Alkaloid production in Catharanthus roseus (L.) G. Don cell cultures. XIII. Effects of bioregulators on indole alkaloid biosynthesis

A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that 1,1-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.     

Induction of catharanthine synthesis and stimulation of major indole alkaloids production by Catharanthus roseus cells under non-growth-altering treatment with Pythium vexans extracts

A Catharanthus roseus cell line was selected that synthesised catharanthine exclusively under elicitation.From the first day of culture, treatment with very low concentrations of a Pythium extract did not alter the growth of the suspension but, within 24 hours, induced the synthesis of catharanthine and stimulated the production of ajmalicine. Kinetic analysis showed that serpentine then began to accumulate and that all of these effects lasted more than 7 days. Elicitation also induced changes in the cell/medium distribution of the alkaloids. Higher, although non-lethal, concentrations of the fungal elicitor were shown to impair alkaloid production. This cell line will serve as a model to study the conditions for the expression of catharanthine synthesis at the molecular level.Abbreviations gE glucose-equivalent - MS Murashige and Skoog medium - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Promotion of indole alkaloid production in Catharanthus roseus cell cultures by rare earth elements

Production of the indole alkaloids, ajmalicine or catharanthine, in cell suspension cultures of Catharanthus roseus was enhanced by cerium (CeO₂ and CeCl₃), yttrium (Y₂O₃) and neodymium (NdCl₃). The yield of ajmalicine in these treated-cultures reached 51 mg l^–1 (CeO₂), 40 mg l^–1 (CeCl₃), 41 mg l^–1 (Y₂O₃) and 49 mg l^–1 (NdCl₃) while catharanthine production reached to 36 mg l^–1 (CeO₂) and 31 mg l^–1 (CeCl₃). A major portion of increased alkaloids was released into medium in these treatments. But Sm₂O₃, SmCl₃, La₂O₃, LaCl₃, complex of chromium (III)-titanium (IV) and NaSeO₄ treatments had little effect on alkaloid production of C. roseus cell cultures.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Selection of protoclones for high yields of indole alkaloids from suspension cultures ofCatharanthus roseus and the qualitative analysis of the compounds by LC-MS

Summary To produce economically important indole alkaloids by cell culture, we have selected protoclones ofCatharanthus roseus for high yields of catharanthine and ajmalicine. Protoplasts were enzymatically isolated from suspension-cultured cells. Protoclone VPC-10 produced catharanthine at 5.9 μg/g fresh wt of cells after 10 days of culture, although the original cell line did not produce it at a level detectable by HPLC. Under the same conditions, protoclone VPC-15 produced ajmalicine at 133.6 μg/g, which was about 3 times the productivity of the original cell line. In addition, the indole alkaloids were qualitatively confirmed by LC-MS.     

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     

Traditional and non-traditional plant growth regulators alters phytochemical constituents in Catharanthus roseus

The effect of different plant growth regulators (PGR) and elicitor treatments on the alkaloid profile variation of Catharanthus roseus was investigated in the present study. The PGR used were paclobutrazol (PBZ), gibberellic acid (GA₃) and Pseudomonas fluorescens elicitors (PF Elicitors). The estimated alkaloids were ajmalicine, catharanthine, tabersonine, serpentine and vindoline. In roots, the ajmalicine content increased significantly under all the treatments on all sampling days. In roots, the catharanthine contents increased with the age in control and growth regulator treatments, but the increase was not prominent and significant in PGR treatments when compared to controls. The serpentine contents of the plant increased with PGR treatments, but the increase was more prominent in PBZ treatments when compared to other treatments. The increase was in the order PBZ > PF Elicitors > GA₃. C. roseus never showed any significant increase in tabersonine contents in the roots under GA₃ treatments, but it increased significantly under PBZ and PF Elicitors when compared to control plants. The root vindoline contents increased with PBZ and PF Elicitors treatments but the decreased under GA₃ treatments when compared to control plants. Our results have good significance, as these increases the secondary metabolites of this traditional medicinal plant.     

Elicitor-stimulation of Monoterpene Indole Alkaloid Formation in Suspension Cultures of Catharanthus roseus

Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.     

The effect of inoculum density and conditioned medium on the production of ajmalicine and catharanthine from immobilized Catharanthus roseus cells

The effect of the cell-inoculum size and the addition of conditioned medium on ajmalicine and catharanthine production were studied using immobilized Catharanthus roseus cells. Higher specific-uptake rates of ammonium, nitrate, and sugars were observed in the low-inoculum-density cultures (50 g FW/L) compared to the high-inoculum-density cultures (100 g FW/L). Alkaloid production was not correlated with the exhaustion of a particular nutrient from the medium. The high-inoculum-density cultures produced higher ajmalicine concentrations throughout the experiment. Catharanthine production was similar between the two inoculum-density cultures. The addition of conditioned medium to MS-production medium dramatically improved the production of ajmalicine and catharanthine. The addition of conditioned medium enhanced ajmalicine production from immobilized Catharanthus roseus cultures on day 15 by at least two- to fourfold compared to media without the conditioning factors. Catharanthine production was increased by nearly fivefold in cultures with conditioned medium compared to those without conditioned medium. The enhancing effects of conditioned medium on alkaloid production were attributed to an unidentified factor produced and secreted by suspension cultures of C. roseus. The presence of conditioned medium also decreased the sucrose hydrolysis rate. The ajmalicine concentration in these immobilized cell cultures was found to be a function of the fresh-weight concentration, irrespective of the inoculum density or the culture medium. The medium choice and the inoculum density determined how rapidly fresh weight was accumulated and thus, how quickly ajmalicine was produced. Ajmalicine production correlated positively with fresh-weight concentration, but catharanthine production was not correlated with fresh-weight concentration.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

In situ adsorption for enhanced alkaloid production byCatharanthus roseus

Summary A significant fraction (10–40%) of the indole alkaloids produced byCatharanthus roseus was observed to be secreted into the medium. When a neutral polymeric resin, known to adsorb these alkaloids, was added to the cultivation medium, the accumulation of total indole alkaloids and the specific alkaloids, ajmalicine and serpentine were stimulated. Sorbent addition was also observed to result in increased ratios of ajmalicine to serpentine, which suggests the potential of using in situ adsorption to direct metabolism toward a specific product or intermediate within a given pathway.     

The role of strictosidine in monoterpenoid indole alkaloid biosynthesis

In contrast to previous reports that vincoside was the sole precursor for indole alkaloids in Vinca rosea, the 3α epimer strictosidine has been incorporated into tetrahydroalstonine, ajmalicine, catharanthine and vindoline; the anomalous 3β to 3α inversion is no longer required.     

Formation of catharanthine,akuammicine and vindoline in Catharanthus roseus suspension cells

Catharanthine and akuammicine, together with ajmalicine and strictosidine, were isolated from a culture strain of Catharanthus roseus suspension cells. The biosynthetic capability of the cultured cells to produce akuammicine, catharanthine and vindoline was confirmed by feeding experiments with dl-tryptophan-[3-¹⁴C] to yield the radioactive alkaloids.     

Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus

Compact callus cluster (CCC) cultures established from Catharanthus roseus consist of cohesive callus aggregates displaying certain levels of cellular or tissue differentiation. CCC cultures synthesize about two-fold more indole alkaloids than normal dispersed-cell cultures. Our studies here show that additions of KCl, mannitol, and a variety of synthetic precursors and bioregulators to the CCC cultures markedly improved indole alkaloid production and release of these alkaloids into the medium. Treatment with 250 mM mannitol and 4 g/l KCl yielded 42.3 mg l(-1) and 33.6 mg l(-1)of ajmalicine, respectively; these amounts were about four-fold higher than the control. Succinic acid, tryptamine, and tryptophan feedings also significantly increased ajmalicine (41.5 mg l(-1), 36.9 mg l(-1), and 31.8 mg l(-1), respectively) and catharanthine (21.1 mg l(-1), 17.2 mg l(-1), and 18 mg l(-1), respectively) production by the CCC cultures, while geraniol feeding inhibited biomass and alkaloid accumulation. We also found that tetramethyl ammonium bromide could significantly improve ajmalicine production (49.3 mg l(-1)) and catharanthine production (18.3 mg l(-1)) in C. roseus CCC cultures. The mechanisms responsible for these treatment effects are discussed herein.     

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.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. Effect of culture temperature

A three year old, alkaloid producing cell line of Catharanthus roseus, maintained at 25°C, was grown on 2% sucrose at various temperatures from 10° to 45°C. Growth rates were maximal at 35°C but declined rapidly above 35°C and below 25°C. Maximum serpentine yields reached a peak at between 20°C and 25°C and fell sharply above and below these temperatures, while ajmalicine showed a sharp peak of accumulation at 20°C. The variable serpentine/ajmalicine ratio at different growth temperatures suggests that lower temperatures may favour ajmalicine accumulation. Both the growth rate and the rate of alkaloid accumulation at 25°C were therefore sensitive to small changes in average culture temperature.     

Stable endosperm cell suspension cultures from wild-type and opaque-2 maize

Stable cell suspension cultures have been established from immature endosperms of A69Y wild-type and opaque-2 maize (Zea mays L.). Cultured cells are capable of storage protein (zein) synthesis and accumulation throughout the growth period. Electrophoretic patterns of zeins show, for opaque-2 cells, the preferential inhibition of the accumulation of 22 kDa peptides typical of the mutation. Viable protoplasts, able to regenerate cell walls, as well as to divide and to express foreign DNA in transient expression experiments, can be obtained with high yields from cultures of both genotypes.Abbreviations 02 opaque-2 - wt wild-type - DAP days after pollination - PCV packed cell volume - f.w. fresh weight - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - CAMV cauliflower mosaic virus - CAT chloramphenicol-acetyl-transferase     

Effects of bioregulators on indole alkaloid biosynthesis in Catharanthus roseus cell culture

Several carotenoid-inducers are effective in promoting indole alkaloid formation in Catharanthtus roseus cell culture. Among the five compounds tested, viz. 1,1-dimethylpiperidine, 2-diethylaminoethyl-3,4-dichlorophenylether, 2-diethylaminoethyl-2,4-dichlorophenylether, 2-diethylaminoethyl-β-naphthylether and 2-diethylaminoethyl-3,4-dimethylphenylether, 1,1-dimethylpiperidine, 2-diethylaminoethyl-2,4-dichlorophenylether and 2-diethylaminoethyl-β-naphthylether at 5 ppm concentration increased total alkaloid production by up to ca 20 % with concomitant increases in ajmalicine and catharanthine. Concentrations of 2-diethylaminoethyl-2,4-dichlorophenylether higher than 5 ppm caused growth inhibition and decrease in alkaloid synthesis.     

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