroxidase activity and the differentiation status of callus cultures, especially chloroplast development. Some possible relationships between serpentine or vindoline biosynthesis and peroxidase activity are proposed.     

Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures.

The effect of different concentrations of growth regulators and sucrose on anthocyanin production in cell suspension cultures of Camptotheca acuminata Decaisne (Nyssaceae) was described for the first time and qualitatively and quantitatively evaluated. Anthocyanin production was significantly greater in the presence of kinetin, compared to benzyladenine, with the greatest concentration observed in the presence of 2 microM kinetin. No significant differences in anthocyanin production were observed when comparing 2,4-dichlorophenoxyacetic acid to alpha-naphthaleneacetic acid, except when using 2 microM, 2,4-dichlorophenoxyacetic acid, which resulted in greater anthocyanin production. High sucrose concentration enhanced the production of anthocyanins. Based on the absence of anthocyanin production in the dark, we concluded that light was essential for stimulating anthocyanin production. The optimised medium consisted of: 2 microM kinetin, 2 microM 2,4-dichlorophenoxyacetic acid and 292 mM sucrose. HPLC/DAD and HPLC/MS analyses revealed that the main anthocyanin was Cy 3-O-galactoside and that the minor derivative was Cy 3-O-glucoside.     

The production of cytotoxic lignans by plant cell cultures

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.     

Effects of plant growth regulators on three wood-rotting polypores

The results of the effects of plant growth regulators, kinetin, gibberellic acid, 3-indoleacetic acid and ethrel (2-chloroethylphosphonic acid) on three wood-rotting polypores,viz. Polyporus palustris, Daedalea flavida andTrarnetes badia, are given. 3-Indoleacetic acid, kinetin and gibberellic acid increased the dry mass and protein, and decreased free amino acids, while ethrel decreased dry mass and protein, and increased free amino acids inP. palustris. All the treatments decreased dry mass and protein, and increased free amino acids inD. flavida. Kinetin decreased dry mass and protein, and increased free amino acids whereas 3-indoleacetic acid, gibberellic acid and ethrel increased dry mass and protein, and decreased free amino acids inT. badia over the control. The effects were most pronounced in the three species upon treatment with 50 μm kinetin, 50 μm gibberellic acid, 50 μm 3-indoleacetic acid and 25 μm ethrel. Among the treatments, the effects of 3-indoleacetic acid were most marked in enhancing growth, measured by dry mass ofT. badia out of the three species studied.     

Biotransformation of ergot alkaloids by plant cell cultures with high peroxidase activity

Ergot alkaloids agroclavine and elymoclavine have been modified using plant cell cultures exhibiting high peroxidase activity. Setoclavine and isosetoclavine have been isolated from media after transformation of agroclavine on a semipreparative scale. 10-Hydroxyelymoclavine resulted from similar treatment of elymoclavine.     

Effects of growth regulators on fatty acids of soybean suspension cultures

Soybean suspension cultures were grown for 24 weeks in the absence of plant growth regulators and in the presence of 1 ppm levels each of an auxin (indole-3-butyric acid), a cytokinin (kinetin) and a gibberellin (gibberellic acid), individually and in all possible combinations. Cells grown in the presence of the auxin with and without gibberellin contained relatively greater amounts of palmitic and smaller amounts of polyunsaturated acids than did cells grown under other regimens. The combination of cytokinin and gibberellin caused a higher proportion of linoleic and a lower proportion of linolenic acids than in cells of the other groups. Neither of these regulators by itself produced the effect, and addition of auxin to the other two diminished the effect.     

Effects of plant growth regulators in soil on clover growth and nitrogen fixation

Effects of the growth regulators Barleyquat B, New 5c Cyocel, Cerone, Terpal and paclobutrazol on Rhizobium trifolii in vitro, on growth and root nodulation of clover grown in pots and on symbiotic nitrogen fixation were measured.Paclobutrazol in soil markedly reduced the weight of clover plants but had no effect on nodulation in relation to plant size. Paclobutrazol decreased the amount of nitrogen fixed per plant (measured by acetylene reduction) when present in soil at a concentration which would result from a single direct application at 0.25 kg a.i. ha^–1 remaining unchanged and evenly distributed in the top 5 cm of soil. A concentration in soil equal to that from an application at 0.125 kg a.i. ha^–1 had no significant effect on the rate of nitrogen fixation per plant and the rate relative to plants weight increased. It was evident that the inhibitory effect on plant growth was greater than on symbiotic nitrogen fixation. The other PGR's had virtually no effect on clover.None of the compounds seems likely to affect nitrogen fixation by a subsequent clover crop in the field if previously applied to a cereal crop once at typical rates of application.

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Zusammenfassung Die Einflüsse der Wachstumsregulatoren Barleyquat B, New 5c, Cyocel, Cerone, Terpal und Paclobutrazol auf das Wachstum von Rhizobium trifolii in vitro, auf das Wachstum und Knöllchenbildung des Klees und auf die symbiotische N₂-Bindung wurden bestimmt.Paclobutrazol im Boden führte zu einer bedeutenden Gewichtsverminderung der Kleepflanzen, blieb aber ohne Einfluß auf die Knöllchenbildung in bezug auf die Pflanzengröße. Paclobutrazol reduzierte die Menge Stickstoff gebunden pro Pflanze, (gemessen mittels Acetylenreduktion) wenn er im Boden in einer Konzentration enthalten war, die anwesend wäre, wenn eine einzige Applikation von 0,25 kg Wirkstoff.ha^–1 unverändert und gleichmäßig in den oberen 5 cm des Bodens verteilt bleiben würde. Eine Bodenkonzentration gleich jener von einer Applikation von 0,125 kg Wirkstoff.ha^–1 hatte keine signifikante Wirkung auf die N₂-Bindungsleistung pro Pflanze, und das Bindungstempo in bezug auf das Pflanzengewicht nahm zu. Offensichtlich war die Hemmwirkung auf das Pflanzenwachstum größer als auf die symbiotische N₂-Bindung.Wahrscheinlich wird keine der Verbindungen die N₂-Fixierung des Klees im Freiland beeinträchtigen, wenn sie einmal vorher auf eine Getreidearte in der üblichen Aufwandmenge appliziert werden.

     

The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures

We have studied the biomass and alkaloid production of geneticallytransformed hairy root cultures of Lobelia inflata L. Thehairy root clone 8009/h7 transformed with Agrobacteriumrhizogenes strain R 1601 was cultivated on B5 solid media containingdifferent amounts of the growth regulators KIN, IAA or NAA. KIN significantlydecreased growth and lobeline production and strongly inhibited biomassformation at 5 mg/l. IAA and NAA had characteristic morphologicaleffects on growth, in increasing the number of the lateral roots. However theyrestricted linear growth. Addition of IAA or NAA into the culture mediumincreased the biomass formation and lobeline production of hairy roots. It wasfound that the greatest amount of lobeline was obtained at the 0.2mg/l IAA concentration, similar to the effect of NAA.     

Anthocyanin production by plant cell cultures on media based on milk whey

Summary Selected callus cultures ofAjuga reptans produce anthocyanins in the dark on Murashige-Skoog medium with sucrose as carbon source. From these cultures we could isolate, by a two-stage selection procedure, new lines, which produce anthocyanins on media based on milk whey with lactose as the only carbon source. The anthocyanin production of the cell lines on milk whey is comparable with the production of the cell lines on MS-medium. Our results prove that it is possible to lower medium costs of plant cell culture by using cheap raw materials.     

Improving alpha-tocopherol production in plant cell cultures

Suspension cell cultures of Helianthus annuus L. were previously established for the production of the most active component of vitamin E, alpha-tocopherol, by optimizing medium composition and culture conditions. In the present work, the possibility of enhancing alpha-tocopherol production by the addition of jasmonic acid to the culture medium was investigated both in sunflower and Arabidopsis cell cultures. A considerable increase (49% and 66%, respectively) of alpha-tocopherol production was obtained in both, after a 72-h treatment with 5 microM jasmonic acid. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effects of rheological properties and mass transfer on plant cell bioreactor performance: production of tropane alkaloids

Volumetric mass transfer coefficients, K(L)a were measured over an aeration rate range from 0.1 to 1.0 vvm in a 1.2-L draft-tube-type airlift bioreactor for different Datura stramonium cell concentrations and correlated with superficial air velocity and rheological properties of the cell suspension. The measured K(L)a values (17-40 h(-1)) for a cell volume fraction of 0.2 (v/v) were approximately 2 times higher than those for the highest cell concentrations tested (cell volume fraction 0.7-0.8 v/v). Cell suspensions exhibited yield stress and pseudoplastic behavior. This behavior was described by the Casson model. The estimated yield stress values depended upon cell concentration with an exponent of 4.0. An empirical correlation based on the data for plant cell suspensions exhibiting yield stress was developed in order to determine aeration strategy for the plant cell cultivation in draft-tube-type airlift bioreactors: \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm K}_{\rm L} {\rm a} = {\rm A}({\rm U}_{{\rm gr}});{0.3} ({\rm \eta }_{{\rm eff}});{ - 0.4} $$\end{document} Aeration rates above 1.0 vvm caused a significant drop in cell yield and product content. Maximum growth and production were obtained at 0.6 vvm aeration. The cell and product yields obtained at 1.7 vvm were 2.8 times lower than the maximum values (25 g cell DW/L and 73.8 mg tropane alkaloid/L). The effects of the increased aeration rates on cell yield were also evaluated in terms of Reynolds stress. It was found that there was a relation between cell damage and the estimated Reynolds stress. The Reynolds stress estimated for the same aeration rate decreased with increasing cell concentration, suggesting that cells in the cultures at low cell concentrations are subjected to hydrodynamic damage. In the experiments with the cell cultures having a cell concentration of 0.3 (v/v), approximately 70% reduction in cell concentration was observed when the Reynolds stress was increased from 10 to 50 dyn/cm(2). (c) 1993 John Wiley & Sons, Inc.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Physiology of,an enzyme production by,plant cell cultures

Red kidney bean (Phascolus vulgaris) cells, derived from roof, callus, were grown in suspension culture in shake flasks and in laboratory fermentors using batch and continuous batch culture techniques. The medium contained casein hydrolysate, sucrose, inorganic salts, vitamins, and growth hormones. In continuous batch culture yields of up to 171 g wet weight, (8.5 g dry weight) per liter were obtained in 7 days. Organic nitrogen was used preferentially. Growth on nitrate was considerably slower than on organic nitrogen sources. Indole acetic and naphthalene acetic acids were not essential for good growth of the cells whereas kinetin and 2, 4-D were. The optimum pH for growth was about p11 4.5. The presence of amylase and peroxidase was detected in culture filtrates. Amylase activity was low in either the presence or the absence of starch in the medium. Peroxidase production could be related directly with growth of the culture. Maximum peroxidase yield, as measured by the guaiacol method and expressed as horse radish peroxidase, was 1.25 × 10^?8 M.