Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine

Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in the accumulation of catharanthine, tabersonine and condylocarpine. Condylocarpine is not an intermediate in the pathway to catharanthine or tabersonine when it is fed to the cultures. The results support the hypothesis that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine.     

Serpentine release by suspension cultures of Catharanthus roseus

Summary Suspension cultures ofCatharanthus roseus filtered (with or without a vacuum) and resuspended in fresh or spent medium will release serpentine into the medium. This treatment is associated with small increases in pH and conductivity of the medium. The released serpentine quickly disappears, and is probably taken up by the cells.     

Dissimilation of methionine in cell suspension cultures from Catharanthus roseus L.

Plant cell suspension cultures from Catharanthus roseus were investigated for their capability to dissimilate methionine or its analogs in order to reutilize the sulphane group for cysteine biosynthesis. Three steps have been described as prerequisites of this process: (a) oxidative degradation by the amino-acid oxidase of methionine giving rise to methanethiol production; (b) demethylation by methyltransferases leading to homocysteine and S-methylmethionine (c) replacement of the homocysteine sulphane sulphur by alkylthiol yielding methionine and free hydrogen sulphide. A reversal of the cystathionine pathway as a source of cysteine was ruled out because the cells lack cystathionine γ-lyase. The absence of this enzyme is compensated by the S-alkyl exchange of homocysteine with methylmercaptan. Hydrogen sulphide thus liberated is used for de novo synthesis of cysteine. The complete pathway can be catalyzed by the constitutive set of enzymes present in the higher plant.     

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.     

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.     

Biotransformation of anhydrovinblastine to vinblastine by a cell-free extract of Catharanthus roseus cell suspension cultures

A crude enzyme preparation obtained from cell suspension culture of Catharanthus roseus converted 3′,4′-anhydrovinblastine to vinblastine, an anticancer agent. NADH and MnCl₂ in the reaction mixture enhanced vinblastine yields.     

Effect of fungal homogenate,enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures

The addition of Aspergillus niger homogenate to Catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. The use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. Furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. The exposure of the cells to an osmotic stress produced a marked increment (320%) in the total alkaloid content. When both stress treatments were applied sequentially, an additive effect on alkaloid accumulation was observed. It was 300% higher than in cells cultured without treatment, the majority of the alkaloids found in the medium.Abbreviations BAP benzylaminopurine - CM chorismate mutase - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - MS Murashige-Skoog - PAL phenylalanine ammonia lyase - PC Phillips-Collins     

Growth and stoichiometry of a Catharanthus roseus cell suspension culture grown under nitrogen-limiting conditions

The uptake of carbohydrate and nitrate by Catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. Biomass production was similar when using either 6, 12, 18, or 24 mM nitrate as the nitrogen source and 20 g L(-1) sucrose as the carbon source. In all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. Apparent biomass yields, Y(X/S) and Y(X/N) were 0.49 g biomass g(-1) glucose equivalent and 0.23 g biomass mmol(-1) nitrate, respectively. The determination of the cellular carbon-to-nitrogen ration (C/N ration) resulted in the identification of three district growth phases: an active growth phase, and accumulation phase, and a biomass decline phase (endogenous metabolism). The onset of the last two phases was correlated with nitrate and sugar of the last two phases was correlated with nitrate and sugar exhaustion, respectively. Balanced stoichiometric equations describing the active growth and accumulation phases were proposed based on elemental composition and ash content of the biomass. The stoichiometric equation related to the accumulation phase predicts that the available sugars are stored as starch- and lipid-like materials.     

Relationship between cell morphology and indole alkaloid production in suspension cultures of Catharanthus roseus

The relationship between the morphology and indole alkaloid production of Catharanthus roseus cells was investigated. Eleven cell lines were randomly selected from protoplast-derived clones. In each line, most of the cells maintained only one of the two shapes, either spherical or cylindrical. The cell aspect ratio (cell length/width) for most isolates was stable for more than two years of subculture. Cell division patterns of spherical and cylindrical cell isolates were different and patterns of division remained stable in each phenotype and were not considerably affected by auxin or cytokinin levels in the culture media. These observations indicate that cell morphology of our isolates is stable and probably internally determined. Production of the indole alkaloids, ajmalicine and catharanthine was significantly greater when the cell aspect ratio was more than 2.8.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - CPA p-chlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - SH Schenk and Hildebrandt (1972) medium     

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Summary Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL -difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL -difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - DFMA DL -difluoro-methylarginine - DFMO DL -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Physiological responses to salt stress in young umbu plants

Soil salinity affects plant growth and development due to harmful ion effects and water stress caused by reduced osmotic potential in the soil solution. In order to evaluate the effects of salt stress in young umbu plants, research was performed in green house conditions at the Laboratory of Plant Physiology at Federal Rural University of Pernambuco, Brazil. Growth, stomatal behaviour, water relations, and both inorganic and organic solutes were studied aiming for a better understanding of the responses of umbu plants to increasing salinity. Plants were grown in washed sand with Hoagland and Arnon nutrient solution with 0, 25, 50, 75, and 100 mM NaCl. Growth, leaf water potential, transpiration, and diffusive resistance were evaluated. Na⁺, K⁺, Cl⁻, soluble carbohydrates, and free amino acid contents were measured in several plant organs. Most variables were affected with salinity above 50 mM NaCl showing decreases in: number of leaves, plant height, stems diameter, and dry masses, and increases in root-to-shoot ratio. Reductions in ψ_pd were observed in plants grown under 75 and 100 mM NaCl. All salt levels above zero increased Na⁺ and Cl⁻ contents in leaves. However, K⁺ content was not affected. Na⁺ and Cl⁻ in stems and roots reached saturation in treatments above 50 mM NaCl. Organic solute accumulation in response to salt stress was not observed in umbu plants. These results suggest that umbu plants tolerate salt levels up to 50 mM NaCl without showing significant physio-morphological alterations.     

In situ cell differentiation monitoring of Catharanthus roseus suspension culture processes by NIR spectroscopy

Bioprocess and Biosystems Engineering - Plant suspension culture is attracting interest as a promising platform to produce biological medicines due to the absence of virus, prions or DNA related to...     

Interactive effects of triadimefon and salt stress on antioxidative status and ajmalicine accumulation in Catharanthus roseus

The ability of triadimefon (TDM), a triazole group of fungicide, to ameliorate salinity stress was studied in Catharanthus roseus (L.) G. Don. plants subjected to sodium chloride (NaCl) treatment. NaCl treatment at 80 mM level decreased overall growth of this plant and reduced the chlorophyll contents, protein, antioxidant enzymes such as peroxidase (POX), superoxide dismutase (SOD) and polyphenol oxidase (PPO). The root alkaloid ajmalicine got increased under salt treatment. When these stressed plants were treated with TDM at 15 mg l⁻¹ concentration minimized the inhibitory effects of NaCl stress by increasing the root, shoot growth and leaf area and increased dry weight (DW), chlorophyll, protein contents and the activities of antioxidant enzymes like POX, SOD and PPO, thereby paved the way to overcome the salinity injury. The quantity of ajmalicine was again increased with the TDM treatment when compared to both control and NaCl treated plants. From these results, it is proved that the fungicide TDM have great role in the enhancement of plant antioxidative enzymes and the enhanced scavenging of potentially harmful free radicals, as a mechanism of protecting plants against noxious oxidative stress from the environment and also in the enhancement of active principles.     

甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比

研究干旱胁迫和盐胁迫对“芦选一号”。日‘薯愈伤组织可溶性蛋白、可溶性糖、脯氨酸含量、SOD活性等的影响,从而在细胞水平上探讨甘薯抵御渗透胁迫的生理机制。并分析甘薯细胞对干旱处理（PEG-6000）和盐处理（NaCl）的反应差异。结果表明,可溶性蛋白质含量在干旱胁迫下缓慢升高,在轻度和中度盐胁迫的生长前期和中期有较大幅度的上升。但后期下降,表明短时间盐胁迫下,Na^＋可能促进可溶性蛋白的合成;MDA在重度干旱胁迫下的含量显著低于重度盐胁迫,而SOD活性显著高于盐胁迫。表明在盐胁迫下细胞膜透性增加的主要原网是膜脂过氧化作用。干旱处理则是PEG-6000脱水的直接结果;重度干旱胁迫下,可溶性糖含量在短期内迅速升高,然后下降,而脯氨酸含量则在胁迫中后期迅速上升。脯氨酸可能有补偿可溶性糖含量降低的作用。