Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus

Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached 2 mg g(-1) dry wt after 27 d in culture. Maximal vindoline accumulation coincided with maximum activities of deacetoxyvindoline 4-hydroxylase, deacetylvindoline acetyl-CoA acetyl transferase and tryptophan decarboxylase. Shoot exposure to jasmonate shortened the time required for the maximal vindoline accumulation to 14 d.     

Anthocyanins in Catharanthus roseus in vivo and in vitro: a review

The production of anthocyanin in Catharanthus roseus flowers from both field-grown and regenerated by somatic embryogenesis plants and cell cultures was described. The anthocyanins were identified as the 3-O-glucosides, and the 3-O-(6-O-p-coumaroyl) glucosides of hirsutidin, malvidin and petunidin, respectively both in vivo and in vitro. The influence of environmental conditions on in vitro anthocyanin accumulation is described. The relationship between in vivo and in vitro anthocyanin production is discussed.     

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Somatic embryogenesis and indole alkaloid production in Catharanthus roseus

ABSTRACT

A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.     

长春花悬浮培养细胞对天麻素的生物转化

应用长春花(Catharanthus roseus (L.) G. Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究.经过8 d培养形成一个转化产物,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇,为天麻素水解后形成的甙元.     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

Tryptophan decarboxylase from transformed roots of Catharanthus roseus

Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots was purified 80-fold. Antibodies against TDC were obtained and they recognized only one protein of 55 kDa in crude extracts from hairy root cultures. Elicitation of transformed root cultures with macerozyme yielded a marked increase in TDC activity, which was accompanied by a similar increase in the amount of immunoreactive TDC protein. These results suggest that the alkaloid accumulation, produced by elicitation, requires the synthesis of new TDC polypeptide in C. roseus root cultures and establishes important differences in the regulatory control of this enzyme in root cultures compared to developing seedlings, where the posttranslational regulation apparently plays a major role.     

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.     

Enzymic synthesis of corynanthe-type alkaloids in cell cultures of Catharanthus roseus: Quantitation by radioimmunoassay

The soluble enzyme system from Catharanthus roseus cell suspension cultures which synthesizes ajmalicine, 19-epiajmalicine and tetrahydroalstonine from tryptamine and secologanin has been further characterized. The enzymic reaction was followed quantitatively by using a radioimmunoassay (RIA) with antibodies directed against ajmalicine. This RIA proved exceedingly useful in determining the enzymology of the reaction and displayed a sensitivity shown previously only by radio-tracer methods. By this method, the enzyme system was found to function optimally at pH 6.5. Sensitivity of the enzymic synthesis of ajmalicine and its stereoisomers to δ-d-gluconolactone, a β-glucosidase inhibitor, indicated the involvement of a β-glucosidase in the formation of these alkaloids. The enzyme system catalysed the formation of unnatural ajmalicine analogues from ring-substituted tryptamines.     

Production of indole alkaloids by selected hairy root lines of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC-MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine. (c) 1993 John Wiley & Sons, Inc.     

Phenolic compounds in Catharanthus roseus

Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole alkaloid biosynthesis for chorismate, an important intermediate in plant metabolism. The biosynthesis C6C1 compounds is induced by biotic elicitors.     

In vitro micropropagation of Ruscus aculeatus

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid and 1 mg dm⁻³ kinetin.     

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     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

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.     

Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus

A synchronous cell division system was established using the double phosphate starvation method, based on the observation that one of the limiting factors in the growth of a suspension culture of Catharanthus roseus (L.) G. Don cells in the medium of Murashige and Skoog was phosphate. In the system, an increase in cell number took place in a short period of only 4 h, while the cell number remained almost constant during other periods of the cell cycle. The synchrony of the culture was confirmed by changes in mitotic index, which increased sharply prior to the increase in cell number. The S phase was determined by measuring incorporation of [³H]-thymidine into the DNA fraction during the cell cycle and synchrony of DNA synthesis was verified likewise. Synchronization by phosphate starvation is discussed in relation to the function of phosphate as a nutrient. The synchronous system thus established will be useful in biochemical studies of the cell cycle in higher plants.