EP0059729A1

EP0059729A1 - Plant-derived cell aggregate, plant granulate, production and use thereof

Info

Publication number: EP0059729A1
Application number: EP81902475A
Authority: EP
Inventors: David John Bartlett
Original assignee: Individual
Current assignee: Individual
Priority date: 1980-09-12
Filing date: 1981-09-11
Publication date: 1982-09-15
Also published as: IL63809A; DK212082A; IL63809A0; BE890317A; JPS57501360A; WO1982001010A1

Abstract

Agregat de cellules derivees des plantes comprenant une pluralite de cellules non differenciees, granule vegetal comprenant une pluralite de ces agregats. Un procede de production d'un tel agregat consiste a etablir une serie de cultures de cellules pour obtenir une gamme de quantites absolues et relatives de cytocinine et d'auxine, a agiter les cultures et a selectionner la (les) culture (s) presentant l'agregation desiree. Une biosynthese ou biotransformation consistant a utiliser un tel granule vegetal comme catalyseur est egalement decrit. L'invention presente de nombreux avantages par rapport aux techniques de cultures de cellules conventionnelles et l'utilisation des cultures resultantes dans des systemes de biosynthese.Aggregate of cells derived from plants comprising a plurality of undifferentiated cells, plant granule comprising a plurality of these aggregates. One method of producing such an aggregate is to establish a series of cell cultures to obtain a range of absolute and relative amounts of cytokinin and auxin, shake the cultures and select the culture (s) exhibiting the desired aggregation. A biosynthesis or biotransformation consisting in using such a plant granule as a catalyst is also described. The invention has many advantages over conventional cell culture techniques and the use of the resulting cultures in biosynthetic systems.

Description

Title: "Plant-derived cell aggregate, plant granulate, production and use thereof"

Technical Field:

This invention relates to a plant-derived cell aggregate, to a plant granulate, to the production thereof and to the use thereof; more particularly, it relates to a novel aggregated material having characteristic morphology and comprising agglomerates of plant cells, which is robust chemically, physically and biologically and which may be used in biotransformational and biosynthetic reactions as the abilities of the whole parent plants are retained or even enhanced.

Background art:

The production of chemical compounds by biosynthesis, that is by enzymes, cells or organisms, rather than by classical chemical synthesis, has a number of advantages, inter alia complex compounds, even those of which the chemical constitution has not been fully determined, or a particular isomer of a given compound, may be obtained in a single operation.

Currently, to achieve such biosynthesis, the whole machinery of the cell or organism is generally needed or utilised and only rarely may isolated enzymes or enzyme systems be substituted. Where possible, the enzymes may be used in solution, suspension or may be immobilised by being attached, internally or externally, to or embedded in a solid support material. The reactions which may be effected in this way are limited by the stability of the enzymes to such treatment and the difficulty and expense of effecting multi-step reactions, particularly when regeneration of expensive intermediates, such as co-enzymes, is involved.

The use of whole cells from microbial, plant or animal cell cultures either freely suspended or immobilised may overcome the above disadvantages, but, in the case of plant cells, so far only limited success has been obtained with cell cultures. Almost all of the plant products of commerce are still obtained from whole plants either gathered from the wild or grown as crops. Since the technology of microbial cell cultivation is well known, smother approach which is attracting increasing attention is that of so- modifying the genetic constitution of specific micro-organisms, in particular bacteria, that they will biosynthesise a particular desired compound if grown in a medium which supports such synthesis and which allows the expression of the new genetic make-up. So far, this has been limited to one-step processes, that is the transfer of the ability to synthesise one protein. For it to succeed for many pharmaceutical compounds, it will be necessary to transfer several genes, to elicit the expression of each and to produce in the cells a temporally and spatially inter-related biosynthetic system that interacts favourably with the cell's basic metabolic sequences. It may prove easier to transfer such systems from one plant, say an agronomically-difficult crop, to another, which is more convenient to grow, harvest, store and/or process, but in such and other "genetically-engineered" transformations there would be considerable advantage in detecting and selecting the transferred property at the cellular stage, i.e. before the whole plant stage, and in having available a reliable method to re-differentiate the selected cells to whole plants.

In cell cultures, be they of plant or animal origin, those skilled in the art regard any tendency to form aggregates as something to be avoided. As far as is known, no research has been directed to the possibility of producing cell aggregates or granulates or to the application thereof. Such references as have appeared in the literature have not taught or suggested that the production of aggregates or granulates would lead to any useful results and, indeed, have been directed to the suppression rather than to the production of such materials.

Disclosure of invention:

In the most general terms, the present invention relates to cell culture; more particularly, it relates to a technique which produces, in high yields, plant cells in a novel form as undifferentiated large aggregates of cells in which form they may be easily handled and used in culture vessels, bioreactors or columns and in which form over a prolonged period they more fully exhibit the biosynthetic and biotransformational abilities known to exist in the plants from which the cells are derived. The process may be regarded as the production in a single operation of highly competent naturally immobilised cell systems (NICS) in a bead-like form. Also, the present invention is particularly concerned with the production of plant chemicals.

In addition to the industrial production of chemicals by vat-culture, the expression of bio- synthetic capabilities without the need for differentiation to a whole plant makes the present process a useful stage in the genetic engineering of plant cells whether by gene transfer, somatic fusion or haploid culture. Furthermore, the growth of plant cells in this form, allowing as it does the formation of enzymes not active in cell suspensions, provides for industrial use a convenient source of enzymes which would otherwise have to be obtained from the whole plant. It should also be noted that the plant aggregate and granulate in accordance with the present invention may exhibit properties which are not evident in conventional cell suspensions or in the parent plants. These properties may be attributed to the form of the aggregate or granulate and the production thereof, to the relationship between the constituent cells and to the relationship between the cells and the medium. In the course of research undertaken to investigate the conditions necessary to produce cell suspensions from plants, callus tissue was incubated in various media differing only in the contents thereof of growth substances. Much wider ranges of absolute concentrations, in particular from 10 ^-9 to 10^-3 M, and ratios, in particular from 10⁶ : 1 to 1 : 10⁶, of growth substances than those used by previous workers were investigated and these studies were combined with an examination of the effects of variations in illumination and agitation on such cell cultures. Surprisingly, for various species not only were conditions giving cell suspensions identified, but also conditions producing cell aggregates, root-like structures and occasionally shoots. A study of the physiological and biochemical properties of these morphologically-distinct tissue cultures established that they were often in different "physiological states", e.g. photosynthetic or "leal-like" in contrast to heterotrophic aggregates or suspensions. The importance and potential of being able reliably and repeatedly to produce cell cultures which differ morphologically and physiologically are readily apparent to those skilled in the art. Thus, those cultures in which differentiation occurs constitute a useful step in the production of new varieties of plants from variant cells, e.g. natural variants, mutants or transformed cells. Similarly, the ability to produce cell suspensions reliably is a most useful art in the production of mutants or in transforming cells. However, the most unexpected and most useful result was the discovery of conditions which gave rise to nearly spherical macroscopic aggregates of cells not visibly differentiated, except for pigmentation, but which for a particular set or sets of conditions were in specific physiological states. These aggregates were self-sustaining when sub-cultured into medium of the same composition as that in which they were produced. The importance of such a material in biosynthesis will be readily apparent to those skilled in the art. For example, a column filled with a plant granulate comprising a plurality of such aggregates may be regarded as a naturally immobilised enzyme complex capable of biosynthesis from simple nutrients or of biotransformation from added simple or complex substances.

The present invention provides a plant-derived cell aggregate characterized in that it comprises a plurality of undifferentiated, i.e. not visibly differentiated, cells.

The present invention also provides a so-called "plant granulate" characterized in that it comprises a plurality of such plant-derived cell aggregates. The aggregates are approximately spherical and are sufficiently rigid to maintain the shape thereof.

The present aggregates generally comprise sufficient cells to give a cross-section of at least 1 mm, preferably at least 3 mm. A plant granulate in accordance with the present invention generally comprises at least 5, preferably at least 10, such aggregates. In other words, the size of the aggregates is such that the granulate will appear to have a particle size which is at least as great as that of fine sand and is preferably much larger. In a preferred form thereof, the aggregate will have a particle size (per colony) of from 4 to 40 mm. Typically, aggregates according to the present invention are smooth approximately spherical, sometimes hollow, lumps of firm tissue, sometimes appearing as fused spheres if separation of the spheres has not taken place. The pigmentation of the aggregates varies with the culture conditions. A distinction may be drawn between aggregates which contain chlorophyll which are in principle green and those which do not contain chlorophyll and are non-green or "white". Other colours may result from the presence of other pigments which may alter the appearance.

The plant-derived cells are obtained from multi-cellular green plants; more particularly, they may be derived from members of the group Spermato-phyta (formerly Phanerogamia) including Gymnospermae and Angiospermae. i.e. seed plants. The following families are particularly suitable sources of cells: Salicaceae. Leguminosae. Scrophulariaceae. Umbelliferae, Apocynaceae. Solanaceae and Papaveraceae. For example, suitable cells may be derived from the following: Populus alba. Salix purpurea. Medieago sativa, Digitalus purpurea, Apium graveolens, Catharanthus roseus. Atropa belladonna and Papaver somniferum. The present invention further provides a process for the production of such a plant-derived cell aggregate and hence such a plant granulate characterized in that it comprises establishing a series of cell cultures providing a range of absolute and relative amounts of cytokinin and auxin, for example from 10^-9 to 10^-3 M, more particularly from 10^-8 to 10^-4 M, agitating the cultures, preferably by reciprocal shaking, and selecting the culture(s) exhibiting the desired aggregation, for example by visible inspection or by means of biosynthetic ability or chemical analysis.

The cultures are generally subjected to illumination. Variation in the above parameters enables the properties of the granulate to be controlled and varied.

Preferred examples of the growth substances of the cytokinin type include benzyl-amino-purine and kinetin. Preferred examples of the growth substances of the auxin type include naphthalene acetic acid and 2,4-di- or p-choro-phenoxy-acetic acid.

The growth substances may be either natural or synthetic.

The present invention further provides a biosynthesis or biotransformation characterized in that it comprises using such a plant-derived cell aggregate or such a plant granulate as a catalyst. The aggregate or granulate will, under suitable conditions, in growth-supporting and non-growth media, produce by biosynthesis those materials which the plant species from which it is derived normally produces. This aspect of the present invention is particularly important, for example, for the production of complex alkaloids or other naturally- occurring compounds which are produced by rare species of exotic plants and therefore are only available in very small quantities, because, by culturing aggregates from such a plant and producing a plant granulate comprising a significant number of such aggregates, the desired compound may be made, biosynthetically, on a scale and at a location which is completely independent of the location and frequency of occurrence of the plant in nature.

Surprisingly, a marked increase in biosynthetic activity in the aggregates compared with that of the parent plant or cell suspensions derived therefrom has been observed, which characteristic is of considerable importance.

Best mode of carrying out invention:

For the production of the aggregate and hence the granulate in accordance with the present invention, the cell starting material which has been found to be particularly effective is callus tissue. This type of tissue culture is well known and may be obtained and sub-cultured by conventional means. (See, for example, Seabrook, J.E.A., "Laboratory Culture", "Plant tissue culture as a source of biochemicals", Staba, E.J., (Ed.), (1980), C.R.C. Press.)

The production of the aggregates and granulates from such starting materials depends upon a number of parameters, the optimal values of which must first be established for a given species. It has been found for many species that it is sufficient to specify three factors, although others may also have some effect on the process. These three important variables are medium composition, agitation and illumination. A basic culture medium suitable for the production of the aggregates and the plant granulates is that of Schenck and Hildebrandt appearing in Can. J. Bot., (1972), 50, 199, "Medium and techniques for induction and growth of mono- cotyledonous and dicotyledonous plant cell cultures", with the replacement of the growth substances listed therein by the combinations given below and with 0.1M sucrose (in place of 3% w/v sucrose) as the carbon source. The cultures are thus grown heterotrophically, although the possibility of autotrophic nutrition is not to be excluded. Other suitable basic culture media, for example that of Murashige, T., and Skoog, F., (1962), Physiol. Plant., 15, 473, may be used if desired. The most important medium constituents affecting the quality of growth and hence the production of the aggregates and granulates are the growth substances. In general, two different types of growth-promoting substances ("plant hormones") must be present in the culture medium, namely those of the cytokinin type and those of the auxin type. Flasks containing such substances in the basic medium in a wide range of concentrations and in a wide range of ratios are prepared. For example, as suggested above, 49 different combinations may be used as illustrated in the following Tables: molar concentration of auxin

molar concentration of cytokinin

Ratios of auxin: cytokinin

Patterns of

(a) absolute concentrations (horizontal and vertical)

(b) ratios auxin (diagonal) cytokinin

Mola concen tration molar AUXIN concentration CYTOKININ

Callus tissue is placed in these media and incubated for from 1 to 4 weeks or longer if necessary. During this period, growth occurs in some or all media and the cultures as a result of selection, induction or conditioning take on specific morphological and physiological characteristics, sometimes sharply different in each flask, sometimes spread over a range of flasks. As early as this first passage, it is possible to identify the medium constituents which, in combination with the favourable values of the other two important variables mentioned above, give cultures having the desired properties, e.g. green aggregates or unpigmented suspensions.

Thus, the aggregates and granulates may be produced in a single growth period often of less than one month and on sub-culture the cells continue to grow, divide and maintain the roughly spherical shape thereof. In some cases, cells or groups of cells may slough or break off, continue to grow and divide and so increase the number of aggregates.

The aggregates may be sub-cultured, at least 25 times or for more than one year, by division and transfer to new medium. No distinct centres of growth, "meristems", have been identified. The tissue of each colony consists of closely packed cells having apparently normal cell walls, including plasmodesmata, and without the marked variation in cell size and shape normally associated with single cell suspensions. Groups of the different combinations for a given species are exposed to different intensities of illumination and the optimal medium composition for a given property, e.g. green aggregates will vary with the illumination. These factors, medium composition and illumination are so inter-active that it is not desirable first to establish an optimal composition and then to vary illumination for callus material in that medium only. For any change in illumination, it is necessary to re investigate the medium composition. This is an important innovation in that previous studies have concentrated on optimising media and then studying the effects of other variables only in the so-called optimal medium. It is equally important that a desired property of the culture should be monitored throughout the "optimising" as it has been shown that the optimal conditions for growth, for greening, for aggregation and for biosynthesis may differ markedly. Previously, media and growth condition, assessed sequentially, have been optimised for fastest biomass production on the assumption that this would also give maximal chemical production.

The third factor which must be investigated ia. the production of aggregates and granulates is agitation, the type and vigour of which affects the size, shape and rigidity of the aggregates and granulates.

It might be expected that high rates of agitation, producing high shear, would prevent aggregation and cause cell rupture, but, surprisingly, it is the higher rates of agitation, both reciprocal and orbital, which in general favour good growth, fresh weight increase and produce well-rounded firm aggregates, while the lowest rates of shaking give very little growth. Thus, a minimal shaking speed is necessary and above that the optimal shaking speed for any particular property will need to be established. In this case the interaction with the other factors may be less pronounced and so the optimal agitation may be investigated after the other two more interacting variables have been specified. It may be this production of the aggregates and granulates under conditions of vigourous agitation which results in the remarkable stability and robustness thereof enabling them to be handled in columns or stirred vats and to retain their biosynthetic abilities even when treated with chemical agents which often totally inhibit such activity in micro-organisms or freely suspended cells. This latter property is of industrial importance since detergents may be used to render the membranes of the plant granulate permeable to substances which do not normally pass therethrough, thereby permitting the unexpected use in columns or other flow-through systems.

By surveying a required property, e.g. aggregate formation, at different growth substance ratios, illuminations and agitations, cultures having desirable commercial properties may be established.

It will be appreciated that, given the teaching herein, such optimisation is routine for those skilled in the art. The following Examples illustrate the present invention. Throughout aseptic precautions were observed where appropriate and most operations were carried out in a laminar flow cabinet. EXAMPLE 1 Production of cells in particular physio logical states. Callus cultures were established using conventional techniques and media. In general, the callus was cultured on media containing 1.2 x 10^-5M auxin (2,4-dichloro-phenoxy acetic acid plus p-chloro-phenoxy- acetic acid) and 5.0 x 10^-7M cytokinin (kinetin (K), 6-furfuryl-amino-purine). Such material, without regard for pigmentation and both initially and after many sub-cultures, formed suitable inocula for subsequent liquid cultivation. In this study, twenty-five flasks were taken and filled with a standard medium minus its growth substances and with 0.1M sucrose as the carbon source. Each group of five flasks contained a different concentration of auxin (naphthalene, more correctly naphthyl-1, acetic acid, NAA), viz 10^-4, 10^-5, 10^-6, 10^-7 and 10^-8M, and to one flask in each group of five one of five different concentrations (over the same range as the auxin) of cytokinin (6-benzylaminopurine, BAP) was added so that the effect of varying combinations between auxin and cytokinin over the whole range was studied, all twenty-five combinations being observed. The flasks after being plugged with cotton wool and autoclaved at about 1 kg/sq. cm. g (15 lbs per sq. in g) steam pressure for from 15 to 20 minutes, were allowed to cool and were innoculated with callus tissue, were reciprocally shaken at 150 strokes per minute and exposed, at a temperature of 25°C ± 1 C°, to illumination from natural fluorescent tubes at intensities of from 0.2 to 10 Klux. Cells in various states (suspended or aggregated, white or green) were specifically and consistently formed and the conditions for the production thereof could therefore be defined.

For example, the results obtained in the case of Medicago sativa at low light intensities are illustrated in the following Tables:

Fresh weight relative to 6N6B = 100

% Fresh weight present as aggregates >1.5mm

μg Chlorophyll/g fresh weight relative to 6N6B = 100

Clearly, areas within the Tables may be delineated where green aggregates or white aggregates will be formed.

By such experiments and by visual, physical or chemical examination, it is possible to produce Tables from which may be determined the favourable conditions for whatever state is sought. For any given parameter or desired set of properties, there will often be an area rather than a unique combination within the Table and the values may vary with the other physical quantities, such as illumination, agitation and temperature, being manipulated, but the results are determinate and reproducible and may be defined for a given species.

As a further example, by such a survey it may be shown that for Populus alba at low light intensities and with constant reciprocal shaking (150 rpm) at 25°C, optimum values are as follows: for green aggregates - NAA 10 ^-6M, BAP 10^-8M for white aggregates - NAA 10^{-4/ 5}M, BAP 10^-6M for cell suspensions - NAA 1^0-8M, BAP 10^-6M At higher light intensities, an increase in cytokinin to 10 'M is desirable and the range of growth substance ratios and concentrations in which some green aggregates are formed is increased. These surveys have been shown to be applicable to a wide range of plants including those mentioned above.

EXAMPLE 2 Biochemical properties of cells in relation to particular physiological states. The rate of biosynthesis of salicylic acid in Populus alba cultures was studied and was found to be dependent on the physiological state as defined by the growth substances in which the cells were grown and/or used. Thus, production of salicylic acid could not be detected in cell suspensions, but green aggregates not only synthesised signi ficant amounts, but, in a medium containing 10 ^-9M

NAA, the synthesis varied with the concentration of kinetin.

ι Similarly for Salix purpurea grown in media containing 10^-6M NAA and with varying amounts of cytokinin, in this case BAP, the glucosylation of added salicylic acid varies as follows:

The efficacy of the plant granulate is seen when the glucosylation of salicylic acid by cultures in different degrees of aggregation or of other phenols by green aggregates is considered:

all the substrates were at 2mM concentration in the presence of Populus alba aggregates at 100 g fresh weight per litre.

The commercial importance of such biotransformations may be further illustrated by the specific glucosylation of one of the two hydroxyl groups of hydroquinone leaving the second unsubstituted, a process which is difficult to effect chemically. EXAMPLE 3 The stability of the granulate and the ability thereof to effect biosynthesis and biotransformation.

The plant granulate and the component aggregates are stable over long periods, are resistant to mechanical damage and retain the biosynthetic potential thereof even under non-growth conditions.

Thus, Populus alba granulate fed with salicylic acid each day for six days had an undiminished rate of formation of glucoside on the sixth day, though progressively more of the product appeared in the medium from about the third day. By the sixth day, some 25% of the salicyl glucoside had leaked into the medium and over 90% of the added phenol had been transformed. The release of products into the medium may be facilitated by the use of substances (pore- formers), such as toluene or detergents, which react with the cell membrane. Surprisingly, unlike microbial systems, the plant granulate's biosynthetic abilities are not destroyed by these substances and so by the use thereof flow systems may be established enabling the plant granulate to be used in a continuous mode. Two particular examples of pore-former use are given, firstly release of product and secondly facilitated entry of substrate.

1. Populus alba granulate incubated with salicylic acid for twenty-one hours

Salicyl glucoside production relative to that of granulate alone

= 100 in granulate in medium Granulate alone 100 0

Granulate + 1% toluene 7 0 Granulate + 0.1% Triton (Registered Trade Mark) X100 22 Granulate + 0.1% Cetavlon (Registered Trade Mark) 64 36

2. Populus alba granulate incubated with warfarin (3-α-acetonyl-benzyl-4-hydroxy-coumarin) for twenty-seven hours Uptake as % % conversion of added to glucoside substrate Granulate alone <10% 0

Granulate + 0.1% Cetavlon >90%* 25% * this uptake occurs within the first 10 hours Industrial applicability:

As indicated above, the present aggregates and granulates have biosynthetic and biotransformational abilities.

Commercially, the plant aggregates and granulates have potential in the production of pharmaceuticals, flavourings, for example Capiscum frutescens, colourings and foods. Generally, the plant aggregates and granulates would be used in liquid media, either agitated or flowing.

For example, in the case of pharmaceuticals, the present aggregates and granulates may be used in the production of useful alkaloids, for example from Catharanthus, such as vinblastine and vincristine, from Digitalis, such as digitalin, from Dioscurea, such as diosgenin, and from Podophyllum, such as podophylotoxin. Similarly, the group of antibiotics known collectively as phytoalexins may be obtained in this way from a variety of species.

The techniques outlined above are suitable for industrial application.

Claims

Claims :

1. A plant-derived cell aggregate characterized in that it comprises a plurality of undifferentiated cells.

2. A plant-derived cell aggregate according to claim 1 characterized in that it comprises cells derived from a member of the group. Spermatophyta

3. A plant-derived cell aggregate according to claim 2 characterized in that it comprises cells derived from a member of one of the following families: Salicaceae, Leguminosae, Scrophulariaceae, Umbelliferae. Apocynaceae. Solanaceae and Papaveraceae.

4. A plant-derived cell aggregate according to claim 3 characterized in that it comprises cells derived from one of the following: Populus alba. Salix purpurea. Medicago sativa, Digjtalus purpurea. Apium graveolens, Cathoranthus roseus. Atropa belladonna and Papaver somniferum.

5. A plant-derived cell aggregate according to claim 1 characterized in that it comprises sufficient cells to give a cross-section of at least 1mm.

6. A plant-derived cell aggregate according to claim 5 characterized in that it comprises sufficient cells to give a cross-section of at least 3mm.

7. A plant granulate characterized in that it comprises a plurality of plant-derived cell aggregates according to claim 1.

8. A plant granulate according to claim 7 characterized in that it comprises at least 5 aggregates.

9. A plant granulate according to claim 8 characterized in that it comprises at least 10 aggregates.

10. A process for the production of a plant- derived cell aggregate according to claim 1 characterized in that it comprises establishing a series of cell cultures providing a range of absolute and relative amounts of cytokinin and auxin, agitating the cultures and selecting the culture(s) exhibiting the desired aggregation.

11. A process according to claim 10 characterized in that the agitation is reciprocal

12. A process according to claim 10 characterized in that the cultures are subjected to illumination.

13. A process according to claim 10 characterized in that the amounts of cytokinin and auxin range from 10^-9 to 10^-3 M.

14. A process according to claim 13 characterized in that the amounts of cytokinin and auxin range from 10^-8 to 10^-4 M.

15. A process according to claim 10 characterized in that the cytokinin is benzyl-amino-purine or kinetin.

16. A process according to claim 10 characterized in that the auxin is naphalene acetic acid or 2,4-di- or p-chloro-phenoxy-acetic acid.

17. A biosynthesis or biotransformation characterized in that it comprises using a plant-derived cell aggregate according to claim 1 or a plant granulate according to claim 7 as a catalyst.