P. A. A. Harkes

Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spec.

The addition of autoclaved mycelia of Aspergillus niger and the known phytopathogenic fungus Phytophtora cinnamomi to cultured cells of Cinchona ledgeriana Moens. caused a marked increase in the anthraquinone content of the plant cells. This finding in combination with the antimicrobial activity of the anthraquinones isolated from calli of Cinchona pubescens Vahl. led to the conclusion that anthraquinones are phytoalexins.

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.

Induction of triterpene biosynthesis by elicitors in suspension cultures of Tabernaemontana species.

Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae.

Effect of auxin on cytodifferentiation and production of quinoline alkaloids in compact globular structures of Cinchona ledgeriana

Fine cell suspension cultures of Cinchona ledgeriana produce only very low amounts of quinoline alkaloids. These cultures formed self-propagating compact globular structures (CGS) on medium containing 2,4-D and BAP. These CGS could be induced to produce significant amounts of quinoline alkaloids by replacing 2,4-D by low amounts of 1-NAA, which was accompanied by histological changes of the CGS. A few high producing CGS clones could be selected. The stability of this trait was studied over a period of about one year of culture in maintenance medium.

The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkloids and anthraquinones

The influence of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth and the production of alkaloids and anthraquinones in cell suspension cultures of Cinchona ledgeriana Moens was studied. It was found that maximum growth and maximum alkaloid yield were obtained with a B5-medium containing the normal level of nitrate and 4% sucrose, whereas the anthraquinone yield was maximum at 8% sucrose at the normal level of nitrate. Maximum contents of secondary metabolites, expressed as μg.gDW-1, were found using a medium containing 2% sucrose, but four times the normal nitrate concentration.

Plant Cell and Tissue Culture of Cinchona Species

The genus Cinchona, belonging to the family Rubiaceae, is still esteemed for its anti-malaria activity. Several species, e.g. C. pubescens (syn. C succirubra) and C. ledgeriana,, have been cultured in plantations during the past 130 years for the production of Cinchona bark, the raw material for the alkaloids quinine and quinidine. Cinchona plantations are mainly found in Middle-America, Central Africa, India, Phillipines, and Indonesia. After 7–12 years of growth the bark of the trees is harvested. The alkaloid content is then about 10%–15% (Smit 1984).

Conditioning of Media: An Elaborate Method of Optimizing Initial Growth Hormone Concentration

The conditioning of media is a well-known method for growing plant cells at low density (Street 1977). Stuart and Street (1969) studied the conditioning effect in more detail. They were able to prove a conditioning effect for Acer cell cultures. The critical initial cell density of such cultures could be lowered by a factor of at least 10 by using a conditioned medium. It was also attempted to define the chemical basis of the conditioning effect (Stuart and Street 1971). From experiments measuring the cell numbers after 4 weeks of culture of different initial cell densities on various (conditioned) media, the authors concluded that the carbon course, the vitamins and other growth factors as well as the growth hormones (2,4-D and kinetin) were probably not involved in the conditioning effect. Analysis of a conditioned medium showed the presence of a series of amino acids in small amounts. Addition of these compounds to a non-conditioned medium indeed resulted in a lowering of the critical initial cell density, however, not to the same level as a conditioned medium. Furthermore, a volatile factor was proven ro be involved in the conditioning effect.

Tabernaemontana spp.: In Vitro Production of Indole and Biogenetically Related Alkaloids

The family Apocynaceae is probably one of the richest sources of drugs in the plant kingdom. Both alkaloids, e.g. reserpine, vincristine, vinblastine, ajmalicine and serpentine, and steroids, e.g. strophantidine, are found in species of this family. One of the larger indole alkaloid-bearing tribes within this family is Tabernaemontaneae. The tribe Plumerieae comprises well-known genera such as Catharanthus, Vinca, Amsonia, Rhazya and Alstonia.

Production of secondary metabolites in cell cultures of some terpenoid-indole alkaloids producing plants

Within the group of terpenoid-indole alkaloids and related compounds quite a few have pharmaceutical interest. As most of them have quite high prices on the market, the production by means of biotechnological processes seems attractive. Thus in the past years a number of studies have been dealing with plant cell, tissue and organ cultures of terpenoid-indole alkaloids producing plants from genera like Rauwolfia, Catharanthus, Vinca, Tabernaemontana, Voacanga, Amsonia and Cinchona. Three of these are at present studied in our Biotechnology Delft Leiden (BDL) project group, viz. Catharanthus, Tabernaemontana and Cinchona with the aim to improve yields of alkaloids in the cell cultures. Recently we have reviewed the work on Cinchona cell cultures (Verpoorte et al., 1985; Wijnsma and Verpoorte, 1986 e). Here we shall confine ourselves to a brief review of some of the results recently in our laboratories.

Secondary Metabolism in Cell Cultures of Some Terpenoid-Indole Alkaloid Producing Plants

In the past years it has been proven that large scale culturing of plant cells is feasible. The production of shikonin (1) and of ginseng biomass (2) are examples of a commercial scale production with plant cell cultures.