CA1283874C - Process for inducing secondary metabolite production in plant cultures and means therefor - Google Patents
Process for inducing secondary metabolite production in plant cultures and means thereforInfo
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- CA1283874C CA1283874C CA000543676A CA543676A CA1283874C CA 1283874 C CA1283874 C CA 1283874C CA 000543676 A CA000543676 A CA 000543676A CA 543676 A CA543676 A CA 543676A CA 1283874 C CA1283874 C CA 1283874C
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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Abstract
ABSTRACT OF THE DISCLOSURE
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 abscisic acid which is a plant growth regulator. Yields of alkaloids are consequently increased.
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 abscisic acid which is a plant growth regulator. Yields of alkaloids are consequently increased.
Description
This invention relates to plant-derived secondary metabolites and, more particularly, to a method and means for inducing plant cells to produce these metabolites.
A number of chemicals produced by plants are classified as primary metabolites since they are essential to cell function. While primary metabolites ar2 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 antlbiotics, of which the indolic alkaloids are Q~.
7~
A number of chemicals produced by plants are classified as primary metabolites since they are essential to cell function. While primary metabolites ar2 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 antlbiotics, of which the indolic alkaloids are Q~.
7~
~ ~3~
of particularly great interest. The alkaloids include many compounds, at least some of which are known to have therapeutic value. For example, tile indolic alkaloid ajmalicine is known to function as an antihypertensive/tranquilizer. Furtherl 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 rnost commonly from Catharanthus roseus (also known as Madagascar periwinkle, Vinca rosea and Lochnera rosea). Further, morphinan alkaloids e.gO
morphine and codeine, are extracted from poppy plants (Papaver somniferurn) and Cinchona alkaloids e.g. quinine and related quinoline alkaloids, are derived from Cinchona succirubra and related species. Obviously, the plant frorn 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 33~7~
extraction process both te~ious and lengthy as reflected by the relatively hi~h cost of some of these compounds. For example, indolic alkaloids are typically present in the dried Catharanthus roseus plants, from which khey 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 technologyO By use of plant tissue ~ulture (ptc) procedures, one may manipulate the capacity of plant cells to produce the secondar~y ~etabolite 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 ~2~33~
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 "Phytochemi,try", vol. 20, No. 8, pp 1841-1843 (1981), Lee et al. disclose the effect of specific amines, including 2-diethylaminoethyl-2,4-~clllorophenylether, on the ability of C. roseus cultures to produce certain indolic alkaloids. An increase in production of ajmalicine and catharanthine was notedO 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 oE 5ppm 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,Ru~a ~rav~olens~ By addition of a suspension of either living free or immobilized yeasts or of dead ~odotorula r~bra 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 (1385), Hattori and Ohta disclose the results of experiments in which suspension cultures of Red Bean (Viqna angularis) are grown, in the presence of sodium vanadate (Na3V04) 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.
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 compoun~s are pre~erably those halide salts able to ionize in solution. ~ore 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, phytic 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 com~ination 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 ~3~
anti-gibberellin compounds such as 2'-isopropyl-4'-(trimethylamrnonium chloride)-s'-methylphenyl piperidine carboxylate, ~-chloroethyltrimethylarnmonium chloride and tributyl-2,4-dichlorobenzyl phosphonium chloride. Other ABA-related compounds useful herein include ~-(dimethylamino) 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 o, 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 compo~nd. Preferably the plant cells are cultured in a suspension supplemented with the regulator.
Once the plan~ 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.
~ ~3~
Since some metabolites are secreted by plant cells into the growth medium, these metabolites may simply be separated from the cells ~ 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 ~xtraction 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 ~rushed. 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 ;~7~
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 fro~ parsley cell cultures; and mevalonic acid-derived chemicals such as saponins e.g. digitalis from Digitalis ~urpurea or _ 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 abscisic acid used herein is a known compound having the structural formula appearing below:
~3C c~3 C~3 X.~,b~
l OH
O ~ \ c~3 ~3~
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 dorrnin whose commercial nomenclature is 5-(1-hydroxy-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. lea' 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 susper,sion 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.
of particularly great interest. The alkaloids include many compounds, at least some of which are known to have therapeutic value. For example, tile indolic alkaloid ajmalicine is known to function as an antihypertensive/tranquilizer. Furtherl 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 rnost commonly from Catharanthus roseus (also known as Madagascar periwinkle, Vinca rosea and Lochnera rosea). Further, morphinan alkaloids e.gO
morphine and codeine, are extracted from poppy plants (Papaver somniferurn) and Cinchona alkaloids e.g. quinine and related quinoline alkaloids, are derived from Cinchona succirubra and related species. Obviously, the plant frorn 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 33~7~
extraction process both te~ious and lengthy as reflected by the relatively hi~h cost of some of these compounds. For example, indolic alkaloids are typically present in the dried Catharanthus roseus plants, from which khey 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 technologyO By use of plant tissue ~ulture (ptc) procedures, one may manipulate the capacity of plant cells to produce the secondar~y ~etabolite 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 ~2~33~
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 "Phytochemi,try", vol. 20, No. 8, pp 1841-1843 (1981), Lee et al. disclose the effect of specific amines, including 2-diethylaminoethyl-2,4-~clllorophenylether, on the ability of C. roseus cultures to produce certain indolic alkaloids. An increase in production of ajmalicine and catharanthine was notedO 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 oE 5ppm 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,Ru~a ~rav~olens~ By addition of a suspension of either living free or immobilized yeasts or of dead ~odotorula r~bra 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 (1385), Hattori and Ohta disclose the results of experiments in which suspension cultures of Red Bean (Viqna angularis) are grown, in the presence of sodium vanadate (Na3V04) 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.
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 compoun~s are pre~erably those halide salts able to ionize in solution. ~ore 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, phytic 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 com~ination 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 ~3~
anti-gibberellin compounds such as 2'-isopropyl-4'-(trimethylamrnonium chloride)-s'-methylphenyl piperidine carboxylate, ~-chloroethyltrimethylarnmonium chloride and tributyl-2,4-dichlorobenzyl phosphonium chloride. Other ABA-related compounds useful herein include ~-(dimethylamino) 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 o, 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 compo~nd. Preferably the plant cells are cultured in a suspension supplemented with the regulator.
Once the plan~ 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.
~ ~3~
Since some metabolites are secreted by plant cells into the growth medium, these metabolites may simply be separated from the cells ~ 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 ~xtraction 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 ~rushed. 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 ;~7~
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 fro~ parsley cell cultures; and mevalonic acid-derived chemicals such as saponins e.g. digitalis from Digitalis ~urpurea or _ 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 abscisic acid used herein is a known compound having the structural formula appearing below:
~3C c~3 C~3 X.~,b~
l OH
O ~ \ c~3 ~3~
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 dorrnin whose commercial nomenclature is 5-(1-hydroxy-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. lea' 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 susper,sion 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.
3~d~
Abscisic acid may be mixed with water and a few ~rops of base such as KOH to encourage dissolution if necessary.
While trace amounts c~r 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 presen_e of the subject inducers increases the amount of secondary metabolite produced and can significantly decrease the length`~f time required to obtain that particular amount.
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 no~ ~o de~ract 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 ~M*, JOH
and LBE-l. Lines JWM* and JOH were developed at the Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. Line LBE-l 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 o~ its components in Phys. Plantarum ~18) 1964 pp.
lQ0-127. The components of SH medium (Shenck-Hildebrandt), preferred for use herein, are described in Can. J. Bot. 50:
195 ~04 (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 oC
-naphthaleneacetic acid, in a concentration of 2mg/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 NaC1, KCl ~and FeC13 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 O.lM, 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 ~hich 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 O.OlM to l.OM and, ideally, at around 0.5M.
Ferric chloride is suitably added to the suspension medium to achieve a final concentration of from 10-500ppm, more preferably from 20-20Oppm and, ideally, around 5Oppm.
Broth concentrations of sorbitol range from about 0.05M
to a maximum concentration just slightly below the concentration 33~
at which plasmolysis occurs, as c~iscussed wi~h respect to NaCl or KCl addi~ion. Preferred broth concentrations range from O.lM
to O~M and ideally at around 0.2M.
Under the experimental conditions described hereinafter, the ideal concentration of abscisic acid ranges from O.lmg - 0.5mg per 60mls of suspensionO Concentrations as low as O.Olmg may be used although the induction response may be unfavourably low. Concentrations higher than 0.5mg/60mls may also be used although there appears to be little enhancement of the efect seen at 0.5mg/60m1s.
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 hereinafte~
with reference to the following examples. In these examples, 60ml cell suspension cultures are maintained in a basic MS
growth medium containing 3%(w/v) sucrose, 2mg/1 ~ -naphthalenic acid and O.lmg kinetin. The cultures are maintained in 250ml Erlenmeyer flasks on an illuminated rotary shaker (c.120rpmj and subcultured weekly by a one to five dilution.
Abscisic acid and D-sorbitol were obtained from Sigma Chemical Company and NaCl, KCl and FeC13 from Fisher Scientific Limited.
All inducers were prepared as stock solutions dissolved in distilled water at such a concentration that when lml 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 ceric ammonium sulphate following TLC
separation.
~xr~
Example 1 - ABA Induction Synthetic 99% pure abscisic acid was introduced in a range of concentrations from 0.1 - 2.0mg/60ml culture. ~rhe results generated appear in Tables 1 - 4 below. Table 1 shows the effect which the type and concentration of ABA has on tAe induction process as applied to cell line JOH. While the 99~
pure (+)cis-trans synthetic isomer exhibited superior results, all forms of ABA showed desirable inducing properties.
Effects of diferent grades/types of ABA
CatharanthineAjmalicine (m~/l) (mg/l) 1. natural ABA 29.7 23.0 (~)cis-trans isomer 2. synthetic ABA
a) 99% + pure 35.8 8.9 (+~cis-trans isomer b) 99~ pure 28.6 9.1 (+)cis-trans isomer c) 90% pure 31.0 8O3 (mixed isomers) Control 17.4 1.8 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.Smg/60ml culture with only minor variation with increased concentration.
~ ~2~33l3~L
Conc. ABACatharanthine Ajmalicine (m~60ml _lture) (mg/l) _ (m~/l) 0.1 5.8 2.6 0.5 7.3 4.5 1.0 7.5 6.3 2.0 6.7 8.0 Control 4.2 1.6 In Table 3 below, the relationshi2 of cell age and exposure to ABA is shown. It will be noted that a'c least 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 ~OH and an induction period of 4 days.
Cell age at induction Fresh Weight Catharanthine Ajmalicine (day) _ (g)(mg/l) (mg/l) 17.823.1 12.2 Control 18.514.3 1.5 2 14.812.1 2.8 Control 15.48.1 1.1 3 17.926.4 38.8 Control 23.116.1 2.3 4 17.527.3 28.2 Control 21.813.5 6.2 19.720.3 20.6 Control 24.413.6 3.0 6 23.030.1 9.2 Control 22.714.2 ~2~33~7~
Table 4 below shows the response to lrng AB~ 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 ~hat lines JOH and LBE-l are preferred.
Linelmg ABA? Catharanthine Ajmalicine - (m~ (mg/l) JWM C - 1.9 0.5 + 5.1 6.7 C - 9.5 27.7 + 10.9 25.2 JOH ("M") - 1.0 0.9 + 35.1 25.6 JWO - 6.6 27.4 + 12.5 18.5 J~M - 5.1 30.2 + 12.0 16.5 LD-2 - 27.9 15.5 + 7.0 15.9 CPD-l - 7.1 6.4 + 7.2 6.8 LBE-l - 20.3 13.2 ~ 38.0 31.4 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 cell.s once some increase in kiomass has occurred.
Example 2 - Induction by NaCl A range of sodium chloride concentrations (from 0.1 -1.0 g/60ml 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.
Conc. NaC1Fresh WeightCatharanthine Ajmalicine (g/60ml culture)~g) (mg/l) (mg/l)_ loO 5.1 3.4 1.3 0~5 12~5 7~1 5~6 0~2 16~4 16~6 8~4 0~1 18~2 21~6 7~2 Control 21~ 8 5.4 6 ~ 2 Example 3 - Induction by Potassium Chloride 0.1 or 0. 29 of KCl were added to the cells of line JOH. The effects on alkaloid yields are shown in Table 6~
r Conc. KCl Fresh WeightCatharanthine Ajmalicine (g~60ml culture) (g) (mg/l) (m~/l) 0~2 24~1 42~3 1~8 0~1 24~3 8~6 0~9 Control 20~ 8 3 ~1 0~ 3 ~ 22 ~
-- ~L2~3~
Example 4 - Induction by Ferric Chloride The stimulatory effect of 3mg/1 FeC13 on alkaloid accumulation is shown in Table 7. Line JOH was used.
Fresh Weight Catharanthine Ajmalicine ~g) (my/l) (mg/l) 3mg/6Oml culture 15.3 28.0 7.9 Control 23.6 17.4 1.8 Example 5 - Induction by Sorbitol A range of concentrations of sorbitol were tested for their effects on alkaloid accumulation (0.1 - 0.5M final concentration; 1.1 - 5.5g/60ml culture). ~heir effects on line JOH are shown in Table 8 below.
SorbitolFresh ~eiqht Catharanthine Ajmalicine (M) (g) (mg/l) (m~/l) 0.5 14.5 6.1 0.4 14.6 13.1 0.6 0.3 18.3 14.9 1.7 0.2 15.4 29.2 2.4 0.1 18.1 30.7 0.6 Control 27.7 18.9 The major advantaye obtained by induciny cell cultures 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 lmg/l ~-napthalene acetic acid and O.lmg/l kinetin. All lines and chemicals remain the same.
Example 9 - Larger Scale Induction Process For 30 litre batches using ABA, solutions are preferably prepared to give 8.33mg/1 in the final culture fluid. Solutions are prepared in 1 litre of distilled water, filter sterilized and injected into the fermenter from a sterile bottle. Solutions of NaCl are prepared in 1 litre of distilled water to give a final concentration in the culture of 33.33g/1.
Solutions are filter sterilized and added to the culture from a sterile bottle.
Inductions usually take place on day S to 7 of a culture period. Stimulations usually occur within 24 hours and can continue for up to 7 days.
. - 24 -Detection techniques are identical to those for small-scale studies.
F~pt 30 litres. Line JOH - C. roseus Induction at 5 days with ABA
Inoculated with 3 litres of 13 day cells Fresh weight gl~l Catharanthine mgl~
Day 1 21.42 --Day 2 53.29 --Day 5 89.85 0.1 Day 6 109.29 0.205 Day 7 122.98 7.5 Day 9 2~0.2 13.22 Day 12 340 26~25 Expt. 3OL JOH (M) Day 7 induction with ABA
Catharanthine (mgl~l) ~resh weight (gl~l) Day 5 0.23 mgl~l 127 Day 6 0.622 206.9 Day 7 0.649 239.3 Day 8 44.22 320.83 Day 9 84.86 333.58 Day 10 85.25 409.75 Day 11 43.53 442.29 Day 12 38.53 550.61 ~L2~33~7~
__ Expt 10 litres. Line JOH* - C. roseus Induction at 6 days with ABA
Inoculated with 1 litre of 8 day old cells Fresh weight gl-l Catharanthine mgl~
Day 0 18 . 6 -- -Day 2 29. 71 ---Day 4 62.74 ---Day 5 105.88 ---Day 6 190.00 ---Day 8 257.45 6.3 Day 10 249.8 4.6 Day 11 217.1 4. 5
Abscisic acid may be mixed with water and a few ~rops of base such as KOH to encourage dissolution if necessary.
While trace amounts c~r 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 presen_e of the subject inducers increases the amount of secondary metabolite produced and can significantly decrease the length`~f time required to obtain that particular amount.
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 no~ ~o de~ract 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 ~M*, JOH
and LBE-l. Lines JWM* and JOH were developed at the Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. Line LBE-l 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 o~ its components in Phys. Plantarum ~18) 1964 pp.
lQ0-127. The components of SH medium (Shenck-Hildebrandt), preferred for use herein, are described in Can. J. Bot. 50:
195 ~04 (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 oC
-naphthaleneacetic acid, in a concentration of 2mg/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 NaC1, KCl ~and FeC13 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 O.lM, 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 ~hich 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 O.OlM to l.OM and, ideally, at around 0.5M.
Ferric chloride is suitably added to the suspension medium to achieve a final concentration of from 10-500ppm, more preferably from 20-20Oppm and, ideally, around 5Oppm.
Broth concentrations of sorbitol range from about 0.05M
to a maximum concentration just slightly below the concentration 33~
at which plasmolysis occurs, as c~iscussed wi~h respect to NaCl or KCl addi~ion. Preferred broth concentrations range from O.lM
to O~M and ideally at around 0.2M.
Under the experimental conditions described hereinafter, the ideal concentration of abscisic acid ranges from O.lmg - 0.5mg per 60mls of suspensionO Concentrations as low as O.Olmg may be used although the induction response may be unfavourably low. Concentrations higher than 0.5mg/60mls may also be used although there appears to be little enhancement of the efect seen at 0.5mg/60m1s.
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 hereinafte~
with reference to the following examples. In these examples, 60ml cell suspension cultures are maintained in a basic MS
growth medium containing 3%(w/v) sucrose, 2mg/1 ~ -naphthalenic acid and O.lmg kinetin. The cultures are maintained in 250ml Erlenmeyer flasks on an illuminated rotary shaker (c.120rpmj and subcultured weekly by a one to five dilution.
Abscisic acid and D-sorbitol were obtained from Sigma Chemical Company and NaCl, KCl and FeC13 from Fisher Scientific Limited.
All inducers were prepared as stock solutions dissolved in distilled water at such a concentration that when lml 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 ceric ammonium sulphate following TLC
separation.
~xr~
Example 1 - ABA Induction Synthetic 99% pure abscisic acid was introduced in a range of concentrations from 0.1 - 2.0mg/60ml culture. ~rhe results generated appear in Tables 1 - 4 below. Table 1 shows the effect which the type and concentration of ABA has on tAe induction process as applied to cell line JOH. While the 99~
pure (+)cis-trans synthetic isomer exhibited superior results, all forms of ABA showed desirable inducing properties.
Effects of diferent grades/types of ABA
CatharanthineAjmalicine (m~/l) (mg/l) 1. natural ABA 29.7 23.0 (~)cis-trans isomer 2. synthetic ABA
a) 99% + pure 35.8 8.9 (+~cis-trans isomer b) 99~ pure 28.6 9.1 (+)cis-trans isomer c) 90% pure 31.0 8O3 (mixed isomers) Control 17.4 1.8 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.Smg/60ml culture with only minor variation with increased concentration.
~ ~2~33l3~L
Conc. ABACatharanthine Ajmalicine (m~60ml _lture) (mg/l) _ (m~/l) 0.1 5.8 2.6 0.5 7.3 4.5 1.0 7.5 6.3 2.0 6.7 8.0 Control 4.2 1.6 In Table 3 below, the relationshi2 of cell age and exposure to ABA is shown. It will be noted that a'c least 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 ~OH and an induction period of 4 days.
Cell age at induction Fresh Weight Catharanthine Ajmalicine (day) _ (g)(mg/l) (mg/l) 17.823.1 12.2 Control 18.514.3 1.5 2 14.812.1 2.8 Control 15.48.1 1.1 3 17.926.4 38.8 Control 23.116.1 2.3 4 17.527.3 28.2 Control 21.813.5 6.2 19.720.3 20.6 Control 24.413.6 3.0 6 23.030.1 9.2 Control 22.714.2 ~2~33~7~
Table 4 below shows the response to lrng AB~ 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 ~hat lines JOH and LBE-l are preferred.
Linelmg ABA? Catharanthine Ajmalicine - (m~ (mg/l) JWM C - 1.9 0.5 + 5.1 6.7 C - 9.5 27.7 + 10.9 25.2 JOH ("M") - 1.0 0.9 + 35.1 25.6 JWO - 6.6 27.4 + 12.5 18.5 J~M - 5.1 30.2 + 12.0 16.5 LD-2 - 27.9 15.5 + 7.0 15.9 CPD-l - 7.1 6.4 + 7.2 6.8 LBE-l - 20.3 13.2 ~ 38.0 31.4 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 cell.s once some increase in kiomass has occurred.
Example 2 - Induction by NaCl A range of sodium chloride concentrations (from 0.1 -1.0 g/60ml 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.
Conc. NaC1Fresh WeightCatharanthine Ajmalicine (g/60ml culture)~g) (mg/l) (mg/l)_ loO 5.1 3.4 1.3 0~5 12~5 7~1 5~6 0~2 16~4 16~6 8~4 0~1 18~2 21~6 7~2 Control 21~ 8 5.4 6 ~ 2 Example 3 - Induction by Potassium Chloride 0.1 or 0. 29 of KCl were added to the cells of line JOH. The effects on alkaloid yields are shown in Table 6~
r Conc. KCl Fresh WeightCatharanthine Ajmalicine (g~60ml culture) (g) (mg/l) (m~/l) 0~2 24~1 42~3 1~8 0~1 24~3 8~6 0~9 Control 20~ 8 3 ~1 0~ 3 ~ 22 ~
-- ~L2~3~
Example 4 - Induction by Ferric Chloride The stimulatory effect of 3mg/1 FeC13 on alkaloid accumulation is shown in Table 7. Line JOH was used.
Fresh Weight Catharanthine Ajmalicine ~g) (my/l) (mg/l) 3mg/6Oml culture 15.3 28.0 7.9 Control 23.6 17.4 1.8 Example 5 - Induction by Sorbitol A range of concentrations of sorbitol were tested for their effects on alkaloid accumulation (0.1 - 0.5M final concentration; 1.1 - 5.5g/60ml culture). ~heir effects on line JOH are shown in Table 8 below.
SorbitolFresh ~eiqht Catharanthine Ajmalicine (M) (g) (mg/l) (m~/l) 0.5 14.5 6.1 0.4 14.6 13.1 0.6 0.3 18.3 14.9 1.7 0.2 15.4 29.2 2.4 0.1 18.1 30.7 0.6 Control 27.7 18.9 The major advantaye obtained by induciny cell cultures 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 lmg/l ~-napthalene acetic acid and O.lmg/l kinetin. All lines and chemicals remain the same.
Example 9 - Larger Scale Induction Process For 30 litre batches using ABA, solutions are preferably prepared to give 8.33mg/1 in the final culture fluid. Solutions are prepared in 1 litre of distilled water, filter sterilized and injected into the fermenter from a sterile bottle. Solutions of NaCl are prepared in 1 litre of distilled water to give a final concentration in the culture of 33.33g/1.
Solutions are filter sterilized and added to the culture from a sterile bottle.
Inductions usually take place on day S to 7 of a culture period. Stimulations usually occur within 24 hours and can continue for up to 7 days.
. - 24 -Detection techniques are identical to those for small-scale studies.
F~pt 30 litres. Line JOH - C. roseus Induction at 5 days with ABA
Inoculated with 3 litres of 13 day cells Fresh weight gl~l Catharanthine mgl~
Day 1 21.42 --Day 2 53.29 --Day 5 89.85 0.1 Day 6 109.29 0.205 Day 7 122.98 7.5 Day 9 2~0.2 13.22 Day 12 340 26~25 Expt. 3OL JOH (M) Day 7 induction with ABA
Catharanthine (mgl~l) ~resh weight (gl~l) Day 5 0.23 mgl~l 127 Day 6 0.622 206.9 Day 7 0.649 239.3 Day 8 44.22 320.83 Day 9 84.86 333.58 Day 10 85.25 409.75 Day 11 43.53 442.29 Day 12 38.53 550.61 ~L2~33~7~
__ Expt 10 litres. Line JOH* - C. roseus Induction at 6 days with ABA
Inoculated with 1 litre of 8 day old cells Fresh weight gl-l Catharanthine mgl~
Day 0 18 . 6 -- -Day 2 29. 71 ---Day 4 62.74 ---Day 5 105.88 ---Day 6 190.00 ---Day 8 257.45 6.3 Day 10 249.8 4.6 Day 11 217.1 4. 5
Claims (21)
1. A process for stimulating the production of secondary metabolites by plant cells which comprises:
(1) growing the cells in a nutrient suspension until the cell population reaches the logarithmic or early stationary growth phase;
(2) adding to the nutrient suspension a sufficient amount of chemical compound which is non-toxic to the cells to produce an osmotic gradient between the cells and the suspension;
(3) continuing the growth of the cells to accumulate a concentration of the secondary metabolites which is higher than the concentration which could be achieved in the absence of said osmotic gradient, the maximum osmotic pressure of the osmotic gradient being insufficient to cause plasmolysis of the cells.
(1) growing the cells in a nutrient suspension until the cell population reaches the logarithmic or early stationary growth phase;
(2) adding to the nutrient suspension a sufficient amount of chemical compound which is non-toxic to the cells to produce an osmotic gradient between the cells and the suspension;
(3) continuing the growth of the cells to accumulate a concentration of the secondary metabolites which is higher than the concentration which could be achieved in the absence of said osmotic gradient, the maximum osmotic pressure of the osmotic gradient being insufficient to cause plasmolysis of the cells.
2. The process according to claim 1 wherein the cells are Catharanthus roseus cells.
3. The process according to claim 2 wherein said chemical compound is selected from those compounds which create osmotic stress ionically and those non-ionic organic compounds which create osmotic stress.
4. The process according to claim 1 wherein said chemical compound is a halide salt.
5. The process according to claim 4 wherein said chemical compound is a halide salt of a Group I or Group II element.
6. The process according to claim 4 wherein the compound is selected from KCl, NaCl and FeCl3.
7. The process according to claim 1 wherein said chemical compound is a sugar acid or a sugar alcohol.
8. The process according to claim 7 wherein the chemical compound is selected from mannitol, xylitol and sorbitol.
9. The process according to claim 8 wherein the chemical compound is sorbitol.
10. The process according to claim 3 where in the compounds which create osmotic stress ionically have a concentration of at least about 0.01 M and the non-ionic, organic compounds which create osmotic stress have a concentration of at least about 0.05 M.
11. The process according to claim 10 wherein the compounds which create osmotic stress ionically have a concentration of 0.5 M and the non-ionic, organic compounds which create osmotic stress have a concentration of 0.2 M.
12. A process for stimulating the production of secondary metabolites by plant cells which comprises:
(1) growing the cells in a nutrient suspension until the cell population reaches the logarithmic or early stationary growth phase;
(2) adding to the nutrient suspension a sufficient amount of a plant growth regulator which is non-toxic to the cells to produce an osmotic gradient between the cells and the suspension;
(3) containing the growth of the cells to accumulate a concentration of secondary metabolites which is higher than the concentration which could be achieved in the absence of said osmotic gradient, the maximum pressure of the osmotic gradient being insufficient to cause plasmolysis of the cells.
(1) growing the cells in a nutrient suspension until the cell population reaches the logarithmic or early stationary growth phase;
(2) adding to the nutrient suspension a sufficient amount of a plant growth regulator which is non-toxic to the cells to produce an osmotic gradient between the cells and the suspension;
(3) containing the growth of the cells to accumulate a concentration of secondary metabolites which is higher than the concentration which could be achieved in the absence of said osmotic gradient, the maximum pressure of the osmotic gradient being insufficient to cause plasmolysis of the cells.
13. The process according to claim 12 wherein the plan growth regulator is selected from anti-gibberellin compounds and abscisic acid.
14. The process according to claim 13 wherein the plant growth regulator is abscisic acid.
15. The process according to claim 12 wherein the plant cells are Catharanthus roseus cells.
16. The process according to claim 1 including the step of extracting the secondary metabolites from the plant cells.
17. The process according to claim 16 wherein the secondary metabolites are catharanthine and ajmalicine.
18. The process according to claim 12 including the step of extracting the secondary metabolites from the plant cells.
19. The process according to claim 18 wherein the extracted secondary metabolites are catharanthine and ajmalicine.
20. The process according the claim 14 wherein the abscisic acid concentration is at least 6 x 10-6 M.
21. The process according to claim 20 wherein the abscisic acid concentration is 3.14 x 10-5 M.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89293886A | 1986-08-04 | 1986-08-04 | |
| US892,938 | 1986-08-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1283874C true CA1283874C (en) | 1991-05-07 |
Family
ID=25400744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000543676A Expired - Lifetime CA1283874C (en) | 1986-08-04 | 1987-08-04 | Process for inducing secondary metabolite production in plant cultures and means therefor |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0277250A1 (en) |
| JP (1) | JP2845443B2 (en) |
| KR (1) | KR900004437B1 (en) |
| CA (1) | CA1283874C (en) |
| FI (1) | FI881491A0 (en) |
| WO (1) | WO1988000968A2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0378921A3 (en) * | 1988-12-21 | 1990-09-19 | Idaho Research Foundation, Inc. | Enhancement of plant metabolite production by timed elicitation |
| AU2003215781A1 (en) | 2002-03-20 | 2003-09-29 | Rachid Ennamany | Method for production of phytoalexins |
| WO2012074502A1 (en) * | 2010-11-29 | 2012-06-07 | Chayil Technologies, Llc | Secondary metabolite stimulation in photoautotrophic cultures |
| ES2638888T3 (en) * | 2011-03-27 | 2017-10-24 | Arch Chemicals, Inc. | Modulation of epigenetic DNA methylation to cause cells to adopt DNA methylation patterns associated with young cells |
| WO2016120713A2 (en) | 2015-01-30 | 2016-08-04 | Naolys Sarl | Topical composition and method for preparing same |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2300131A1 (en) * | 1975-02-04 | 1976-09-03 | Synthelabo | Alkaloid prodn by culturing Vinca minor cells - in undifferentiated state in liq medium (BE030876) |
| FR2470603A1 (en) * | 1979-12-07 | 1981-06-12 | Synthelabo | Anti-mitotic extracts of Catharanthus roseus G-Don - two new strains cr-1 and cr-2 produce extracts of high activity and low toxicity, whose activity is compared with that of vinblastine |
-
1987
- 1987-08-03 EP EP87904973A patent/EP0277250A1/en not_active Withdrawn
- 1987-08-03 KR KR1019880700360A patent/KR900004437B1/en not_active IP Right Cessation
- 1987-08-03 JP JP62504568A patent/JP2845443B2/en not_active Expired - Lifetime
- 1987-08-03 WO PCT/JP1987/000583 patent/WO1988000968A2/en not_active Application Discontinuation
- 1987-08-04 CA CA000543676A patent/CA1283874C/en not_active Expired - Lifetime
-
1988
- 1988-03-30 FI FI881491A patent/FI881491A0/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0277250A1 (en) | 1988-08-10 |
| WO1988000968A2 (en) | 1988-02-11 |
| KR880701774A (en) | 1988-11-05 |
| FI881491A (en) | 1988-03-30 |
| JP2845443B2 (en) | 1999-01-13 |
| WO1988000968A3 (en) | 1988-03-24 |
| FI881491A0 (en) | 1988-03-30 |
| JPH01500401A (en) | 1989-02-16 |
| KR900004437B1 (en) | 1990-06-25 |
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