Alkaloid production in Catharanthus roseus cell cultures

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.Abbreviations IAA indolyl-3-acetic acid - IIAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine NRCC No. 20087     

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     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

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     

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.     

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.     

Alkaloid production in Catharanthus roseus cell cultures: Isolation and characterization of alkaloids from one cell line

A detailed study of one cell line (coded as 943) of Catharanthus roseus cell cultures has revealed the presence of the following alkaloids: ajmalicine, vallesiachotamine, hörhammerinine, hörhammericine, vindolinine, 19-epi- vindolinine, 19-acetoxy-11-methoxytabersonine, 19-acetoxy-11 -hydroxytabersonine, 19-hydroxy- 11-methoxytabersonine, yohimbine and isositsirikine, together with dimethyltryptamine and a new strychnos-type alkaloid.     

Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the -glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Alkaloid production in Catharanthus roseus cell cultures: Initial studies on cell lines and their alkaloid content

Several hundred serially cultured cell suspensions derived from three cultivars of periwinkle (Catharanthus roseus) were established in Gamborg's B5 medium and then transferred to Zenk's alkaloid production medium. Total alkaloid concentration ranged from 0.1 to 1.5% of dry weight. Alkaloids present were of the corynanthe, strychnos and aspidosperma types, with the greatest diversity arising during the third to the fifth week of subculturing. The alkaloid content appeared both specific for, and reproducible in, individual cell lines.     

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     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

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.     

Alkaloid production in Catharanthus roseus (L.) G. Don.

Analysis of 76 cell clones derived from one leaf of a periwinkle plant (Catharanthus roseus (L.) G. Don) showed the occurrence of Corynanthe-, Strychnos-, and Aspidosperma-type alkaloids. The majority of clones (62%) displayed compounds of all three types. Variation of the alkaloid spectra of the cell clones was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants.NRCC # 19100     

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.     

Alkaloid metabolism in wounded Catharanthus roseus seedlings.

The effect of mechanical wounding on alkaloid metabolism was analyzed in Catharanthus roseus seedlings. Wounding induced an increase in ajmalicine accumulation, whereas catharanthine remained unaffected. A positive dual effect on vindoline was noticed. Short and mid-term effects were detected between 12 and 24 h after mechanical damage was inflicted, and apparently resulted from the accelerated transformation of the tabersonine intermediaries to vindoline. Long-term effects involved a generalized increase in carbon flux towards alkaloid synthesis. Exposure to ethylene (1 ppm) produced similar results to those observed in wounded seedlings, suggesting that it might be mediating the wound-induced increase in alkaloid synthesis. No synergistic or additive effects were observed when wounded seedlings were exposed to ethylene or jasmonate.     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

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