GB1595059A

GB1595059A - Tropane alkaloids

Info

Publication number: GB1595059A
Application number: GB46866/76A
Authority: GB
Original assignee: Saint Firmin A R
Current assignee: Saint Firmin A R
Priority date: 1976-11-10
Filing date: 1976-11-10
Publication date: 1981-08-05

Description

(54) TROPANE ALKALOIDS (71) I, ANNETTE RENE SAINT-FIRMIN, a citizen of Haiti, of 44 Underwood Place, N.W. Washington D.C. 20012, United States of America, do hereby declare the invention for which I pray that a Patent may be granted to me, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to the production of tropane alkaloids.

Tropane alkaloids are found in nature in various members e.g. of the plant family Solanaceae, from which the alternative designation of solanaceous alkaloids is derived. The three main tropane alkaloids are atropine, hyoscine (scopolamine), and hyoscyamine.

The alkaloid atropine can be isolated from the plant deadly nightshade (Atropa belladonna). The related alkaloid scopolamine (hyoscine) which has "truth drug" properties can be isolated from the plant species Scopolia spp. The third main member of the tropane group of alkaloids is hyoscyamine and this can be isolated from plants of the species Hyoscyamus spp.

These solanaceous alkaloids are also found in the plant Hyoscyamus niger, which grows in Europe, together with various other chemically related alkaloids of the group in minor amounts. However the alkaloid content of Hyoscyamus niger is too low for commercial extraction.

The tropane alkaloids have well known medical properties. Indeed it is related that, as long ago as 1500 BC, plants producing alkaloids of the atropine group were used for criminal, magic or medicinal purposes. The tropane alkaloids are in demand not only by virtue of their own medical properties but also as starting materials for preparing other active compounds for use in medicine and in biological research.

Various members of the plant family Solanaceae are known to contain atropine, scopolamine and hyoscyamine and related tropane alkaloids, more particularly plants of the species Hyoscyamus spp. and Duboisia spp. Besides Hyoscyamus niger mentioned above, there can also be mentioned Hyoscyamus muticus L. and Hyoscyamus aureus L.

Since the plants which are known to be rich in tropane alkaloids mostly originate from tropical or subtropical regions, current commercial methods of extracting atropine group alkaloids from natural sources rely on dried plant material as the raw material. Such dried material can be produced by oven-drying, for example at approximately 40"C, or by freeze-drying. However this is a relatively inefficient and expensive method of obtaining the desired alkaloids since one must wait for the plant to reach maturity before harvesting and drying of the plant can take place. The yield of alkaloids is affected by the climatic and edaphic factors under which the crop is grown. In particular the crop is prone to storm damage, storms being generally more frequent in the tropics than in the more temperate regions such as Europe, and such storm damage may tend to reduce the alkaloid content of the plants. Furthermore, the percentage of alkaloids in different crops of the same plant may vary considerably from year to year and from location to location due to differences, for example, in- rainfall and in soil conditions under which the crop is grown. Moreover, the percentage of total alkaloids in the dried plant material may vary considerably at the time of extraction due to differences between chronological and physiological age, as well as to variations in storage conditions and in the conditions under which the harvested plants are transported.

I have now discovered a process whereby tropane alkaloids can be produced via a cell culture which can be grown reproducibly under controlled environmental conditions to give as high alkaloid content, for example, as can be obtained in good dried plant material. In many cases the alkaloid contents are higher than are normally found in dried plant material.

Accordingly the present invention provides a process for the production of tropane alkaloids from plant material of a tropane alkaloid-producing plant of the family Solanaceae, comprising (a) inducing mitotic cell formation from said plant material; (b) providing a cell culture therefrom and (c) inducing differentiation of the cells in the culture under controlled light and temperature conditions, said cell culture being irradiated with light that is richer in the far red than in the red, the spectral range being not greater than 730nm and at energy levels within the range 2000 to 400,000 ergs per sq cm per second and temperatures in the range 12-27"C.

Preferably the plant material is from a plant of one of the following tribes of plants: Solanae, Daturae, Salpiglossidae, Nicandrae, and Cestrae (Classification of Wettstein (1887), Emberger (1960), Melchior (1964) and especially one of the genera Hyoscyamus and Duboisia. Worthy of particular mention are Hyoscyamus muticus L. Hyoscyamus aureus L., Duboisia australica, and Duboisia myoporoides The plant material may take a variety of explant forms, for example, leaf, stem root, anther, pollen grain, petal, or hair material. Thus suitable plant material includes explant inter-nodal stem material, preferably from floral stems. Altneratively, and preferably, the plant material comprises seeds or seedlings.

Mitotic cell formation can be induced by various methods. One method for inducing mitotic formation e.g. callus formation comprises cutting material from a whole plant, and contacting the resulting explant with a suitable culture medium, preferably under controlled aseptic environmental conditions. This method is suitable for use, for example, with stem segment explants. Preferably inter-nodal segment explants are taken and contacted with the culture medium. Inter-nodal floral stem segments are particularly suitable for this purpose.

Under favourable conditions callus cells form at the cut ends of the explants. Using floral stem explants it is not necessary to add any auxin, cytokinin or gibberellin to the culture medium. If the explant originates from another part of the plant it will usually be desirable to incorporate in the cell culture medium during callus cell formation a mixture of an auxin and a cytokinin in low concentration.

For best results the mother plant from which the explant is taken should be in good physiological condition. I prefer to grow the mother plant by hydroponic culture using a conventional mineral solution of controlled pH, i.e. of pH about 5.1 to about 5.4. 1 also prefer to control the temperature and the light conditions. Humidity conditions of about 70% relative humidity are suitable.

An alternative, and preferred method, for inducing, e.g. callus formation involves hormonal treatment of seedlings of the chosen plant. According to this method, seeds of the plant are allowed to germinate and produce seedlings whose growth is modified by a first auxin. After a period of growth under the influence of the chosen first auxin, the seedlings are then subjected to the influence of a second auxin. If the two auxins are appropriately chosen, callus cell formation is induced within a further period of growth after the change to the second auxin without any need to cut the seedling.

A preferred process in accordance with the invention thus comprises treating seedlings with a first auxin, allowing roots to develop under the influence of the first auxin, treating the thus-treated seedlings with a second auxin different from the first auxin, to cause mitotic cells to develop, and then culturing the mitotic cells thus produced. The cell culture may then be worked up to recover the tropane alkaloids therefrom.

I have found that it is best to work up a cell culture that contains at least a substantial proportion of re-differentiated plantlets. Thus the culture can be grown and, if need be propagated, until such time as re-differentiated plantlets develop spontaneously. However I prefer to induce re-differentiation by appropriate control of the environmental factors under which the cell culture is grown as will be described hereafter.

Using this preferred dual auxin treatment process I have found that the period required for obtaining alkaloids starting from seeds is shorter than if I use the route which involves inducing mitotic cell formation from an explant (using, for example, an inter-nodal floral stem segment explant).

The auxin may be a naturally occurring auxin but is preferably a synthetic auxin. Typical synthetic auxins include but are not limited to 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4,5-trichlorophenoxyacetic acid (2 ,4,5-T), 4-(2,4-dichlorophenoxy)butyric acid (4-(2,4 DB)); 2-(2,4-dichlorophenoxy)propionic acid (2-(2,4-DP)); sodium 2,3,6- trichlorophenylacetate; sodium 2-(2,4,5-trichlorophenoxy)ethyl sulphate; sodium 2-(2,4dichlorophenoxy)ethyl sulphate; sodium 3-(2,4,5-trichlorophenoxy)propionate, 3-indole butyric acid; 1-naphthaleneacetamide and 1-naphthalene acetic acid (NAA). As an example of a naturally occurring auxin there may be mentioned 3-indoleacetic acid (IAA).

When treating seedlings with an auxin I prefer to use also a cytokinin. Examples of suitable cytokinins are kinetin, zeatin and benzyladenine. Typically an amount of cytokinin is used in the range of from about 0.0001 micrograms up to about 0.01 mg per litre. Usually it is satisfactory to use amounts of cytokinin in the range of from about 0.001 mg to about 0.004 mg per litre.

It is also possible to include, in addition to the chosen auxin and the chosen cytokinin, a gibberellin such as gibberellic acid. However, if a gibberellin is included, then lesser amounts of auxin and cytokinin should be used than in the absence of the gibberellin.

If a cytokinin, and possibly also a gibberellin is included in the growth medium together with the first auxin, than I have found it desirable to include it or them also in the growth medium containing the second auxin. .In this way the only change made to the medium during callus cell formation is the change of auxin.

When the chosen plant material comprises seeds, these are preferably first disinfected by any of the known techniques, e.g. by contact with bromine water or with a dilute solution of sodium hypochlorite. The seeds are then allowed to germinate under sterile conditions, preferably under controlled environmental conditions. In one technique the resulting seedlings are then preferably transplanted on to a suitable solid medium. The controlled environmental conditions are preferably maintained after transplantation. The medium used should contain those mineral salts required for development of the plant in appropriate quantities and also the first auxin, e.g. NAA. The medium preferably also contains traces of vitamins. Any conventional solid plant growth medium can be used to which the first auxin is added.

The period of time during which the seedlings are exposed to the first auxin may vary from about 1 to about 20 days but preferably is in the region of from about 5 to about 15 days. From about 8 to about 12 days is generally suitable under appropriately controlled environmental conditions, e.g. about 10 days. During this period of growth vigorous development of roots is generally observed.

After this period of growth in the presence of the first auxin the seedlings are exposed to the second auxin, e.g. 2,4-D. This can be effected by transplanting the seedling onto a similar solid medium which is essentially identical to that previously used except for the auxin.

Liquid culture techniques can be used in place of solid culture techniques. For example the seeds can be germinated in a test tube on a filter paper "bridge" dipping into water.

After the seedling has grown to a suitable size the water is replaced by a suitable liquid medium containing the first auxin. The change of auxin is particularly simply achieved when a liquid culture technique is employed since in this case it is merely necessary to drain off the liquid medium containing the first auxin and introduce a similar liquid medium containing the second auxin.

The first and second auxins can be used in amounts of, for example, about 0.0001 mg up to about 1 mg per litre, more usually in the region of about 0.0005 mg to 0.2 mg per litre.

Frequently it is sufficient to use amounts of auxin within the range of from about 0.0005 mg to about 0.1 mg per litre. Less than about 0.0005 mg per litre of auxin does not usually produce any significant effect. Since auxins differ in potency the optimum amount to be used will vary with the type of plant and from auxin to auxin. Care must be taken not to use too much auxin because many synthetic auxins have a herbicidal action at higher concentrations.

Mitotic cell formation generally becomes evident amongst at least a proportion of the seedlings after a further period of growth under the influence of the second auxin. This period may vary according to the environmental conditions under which the seedlings are grown but under favourable conditions may be within the range of from about 5 days to about 10 days. After the first observance of e.g. callus formation growth in the presence of the second auxin may be continued for a further period of, for example, about 5 days to about 10 days until a convenient stage for the next process step is achieved.

In this next process step at least some of the callus cells, whether induced from an explant or from seedlings, are transferred to a liquid cell culture medium and incubated, preferably with shaking or some other suitable form of agitation in order to prevent settling or "clumping" and to facilitate gas uptake and release. Incubation is again preferably accomplished under controlled environmental conditions. In this way a single cell culture of plant cells from the chosen plant is produced. Incubation conveniently proceeds for a period of time of from about 10 days to about 14 days following which the cells may be harvested for extraction of their alkaloid content. Alternatively the culture may be propagated by repeated subculturing.

Differentiation of the cells may or may not occur spontaneously during the primary incubation period or during one of the sub-culturing steps. Thus a few of the cells may become differentiated, particularly if cell culture is not wholly effected in the dark, leading to development of a greenish or reddish tint to the cell. Upon further culture embryoids develop from such differentiated cells. However, I have discovered that cell differentiation can be positively induced by appropriate control of the environmental conditions, and particularly of the lighting conditions.

I have found that the callus cells contain only traces or tropane alkaloids. However, cells can synthesise tropane alkaloids after re-differentiation has occurred. Thus redifferentiated cells will usually be found to have alkaloid contents of between about 0.8% and about 1% by weight based upon the harvested dried cell culture material, i.e. of the same order of magnitude as the alkaloid content of whole plant material. Further, I have found that, if the re-differentiated cells develop to form embryoids, the alkaloid content of the harvested material normally increases to a somewhat higher percentage than this. If the development of the new plantlets is allowed to continue beyond the embryo stage the alkaloid content of the harvested material falls to a lower percentage again. Starting from Hyoscyamus muticus L. or Hyoscyamus aureus L. I have obtained from embryoidcontaining cell cultures yields or alkaloid corresponding to between about 1% and about 5% by weight based on dried harvested plant cell material. In the case of embryoidcontaining cultures obtained starting from Duboisia australica or Duboisia myoporoides the alkaloid yield may be as high as about 8% to about 10% by weight based on dried harvested cell material.

Harvesting is thus best effected soon after re-differentiation has occurred.

Tropane alkaloids are usually most easily obtained when the majority of the cells are at the embryoid stage, that is to say when torpedo shaped embryoids are visible, but before the full development of an embryo (i.e. before the formation of two cotyledons). If the majority of the re-differentiated cells are allowed to develop beyond the embryo stage and to produce plast-containing leaflets or leaves the recovery of alkaloids generally becomes somewhat more complicated. Usually the pattern of alkaloids formed changes somewhat as the plantlets develop. Atropine and hyoscyamine predominate at the early stages of development with other tropane alkaloids appearing as the plantlet grows further.

Additionally, secondary metabolites appear in the embryos and at later stages which tend to interfere somewhat with the extraction of the alkaloids. Extraction of alkaloids is thus simplest at the embryoid stage. However, the optimum moment for harvesting is to some extent dependent on the particular alkaloid of interest. It may be better of delay harvesting beyond the embryoid stage if the alkaloid of interest is other than atropine or hyoscyamine.

I have found in my work on the plants of the species Hyoscyamus spp. and Duboisia spp.

that the growth of the plants is greatly influenced by light and to a lesser extent by temperature and humidity. In a similar way light has an influence on callus cell formation and growth and on the growth and development of cells in liquid cell culture. Temperature is also of significance in this respect. In particular differentiation of the cells can be induced by appropriate control, inter alia, of the lighting conditions.

In considering the lighting conditions used in my process, three parameters are of particular importance, viz. the spectrum, the energy level and the day length.

The overall spectrum of the light to which the plants, seeds, seedlings, callus cells or embryos are exposed can be controlled by selecting the type of lamps used for irradiation, as is well known, using for this purpose the spectral data published by the manufacturers.

Suitable lamps can be selected from fluorescent tubes of various types, and incandescent lamps, particularly tungsten filament, xenon, and iodine vapour lamps.

Filters can be used to adjust the spectrum if desired.

During plant growth and explant growth (as well as during callus cell culture when this is carried out with illumination) I prefer to use light that approximates as closely as possible to the light conditions that prevail in the plants normal habitat. Thus for the plant Hyoscyamus muticus L. I prefer to use light that approximates to tropical sunlight such as is found in the Sahara and similar sub-desert regions. Such light has an infra red to red ratio of approximately 1.0 and results in normal phytochrome action in the plant and in explants and callus cells derived thereform.

I have found that if the active form of the phytochrome is excited, in the case of these plants, growth is regulated, while in the case of callus cells re-differentiation is induced.

Thus by irradiation with light richer in far red than red (maximum of action at 730 nm) the active form of the phytochrome is excited and the corresponding physiological effect is observed in the plant or cell culture.

Light which approximates to tropical sunlight can be obtained using a mixture of fluorescent and incandescent tungsten filament lamps.

Light that is richer in far red than red can be produced by using incandescent tungsten filament lamps alone, by using xenon lamps, or iodine vapour lamps, or by using "Grolux" tubes.

Although continuous lighting can be used at all stages of my process and although callus cell culture can be effected in total darkness, I prefer to use lighting regimes that include both days and periods of darkness.

In a first preferred regime, irradiation is carried out so as to simulate long days of tropical sunlight, e.g. irradiation with simulated tropical sunlight for periods of at least about 13 hours preferably up to about 16 hours or more, interspersed with periods of night lasting, for example, from about 11 hours down to about 8 hours.

In an alternative second regime, long days can be simulated by irradiation with simulated tropical sunlight for periods of, for example, 9 hours followed by 7 hours of light, which is richer in far red than red and is provided by tungsten filament lamps, followed by an 8 hour night.

In a third regime a 9 hour period of simulated tropical sunlight is followed by 15 hours of the infra red rich light (and no periods of darkness).

In a fourth regime a 16 hour day provided by a ceiling consisting of a mixture of fluorescent "Grolux" tubes and incandescent tungsten filament lamps is followed by 8 hours night.

The particular periods mentioned can of course be varied and my invention is not limited to the particular periods mentioned above. Other combinations of lighting and darkness can be used as will be apparent to those skilled in the art.

The choice of lighting regime will be chosen so as to be appropriate for the stage of the process. Thus, for example, the first above-mentioned lighting regime can be maintained up to and including callus cell formation. However, thereafter cell culture is preferably effected using the second or third above-mentioned lighting regime in order to induce re-differentiation of the cells.

The energy level can be varied within wide limits e.g. from 2,000 up to 400,000 ergs per sq. cm. per second or more. However, I have found that energy levels within the range of 100,000 to 200,000 ergs per sq. cm. per second, e.g. about 140,000 ergs per sq. cm. per second, are preferable for plants (and plant material derived therefrom) such as members of the species Duboisia spp. and Hyoscyamus spp.

During each of the stages of my process the temperature may either by constant (at a value of, for example, 25"C or 27"C), or the temperature may be alternated (e.g. 27"C by day/17 C by night). According to a preferred regime using plant material from the species Hyoscyamus spp. a temperature of 27"C is maintained during periods of irradiation and a temperature of 17"C during night-time. For plant material from the species Duboisia spp.

the corresponding daytime and night-time temperatures are preferably 22"C and 12"C respectively.

The humidity should desirably be maintained during growth of plants and of explants at a level of about 70% relative humidity. However the humidity can vary from about 40% up to about 70% relative humidity.

Harvesting of the cells or re-differentiated plantlets can be by any convenient method. In one technique the cells are killed with methanol. The cell debris is then removed by filtration or by centrifugation and the supernatant evaporated to dryness. The residue is then taken up on dilute acid (e.g. 1% H2SO4) and extracted with a suitable organic solvent, such as chloroform. After adjustment of the pH of the aqueous layer to 8.5. or higher, further solvent extraction e.g. with chloroform yields the desired tropane alkaloids, which can be separated, and purified e.g. by chromatographic techniques, for example by silica gel or activated alumina chromatography.

The invention will be illustrated by reference to the following examples.

The standard synthetic medium used in the Examples (modified Wood and Braun's Medium) is made up from the following stock solutions: (a) White's I solution (amounts in 2 litres of solution) Ca(NO3)2.4H2O 5.96g Na2SO4.1 OH2O 9. 16g KCI 1.30g NaH2PO4.2H2O 0.628g MgSO4.7H2O 14.96g MnSO4.4H2O 0.132g ZnSO4.4H2O 0.054g H3BO3 0.030g KI 0.015g H2O To 2 Litres.

(b) White's II solution (amounts in 2 litres of solution) Glycine 0.30g Nicotinic acid 0.05g Thiamine hydrochloride 0.01g Pyridoxine hydrochloride 0.01g H2O To 2 litres (c) Wood and Braun's solution (amounts in 2 litres of solution) KCI 16.90g NaNO3 36.00g MgSO4.7H2O 20.00g NaH2PO4.2H2O 6.932g H2O To 2 litres To make up 1 litre of the standard synthetic medium the following constituents are mixed: White's I solution 100 ml White's II solution 10 ml Wood and Braun's solution 100 ml (NH4)2SO4 (0.79 g/litre) 10 ml Glucose 30g (or Sucrose 20g) Agar 10g 1 M inositol solution 10 ml Fe EDTA complex solution 1 ml H2O To 1 litre (EDTA is ethylene diamine tetraacetic acid; the Fe EDTA complex solution contains 7.84 mg of the complex per litre).

Example 1 Hyoscyamus muticus L. plants are grown from seed by hydroponic culture using a mineral salt solution (see P. Chouard and M. Tran Thanh Van, C.R. Acad. Sc. Paris, 259 (1964), p4783-4786), at a pH of 5.1 to 5.4 under specified light, temperature and humidity conditions. The spectrum of the artificial light used is as complete as possible so as to simulate tropical sunlight. The infra-red/red ratio is approximately 1. The day length is controlled to be 16 hours per day of 24 hours. No illumination is used during the nights. The light is furnished by an artificial lighted ceiling or phytotronic type made up of a mixture of fluorescent tubes, i.e. "Atlas" daylight tubes, and of incandescent tungsten filament lamps.

The energy level is controlled at about 140,000 ergs per square centimetre per second. The temperature is held at 27"C during the days and at 17"C during the nights. The relative humidity is 70%.

When the plants have grown to a suitable stage, floral stem explants are taken and placed in a capped 250 ml Erlenmeyer flask containing about 50 ml of the solid medium consisting of a standard synthetic medium (modified Wood and Braun's Medium) to which is added 7 to 10% by weight of agar. The same lighting and temperature conditions and the same day length are maintained. In due course callus formation can be observed.

Some of the callus cells are transferred to another capped 250 ml Erlenmeyer flask containing a further similar quantity of solid medium. The callus cells continue to grow under the same controlled lighting and temperature conditions.

Portions of the resulting callus cells are transferred to a 250 ml Erlenmeyer flask containing about 50 ml of the liquid culture medium (modified Wood and Braun's Medium). The flasks are capped and shaken continuously to encourage gas exchange or release and to prevent "clumping" or settling of the cells and maintained at a constant temperature of 25"C. These are then subjected to different procedures.

(a) One flask is maintained in the dark and cell culture is effected in the dark for 10 days. Cell culture is not limited during this period by the supply of oxygen.

After 10 days' growth the cells are harvested in the following manner. Approximately 100 ml of methanol are added to the flask and the contents are mixed and boiled. Upon cooling to room temperature the mixture is centrifuged and the cells discarded. The supernatant liquor is evaporated to dryness and the residue triturated with 1% H2SO4 (approximately 25 ml), the resulting solution being filtered to remove any insoluble material before being extracted with several 20 ml portions of chloroform. The chloroform extract of the acid layer is discarded and the pH of the aqueous layer is then adjusted to about 8 to 9 by addition of freshly prepared NH4OH solution. The solution is extracted several times with 20 ml aliquots of chloroform. On evaporation of the resulting chloroform extracts a very small amount of a mixture of tropane alkaloids is obtained which can be identified by conventional methods, e.g. by thin layer chromatography on activated alumina. The yield of alkaloids corresponds to less than 0.1% by weight based on dried harvested cell material and consists mainly of atropine and hyoscyamine.

(b) A second flask is also kept 10 days in the dark and the resulting callus cell culture is multiplied four times by sub-culturing at intervals of 10 days. A similar yield of tropane alkaloids is obtained from the final subculture upon harvesting by the method described in Example 1(a) above.

(c) A further flask is subjected to the same lighting regime (i.e. simulated tropical sunlight and a 16-hours day length) as is used during plant growth. The energy level is again 140,000 ergs per sq. sm. per second. Cell growth is somewhat faster than when following the procedure of Example 1(a) and a few of the cells spontaneously undergo differentiation, which can be detected by the reddish or greenish tinge of such cells. Upon harvesting by the method described in Example 1(a) after 10 days' growth, the yield of tropane alkaloids is again about 0.1% by weight based on dried harvested material.

(d) Yet another flask is subjected to the following lighting regime: 9 hours simulated tropical sunlight, 7 hours irradiation from tungsten filament lamps alone, 8 hours

Example 2 (a) - (e) Examples 1(a) to 1(e) are repeated using seeds of the plant Hyoscyamus aureus L.

Similar yields of tropane alkaloids are obtained in each case but consisting predominantly of scopolamine.

Example 3 (a) - (e) Examples 1(a) to 1(e) are repeated, using seeds of the plant Duboisia australica or Duboisia myoporoides, except that day and night temperatures of 22"C and 12"C are used in place of the specified temperatures of 27"C and 17 C. The (a) to (c) procedures give minimal alkaloid yields; the (d) and (e) procedures give about 9% each.

Example 4 The procedure of Example 1(e) is repeated on a larger scale using 10 litres of the liquid culture medium. The yield of alkaloids is about 50 grams.

Example 5 (a) - (e) Seeds of Hyoscyamus mucus L. are sterilized by brief immersion in 10% bromine water.

They are then transferred to damp filter paper contained in a Petri dish, which is then placed in a sterile cabinet maintained at 25"C, and irradiated for periods of 16 hours in simulated tropical daylight (infra red to red ratio approximately 1) interspersed with 8 hour periods of darkness. The light used is provided by an artificial lighted ceiling of phytotronic type made up of "Atlas" daylight fluorescent tubes and incandescent, tungsten filament lamps. The energy level is about 140,000 ergs per square centimetre per second. Within 3 days of commencing this treatment most of the seeds germinate. After 7 days the seedlings are large enough for transplantation.

The seedlings are transplanted, still under sterile conditions, onto a synthetic solid medium made up from the standard mineral salt medium used in Example 1 and containing 10% by weight of agar, to which is added 0.001 mg per litre of NAA and 0.002 mg per litre of kinetin. Illumination of the seedlings is continued as before and the temperature is maintained at 270C during periods of illumination, being allowed to fall to 17"C during dark periods.

After 10 days of this regime the seedlings have developed a vigorous root growth with many more roots than seedlings grown under similar conditions except for the auxin.

The seedlings are then transplanted to a similar solid medium but containing 0.04 mg per litre of 2,4-D instead of the 0.001 mg per litre of NAA. After 7 days of further growth still under the same environmental conditions callus formation is visible. Callus cell growth is continued for 7 days. At this stage the roots of the plantlet are covered by an off-white callus. If some of the callus cells are transferred onto another portion of the same 2,4-D-containing solid medium they continue to grow very fast.

Some of the callus cells are thereafter transferred to an auxin-free synthetic liquid medium (the modified Wood and Braun's Medium) and cultured by the methods described in Examples 1(a) to 1(e). Similar results are obtained.

Example 6 (a)-(e) The procedure of Example 5 is repeated using seeds of Hyoscyamus aureus L. Similar results are obtained.

Example 7 (a)-(e) The procedure of Example 5 is repeated using seeds of Duboisia australica and Duboisia myoporoides. In each case the yield is as follows based on dried harvested cell or embryoid material: (a) Procedure of Example l(a) 0.1% approximately (b) Procedure of Example 1(b) 0.1% approximately (c) Procedure of Example 1(c) 0.1% approximately (d) Procedure of Example 1(d) 9% approximately (e) Procedure of Example l(e) 9% approximately Example 8 (a)-(e) Seeds of Duboisia myoporoides which have been steriiised by dipping briefly in 10% bromine water are placed invididually on filter paper "bridges" arranged to dip into distilled water in corresponding test tubes which are then capped. The seeds are irradiated at 22"C at an energy level of 140,000 ergs per square centimetre per second with artificial tropical daylight of the type described in Example 1 for 16 hour periods, interspersed with 8 hour periods of darkness during which the temperature is allowed to fall to 120C. Seedlings develop and after 7 days the distilled water in each tube is poured out and replaced by a quantity of synthetic liquid medium (modified Wood and Braun's Medium) containing 0.0001 mg per litre of NAA and 0.002 mg per litre of kinetin. The same temperature and lighting conditions are maintained for a further 7 days, following which the medium is replaced by a similar liquid medium containing 0.04 mg per litre of 2,4-D in place of the NAA but otherwise identical with the NAA-containing medium. Callus cells form and growth is maintained under the same conditions for a further 14 days.

The callus cells are then cultured and the resulting cell culture harvested by the methods described in Examples 1(a) to 1(e). The yields are similar to those obtained in Example 7.

WHAT I CLAIM IS: 1. A process for the production of tropane alkaloids from plant material of a tropane alkaloid - producing plant of the family Solanaceae, comprising (a) inducing mitotic cell formation from said plant material; (b) providing a cell culture therefrom and (c) inducing differentiation of the cells in the culture under controlled light and temperature conditions, said cell culture being irradiated with light that is richer in the far red than in the red, the spectral range being not greater than 730nm and at energy levels within the range 2000 to 400,000 ergs per sq cm per second and temperatures in the range 12-27"C.

2. A process according to claim 1 wherein the energy level is within the range 100,000 to 200,000 ergs per sq. cm per second.

3. A process according to claim 1 or 2 wherein periods of irradiation by light are interspersed with periods of darkness (non-irradiation).

4. A process according to any one of claims 1 to 3 wherein the plant material is inter-nodal floral stem material.

5. A process according to any one of claims 1 to 3 wherein the plant material is a seed or seedling.

6. A process according to claim 5 wherein the seedlings are treated with a first auxin, roots are allowed to develop under the influence of said first auxin the thus treated seedlings then treated with a second auxin different from the first auxin so as to cause mitotic cells to be produced and the mitotic cells then cultured.

7. A process according to claim 6 wherein the first and/or second auxin is used together with a cytokinin with or without a gibberellin.

8. A process according to claim 6 or claim 7 wherein at least one auxin is a synthetic auxin and is, 2,4-dichlorophenoxy acetic acid; 2,4,5-trichlorophenoxy acetic acid; 4-(2,4dichlorophenoxy) butyric acid; 2-(2,4-dichlorophenoxy) propionic acid; sodium 2,3,6trichlorophenylacetate; sodium 2-(2,4,dichlorophenoxy)ethyl sulphate; sodium 2, (2,4,5, trichlorophenoxy)-ethyl sulphate; sodium 3,(2,4,5 trichlorophenoxy) propionate; 3-indole butyric acid; 1-naphthaleneacetamide or 1-naphthalene acetic acid.

9. A process according to claim 6 or claim 7 wherein one auxin is a naturally occuring auxin and is 3-indole acetic acid.

10. A process according to any one of claims 6 to 9 wherein the concentration of the first and second auxins in their respective culture media are 0.005 to 0.2 milligram per litre.

11. A process according to any one of claims 1 to 10 wherein the plant material is from a plant of the genus Hyoscyamus or Duboisia.

12. A process according to claim 11 in which the plant material is from a plant of the species Hyoscyamus muticus L. H. aureus L; Duboisia australica, or D. myoporoides.

13. A process according to claim 12 in which the temperature of the culture is 27 during period of irradiation and 17 during periods of non-irradiation using plant material from a species of Hyoscyamus or 22"C during periods of irradiation and 12" during periods of non-irradiation using plant material from a species of Duboisia.

14. A process according to claim 1 substantially as described with reference to Example 1(d); 1(e); 2(d); 2(e); 3(d);3(e); 4; 5(d); 5(e); 6(d); 6(e); 7(d); 7(e); 8(d); 8(e).

15. A process for the production of tropane alkaloids according to any one of claims 1 to 4 including the further steps of extracting the alkaloid from the differentiated cells.

16. A process according to claim 15 substantially as herein described.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. tropical daylight of the type described in Example 1 for 16 hour periods, interspersed with 8 hour periods of darkness during which the temperature is allowed to fall to 120C. Seedlings develop and after 7 days the distilled water in each tube is poured out and replaced by a quantity of synthetic liquid medium (modified Wood and Braun's Medium) containing 0.0001 mg per litre of NAA and 0.002 mg per litre of kinetin. The same temperature and lighting conditions are maintained for a further 7 days, following which the medium is replaced by a similar liquid medium containing 0.04 mg per litre of 2,4-D in place of the NAA but otherwise identical with the NAA-containing medium. Callus cells form and growth is maintained under the same conditions for a further 14 days. The callus cells are then cultured and the resulting cell culture harvested by the methods described in Examples 1(a) to 1(e). The yields are similar to those obtained in Example 7. WHAT I CLAIM IS:

1. A process for the production of tropane alkaloids from plant material of a tropane alkaloid - producing plant of the family Solanaceae, comprising (a) inducing mitotic cell formation from said plant material; (b) providing a cell culture therefrom and (c) inducing differentiation of the cells in the culture under controlled light and temperature conditions, said cell culture being irradiated with light that is richer in the far red than in the red, the spectral range being not greater than 730nm and at energy levels within the range 2000 to 400,000 ergs per sq cm per second and temperatures in the range 12-27"C.

16. A process according to claim 15 substantially as herein described.

17. A process according to claim 15 as dependent on claim 16 wherein the species is

Hyoscyamus Muticus L. and the alkaloid is atropine and hyoscyamine.

18. A process according to claim 15 as dependent on claim 16 wherein the species is Hyoscyamus aureus L and the alkaloid is Scopolamine.