GB2113714A - Callus culture in nutrient flow - Google Patents

Abstract

Particulate plant tissue biocatalysts are prepared from particulate non-phylotoxic solids such as foamed polymers or fibrous materials by contact with an aqueous suspension culture of a callus forming plant.

Description

SPECIFICATION Callus culture in nutrient flow This invention provides method and apparatus for preparing plant tissue cultures and for using them in the production of secondary metabolites and novel biocatalysts comprising plant tissues for use in biochemical transformations. As used herein, the term "plant" is used to mean those plants capable of forming callus tissue, especially spermatophyta. The invention is particularly applicable to tissue cultures of Angiosperms. It is known that plant cells isolated from the parent plant can be induced to grow and multiply in or on a suitable medium to form a sustainable growth of plant callus tissue.A callus is an aggregate of substantially undifferentiated, unspecialised plant cells ("callus cells") which are capable, depending upon the physical or chemical environment in which they are placed, of either multiplying or of developing into any of the more specialised plant cells, and, ultimately into a complete plant. The term "callus" as used herein includes partially differentiated callus.

Plant cells may be grown in suspension in an aqueous nutrient solution as a cell culture, or allowed to develop into large agglomerates when left undisturbed on a bed of a nutrient substrate such as agar jelly (tissue culture). Suspension cell culture has the advantage of permitting close control over the cells' environment and is particularly suitable for maintaining the cells in an undifferentiated and rapidly multiplying state. The alternative method has the disadvantage that the environment of the growing cells cannot be closely controlled, and toxic metabolic products accumulate in the substrate. Cultures grown under the latter conditions form coherent pieces of callus tissue, which in time begin to exhibit signs of differentiation and a slowing of growth.

A recent variation of the plant cell culturing method involves the immobilisation of the cells by precipitation with a polymer or gel which encapsulates the cells, or small cell aggregates, in the pores of the polymer.

This third variation is described by Brodelius, Deus, Mosbach and Zenk in FEBS Letters Vol. 103 No. 1 (July 1979) Pg 93-97 and in their European Patent Application No. 22,434.

Immobilised plant cell cultures are characterised by single cells, or at most small cell clusters, encapsulated within the closed pores of polymer beads. These closed pores prevent or restrict contact between the cells and the nutrient medium and also prevent or restrict interactions between the individual cells or small clusters and their neighbours. The method has the advantage of providing particulate plant cell biocatalysts in a physical form suitable for large scale biochemical engineering projects. It has the disadvantages that it requires several steps to immobilise the cells, creating problems in maintaining sterility, and that it exposes the plant cells to a potentially hostile chemical medium.Most polymerising systems contain phytotoxic monomers or polymerisation catalysts, while immobilisation by solidification or precipitation of a preformed polymer generally requires exposure of the cells to high temperatures or toxic precipitants.

The primary products of plant metabolism are proteins and carbohydrates. However, there are many important plant products which fall into neither of those categories, and they are conveniently referred to as secondary metabolites.

For many years attempts have been made to recover secondary metabolites from plant cell cultures. Plant secondary metabolites include a very wide range of galenicals, essential oils and resins which are of great commercial importance in the pharmaceutical, perfumery and flavouring industries. However, the great majority of attempts to extract such metabolites from cultures of plant cells have hitherto been unsuccessful. It has appeared that most plants do not provide cell cultures capable of forming useful secondary metabolites, at least under the conditions of culture that have so far been employed. In this context, the assumption has hitherto been made that plant cell cultures can be grown and treated in exactly the same way as bacterial cell cultures.

We have now discovered that this assumption is, in general, fallacious. We believe that a fundamental difference exists between bacterial and plant cell cultures in that, in the former, the individual bacteria are whole and viable organisms in which all the potential metabolic pathways are normally operative, when they are suspended or immobilised in the presence of a suitable nutrient medium, whereas the latter are not complete organisms, but only detached parts of plants, which do not, in isolation, express to the full all the metabolic pathways which are available to a whole plant.

We have now discovered that when plant tissue cultures are grown as calli supported on a substrate and in direct contact with a nutrient medium, the available metabolic pathways are more fully expressed leading to the production of very much greater proportions of many of the more desirable secondary metabolites compared with cell cultures grown in suspension, or immobilised according to the method of Brodelius et al. We have further discovered that plant tissue cultures may be efficiently grown under controlled nutrient conditions and with enhanced yields of secondary metabolites when callus tissue is maintained substantially stationary with respect to the substrate and is contacted with a circulating aqueous nutrient solution.

We have particularly found that when particles of an inert (e.g. non phytotoxic) substrate having an open cellular structure of suitable dimension (e.g. having interconnecting pores of a size sufficient to permit at least small, single plant cells to pass freely through their internal voidage) are contacted with suspension cultures of plant cells, spontaneous invasion of the substrate by the cells occurs leading to the formation and retention of callus tissue throughout the voidage of the substrate. We thereby provide a particulate callus tissue biocatalyst. This is surprising to those experienced in plant cell culture, who have hitherto assumed that chemical immobilisation of the cells was necessary in order to obtain particulate plant biocatalysts, or the adhesion of plant cells to particulate supports, due to the known, poor adhesive character of callus tissue.

Our invention provides a method for the preparation of particulate plant tissue biocatalysts, which comprises contacting an aqueous suspension culture of plant cells with a particulate, inert solid substrate having an open interconnecting system of internal voids whose dimensions are sufficiently large to permit at least the smallest cells of said culture to pass freely through said internal voids and sufficiently small to entrap aggregates of said plant cells within said substrate, thereby establishing a culture of plant callus tissue immobilised with respect to said substrate, and recovering said immobiiised callus tissue from said aqueous suspension.

According to a preferred embodiment our invention provides a novel, particulate, plant tissue biocatalyst consisting essentially of an inert particulate, solid support having an open, interconnecting system of internal voids, and a plant callus tissue penetrating and at least partly filling said voids.

According to a second preferred embodiment, the invention provides a process for the production of a plant metabolite which is exported from plant callus tissue, wherein a particulate plant tissue biocatalyst of the invention, as aforesaid, is contacted with an aqueous nutrient medium adapted to promote the formation of said metabolite, and said metabolite is recovered from the aqueous medium.

According to a third preferred embodiment, our invention provides a process for the production of a plant secondary metabolite which is retained within plant callus tissue, which process comprises containing a particulate plant tissue biocatalyst of the invention as aforesaid with an aqueous nutrient medium adapted to promote the formation of said metabolite, separating said biocatalyst from said medium and extracting said metabolite from said biocatalyst.

The preferred embodiments hereinbefore described have the particular advantage of providing a biocatalyst which is in a particularly suitable physical form for biochemical engineering applications, being substantially stronger and less friable than unsupported callus tissue. The particles may, for example, conveniently be used to pack column reactors or may be liquid or gas fluidised in fluid bed reactors.

Compared with alternative plant cell biocatalysts hitherto proposed by Brodelius as hereinbefore described our preferred biocatalysts have three major advantages as follows:- 1. Ease and cheapness of manufacture -- the plant cells may be immobilised in a single step compared with the multistep operations required for chemical immobilisation. This is particularly important in minimising the risk of biological contamination.

2. Greater viability of immobilised cell population:-- because conditions inimical to plant cell growth and survival, such as elevated temperatures or potentially phytotoxic chemicals may be substantially eliminated, our preferred method provides a much higher percentage of viable cells in the biocatalyst.

3. Greater production of secondary metabolites:-- the open structure of our biocatalyst permits greater contact and interaction between the cells and both the nutrient medium and one another. The former is of importance in promoting up-take of nutrients and export of metabolites, and the latter in establishing substantial volumes of continuous callus tissue within which significant chemical gradients and partial differentiation can occur, thereby maximising the number metabolic pathways available for biochemical synthesis.

It is preferred that the nutrient medium in which the cell culture is grown, when contacted with the particulate support, according to the preferred embodiment of our invention, should be adapted to the promotion of cell growth and replication.

The desirable pore dimension of a porous substrate will depend upon the size of the cells present in the suspension culture. Typically, a plant cell suspension culture comprises single cells, of quite widely varying sizes, together with small aggregates of cells. The average number of cells present in the small, suspended, cell aggregates (i.e. the "lumpiness" of the culture) varies according to conditions of growth, including the nutrient regime and degree of agitation applied. Conventional methods of suspension culture attempt to minimise lumpiness.

Cell immobilisation by the method of Brodelius et al. encapsulates the particles present in the suspension culture and inhibits their aggregation into substantial pieces of callus tissue.

Preferably, the particulate substrates for use according to the preferred embodiments of our invention have a pore size which permits at least the smallest cells of the culture to pass freely through their internal voidage, but not large enough to permit all the aggregates in the culture to pass freely through their internal voidage. Thus it is preferred that at least 5%, preferably at least 10% more preferably at least 20% of the particles in the suspension culture can pass through the internal voidage of the support particle. Generally.

we prefer that at least 5%, preferably at least 1 0% of particles in the suspension culture are too large to pass freely through the internal voidage of the substrate.

For example, a foamed polymer, such as foamed polyurethane, having from 10 to 60 pores per inch, preferably 20 to 30 pores per inch, has been found especially suitable. If the pore size is smaller, or larger, the rate of non-reversible invasion by the particles is proportionally slower.

Eventually, a pore size is reached which is too small to permit invasion by even the smallest cells, or too large to permit entrapment and retention of even the largest cell aggregates.

Preferably for commercial production, a column may be packed with the particulate substrate, and the callus tissue grown thereon. Aerated nutrient solution may be passed through the column, e.g.

nutrient solution may be passed down the column and air bubbled up through the column.

It is particularly preferred to maintain a stirred, air agitated or liquid fluidised system in which callus tissue supported by a particulate substrate as aforesaid is maintained in suspension in the nutrient fluid, in a reactor or tank through which nutrient may be circulated.

Preferably the system includes means for circulating the nutrient solution from a reservoir to the bed, column or reactor and for returning the solution withdrawn therefrom to the reservoir.

Preferably means are provided for introducing fresh nutrients to the system or for withdrawing part of the solution, for dumping, cleaning or recovery of secondary metabolites. Circulation of the nutrient solution may conveniently be affected by a peristaltic pump.

It is possible to pump liquor both to and from the bed, reactor or column, for example using two channels of a multichannel pump. Preferably, however, flow in one direction is by gravity, maintaining the substrate and upper level of liquid in the reservoir at different levels and establishing a pumped flow from the lower of the two to the higher and a gravity flow in the reverse direction via an overflow or constant head outlet. According to one option, reservoir is at a higher level than the substrate and liquid flows from the reservoir through a substantially upright or upwardly slanting outlet tube, the upper end of which is situated within the reservoir, and the height of which can be adjusted to control the volume of liquid in the reservoir, and hence the proportion of liquid in contact with the substrate.

The substrate is desirably constructed of a water-permeable and, preferably, biologically inert material. Preferably the substrate is of a material that will withstand autoclaving, for easy sterilisation. The substrate may comprise a fibrous material such as glass fibre, cellulose or synthetic organic fibres, wire mesh bundles such as those described in UK Patent No. 2,006,181 or foamed polymer such as foamed polyurethane.

An important advantage of the present method is that it permits controlled variations of the nutrient regime supplied to the culture. This has been found valuable in inducing the production of secondary metabolites by cultures which do not normaily yield them when grown under the normal conditions of suspension culture.

Typically, cells are first grown by conventional suspension culture techniques to promote optimum reproduction of a healthy cell population.

The substrate is then innoculated with the cells and culture in accordance with the invention is commenced.

Preferably the culture is initially fed a solution adapted to promote optimum growth of the unspecialised cells, until a healthy growth has been established, and the composition of the nutrient solution is then gradually altered to optimise production of the desired secondary metabolite. For example, precursors of the desired metabolite or development modifiers such as auxins, cytokinins and gibberellins may be added to the solution, and/or the concentrations of selected nutrients or development modifiers in the solution may be reduced to inhibit competing biochemical pathways.

The supply of air may be controlled in various ways. In general callus culture cannot be conducted in totally anaerobic conditions. Often sufficient aeration is achieved by permitting some contact between the callus and the atmosphere, or by bubbling air through the nutrient solution or even allowing the nutrient to absorb some air from the atmosphere prior to contacting the calli.

Production of secondary metabolites may be favoured by restricting the air supply to the culture.

Illumination can profoundly affect the development of plant tissue cultures. It is sometimes preferred that the vessel containing the culture should be transparent to permit illumination of the growing cells. Normally cultures will be grown in artificially simulated white light, but illumination by selected wavelengths or growth in darkness or under cycles of illumination may in some instances be preferred in order to favour production of specific metabolites. Preferably means are provided to control the temperature of the culture, which also affects the rate of growth and the yield of secondary metabolites.

We have found that our invention is useful for the production of a number of plant secondary metabolites. For example, cells of certain Capsicum species e.g. Capsicum frutescens or Capsicum annum can be grown according to our invention and induced to form significant yields of capsaicinoids, the principle active compounds in the flavour of chilli pepper. The same cells do not yield capsaicinoids in suspension cultures.

According to a further embodiment therefore our invention provides a method for the production of capsaicinoids which comprises forming a culture of callus tissue from a capsaicinoids producing species of Capsicum, supporting the culture on a substrate in contact with air and with an aqueous nutrient solution and removing capsaicinoids from the culture. The term "Capsaicinoids" is used herein generically to include the various components of commercial capsaicin including the compound capsaicin itself, dihydrocapsaicin, nor-dihydrocapsaicin homo-capsaicin, homodihydrocapsaicin.

Preferably, to stimulate production of capsaicinoids, precursors such as valine and phenylalanine are added to the nutrient solution, once a healthy growth has been established, and competing protein synthesis is checked by progressively reducing the nitrate and/or carbohydrate content of the solution. This may be achieved by allowing the culture to deplete the solution naturally, and then adding the precursor.

We have found that particularly high yields of capsaicin are obtained when iso-capric acid is added as precursor to the nutrient medium. Water soluble salts such as sodium or potassium iso-caprate may also be used. Alternatively, or additionally, other precursors of capsaicin such as vanillin, ferulic acid, trans cinammic acid, p.hydroxy benzoic acid, protocatechuic aldehyde, p.coumaric acid, m.coumaric acid, gallic acid, caffeic acid may be used.

The invention may also be used to stimulate the production of diosgenin by tissue cultures obtained from diosgenin-forming strains of yam of the genus Dioscorea. Diosgenin is widely used industrially for the synthesis of certain commercially valuable steroids.

Other secondary metabolites which may be prepared in accordance with the invention include L-DOPA, the constituents of rose oil, various alkaloid such as opium alkaloids and derivatives of datura.

Secondary metabolites may be recovered from cultures grown according to our invention, either from the circulating solution (e.g. by filtering off suspended dead cells, or by evaporation, selective absorption, precipitation, dialysis, reverse osmosis, solvent extraction or any other convenient separatory method) or by harvesting the cultures and extracting the desired products, depending upon the extent to which they are excreted. In many cases the metabolites may be removed from the callus by plasmolysis, which involves contacting the cell with an aqueous solution of a non-toxic, nonionic compound at a concentration sufficient to promote osmotic shrinkage of the cell, which may be accompanied by excretion of secondary metabolites.

The invention is particularly useful for systems in which the desired metabolite is excreted into the nutrient medium, and we prefer in such instances to recover the metabolite continually or intermittently from the nutrient medium while it is recycling. This reduces the inhibition of the desired metabolic pathways by feedback mechanisms caused by excessive build up of the metabolite in the nutrient.

Suitable nutrient solution for the growth of callus cultures are well known in the art. A typical example is described by Marashige and Skoog in Physicologa Plantarum Vol. 1 5 pp 473-497.

Such solutions may be supplemented by additions of natural plant fluids such as coconut milk, or by yeast or malt extracts.

Growth of plant tissue cultures should, of course, be conducted in sterile conditions to avoid contamination of the culture and its nutrient medium.

EXAMPLE I Suspension cultures of Capsicum frutescens were grown in 250 ml Erlenmeyerflasks containing 60 my of liquid medium comprising 4.71 g 1-1 Murashige and Skoog's medium supplemented with 2 Jttg l-1 (10-8 M) indolyl-3 acetic acid (lAA), 20 ,ug 1-1 (10-7 M) kinetin, 20 g l-1 sucrose, and 50 ml 1-' coconut milk, at pH 6.0.

Carrot (Daucus carota) suspended cells were similarly cultured in liquid medium containing 4.71 g 1-' Murashige and Skoog's medium supplemented with 0.2 mg 1-1(10-6 M) 2,4dichlorophenoxyacetic acid (2,4-D), 0.1 mg 1-' (10-1 M) kinetin, and 30 g 1-' sucrose, pH 5.8 (carrot medium). The cultures were agitated on a rotary shaker of orbital diameter 2.5 cm at 96 rpm.

1 g fresh weight cells was transferred to fresh medium at intervals of 2 weeks, and maintained at 24 + 1 OC in continuous fluorescent illumination of an illuminance of 20 ,umol m-2 sec-l.

Three (for carrot) or six for pepper foam particles, each of dimensions 1.0 cm3 and of 1 8 pores per cm, were added to newly sub-cultured flask cultures. At intervals flasks were harvested and determinations were made of the fresh and dry weight and, for the pepper cultures, the number of immobilised cells per cultures and the fresh and dry weight of suspended cells per culture. Loaded foam particles (i.e. containing cells) from each flask were resuspended in 100 ml Murashige minimal organics medium in the absence of sucrose or growth substances, to limit cell growth; after 3 or 4 days' agitation on a rotary shaker the % cell retention was determined.

The fresh weight of suspended and immobilised cells was determined after the removal of excess medium on a Buchner funnel. The dry weight of suspended immobilised cells was determined after drying at 900C for 24 hrs in a hot air oven.

During the first 6 to 9 days of the carrot cell culture period, there was a lapse phase in which the total cell fresh and dry weight increased only very slowly. However, by day 3, cells became entrapped in the foam particles, followed by a continued uptake of cells until about day 1 5. After this time, there was a sharp reduction in the rate of increase of the dry weight of suspended cells, and no further increase in the dry weight of immobilised cells. Nevertheless, the fresh weight of the immobilised and suspended cells continued to increase to day 21, presumably due to cell expansion. By day 21, cells had grown out through the surface of the foam particles, were extremely compact, and displayed a variety of shapes and sizes, usually between 20 and 100 cm in length, and were occasionally organised into globular structure. By the end of the 21 day culture period, each foam particle contained approximately 2.9-3.0 x 106 carrot cells.

Unlike the carrot cells, the growth and immobilisation process of the pepper cells had no lag phase. The fresh and dry weights and numbers of immobilised cells increased in an approximately linear manner for the first 12 days of the culture period; after this time, the rate of increase became greater, and, unlike the case for the carrot cultures, there was no indication of a stationary phase of growth by day 21. By day 3, the fresh weight of the immobilised pepper cells was greater than that of the suspended cells, and this situation was sustained throughout the period of culture. The total cells immobilised by day 21 was 4.8 x 10-6 cells per particle.

The % retention of both carrot and pepper cells on transfer to, and agitation in, fresh medium, was usually about 99%, and never less than 95%.

Throughout the growth cycle the viability of the immobilised cells remained high, at between 70 and 80%. Sectioning of foam support particles demonstrated that, as early as day 1 in the culture period, cells of either species became irreversibly entrapped in the centre of the foam support particles.

EXAMPLE II Particles of foam supported pepper callus prepared according to Example I were placed in a 20 cm by 2.5 cm glass column. 50 mls of Marashige and Skoog nutrient solution were circulated through the column for 5 days under constant artificial illumination at 250C. During this period air was blown through the column.

5 mm of ferulic acid was added to the nutrient and circulation resumed for two weeks, without air supply to the column other than that absorbed by the nutrient on contact with the atmosphere.

The circulating medium was passed through a kieselguhr column which accumulated a high concentration of absorbed capsaicin. The latter was subsequently eluted and recovered.

Claims (14)

1. A method for the preparation of particulate, plant tissue biocatalysts, which consists essentially in the steps of (i) forming an aqueous suspension culture which culture consists essentially of cells and cell aggregates of a callus forming plant, suspended in an aqueous nutrient medium; (ii) contacting said suspension culture with a particulate non phytotoxic solid substrate which has an open interconnecting system of internal voids whose dimensions are sufficiently large to permit at least the smallest of said cells to pass freely through said internal voids and sufficiently small to entrap at least the largest of said aggregates; and (iii) permitting said cells to invade said substrate and to establish a culture of plant callus tissue immobilised with respect to said substrate.

2. A method according to claim 1, wherein said support consists essentially of a foamed polymer.

3. A method according to claim either of claims 1 or 2 wherein said support consists essentially of a three dimensional reticular structure formed from a material selected from metal wire and natural and synthetic fibres.

4. A method according to any of claims 1 to 3, wherein said cells are selected from cells of capsaicin forming strains of capsicum.

5. A particulate, plant tissue biocatalyst consisting essentially of a non-phytotoxic, solid support, having an open, interconnecting system of internal voids, and a plant callus tissue penetrating and at least partly filling said voids.

6. A biocatalyst according to claim 5, wherein said support consists essentially of a foamed polymer.

7. A biocatalyst according to either of claims 5 or 6, wherein said support consists essentially of a three dimensional reticular structure formed from a material selected from metal wire and natural and synthetic fibres.

8. A method for the preparation of plant secondary metabolites which consists essentially in contacting a biocatalyst as claimed in claim 5 with an aqueous nutrient medium adapted to the formation of secondary metabolites.

9. A method according to claim 8, wherein the secondary metabolites are exported from the callus tissue and are continuously or intermittently recovered from the aqueous medium.

10. A method according to either of claims 8 or 9, wherein the secondary metabolites are retained in the callus tissue, the biocatalyst is recovered from the aqueous medium and the secondary metabolites are extracted from the biocatalyst.

11. A method according to any of claims 8 to 10, wherein the biocatalyst comprises tissue selected from capsaicin producing strains of capsicum.

12. A method according to any of claims 8 to 11, wherein said biocatalyst is contacted with said nutrient medium by fluidising a bed of said catalyst with said medium.

1 3. A method for the preparation of particulate plant tissue biocatalysts according to any of claims 1 to 4 substantially as hereinbefore described with reference to the foregoing examples.

14. A biocatalyst according to any of claims 5 to 7 substantially as hereinbefore described with reference to the foregoing examples.

1 5. A method for the preparation of plant secondary metabolites according to any of claims 8 to 12 substantially as hereinbefore described with reference to the foreqoing examples.