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 fungal elicitors to increase indole alkaloid accumulation in catharanthus roseus suspension cell culture

Various fungal elicitors derived from 12 fungi were tested to improve indole alkaloid production in Catharanthus roseus cell suspension cultures. Results show that different fungal mycelium homogenates stimulate different kinds of indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which ranged from 2- to 5-fold higher than the control. Some fungal culture filtrates also efficiently elicited the biosynthesis of different indole alkaloids. The optimal elicitor addition and exposure time for the maximal alkaloid production were on day 7 after subculture and for 3 days of treatment but different fungal elicitors showed the different optimal treatment dosages. Additions of elicitor at the doses ranging from 5 mg/l to 30 mg/l of carbon hydrate equivalent resulted in varieous amounts and kinds of indole alkaloid accumulation. Exposed to a same fungal elicitor, several different cell lines generated the different responses regarding as growth rate, culture color and alkaloid production.     

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     

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.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Transgenic periwinkle tissues overproducing cytokinins do not accumulate enhanced levels of indole alkaloids

Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - DW dry cell mass - ipt isopentenyl transferase gene     

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.     

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     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

外源钾水平对长春花生长和生物碱积累的影响

钾营养对于植物的生长发育具有重要作用,通过对长春花施以不同水平的外源钾,以探讨在不同浓度的钾素营养条件下,长春花生物量和生物碱的积累特点。结果表明,外源钾素营养水平的增加显著促进长春花的株高、根长、生物量积累,其中以10和15 mmol·L^-1浓度下的作用效果最显著。同时,外源的钾素营养水平提高还显著增加长春花的吲哚类生物碱含量,其中长春质碱的含量随着钾素营养浓度增大而增加;文多灵和长春碱随着钾素营养浓度增大都是先增加后减小,在15 mmol·L^-1浓度下达到最大值,过高的钾素营养不利于两者的合成。     

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.     

Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P-450 dependent monooxygenase.

An end-product indole alkaloid, catharanthine, inhibits a membrane-bound cytochrome P-450 dependent monooxygenase of the higher plant, $\text{Vinca rosea}$. Kinetic analysis revealed the alkaloid to be a reversible, linear, non-competitive inhibitor (K_i=1 mM) with respect to its substrates, geraniol and NADPH. Comparable inhibition of the solubilized monooxygenase by catharanthine tends to exclude a mechanism based upon disruption of membrane organization. On the basis of its inhibition of solubilized hydroxylase in the presence and absence of sodium cholate, it is also unlikely that catharanthine competes for putative phospholipid binding site(s). Two additional end-product alkaloids, vinblastine and vindoline were less inhibitory. Since the hydroxylase catalyzes one of the first committed steps in the biosynthesis of indole alkaloids, these observations suggest feedback control of the pathway by catharanthine.     

Development of two stage culture process by optimization of inorganic salts for improving catharanthine production in hairy root cultures of Catharanthus roseus

The effects of the concentrations of inorganic salts in Schenk and Hildebrandt (SH) medium on catharanthine production in hairy root cultures of Catharanthus roseus were investigated. The inorganic salt components could be categorized into four groups. The first group (nitrate) supported both the growth of and catharanthine production by hairy roots with incremental increases in the concentration. The second (ammonium and phosphate) yielded contradictory effects with respect to growth and production. The third (borate and molibdate) inhibited both growth and production, while the fourth (potassium iodide, sulfate, and iron) did not exhibit any significant effects. Through optimization of the concentrations of inorganic salts in the medium, a two stage process of hairy root cultures with different media for growth and production was developed which enabled us to enhance the volumetric yield of catharanthine up to 60.5 mg/l. This productivity was 5.4 times higher than that of a one stage culture in the original SH medium.     

植物生长调节物质对长春花细胞中吲哚生物碱积累的影响

适当浓度的脱落酸(ABA)、乙烯利和乙酰水杨酸(ASA)均对长春花悬浮细胞中吲哚生物碱的积累有较明显的促进作用.2mg·L-1ABA对吲哚生物碱的诱导效果最好,乙烯利的最适剂量为1 g·L-1,1 mg·L-1ASA有利于阿玛碱积累,而2 mg·L-1ASA则有利于长春质碱积累.     

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

A differential response to chemical elicitors in Catharanthus roseus in vitro cultures

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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