Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0025—Culture media for plant cell or plant tissue culture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/16—Enzymes or microbial cells immobilised on or in a biological cell
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

• 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.
• the whole machinery of the cell or organism is generally needed or utilised and only rarely may isolated enzymes or enzyme systems be substituted.
• 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 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.
• the present invention is particularly concerned with the production of plant chemicals.
• 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.
• 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.
• 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.
• 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.
• 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.
• the aggregate will have a particle size (per colony) of from 4 to 40 mm.
• 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.
• suitable cells may be derived from the following: Populus alba. Salix purpurea. Medieago sativa, Digitalus purpurea, Apium graveolens, Catharanthus roseus.
• 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.
• 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.
• 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.
• plant hormones 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
• 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.
• 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.
• 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.
• 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.
• 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.
• Such material without regard for pigmentation and both initially and after many sub-cultures, formed suitable inocula for subsequent liquid cultivation.
• 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 -8 M, 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.
• 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 -9 M
• NAA the synthesis varied with the concentration of kinetin.
• 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.
• Salicyl glucoside production relative to that of granulate alone
• the present aggregates and granulates have biosynthetic and biotransformational abilities.
• the plant aggregates and granulates have potential in the production of pharmaceuticals, flavourings, for example Capiscum frutescens, colourings and foods.
• the plant aggregates and granulates would be used in liquid media, either agitated or flowing.
• 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.
• useful alkaloids for example from Catharanthus, such as vinblastine and vincristine
• Digitalis such as digitalin
• Dioscurea such as diosgenin
• Podophyllum such as podophylotoxin.
• the group of antibiotics known collectively as phytoalexins may be obtained in this way from a variety of species.

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• 1981
  • 1981-09-11 EP EP81902475A patent/EP0059729A1/de not_active Withdrawn
  • 1981-09-11 WO PCT/GB1981/000186 patent/WO1982001010A1/en not_active Application Discontinuation
  • 1981-09-11 JP JP56502936A patent/JPS57501360A/ja active Pending
  • 1981-09-11 BE BE0/205936A patent/BE890317A/fr not_active IP Right Cessation
  • 1981-09-13 IL IL63809A patent/IL63809A/xx unknown
• 1982
  • 1982-05-11 DK DK212082A patent/DK212082A/da unknown

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