DE2224336A1 - Production of metabolites by synthesis in plant cells - Google Patents

Description

, EÄTENTÄNW / fCTE

DR. I. MAAS
DR. F. VOITHENLEITNER

8 MUNICH 40
SCHLEISSHEIMERSTR. 299-TEL 3592201/205

24 154

American Cyanamid Company, Wayne, New Jersey, V.St.A.

Production of metabolites by synthesis in plant cells

The invention relates to the production of chemical substances by growing plant tissues under submerged aerobes Conditions in liquid culture and relates in particular to the production of chemical plant metabolites.

It has been known for many years that plant tissue can be grown independently of the whole plant by growing the tissue can be propagated under conditions generally similar to the conditions used to grow microorganisms for the production of antibiotics and the like. It has been recognized that such plant tissue cultures are a potential source of valuable plant chemical metabolites, i.e. chemical Substances generated during the normal growth of plants. So far, however, this has been Possibility, despite the obvious advantage of fully regulated growing conditions, the vaulted plants compared to common agricultural methods,

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not realized. It has already been proposed to produce valuable plant metabolites by growing whole root organs under submerged aerobic conditions in a suitable aqueous nutrient medium following a procedure similar to that used for the production of antibiotics is exactly analogous to generate. Although this technique has achieved moderate success in the laboratory, it appears however, it is unlikely to be commercially useful mainly because of mechanical problems the cultivation and processing of a large mass of coarse fibrous root material. Another attempted solution was based on the cultivation of undifferentiated individual cells of the plant. There even one single cell of a plant contains all of the genetic information necessary for the plant as a whole is characteristic, the expectation appears to be justified that the cultivation of individual plant cells under suitable conditions Conditions leading to the production of the metabolites normally found in the plant concerned. In In fact, some reports have been published that normal plant metabolites are nominally dispersed undifferentiated cell growth systems were detected. Even if, however, the reports of this earlier work are taken at face value, the metabolite concentrations achieved are significantly below those which are achieved during normal plant development. Such a cultivation of undifferentiated cells has therefore found no practical application.

Surprisingly, it has now been found that plant metabolites can be obtained in advantageous amounts by suspension culture by organ primordia a plant known to develop the metabolite by decreasing its concentration

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Auxin activity in an aqueous aerobic nutrient medium with growing undifferentiated cells or cell clusters of the plant submerged therein and the organ primordia obtained are cultivated until the advantageous amounts of plant metabolites can be obtained.

This is achieved / by using an aqueous nutrient medium with vigorously growing undifferentiated cells of the Plant known to develop the metabolite is inoculated. The undifferentiated cells are in the medium for some time under submerged aerobic conditions grown in the presence of a substance that imparts auxin activity to the medium. Then it will be the concentration of auxin activity decreases, causing a redifferentiation of the plant cells under Occurrence of plant organ primordia and the simultaneous Development of the metabolite takes place. The redifferentiated plant cells become in the medium grown for some time under submerged aerobic conditions, creating a mass called "mature." Whole mash ". This growth leads to the accumulation of beneficial amounts of the metabolites, which then isolated and recovered by conventional methods.

The term "primordium" as used herein denotes a rudimentary organ, the analogous microscopic one Contains structural elements as they are in the corresponding Organ of the normal plant embryo can be seen in part of its development. So they can Primordia root primordia, "the embryonic root tissue recognizable, are similar, cotyledon primordia, which the Embryonic stages of cotyledon development are similar, and Stem and shoot primordia, which in turn are those parts of the normal embryo in a section of its development are analog, be. Under certain circumstances you can

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more complex primordia occur, the elements of more as an embryonic component, and in some cases even whole embryoid structures appear, which are then regarded as primordia of the whole plant.

It is essential for the method according to the invention that the undifferentiated cells or cell clusters further bred under conditions that are favorable for redifferentiation and the formation of primordia will. So it was found that in those cases where no cell differentiation has occurred or her Occurrence has been hindered, no formation of normal chemical metabolites takes place. These results conflict with some of the published work on single cell cultures and that of the invention On the contrary, our own underlying experiments support the theory that the formation of chemical metabolites, the to a lesser extent in nominally undifferentiated cell systems was observed by previous editors, probably differentiated by the presence of '/ on Centers in cell clusters of the systems was conditioned, even if this was not recognized. In any case, invention applied according to growth conditions that lead to the formation of primordia to a considerable extent, while so far Growth conditions were chosen which were favorable for the maintenance of undifferentiated cells, i. the formation of primordia was intentionally prevented.

As examples of the types of chemical plant metabolites obtained by the route according to the invention are to be mentioned: alkaloids, for example strychnine and brucine from Strychnos species; the Rauwolfia alkaloids from Rauwolfia or Alstonia species;

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Vinblastine from the species Catharanthus (formerly Vinca); Atropa alkaloids from the species Beta vulgaris and Malva; Dyes from the species Indigofera, Rhus, Beta and Lawsonia; essential oils of the species Mentha, Lavandula and Rosa; Pesticides from the species Chrysanthemum and Derris and lonchocarpus; Resins made from Pinus and Juniperus species; Rubber from the species Parthenium argentatum, Heva, Ficus and Taraxacum; Flavorings from the sassafras species, Smilax and Betula; Cardiac glycosides from Convaleria and digitalis; etc. A detailed list of useful ones Plant metabolites can be found in handbooks. The nature of plant chemical metabolites, which are obtainable according to the invention, however, cannot be made with the aid of individual plant genera (genera) or - Families (familiae) and even less with the help of the actual products are defined since the invention general applicability to the entire seed-forming part of the plant kingdom, i.e. to the stem (phylum) Spermatophyta, which includes some 300 QOO species.

For the implementation of the submersed breeding process According to the invention, an aqueous medium is wsrweadet, the one or more metabolizable carbon sources, for example carbohydrates or fatty acid derivative ©, and one contains an assimilable source of nitrogen, for example nitrate ions, ammonium ions, glycine or urea. The individual needs of the different types of plant tissue are somewhat different. Fabrics like However, glucose, sucrose, corn syrup, starch, dextrose and other substances of this type may be preferred Carbon source can be used. Also includes the Nutrient medium Mineral salts, including phosphates, chlorides, sulfates and metals, including sodium, potassium, calcium, Magnesium and those elements that are required in trace amounts. The trace metals are usually as

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Contains impurities in all but the most highly purified inorganic compounds. When cleaned Chemicals used in the media can be added specifically. The optimal mineral requirement the culture used in each case can be determined by routine experiments. Mixtures of metal salts, as they are used in media for mushroom fermentations, very suitable. Another component of the basic media besides a carbon source, a nitrogen source and mineral salts are vitamins. In general, at least one or more of the common vitamins for an optimal growth of the plant tissue may be required in the method according to the invention. To this Vitamins include thiamine, riboflavin, pantothenic acid, and niacin. Mixing these vitamins creates a satisfactory growth guaranteed. A raw source of vitamins, such as yeast extract, is particularly beneficial. As in the case of mineral salts, the vitamin requirements of the tissue used can be determined relatively easily will. In general, a carbohydrate solution fortified with minerals and vitamins is used for breeding the plant tissue used. Suitable ranges for the nutrients in milliequivalents per liter are potassium, 2 to 20; Ammonium, 5 to 50; Magnesium, 0.2 to 2; Calcium, 0.5 to 5; Phosphorus, 0.25 to 2.5; and nitrate, 0 to 50. Carbohydrates are advantageously used in amounts of 5 to 50 g / l and preferably 10 to 30 g / l. If an auxinactive Substance is used to promote the proper growth of an undifferentiated plant material, should it be used in solid or liquid media in a very dilute concentration. One is suitable

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1 χ 10 to 0.5 χ 10 'and preferably about 5 χ 10 molar concentration, but can be slightly higher or lower concentrations are used «

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The term "auxin" as used herein is a general term for those that promote growth Phytohormones or growth regulating components of plants. To natural auxins, i.e. substances which exhibit growth regulating activity and are found in living whole plants will generally be 3-indole acetic acid and certain derivatives thereof, for example their esters, glycosides, etc. include auxin-active substances the natural auxins as well as man-made chemical compounds that promote growth have phytohormonal activity. Auxin active substances are sometimes referred to as embryonic cell growth stimulants. Typical auxinactive Substances which are suitable for the purposes according to the invention are generally known. Examples of this are 3-indole acetic acid, 3-indole butyric acid, 1-naphthalene acetic acid, p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and the like. Also known are "antiauxin" compounds, i.e. compounds which inhibit or neutralize the phytohormonal activity of the auxin-active substances can. Typical known antiauxins are, for example, 1-naphthoxyacetic acid, cinnamic acid, tropic acid, 2,3,5 = Triiodobenzoic acid and 2,4,6-trichlorophenoxyacetic acid »

Different culture media for the cultivation of undifferentiated Plant cells have been described in the literature. For example, there is an adapted Approach one given by P.R.White in the literature Medium from the following components:

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"• ft -

222A336

Concentration component mg / 1

Calcium nitrate 200

Magnesium Sulphate 360 Potassium Nitrate 80

Potassium chloride 65

Sodium Sulphate 200 Sodium Dihydrogen Phosphate 16.5

Ferrous sulfate 2.5

Potassium iodide 0.75

Manganese sulfate 4.50

Zinc sulfate 1.50

Boric acid 1.50

Thiamine 0.1

Pyridoxine 0.1

Nicotinic acid 0.5

Glycine 3.0

Sucrose 20,000

Naphthalene acetic acid as required

The well-known basic nutrient medium according to Heller, which contains the following components, is also suitable:

component

Potassium Chloride Sodium Nitrate Magnesium Sulphate Heptahydrate Sodium dihydrogen phosphate hydrate Calcium chloride dihydrate Sucrose

plus the following micronutrients:

Ferric chloride zinc sulfate boric acid manganese sulfate Cupric sulfate Aluminum chloride Nickel chloride Potassium iodide Sodium molybdate

concentration , 0 mg / 1 , 0 750 , 0 600 ,1 250 , 03 125 , 03 75 , 03 20,000 , 01 1 , 03 1 1 0 0 0 0 0 0

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These media, as well as the various others that are also described in the literature, are described in the required Way for the respective purpose with auxinactive compounds or antiauxins and / or cytokinenes added. However, the respective basic medium is generally of no great importance, but only serves the purpose to optimize the growth of a certain plant material. It was found that the medium according to Linsmaier and Skoog (cf. Example 29) or that according to Gambory (cf. Example 10) is an appropriate basic medium from which optimal media can be developed for plant material of all types.

For the practical application of the invention it is necessary first to produce a vigorously growing undifferentiated cell culture of the desired species, and knowledge in the field of plant tissue culture has now developed to such an extent that this can be done at will through simple experiments. For example, a single cell culture of Catharanthus roseus can be obtained by inoculating the surface of agar slants, which is prepared from an iron nutrient medium containing an auxin-active substance, with aseptic living stem sections of Catharanthus roseus. Advantageously, a cytokine, for example, kinetin or zeatin "as cell division auxiliary factor added The Währmedium contains the usual main mineral = and trace elements _g assimilable source of carbon and nitrogen, for example ammonium or Mitra functions, and preferably one or more vitamins of the B Group. After an incubation period of a few days to weeks at 15 to 35 ⁰ C is observed, that has formed on the original Inoculumsegment "Kailus" fabric, consisting of a bandage or disorganized heap of new cell growth. This new growth will be cut off

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and aseptically transferred to fresh agar slant or preferably into liquid medium with the same composition (without the agar) and incubated ^{on a shaking device, again at 15 to 35 ° C.} If further growth takes place, the cell material is subdivided and further transferred to fresh medium, with particular care that preferably smaller aggregates which have developed in the previous incubation stage are transferred. After several such transfers to fresh medium it appears that a transformation of the material into a more dispersed growth form has taken place, and by working properly it is possible to arrive at a system which is practically entirely composed of a heap of undifferentiated cells or individual undifferentiated cells Cells that are dispersed in the liquid medium. This is the ideal condition for maintenance breeding of the parent clone. With this material, selections or mutations of the plant organism can be carried out in exactly the same way as such measures are carried out with microorganisms. A® selection of new cell culture lines (cell strains) from such a population can be carried out either with or without accompanying mutation measures based on cell morphology, pigment formation (or lack thereof), rate of growth, viability on a particular medium or, more generally, on the basis of any of the factors that are responsible for the classic selection of industrial microbial cultures can be used. Most expediently, the selection is made on the basis of the ability to produce an increased amount of the desired metabolite in the productive differentiated stage and this selection is made on the basis of test results.

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The medium and other growth conditions that are necessary for the undifferentiated individual plant cells or clusters of cells for redifferentiating to the point of formation of primordia and thus entry into the Induction phase of the production of chemical metabolites are also determined through experiments. the Redifferentiation phase of growth can be on solid media or in semi-solid pasty or liquid media but the latter media are preferred. Preferably the undifferentiated Cells and clusters of cells, which may be referred to as "pre-embryos", in a submerged aerobic liquid system grown in exactly the same way as if they were microorganisms, what they in a sense called free cells also are. For a submerged aerobic culture, the medium is not solidified by a gelling agent, low concentrations However, thickening agents like agar or methyl cellulose can be useful to increase viscosity and thus to modify the shear action in the stirred system. Otherwise the growth medium contains again the usual main mineral and trace element ions, available carbon, available nitrogen, sulfur and sources of phosphorus and one or more B vitamins. Generally speaking, the transition is from the undifferentiated Cell state "into the redifferentiated phase or metabolite production phase by reducing the concentration of the growth medium has reached auxin activity »This reduced concentration of auxin activity can by transferring the undifferentiated cells to a medium with a reduced concentration of auxin activity or by diluting the medium of the undifferentiated growth phase with fresh medium that does not have auxin activity contains, or by adding an antiauxin compound to the undifferentiated growth phase medium, after a useful level of undifferentiated growth is achieved. In those cases where

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the auxinactive substance is metabolically fickle, for Example 3-indole acetic acid, you can see the concentration simply let the auxin activity decrease naturally by means of an extended incubation period. It's tough or impossible to give an absolute value for the auxin activity with undifferentiated growth is retained because this value is different for different auxinactive compounds and also for cells is different from different plant species. This interdependence of species and concentration of auxin activity for sustained undifferentiated growth is likely to be a consequence of different ones Sensitivity of different species to certain auxinactive compounds and can also depend on the amount endogenously produced auxin, which also changes from one species to another. That's why the optimal concentration for a given auxin-active substance to maintain undifferentiated cell growth of a given plant species determined by experiments. Through such experiments with the most common auxinactive Substance, namely 2,4-dichlorophenoxyacetic acid, it has been found that about 2 to 20 χ 10 mol per liter of the auxinactive compound are sufficient to undifferentiated To maintain cell growth for many types of plant material. If the concentration of auxin active substances is too high Substance show signs of toxicity, for example poor growth or even cell death. If the concentration of auxin-active compound is too low, the desired undifferentiated growth habit remains not received. More active auxinactive compounds, for example 2,4,5-trichlorophenoxyacetic acid, must be in lower concentrations are applied than they

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mentioned above for 2,4-dichlorophenoxyacetic acid. Vice versa need less active auxin substances, for example 1-naphthaleneacetic acid, in slightly higher concentrations than those mentioned above for 2,4-dichlorophenoxyacetic acid.

When there is a redifferentiation of a given plant material by reducing the concentration of auxin activity is to be brought about, it is of course useful if the concentration of auxinactive Compound in the medium of the undifferentiated phase is not significantly higher than the amount that is needed to just to maintain growth in the undifferentiated form. Then there is only a minimal dilution with auxin-free agents Medium required to reduce the concentration of auxin activity to the level at which redifferentiation occurs can take place. In general, dilution to at least twice the volume with fresh is recommended auxin-free medium is required to increase the auxin activity from the concentration for undifferentiated growth to reduce the value that allows redifferentiation. Greater dilutions make this possible Use of less critical higher concentrations of auxin activity in the undifferentiated phase. If the Auxin activity is removed by washing the undifferentiated cells, then of course there is no increase of the final volume required. This also applies (i.e. no volume increase is required) if the An antiauxin is added to reduce the effective concentration of auxin activity.

The appropriate medium (with lower auxin activity) is obtained to initiate differentiated growth to its volume, inoculated with 5 to 50% of the disperse (undifferentiated) growth phase, as above for

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Catharantus roseus described is obtained, and then incubated in a shaken, rotated, or stirred vessel at 15 to 35 ⁰ C and preferably 20 to 30 C until organized differentiated growth has taken place to the desired extent. Under these conditions, organized growth takes the form of small rounded formations, usually 0.5 to 4 mm in their largest dimension, showing a microscopic or even macroscopic structure corresponding to those found in an organ or organs of the intact plant can be seen.

When satisfactory growth is achieved in the differentiated growth phase, i.e. when considerable quantities of the desired metabolite have arisen, usually after 3 days to 3 weeks, the slurry of Plant primordia are harvested and then normally processed to obtain the desired metabolite product. Depending on the nature of the metabolite produced, this can be done by solvent extraction or by short boiling followed by filtration to remove the exhausted solid plant parts and specific ones Precipitation of the desired product or by separation of the product by adsorption or by ion exchange take place. The isolation method generally follows the procedure used to isolate the metabolite in question is applied from whole plants, and the invention does not lie in the isolation method used in the individual case.

In some cases it is not necessary to obtain the desired metabolic product from the bulk of the plant primordia to isolate, but this mass itself can be the desired product. For example, the inventive

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Method to be applied to the cultivation of corn with high lysine content, so that cheap sucrose and as a result Ammonium or nitrate ions are converted into high quality protein.

Through the following examples in which all parts and percentages are by weight if nothing otherwise indicated, the invention is explained in more detail.

example 1

Initiation and maintenance of callus growth from Beta vulgaris on normal plant material on solidified medium

Seeds of the common beetroot Beta vulgaris, variety Detroit Select Globe, are germinated on damp filter paper and when fully germinated, the germinated seedlings are dipped in 1 percent sodium hypochlorite solution for 5 minutes sterilized. (From this point on, all processes in which living tissue are involved is carried out under strictly aseptic conditions) »The seedlings are placed on a moist sterile round Filter paper transferred ^ and into root segments, hypoeotyl segments and cut up cotyledon segments. Each of these segments is placed in separate 10 cm (4 ") Petri dishes transferred, each containing 30 ml of the following medium.

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MS medium

component Conc
tration
mg / 1 (Potassium nitrate 950 Main mineral (Ammonium nitrate 720 ions and stick (Magnesium sulfate heptahydrate 185 sources of material (Calcium chloride dihydrate 220 (Potassium phosphate 68 (Ferrous sulfate heptahydrate 30th (Manganosulfate hydrate 7th (Zinc sulfate heptahydrate 4th Trace elements (Boric acid, anhydrous 2 (Ammonium molybdate 0.1 (Potassium iodide 0.4 (Cupric sulfate pentahydrate 0.12

Carbon source

Sucrose

Solidifying agent agar

Vitamins

(Biotin

(Choline chloride

(Inositol

(Nicotinic acid

(Pantothenic acid

(Pyridoxine

(Riboflavin

(Thiamine

20000 10000

1000 1 1 1 1 1

auxin-active
link

Cytokinin

2,4-dichlorophenoxyacetic acid

Kinetin

Distilled water up to 1 liter

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The medium is autoclaved for 20 minutes at 1.05 atmospheric pressure (15 psig). The inoculated dishes are ^{incubated at 25 °} C. and subdued light. After 3 weeks, clumps of callus tissue appear on the ends of several of the original inoculum pieces. Some dishes are contaminated by fungal or bacterial growth and are discarded. The callus appearing on the uncontaminated dishes is transferred to 100 ml flasks each containing 30 ml of the same agar medium and incubation is continued for another 5 weeks under the same conditions. After this time sufficient growth has taken place so that subcultures can be transferred to several additional flasks.

Example2

Initiation of disorganized growth in liquid medium and selection of subclones with desired growth characteristics

From each of the several agar subculture flasks of Example 1, a lump of dark brown callus tissue with a diameter of about 8 mm is transferred to a 250 ml Erlenmeyer flask containing 60 ml of medium with the above composition, but without the agar. These flasks, which now contain the inoculated liquid medium, are placed on a rotary shaker, which operates at 120 rpm and a shaking circle of 25 mm , and incubated for ^{4 weeks at 25 °} C. with constant shaking in diffuse light. After this time, the different flasks show different results, which to some extent reflect the unintended selection "of cell types i-yänrend of the previous stages _o Some flasks only show a larger callus clump _t some contain the stem clump and some smaller apparently identical ones

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Fragments that represent sectors that emerged from the trunk lump have grown and broken off during incubation, and some flasks contain broken fragments that can differ even in appearance and probably represent a further segregation of cell types. '

The flasks, which contain smaller fragments, and the flasks, which contain fragments with an appearance different from that of the parent clump are selected for further subculture. After 6 such stages of further selection, transfer and incubation, a cell culture line is obtained which has the desired properties rapid growth in shake flasks and growth in the form of completely unpigmented individual cells and has small aggregates less than 0.5 mm in diameter. These aggregates show differently than the cells of the plant parts in the microscope no fine structure. The growth occurs at such a rate that starting from less than 1 percent by volume of cells in the inoculum the cell volume after 2 weeks of incubation on the rotary shaker 20% of the total liquid volume. These properties remain in the same medium stable.

Example 3

Initiation of differentiated growth in the form of plant primordia in submerged-ae rober liquid culture

The contents of one of the flasks. with dispersed unpigmented disorganized cell material from Beta vulgaris, as obtained in Example 2, is allowed to settle and the supernatant clear liquid is decanted off and discarded. The deposited cells and clusters of cells are washed twice, iaicir jt.wsils 60 ml au :: infreies f liku. "Ic": ■; £ ■ · medium

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(the medium from Example 1, but without the 2,4-dichlorophenoxyacetic acid and also without the agar) can be added and the free liquid is decanted off after the cell material has settled. Equal parts of each of the washed cells are then used to inoculate ten 250 ml Erlenmeyer flasks, each containing 60 ml of the auxin-free liquid medium. The flasks are incubated for ^{three weeks at 25 °} C. on the rotary shaker as above. During this incubation period there is a considerable increase in inoculum cells and clusters of cells, and towards the end of the incubation period redifferentiation takes place, creating a slurry of bright orange and red particles which, as seen in a low-magnification microscope, are almost entirely tiny Root primordia exist, each of which contains in abundance the normal pigment of the original cultivar of Beta vulgaris used.

Example 4 Isolation of the pigment

The slurry of root primordia from the ten flasks of the previous example is filtered. At this point, it appears that all of the pigment is in the cell fraction and none is present in the filtrate which is discarded. The cell mass is restored in 500 ml of water slurried, and the mixture is boiled, whereby, that Pigment is released into the liquid. Used cells that are discarded are removed by filtration a bright red filtrate separated, which for further work-up slowly through a 2.5 cm χ 15 cm (l "χ 6") -

(R) column of cation exchange resin Dowex 50X2 ^v , which retains virtually all of the pigment as a 5 cm (2 ") wide bright red band near the top of the column. The pigment is removed from the column using a

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1 percent sodium bicarbonate solution and eluted from the eluate by acidification to pH 5, freeze-drying the Solution and extraction of the free betanidine pigment isolated from the freeze-dried product with methanol. Betanidin is beneficial as a vegetable dye for coloring food.

Example 5 Larger scale cultivation of Beta vulgaris primordia

Washed inoculum is prepared as in Example 2, and 1 flask of this inoculum is used to inoculate an intermediate culture step consisting of 500 ml of the liquid medium of Example 3 contained in a 1 liter borosilicate glass bottle. After shaking at 120 rpm for 8 days at 25 ^° C., the entire contents of this bottle are used to inoculate 10 liters of the same medium that is contained in a 20 liter borosilicate glass balloon. This medium is then incubated using a magnetic stirrer to mix and bubbling sterile air at a rate of 1 liter per minute through a submerged spray dispenser. The product is a red slurry of root primordia which is fully analogous to the slurry obtained in the laboratory shake flask cultures.

Example 6

Generation of undifferentiated cell cultures of Digitalis purpurea by anther culture

An unopened flower bud is removed from a Digitalis purpurea plant grown outdoors and replaced with a short

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Immersion in 1 percent sodium hypochlorite solution, surface sterilized. The bud is cut up under aseptic conditions and the anthers are opened the agar surface of a Petri dish transferred, the 15 ml of the solidified MS medium, as described in Example 1 is, contains. After 18 days of incubation at 28 ° C, several small lumps of callus can be seen emerging from the transplanted anthers have grown. After another 20 days, the undifferentiated has extensive further growth Callus lump occurred. The callus clusters are separated and transferred to fresh agar plates The same medium transferred and kept on this medium, with a transfer made every 14 days will.

Example 7

Growth of undifferentiated cells of Digitalis pupurea as a disperse culture in shake flasks

Callus clusters about 8 mm in diameter from one Agar plate from Example 6 is used as an inoculum for five Erlenmeyer flasks used, each containing 25 ml of liquid sterile medium with the following composition.

MMS medium

Concentration component mg / 1

Potassium nitrate 950

Magnesium sulfate heptahydrate 185

Calcium chloride dihydrate '220

Potassium dihydrogen phosphate 68

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plus the trace elements, sucrose, vitamins, auxin-active compound and cytokines of the MS medium.

After 18 days of incubation, the contents of the flasks showing the best and most scattered growth are combined, the cell material is allowed to settle, the supernatant clear liquid is poured off and the cell mass is washed with 50 ml of fresh auxin-free MMSR medium with the following composition.

MMSR medium component concentration Potassium nitrate mg / 1 Magnesium sulfate heptahydrate 950 Calcium chloride dihydrate 185 Potassium dihydrogen phosphate 220 Ferrous sulfate heptahydrate 68 Manganosulfate hydrate 30th Zinc sulfate heptahydrate 7th Boric acid 4th Ammonium molybdate 2 Potassium iodide 0.1 Cupric sulfate pentahydrate 0.4 Sucrose O ₇ 12 Biotin 20000 Choline chloride 0.1 meso-inositol 1.0 Nicotinic acid lOOO Pantothenic acid 1.0 Pyridoxine 1.0 Riboflavin 1.0 Thiamine 1.0 Kinetin 1.0 0.06

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The reconstituted cell suspension is shaken for 5 minutes and then dismounted again. The clear supernatant liquid is poured off and discarded. The cell mass is washed one more time with MMSR medium in the same way.

Example 8

Growth of Digitalis pupurea cells under redifferentiation conditions

The washed cell mass from the previous example is used to inoculate 20 flasks (250 ml Erlenmeyer flasks) each containing 25 ml of MMSR medium. The pistons on the rotating chute! Device in subdued light and incubated 28 ⁰ C for 10 days. At this time, each piston contains a multitude of small particles, each particle consisting of a small mass of callus with one or more radiating primordial roots.

At. game 9

Detection of steroid glycosides in root primordial cultures

of Digitalis purpurea

The contents of the flasks with digitalis purpurea root primordia suspension from the previous example is united and under the action of gravity by a folded Filtered paper filter. The collected solid mass is dried at 60 ° C. overnight and yields 14 g dry Solid. 100 mg of this product are mixed with 3 ml of 70 percent aqueous ethanol for one hour at reflux temperature

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held, and the ethanol extract is separated by centrifugation. The ethanol extract is evaporated to a volume of 0.9 ml in a stream of nitrogen, and this aqueous residue is extracted twice with chloroform. The entire chloroform extract is spotted on a silica thin-layer chromatogram plate and the chromatogram is developed with a solvent mixture of 50 parts of cyclohexane, 50 parts of acetone and 2 parts of glacial acetic acid. After development, the thin-layer plate is dried and sprayed with a saturated solution of vanillin in 50 percent aqueous phosphoric acid. ^{The sprayed plate is heated to 120 °} C. for 15 minutes in a hot air oven. At this time, dark blue-gray spots can be seen at Rf 0.32 and Rf 0.50, the position of which exactly corresponds to that of control spots from digitoxin and digoxin on the same plate. Additional steroid glycosides can be seen at Rf 0.0, 0.05 and 0.80, but these were not positively identified.

Example 10 Generation of an undifferentiated cell culture of Datura stramonium

Datura stramonium saplings grown in pots Cut 1 cm above the ground, and the cut shoots are immersed in 1 percent for 1 minute Sodium hypochlorite solution surface sterilized. Then the entire shoot is washed in sterile water, and squared Pieces of several 5 mm leaves are placed on the surface of Agar G (Gamborg) medium in 100 mm petri dishes overplanted.

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G medium component concentration Potassium nitrate mcr / 1 Ammonium sulfate 2500 Sodium dihydrogen phosphate monohydrate 134 Magnesium sulfate heptahydrate 150 Calcium chloride dihydrate 250 Ferroethylene diamine tetraacetate 150 Manganese sulfate monohydrate 28 Boric acid 10 Zinc sulfate heptahydrate 3 Sodium molybdate dihydrate 2 Cupric sulfate pentahydrate 0.250 Cobalt chloride hexahydrate 0.025 Potassium iodide 0.025 meso-inositol 0.750 Nicotinic acid 100 Thiamine 1 Pyridoxine 10 purified agar 1 Sucrose 1000 2,4-dichlorophenoxyacetic acid 20000 Kinetin 1 0.060

The pH is adjusted to 5.5 if necessary.

After five weeks of incubation in the dark at 28 ⁰ C shows vigorous growth on the surface and the edges of the sheet portions of the transplanted Kallushaufen. Some of these callus clumps are separated and each aseptically transferred into 250 ml Erlenmeyer flasks containing 50 ml of liquid G-medium (composition as above, but without the agar). The pistons

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are incubated on the rotary shaker with subdued light and 25 ^{0 C.} As the undifferentiated callus material grows, subcultures are transferred to similar flasks over a 14 day cycle, choosing the more dispersed material where it appears. After three such selections and transfers, a cell culture line is obtained which has the desired growth shape in individual cells and small cell clusters.

Example 11 Redifferentiation of Datura stramonium in suspension culture

to root primordia

A flask with dispersed undifferentiated cell culture of Datura stramonium as obtained in the previous example is selected 6 days after the previous transfer and aseptically gravity filtered on a 100 micron nylon mesh filter. The cell mass is washed with G medium in which 2,4-dichlorophenoxyacetic acid is absent, and the auxin-free cells obtained are used to inoculate a series of 5 flasks, each containing G medium, but in which the respective total concentration of added 2,4-dichlorophenoxyacetic acid is 0.002, 0.004, 0.008, 0.016 and 0.032 mg per liter, respectively. The flasks are incubated on the rotary shaker with subdued light and at 28 ^° C. for 10 days. At this point, the three flasks with the lowest levels of added auxin activity show copious formation of root primordia.

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Example 12

Isolation of hyoscyamine and / or its racemate atropine from cultures of differentiated cells of Datura stramonium

The contents of one of the flasks from the previous example (originally contains 0.004 mg per liter of 2,4-dichlorophenoxyacetic acid) is gravity filtered through a folded paper filter. The collected mass of root primordia is washed with water, and the moist tissue mass obtained (7 grams moist) is 0.1 η with 5 ml triturated with aqueous sulfuric acid. The free liquid is separated by centrifugation with concentrated Ammonium hydroxide made alkaline and extracted three times with 10 ml of chloroform each time. The combined chloroform extracts are back-extracted with 2 ml of 0.1 η sulfuric acid, whereby an aqueous concentrate of alkaloid products is obtained which in turn is made alkaline with concentrated ammonium hydroxide and extracted with 0.6 ml of chloroform will. The chloroform extract is evaporated to about 50 microliters and the entire amount is recorded on the starting point of a silica thin-layer chromatogram plate spotted. The plate is mixed with a solvent developed from 9 parts of chloroform and 1 part of diethylamine. After the developed plate has dried, use Iodine platinum solution (0.5 ml of 10 percent platinum chloride solution are added to 25 ml of 2 percent potassium iodide solution) sprayed, showing several alkaloid stains, of which the strongest at Rf 0.45 in exact agreement with a control spot of atropine on the same Plate appears. The alkaloid commonly found in Datura stramonium plants in the natural state, is the optically active 1-hyoscyamine, this material will however, commonly used in medicine in its racemized form known as atropine. In the chromatogram no distinction is made between the active form and the racemate form.

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222 ^ 336

Example 13

Redifferentiation of Datura stramonium in suspension culture to whole plant primordia

Sterile leaf cuttings of Datura stramonium are used as inoculum for shake flasks which contain G-medium with a content of 2 mg / 1 2,4-dichlorophenoxyacetic acid. Callus forms in 8 days and is transferred to the same medium and incubated for a further 10 days, whereby a disperse undifferentiated suspension of free cells and small clusters of cells is obtained. This cell material is washed with auxin-free G-medium and used as inoculum for shake flasks containing the auxin-free G-medium. After 16 days of incubation on a Pendelschüttelvorrichtung at 25 ⁰ C, the microscopic examination shows the presence of large numbers of whole-plant primordia, that embryo-like structures that correspond in structure to the "heart phase" and "Torpedo" phase of normal plant embryo development.

Example 14

Generating cultures of undifferentiated cells from Rauwolfia serpentina

Freshness commercially available pulp-free Rauwolfia serpentina fruits are made by carefully breaking the hard shell and removing the intact peeled inner seed. The seeds are soaked in 1 percent sodium hypochlorite solution for 5 minutes surface sterilized and with sterile water

2 0 9 8 A 9/0 8 5 8 ORIGINAL

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washed. The sterile seeds are germinated by incubation in sterile moist filter paper in Petri dishes in an incubator at 28 ⁰ C. Germination will start in 8 days, and after about 18 days the shoots will have fully developed from the seed coat. The sterile shoots are transferred whole onto the agar surface of Petri dishes which contain MMS agar medium to which 10 percent by volume of sterile coconut milk has been added. After 2 weeks, callus masses have appeared on the roots, stems and cotyledons of the shoots, and this callus material is cut off and transferred to fresh medium of the same kind in Petri dishes. Two more such transfers are made at 2 week intervals and then selected pieces of kailus are transferred into 50 ml portions of liquid MMS coconut milk medium in 250 ml Erlenmeyer flasks. Disperse undifferentiated cell growth occurs in several flasks and this material is then obtained by transfers to G-medium at 2 week intervals.

Example 15 Growth of Rauwolfia serpentina as redifferentiated

material

Two flasks with the dispersed growth of Rauwolfia serpentina from the previous example are combined, and the cell mass is replaced by decantation with three times G-medium in which the 2,4-dichlorophenoxyacetic acid missing, washed. The auxin-free washed cell mass is used as an inoculum for flasks using auxin-free G medium uses the following additives contains:

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2 2 2 A 3 3 B

1 no add 2 0.1 mg / 1 3 1.0 mg / 1 4th 5.0 mg / 1 5 1.0 mg / 1 6th 5.0 mg / 1 7th 10.0 mg / 1 8th 20.0 mg / 1 9 0.5 mg / 1 10 1.0 mg / 1 11 5.0 mg / 1

Auxin activity added to the flask

1-naphthaleneacetic acid 1-naphthaleneacetic acid 1-naphthalene acetic acid 3-indole acetic acid 3-indole acetic acid 3-indole acetic acid 3-indole acetic acid 2,4-dichlorophenoxyacetic acid 2,4-dichlorophenoxyacetic acid 2,4-dichlorophenoxyacetic acid

After 8 days of incubation on the rotary shaker the first nine pistons (pistons 1 to 9) show different degrees of redifferentiation (at the occurrence of Root primordia visible), those of weak redifferentiation in pistons 5 and 9 up to extensive redifferentiation in pistons 1 and 8. These results are in accordance with the generally accepted relative intensities of auxin activity of the three used Substances. This is because 2,4-dichlorophenoxyacetic acid is much more active than 1-naphthalene acetic acid, and this is the case is itself much more active than 3-indole acetic acid. By transferring Rauwolfia serpentina cells from G-medium containing 1 mg / 1 2,4-dichlorophenoxyacetic acid on G-medium containing no auxin activity, or G medium, which contains 20 mg / 1 3-indoleacetic acid, thus results in a net reduction in auxin activity, and the desired redifferentiation takes place.

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Example 16

Detection of the presence of reserpine in redifferentiated Rauwolfia serpentina material

The liquid in piston 8 in the previous example is decanted, and the root primordia containing solid material is dried in vacuo for 6 hours at 40 ⁰ C dried. The dried mass is ground to a powder in a mortar, moistened with 5 ml of concentrated ammonium hydroxide and triturated with 30 ml of benzene. The benzene phase is separated off by centrifugation and evaporated to dryness at 40 ° C. in a stream of nitrogen. The residue is slurried in 2 ml of 1 η hydrochloric acid, and the aqueous acid is extracted with 2 ml of chloroform (the hydrochloride salt of reserpine is soluble in chloroform). The chloroform extract is evaporated to dryness, the residue is taken up in 0.1 ml of chloroform, and this solution is streaked along the line of origin of a 5 cm × 20 cm (2 "× 8") silica thin-layer chromatogram plate. On development with a solvent mixture of .16 parts of n-butanol, 1 part of acetic acid and 4 parts of water, a chromatogram is obtained which shows seven fluorescent bands under a 365 nm UV lamp, including one with the same Rf value (0, 61) like an authentic sample of reserpine chromatographed in the same way on another plate. The fluorescent band at Rf 0.61 is obviously not pure reserpine, since the fluorescent color is much bluer than that of pure reserpine. Therefore, this band is scraped off the plate, the adsorbent is leached with 2 ml of methanol, and the methanol eluate is examined by mass spectrometry with high resolution. The mass spectrum shows only one group of bands in the mass range from 600 to 650, which is in excellent agreement with those observed in pure reserpine. The determination of the maximum of the main mass band of the group gives exactly a mass of 608.2724 mass units in excellent

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2 2? Λ 3 3 β

Agreement with the expected value for reserpine for that the exact mass calculated for the molecular ion is 608.2733 mass units. So this proves that a redifferentiated suspension culture of Rauwolfia serpentina reserpine produced.

Example 17 Generating cultures of undifferentiated cells from Catharanthus roseus

Commercially available plants of Catharanthus roseus (also known as Vinca rosea) are used as a source of vegetative tissue as follows. A sapling of Catharanthus roseus about 100 mm in height is cut 10 mm above the soil surface, and the entire plant shoot is surface sterilized by immersion in a 1 percent sodium hypochlorite solution for 2 minutes. The plant shoot is washed with sterile water, and parts thereof are aseptically cut off and transferred onto the surface of agar-G medium contained in petri dishes. After 18 days incubation at 25 ⁰ C, the resulting Kallusauswüchse be transferred directly to 50 ml portions of G-medium in 250 ml Erlenmeyer flasks and incubated on the rotary shaker at 25 ⁰ C. Regardless of whether the original piece of plant comes from the stem, the leaf stalk or the leaf blade, a very dispersed, strong, undifferentiated growth quickly becomes apparent. The undifferentiated cell suspension is obtained by transferring it to fresh liquid G medium every 7 days.

Example 18

Growth of Catharanthus roseus as redifferentiated material

A flask with a 7 day old undifferentiated suspension culture of Catharanthus roseus are mixed with 50 ml of fresh G medium in which 2,4-dichlorophenoxyacetic acid is missing.

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By this measure, the originally contained in the piston Auxin activity diluted to half of its original value. Half of the cell suspension thus propagated is discarded, and the other half is placed on the rotary chute for another 7 days brought. During this time a strong new growth takes place. Again 50 ml of auxin-free G medium are added and mixed and half of the resulting suspension is discarded. The other half, which is now a quarter at most of the initial amount of 2,4-dichlorophenoxyacetic acid is placed on the rotary shaker for a further period of 7 days brought. This cycle of dilution with auxin-free medium and incubation is repeated three more times so that the flask ended up with only 1/32 of the original amount of 2,4-dichlorophenoxyacetic acid or in other words contains about 0.031 mg / l of this auxin activity. At this concentration of auxin activity an obvious redifferentiation of the Catharanthus roseus material occurs after a further 4 days of incubation with the appearance of root primordia. Catharanthus roseus is the source of the important anti-tumor agents vinblastine and vincristine.

Example 19 Phytolacca americana grows in the form of undifferentiated cells

in suspension culture

Phytolacca americana seeds are purchased commercially and placed between layers of moist peat moss ^{at 5 ° C. for 3 months.} Then the seed is surface sterilized by immersion in bromine water for 3 minutes, washed with sterile water and placed on sterile filter paper in petri dishes to germinate. Germination occurs promptly and the developed shoots are cut into their parts from root, hypocotyl and cotyledon sections, and these parts are directly divided into 5 ml portions of liquid G-medium containing 6 mg / 1 2,4-dichlorophenoxyacetic acid placed in 2.5 cm x 20 cm (1 "x 8") test tubes on a pendulum shaker (shaking deflection 2.5 cm, 100 strokes per minute) at 28 ° C. After 14 days, callus masses have formed, there are broken pieces of trunk, and vigorous undifferentiated growth is in progress.

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Example 20 Redifferentiation of Phytolacca americana

The transfer of undifferentiated callus from the previous one Example on G-medium that only contains 0.5 mg / 1 2,4-dichlorophenoxyacetic acid contains, leads to redifferentiation with the appearance of cotyledon-like primordia.

The transfer of the undifferentiated callus from the previous one Example on G medium containing 1 mg / 1 1-naphthalene acetic acid contains, leads to prompt redifferentiation, as evidenced by the abundant formation of root primordia shows. Phytolacca americana is the source for the alkaloid phytolaccin and for the research reagent pokeweed mitogen.

Example 21 Growth of Ficus elastica as undifferentiated material

in suspension culture

A leaf from the third internode of a ficus elastica plant grown in a pot is cut off by dipping surface-sterilized in 1 percent sodium hypochlorite solution and a portion measuring 5 cm 5 cm is aseptic removed. This part is cut into square pieces by lern used for inoculating shake flasks G medium can be used. After incubation on the pendulum shaker for 21 days, vigorous growth of undifferentiated callus material observed. Ficus elastica is the source of Indian rubber.

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Example 22

Growth of Ficus carica as undifferentiated material

in suspension culture

A young leaf is cut off from a Ficus carica tree and the surface is sterilized. Parts of the leaf are used to inoculate dishes with Agar-G medium. Vigorously growing lumps of callus appear in 15 days on. These are transferred to liquid G medium in shake flasks, whereupon further growth in well dispersed form. When transferring to G-Medium, which contains 0.5 mg / 1 1-naphthaleneacetic acid finds redifferentiation with the appearance of root primordia instead of. Ficus carica, the common fruit fig, is the source of the milk-coagulating enzyme ficin.

Example 23 Growth of Camptotheca acuminata in suspension culture

Camptotheca acuminata seeds are immersed in 1 percent sodium hypochlorite solution for 10 minutes Surface sterilized, washed with sterile water and placed on moist filter paper in Petri dishes germinated. The sprouted shoots are transferred whole to shake flasks containing G-medium and at Incubated at 28 C on the rotary shaker. There is vigorous callus growth and after transfer a cell culture line containing the desired shows dispersed growth habit. Camptotheca acuminata is the source of camptothecin, an important remedy against Cancer.

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Example 24 Growth of Sassafras alibiduitt in disperse suspension culture

From a Sassafras alibidum tree grown in the forest Green new resting plants removed and by immersion in 70 percent ethanol and then in 1 percent sodium hypochlorite solution surface sterilized. The shoots are cut into 2 to 5 mm thick cross-sections and these sterile sections are placed on agar-G medium to generate callus. When lumps of callus form they are transferred to liquid G-medium in shake flasks on a rotary shaker. Further Incubation leads to dispersed cultures of undifferentiated cells and clusters of cells. Sassafras alibidum is a source for the flavoring sassafras oil and for safrole.

Example 25 Growth of Artemesia absintfaliam ig gadifferentiated cell culture

Sterile seeds of Artemesia absinthium are germinated and the shoots are transferred to liquid G-medium and incubated as described above. Callus tissue forms, which after transfer to G-medium strongly in the form of disperse Cells and clusters of cells grows.

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Example 26

Redifferentiation from Artemesia absinthium

Washed undifferentiated cells from the previous one Examples are used to inoculate flasks with G medium containing only 0.5 mg / 1 1-naphthaleneacetic acid as Contains auxin activity. There is prompt redifferentiation, as the appearance of root primordia shows. Artemesia absinthium is used to flavor vermouth wine.

Example 27 Growth of Nicotiana tabacum in undifferentiated cell culture

Nicotiana tabacum seeds are surface sterilized and germinated on moist filter paper. Stalk parts of the saplings are transferred to agar-MS medium, whereupon callus masses form. These are on liquid MS medium transferred to shake flasks and selected, creating a dispersed growing cell culture line of undifferentiated Material is obtained.

Example 28 Redifferentiation of Nicotiana tabacum into shoot primordia

Washed cells from the previous example are used to inoculate flasks with auxin-free MS medium and after 5 weeks of incubation in dim light on a slowly rotating shaker

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(60 rpm) there is strong formation of green shoot primordia. Nicotiana tabacum is a source of the alkaloid nicotine.

Example 29

Generation and growth of undifferentiated cells of Lavandula officinalis in suspension culture

Commercially obtained seeds of Lavandula officinalis are made by immersion in 1 percent sodium hypochlorite solution surface sterilized and then germinated on damp filter paper. The accrued saplings are divided into 1 cm segments cut and these segments are placed on the surface of agar-MS medium containing 6 mg / 1 2,4-dichlorophenoxyacetic acid contains. After 6 weeks, the resulting piles of callus are transferred to individual shake flasks liquid MS medium and the flasks, which show a more dispersed growth form, are used for further transfer selected. Ten such successive selections and transfers are carried out at approximately 1 week intervals and the strongest cell culture line is then transferred to LS medium, which contains 6 mg / 1 2, 4-dichlorophenoxyacetic acid, transferred, in which an even faster and more vigorous growth takes place.

LS medium component concentration
mg / 1 Ammonium nitrate
Potassium nitrate
Calcium chloride dihydrate 1650
1900
440

2 Π Tk O ■ ' ^r - .'"!■", * '■''■

Magnesium sulfate heptahydrate Potassium dihydrogen phosphate Ferroethylene diamine tetraacetate Boric acid

Manganese sulfate tetrahydrate Zinc sulfate tetrahydrate Potassium iodide

Sodium molybdate dihydrate cupric sulfate pentahydrate cobalt chloride hexahydrate Sucrose

Kinetin

Thi amine hydrochloride meso-inositol

Casein hydrolyzate auxin

370 170 65 6.2 22.3 8.6 0.83 0.25 0.025 0.025 000.0

0.030 0.4 100 1000

concentration

of the auxin-active substance used and the purpose of the medium addicted"

In this LS medium there is the growth of Lavandula © ££ icinalis made entirely of small piles (diameter about OpI mm) of completely undifferentiated cells with a doubling time of the cell volume grow by about 36 hours? until the cell mass fills 100% of the volume «Lavandula officinalis is the source of lavender oil.

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Claims (8)

-40- 222433S claims 1. Process for the production of chemical plant metabolites by suspension culture, characterized in that organ primordia of a plant which the metabolite known to be produced by reducing the concentration of auxin activity in an aqueous aerobic Nutrient medium with undifferentiated growing submersed in it Cells or clusters of cells of the plant are generated and (b) the resulting organ primordia are grown until considerable amounts of the metabolite have been produced. 2. The method according to claim 1, characterized in that (a) an aqueous nutrient medium with vigorously growing undifferentiated cells of a plant known to produce the metabolite is inoculated, (b) growth of the undifferentiated plant cells in the medium for some time under submerged aerobic conditions a substance that confers auxin activity on the medium, (c) the concentration of auxin activity reduced in the medium and thereby a redifferentiation of the plant cells with the appearance of Plant organ primordia and simultaneous production of the metabolite is brought about, (d) the growth of the redifferentiated Plant cells in the medium for some time under submerged aerobic conditions with accumulation of the Maintaining metabolites and thereby producing a ripe whole mash, and (e) the metabolite from the ripe whole mash is obtained. 209849/0858 3. The method according to claim 2, characterized in that the substance that provides auxin activity, 3-indole acetic acid, 3-indolebutyric acid, 1-naphthaleneacetic acid, p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid is used. 4. The method according to claim 2, characterized in that that the concentration of auxin activity in the medium by diluting the medium with at least a portion of fresh Medium without auxin activity is reduced per part of the medium. 5. The method according to claim 2, characterized in that the concentration of auxin activity in the medium by Addition of an antiauxin compound to the medium is reduced. 6. The method according to claim 2, characterized in that that the concentration of auxin activity in the medium by separating the undifferentiated plant cells from the Medium and resuspension of the undifferentiated plant cells is decreased in fresh medium with decreased auxin activity. 7. The method according to claim 2, characterized in that the aqueous «
is held. that the aqueous nutrient medium is at a temperature of 15-35 ^° C 8. The method according to claim 2, characterized in that that the aqueous nutrient medium assimilable sources of carbon, nitrogen, potassium, phosphorus, magnesium and vitamins contains. 209 8 49/085 8