WO1988000968A2

WO1988000968A2 - Process for inducing secondary metabolite production in plant cultures and means thereof

Info

Publication number: WO1988000968A2
Application number: PCT/JP1987/000583
Authority: WO
Inventors: Jane Isobel Smith; Nigel John Smart; Masanaru Misawa
Original assignee: Mitsui Petrochemical Industries, Ltd.
Priority date: 1986-08-04
Filing date: 1987-08-03
Publication date: 1988-02-11
Also published as: EP0277250A1; JP2845443B2; WO1988000968A3; FI881491A; CA1283874C; KR880701774A; JPH01500401A; FI881491A0; KR900004437B1

Abstract

Synthesis of alkaloids by suspension cultured Catharanthus roseus cells is enhanced by stressing the cultured cells osmotically by addition to the culture of such inducers as ionic osmotic stressors such as halide salts e.g. NaCl, KCl and FeCl3 or organic osmotic stressors such as sugar alcohols and sugar acids. Synthesis of alkaloids is also enhanced by addition of anti-gibberellin compounds and other compounds such as abscissic acid which is a plant growth regulator. Yields of alkaloids are consequently increased.

Description

DESCRIPTION

TITLE OF THE INVENTION

Process for Inducing Secondary Metabolite Production in Plant Cultures and Means Thereof TECHNICAL FIELD

This invention relates to plant-derived secondary metabolites and, more particularly, to a method and means for inducing plant cells to produce these metabolites.

BACKGROUND ART

A number of chemicals produced by plants are classified as primary metabolites since they are essential to cell function. While primary metabolites are ubiquitous in the plant population and comprise the majority of plant-produced chemicals, other compounds are classified as secondary metabolites and, because of their various applications, are the focus of both scientific investigation and commercial exploitation.

In some cases, secondary metabolites are produced by some plants in response to stress induced, for example, by microorganism infection, UV irradiation, mechanical wounding as well as by treatment with organic and inorganic substances. The variety of secondary metabolites produced under such stressful conditions is diverse and includes the alkaloids, terpenoids, flavonoids, carotenoids, phenolic compounds and glucosides. Among the more important, pharmaceutically useful secondary metabolites are the alkaloids, steroid hormones, cardiac glycosides and antibiotics, of which the indolic alkaloids are of particularly great interest. The alkaloids include many compounds, at least some of which are known to have therapeutic value. For example, the indolic alkaloid ajmalicine is known to function as an antihypertensive/tranquilizer. Further, vincristine and vinblastine are accepted in the treatment of cancers. Catharanthine, a precursor of both vinblastine and vincristine, is also produced as a secondary metabolite and is valuable as a starting material in preparing those compounds.

Not all secondary metabolites are produced in great quantities by all plants, however. The nature of the metabolite produced depends largely on the plant species. For example, some indolic alkaloids are extracted most commonly from Catharanthus roseus (also known as Madagascar periwinkle, Vinea rosea and Lochnera rosea). Further, morphinan alkaloids e.g. morphine and codeine, are extracted from poppy plants (Papaver somniferum) and Cinchona alkaloids e.g. quinine and related quinoline alkaloids, are derived from Cinchona succirubra and related species. Obviously, the plant from which the secondary metabolite is to be derived must be genetically capable of expressing the metabolite and must be receptive to a particular metabolic stimulus in order to engage in synthesis of the metabolite.

Presently, secondary metabolites are extracted from native plants. Often, the desired metabolites are normally present in low concentrations, however, making the chemical extraction process both tedious and lengthy as reflected by the relatively high cost of some of these compounds. For example, indolic alkaloids are typically present in the dried Catharanthus roseus plants, from which they are normally derived, in concentrations of about 0.0003% by dry weight. The cost of extracting vincristine and/or vinblastine from these plants raises the selling price of these compounds to about $5,000 per gram (1983), Quinine and codeine were valued at about $100/kg and $650/kg, respectively.

While methods have been devised which improve upon the efficiency of the process by which the alkaloids are extracted, the advent of plant tissue culture has provided a useful alternative technology. By use of plant tissue culture (ptc) procedures, one may manipulate the capacity of plant cells to produce the secondary metabolite and hence deemphasize the labour-intensive extraction process based on intact plant harvests.

Plant tissue culture entails growth of a population of plant cells suspended within nutrient broth using a level agitation which strikes a balance between the need to circulate nutrients throughout the culture and the fragility of the plant cells. Not all plants are amenable to this procedure, however, and the media constitution is also a major factor in the success of the method. Advantages of the ptc technique reside in the ability to exercise control over the growth conditions in terms of defined environment, nutrient concentration, and the like. Further, the capacity to circulate a stimulant within the culture broth allows the stimulant to contact a greater number of the individual cells or cell clumps in the broth by comparison with application of the stimulant to a callus whereby only the outermost cells may be effected.

Using ptc, the capacity to induce certain plant species to produce desired secondary metabolites has been investigated. For example, in "Phytochemistry", Vol. 20, No. 8, pp 1841 - 1843 (1981), Lee et al. disclose the effect of specific amines, including 2-diethylaminoethyl-2,4-dichlorophenylether, on the ability of C. roseus cultures to produce certain indolic alkaloids. An increase in production of ajmalicine and catharanthine was noted. It is significant, however, to note that of five closely related chemicals used as alkaloid inducers (i.e. compounds introduced into the culture medium which stimulate or elicit the production of secondary metabolites) only three showed significant utility. Despite their close structural similarities, two of the five elicitors showed virtually negligible results. Moreover, although the amine mentioned above showed good results at concentrations of 5 ppm and lower, greater concentrations caused growth inhibition and decrease in alkaloid synthesis. In Planta Medica (1984), Eilert et al. disclose an attempt to induce production of the antimicrobial alkaloids rutacridone epoxide and hydroxyrutacridone epoxide from a suspension culture of Ruta graveolens. By addition of a suspension of either living free or immobilized yeasts or of dead Rhodotorula rubra cells or a crude cell wall fraction thereof, they were able to show an increase in production of the antimicrobial compounds of interest. Of note is the result that neither chitosan nor alginate were able to induce the desired response despite their having been proven to be useful as elicitors in other systems.

In Plant Cell Physiology 26: 1101 - 1110 (1985), Hattori and Ohta disclose the results of experiments in which suspension cultures of Red Bean (Vigna angularis) are grown, in the presence of sodium vanadate (Na₃VO₄) inter alia. They illustrate results which indicate that the vanadate compound was able to enhance production of the isoflavone glucoside, diadzein diglucoside. Other compounds were unable to generate desired results, however, and still others exhibited a depressing effect on secondary metabolite production.

Thus, while a number of inducers have been investigated in conjunction with a variety of plant species, the results point to the conclusion that no pattern of activity can presently be predicted. This is particularly evident, though not exclusively, in systems in which C. roseus is induced to produce the indolic alkaloids. DISCLOSURE OF THE INVENTION

It is an object of the present invention to identify substances capable of inducing production of secondary metabolites by appropriate plant cells.

It is another object of the present invention to provide a method by which plant cell production of secondary metabolites may be enhanced.

It is another object of the present invention to provide a process for inducing production of and then recovering secondary metabolites from plant cells.

Within the scope of the present invention are those compounds which, when added to the culture suspension, result in osmotic pressure which is sufficient to induce secondary metabolite synthesis in the cultured plant cells. The stress caused by the varied osmotic pressure is believed to be responsible for the increased synthesis. Those compounds which may be added to the plant cell suspension to generate osmotic stress may be selected from a very broad range of compounds. In general, these compounds may be categorized as ionic stress inducing compounds and organic stress inducing compounds. In either case, the end result of their addition to a suspension is the creation of an osmotic gradient between the cell and the surrounding medium.

The ionic, stress-inducing compounds are preferably those halide salts able to ionize in solution. More preferably these salts are Group I or Group II halide salts, the Group numbers referring to the Periodic Table. Especially suitable such compounds include the Group I and Group II chlorides although the bromide salts are also suitable. In a limited sense, the invention extends to such compounds as the chloride salts of sodium, potassium and iron (preferably ferric).

The organic, stress-inducing compounds include the sugar alcohols and sugar acids which include galactinol, xylitol, glycerol, mannitol and inositol and its various derivatives including phosphatidyl inositol, phytie acid and its esters, scyllitol, phytol, aldonic acids, aldaric acids, uronic acids and, notably, sorbitol. Sorbitol in particular has induced significant yields of secondary metabolites and is therefore preferred. Also within the scope of the present invention are compounds which act to induce secondary metabolite production by means other than through osmotic stress. This aspect of the present invention comprises culturing plant cells in a suspension supplemented with any one or a combination of a variety of plant growth regulating compounds and compounds which are related either by chemical structure or be biochemical function. While abscissic acid (ABA) is particularly useful, other related compounds which may be used include the antigibberellin compounds such as 2'-isopropyl-4'-(tri- methylammonium chloride)-5'-methylphenyl piperidine carboxylate, β-chloroethyltrimethylammonium chloride and tributyl-2,4-dichlorobenzyl phosphonium chloride. Other ABA-related compounds useful herein include N- (dimethyl- amino) succinamic acid, 4 ' -dihydrophaseic acid, phaseic acid and lunularic acid. Thus, from one aspect of the present invention, there is provided a process for inducing synthesis of a secondary metabolite by plant cells which comprises osmotically stressing the plant cells.

Another aspect of the invention comprises a process for inducing synthesis of a secondary metabolite by plant cells which comprises growing said cells in the presence of a plant growth regulating compound or related such compound. Preferably the plant cells are cultured in a suspension supplemented with the regulator.

Once the plant cells have been cultured under conditions in accordance with the present invention, the desired secondary metabolite or metabolites may be recovered using chemical procedures currently established in the biochemical extraction art.

Since some metabolites are secreted by plant cells into the growth medium, these metabolites may simply be separated from the cells per se by concentrating the cells using conventional techniques such as centrifugation and filtration. Thereafter, the broth containing the metabolite may either be processed further to concentrate the metabolite by such techniques as dialysis or acted upon directly using standard chemical extraction techniques. Where the desired metabolite is expressed by the plant cell but is not secreted from it, the plant cells are broken open using, for example, increased pressure, either atmospheric or osmotic, or crushed. The metabolite may then be extracted in a suitable solvent and concentrated somewhat using procedures outlined above prior to chemical extraction of the desired secondary metabolite. The specific chemical extraction procedure to be used will depend on the chemical nature of the metabolite to be recovered although, in general, such procedures involve sequential variation of solvent, pH and the like, and are known to those skilled in this art. Thus, a second aspect of the present invention comprises a method of providing a plant-cell derived secondary metabolite which comprises recovering the compound produced by way of the induction process of the present invention. Of the secondary metabolites which may be produced in plant cell culture systems using the process and the inducing substances of the present invention, there may be mentioned: the indolic alkaloids, obtainable from Catharanthus roseus including secologanin and tryptophan-derived strictosidine, ajmalicine, yohimbine, tabersonine, vindoline and catharanthine as well as tryptamine; tyrosine-derived morphine and codeine from Papaver somniferum; phenylalanine-derived chemicals such as coumarins from parsley cell cultures; and mevalonic acid-derived chemicals such as saponins e.g. digitalis from Digitalis purpurea or D. lanata.

This list is not proposed as an exhaustive one. It is likely that production of other secondary metabolites can be induced in plant cells to which they are indigenous. The present invention related in a preferred aspect to the C . roseus derived indole alkaloids catharanthine, ajmalicine, tabersonine and vindoline. The inducing substance abscissic acid used herein is a known compound having the structural formula appearing below:

Abscisic acid is an abscission-accelerating plant hormone i.e. a hormone which promotes separation of plant parts such as leaves from stems during the autumn season. It is a commercially available commodity known also as dormin whose commercial nomenclature is 5-(1hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl)-3- methyl-2,4-pentadienoic acid. Synthetic forms of the drug as well as various cis-trans isomeric forms are also available commercially and may be used herein.

As mentioned, plant cell culturing techniques are known and are used preferably herein. Conventional procedures are therefore employed in which living plant material e.g. leaf material, stem material or meristems are surface sterilized to prevent contamination and smaller masses of individual cells or cell clumps are nursed on agar nutrient base in growth plates until they may be transferred to a suitable liquid nutrient. The suspension is agitated continuously in culture flasks and ultimately transferred to a bioreactor. Agitation is suitably accomplished by the "air-lift" technique in which the culture is mixed by the gentle action of rising air bubbles.

To add the inducer to the medium, a solution of the inducing substance is suitably pre-prepared if desired in order to enhance homogeneity of the substance within the growth-sustaining medium. For example, all substances of the present invention may be mixed with water prior to addition. Abscisic acid may be mixed with water and a few drops of base such as KOH to encourage dissolution if necessary.

While trace amounts or lower levels of the desired secondary metabolite will be generally generated by the cultured plant cells in the absence of the inducing substances of the present invention, the low yield does not allow for an improved extract yield by comparison with procedures already known. Moreover, the period of time required for the production of these metabolites is relatively long, up to 30 or even 45 days, in the absence of the inducers. By contrast, culturing plant cells in the presence of the subject inducers increases the amount of secondary metabolite produced and can significantly decrease the length of time required to obtain that particular amount. BEST MODE OF CARRYING OUT THE INVENTION

While a variety of species of plant cells may be acted upon to generate the secondary metabolite of interest, the preferred embodiment of the invention provides a method by which indolic alkaloids are produced by Catharanthus roseus.

The C. roseus line from which the cultured cells are derived must, obviously, be amenable to plant tissue culturing. Obtaining such a line is well within the skill of persons to whom this disclosure is addressed and it is believed that several such lines presently exist. This is not to detract from the need to provide a plant whose cells have the capacity to endure and function within the cell culture environment. Those characteristics which confer this capacity are not well defined in the art. The presence of cells which are plant tissue culturable is confirmed almost exclusively by the trial and error approach. Thus, while specific cell lines having designations assigned by the inventors is used to exemplify the present invention hereinafter, it is to be acknowledged that the scope of the invention is by no means limited to these particular plant lines. Other lines of the type desired either presently existing or which may easily be created and tested will work with the same general efficacy as reported herein. The preferred C. roseus lines herein are designated JWM*, JOH and LBE-1. Lines JWM* and JOH were developed at the Plant Biotechnology Institute, Saskatoon, Sasketchewan, Canada. Line LBE-1 was developed at Allelix Inc., Mississauga, Ontario, Canada. All lines were originally isolated from C. roseus anthers.

The C. roseus cells may be cultured in any liquid medium containing all necessary metabolites, examples of which include SH medium, LS medium and MS medium, all of which are known in the art. LS medium (Linsmair and Skoog) is described in terms of its components in Phys. Plantarum (18) 1964 pp. 100 - 127. The components of SH medium (Shenek-Hildebrandt), preferred for use herein, are described in Can. J. Bot. 50: 195 - 204 (1972). The components of the MS medium (Murashige and Skoog), the medium preferred herein are familiar to those skilled in the art. To this medium is added a carbon source such as sucrose or lactose. Addition of 3 or 4% sucrose is preferred although similar concentrations of lactose may be used. The sole growth regulator added to the SH medium is α-naphthaleneacetic acid, in a concentration of 2 mg/1. The medium also suitably contains kinetin (6-furfurylaminopurine) which is a synthetic cytokinin which functions in cell division.

The plant cells are grown either in the dark to reduce stress and channel nutrients from light-activated secondary metabolic pathways (e.g. pigment formation) or light preferably until the population reaches the linear or early stationary growth phase at which time they are presumed to be more receptive to the inducing effects of the selected inducing preparation. Thus, the C. roseus cell suspension is grown in the absence of inducer preferably for from 3 to 10 days, more preferably for from 4 to 6 days under the conditions tested. The cell population may be monitored in order to identify the onset of the linear or early stationary growth phase if alternative growth conditions are employed. Moreover, it will be appreciated that it is not absolutely essential that the linear or early stationary phase be reached before adding the inducer since induction may be enhanced by addition in late log phase and in other phases of growth. It is desirable to maintain as large a cell population as possible in order to maximize alkaloid yields. Once the C. roseus suspension has reached the desired growth phase, the inducing substance is introduced in amounts sufficient to establish a desired concentration within the broth.

The inducing substances preferred herein are NaCl, KCl and FeCl₃ as ionic, osmotic stress inducers; sorbitol as organic, osmotic stress inducer and abscissic acid (ABA) as plant growth regulator inducer.

When selected for use, the final broth concentration of either NaCl or KCl is suitably about 0.1 M, as a minimum desirable level. Lower levels may serve the inducing purpose but are unlikely to provide enhanced yields within the shortened time periods available with higher concentrations. The acceptable upper concentration level of NaCl or KCl is dictated by the osmotic pressure which its presence generates within the cultured cells. The upper limit is therefore slightly below that concentration which causes plasmolysis. More preferably, NaCl and KCl may be added to achieve broth concentrations of 0.01 M to 1.0 M and, ideally, at around 0.5 M. Ferric chloride is suitably added to the suspension medium to achieve a final concentration of from 10 - 500 ppm, more preferably from 20 - 200 ppm and, ideally, around 50 ppm.

Broth concentrations of sorbitol range from about 0.05 M to a maximum concentration just slightly below the concentration at which plasmolysis occurs, as discussed with respect to NaCl or KCl addition. Preferred broth concentrations range from 0.1 M to 0.5 M and ideally at around 0.2 M.

Under the experimental conditions described hereinafter, the ideal concentration of abscissic acid ranges from 0.1 mg- 0.5 mg per 60 mis of suspension. Concentrations as low as 0.01 mg may be used although the induction response may be unfavourably low. Concentrations higher than 0.5 mg/60 mls also may be used although there appears to be little enhancement of the effect seen at 0.5 mg/60 mis.

Upon addition of either of the inducers, the C. roseus cells are cultured for a period of time to allow the inducer to interface with the cells and stimulate indolic alkaloid production. A period of 2 - 5 days is preferred although variations of this preferred period are acceptable particularly where conditions are not as specifically described herein. Growth of the culture is preferably continued either in the dark or in light.

After the induction period, the cells are harvested and the alkaloids extracted using conventional techniques. The particular techniques will depend on the specific substance to be recovered, but all are standard in the art.

Embodiments of the invention are disclosed hereinafter with reference to the following examples. In these examples, 60 ml cell suspension cultures are maintained in a basic MS growth medium containing 3% (w/v) sucrose, 2 mg/l α-naphthalenic acid and 0.1 mg kinetin. The cultures are maintained in 250 ml Erlenmeyer flasks on an illuminated rotary shaker (c. 120 rpm) and subcultured weekly by a one to five dilution.

Abscisic acid and D-sorbitol were obtained from Sigma Chemical Company and NaCl, KCl and FeCl₃ from Fisher Scientific Limited.

All inducers were prepared as stock solutions dissolved in distilled water at such a concentration that when 1 ml of stock solution was added to the cell suspension culture, the desired final concentration was obtained.

Unless otherwise indicated, each inducer was added to the cell suspension on day 5 of the growth cycle as a filter-sterilized solution and the cells harvested three days later. Standard solvent extraction techniques were used to extract indolic alkaloids from the cells. Catharanthine and ajmalicine yields were quantified by HPLC analysis. The presence of other alkaloids was determined qualitatively by visualizing with eerie ammonium sulphate following TLC separation. Example 1 - ABA Induction Synthesic 99% pure abscissic acid was introduced in a range of concentrations from 0.1 - 2.0 mg/60 ml culture. The results generated appear in Tables 1 - 4 below. Table 1 shows the effect which the type and concentration of ABA has on the induction process as applied to cell line JOH. While the 99% pure (±)cistrans synthetic isomer exhibited superior results, all forms of ABA showed desirable inducing properties.

1.

Table 2 illustrates the effect of ABA concentration on induction response on line JWM*. It will be noted that a maximum response occurs at 0.5 mg/60 ml culture with only minor variation with increased concentration.

In Table 3 below, the relationship of cell age and exposure to ABA is shown. It will be noted that atleast 3 days of culture growth provided the best results, with ajmalicine concentrations dropping off thereafter. However, catharanthine yields continue to rise even at day 6 of culture growth. The Table 3 results are with cell line JOH and an induction period of 4 days.

Table 4 below shows the response to 1 mg ABA induction of a variety of cell lines each of which is available at either Allelix Inc. or the Plant Biology

Institute in Saskatoon, as indicated. All are C. roseus originated. From these results, it will be apparent that lines JOH and LBE-1 are preferred.

The plant cells have been observed to respond positively to ABA exposure by increased alkaloid production even after only one day of growth, when they are presumably still in lag phase. However, as alkaloid accumulation is to some extent biomass related, it is more appropriate to induce the cells once some increase in biomass has occurred.

Example 2 - Induction by NaCl A range of sodium chloride concentrations (from 0.1 - 1.0 g/60 ml flask) were tested for their effects on alkaloid accumulation in line JOH Table 5 below. High concentrations of NaCl reduce biomass, hence lowering alkaloid yields, whereas the effect on biomass of lower concentrations is offset by the stimulation of catharanthine production.

Example 5 - Induction by Sorbitol

A range of concentrations of sorbitol were tested for their effects on alkaloid accumulation (0.1 - 0.5 M final concentration; 1.1 - 5.5 g/60 ml culture). Their effects on line JOH are shown in Table 8 below.

The major advantage obtained by inducing cell culture in accordance with the present invention is that yields of alkaloids (specifically catharanthine) can reach levels in only 8 days that in non-induced cells would take much longer to achieve, resulting in a considerably saving in terms of both time and cost.

Only minor variations are required to scale up the alkaloid production. For example, the MS medium is enhanced preferably with 4% (w/v) sucrose as opposed to the 3% solution used on small scale, together with 1 mg/l ct-naphthalene acetic acid and 0.1 mg/l kinetin. All lines and chemicals remain the same.

Example 6 - Larger Scale Induction Process For 30 liter batches using ABA, solutions are preferably prepared to give 8.33 mg/1 in the final culture fluid. Solutions are prepared in 1 liter of distilled water, filter sterilized and injected into the fermenter from a sterile bottle. Solutions of NaCl are prepared in 1 liter of distilled water to give a final concentration in the culture of 33.33 g/l. Solutions are filter sterilized and added to the culture from a sterile bottle.

Inductions usually take place on day 5 to 7 of a culture period. Stimulations usually occur within 24 hours and can continue for up to 7 days.

Detection techniques are identical to those for small-scale studies

PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT

(51) International Patent Classification ⁴ (11) International Publication Number : WO 88/ 00 C12N 5/00, C12P 17/18 A3 (43) International Publication Date: 1 1 February 1988 (11.0

(21) International Application Number: PCT/JP87/00583 (74) Agents: AOKI, Akira et al.; Seiko Toranomon Bld 10, Toranomon 1-chome, Minato-ku, Tokyo 105 (

(22) International Filing Date: 3 August 1987 (03.08.87)

(81) Designated States: BE (European patent), DE (

(31) Priority Application Number: 892,938 pean patent), FI, FR (European patent), GB ( pean patent), IT (European patent), JP, KR.

(32) Priority Date : 4 August 1986 (04.08.86)

(33) Priority Country: US Published

With international search report.

Before the expiration of the time limit for amending

(71) Applicant: MITSUI PETROCHEMICAL INDUSclaims and to be republished in the event of the recei

TRIES, LTD. [JP/JP]; 2-5, Kasumigaseki 3-chome, amendments. Chiyoda-ku, Tokyo 100 (JP).

(88) Date of publication of the international search report:

(72) Inventors: SMITH, Jane, Isobel ; 277 St. George Street,

Apartment 903, Toronto, Ontario M5R 2R1 (CA). 24 March 1988 (24.03. SMART, Nigel, John ; 4235 White Spruce Court, Mis- sissauga, Ontario L5C 3X5 (CA). MISAWA, Masana- ru ; 12 Downpatrick Crescent, Weston, Ontario M9R 4A4 (CA).

(54) Title: PROCESS FOR INDUCING SECONDARY METABOLITE PRODUCTION IN PLANT CULTU AND MEANS THEREOF

(57) Abstract

Synthesis of alkaloids by suspension cultured Catharanthus roseus cells is enhanced by stressing the cultured c osmotically by addition to the culture of such inducers as ionic osmotic stressors such as halide salts e.g. NaCl, KCl FeCl₃ or organic osmotic stressors such as sugar alcohols and sugar acids. Synthesis of alkaloids is also enhanced by a tion of anti-gibberellin compounds and other compounds such as abscissic acid which is a plant growth regulator. Yi of alkaloids are consequently increased.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the frontpages of pamphlets publishing international applications under the PCT.

AT Austria FR France ML Mali

AU Australia GA Gabon MR Mauritania

BB Barbados GB United Kingdom MW Malawi

BE Belgium HU Hungary NL Netherlands

BG Bulgaria IT Italy NO Norway

3J ___nin JP -Japan TtO Romania

BR Brazil KP Democratic People's Republic SD Sudan

CF Central African Republic ofKorea SE Sweden

CG Cor.g" KR Republic ofKorea SN Senegal

CH Switzerland LI Liechtenstein SU Soviet Union

CM Cameroon LK Sri Lanka TD Chad

DE Germany, Federal Republic of LL' Luxembourg TG Togo

DK Denmark MC Monaco US United States of America fl finland MG .Madagascar

Claims

1. A process for inducing synthesis of a secondary metabolite by plant cell which comprises osmotically stressing the cells.

2. The process according to claim 1 wherein the cells are suspension cultured and the osmotic stress is created by adding to said suspension an osmotically stressing amount of a chemical compound.

3. The process according to claim 2 wherein the cells are Catharanthus roseus cells.

4. The process according to claim 3 wherein said chemical compound is selected from those compounds which create osmotic stress ionically and organic compounds which create osmotic stress.

5. The process according to claim 3 wherein said chemical compound is a halide salt.

6. The process according to claim 5 wherein said chemical compound is a halide salt of a Group I or Group II element.

7. The process according to claim 5 wherein the compound is selected from KCl, NaCl and FeCl₃.

8. The process according to claim 3 wherein said chemical compound is a sugar acid or a sugar alcohol.

9. The process according to claim 8 wherein the chemical compound is selected from mannitol, xylitol and sorbitol.

10. The process according to claim 3 wherein the chemical compound is sorbitol.

11. A process for inducing synthesis of a secondary metabolite by plant cells which comprises growing said cells in the presence of a plant growth regulator.

12. The process according to claim 11 wherein said plant cells are grown in suspension culture supplemented with said plant growth regulator.

13. The process according to claim 12 wherein the plant growth regulator is selected from anti-gibberellin compounds and abscissic acid.

14. The process according to claim 12 wherein the plant growth regulator is abscissic acid.

15. The process according to claim 12 wherein the plant cells are Catharanthus roseus cells.

16. The process according to claim 3 further comprising the step of extracting the indole alkaloids from the plant cells.

17. The process according to claim 16 wherein said alkaloids are catharanthine and ajmalicine.

18. The process according to claim 15 further comprising the step of extracting the indole alkaloids from the plant cells.

19. The process according to claim 18 whether the extracted alkaloids are catharanthine and ajmalicine.