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WO1990000193A1 - Production autotrophe soutenue de metabolites secondaires de plantes - Google Patents

Production autotrophe soutenue de metabolites secondaires de plantes

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Publication number
WO1990000193A1
WO1990000193A1 PCT/US1989/002800 US8902800W WO9000193A1 WO 1990000193 A1 WO1990000193 A1 WO 1990000193A1 US 8902800 W US8902800 W US 8902800W WO 9000193 A1 WO9000193 A1 WO 9000193A1
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WIPO (PCT)
Prior art keywords
secondary metabolite
cell
precursor
plant
cell line
Prior art date
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Ceased
Application number
PCT/US1989/002800
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English (en)
Inventor
John Omaha
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PLANTOMA Corp
Original Assignee
PLANTOMA Corp
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Filing date
Publication date
Application filed by PLANTOMA Corp filed Critical PLANTOMA Corp
Publication of WO1990000193A1 publication Critical patent/WO1990000193A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells
    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • 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

  • the invention relates to manipulation of plant cell cultures and the sustained, efficient in vitro production of valuable products ordinarily produced in intact plants. More specifically, the invention concerns plant cell transformations and plant cell fusions which are capable of generating valuable secondary products from self-generated precursors.
  • the immortalization of animal cells to sustain the production of a desired product is well established. Since the seminal work of Koehler and Milstein, it has been possible to perpetuate the production of a particular antibody by the descendants of a single B cell by immortalizing the producer to permit continuous culture. Immortalization is accomplished by fusion with a malignant cell which thus shares its ability to undergo continuous passaging with the producing cell. Other methods to immortalize the antibody-producing cells have met with varying success; these include infection with virus and transfection with portions of the viral genome. These viral infection or transfection methods are considered to bring about "trans ⁇ formation" of the cell-in extending its survival.
  • Analogous constructs involving plant cells have not been freguently reported, perhaps because plant cells can be cultured satisfactorily in the presence of cytokinins produced autonomously in plant tumors or plant terato as.
  • Plant teratomas are commonly caused by infec ⁇ tion with soil bacteria, most particularly Agro-bacterium tumefaciens wherein a defined part of a contained Ti plasmid is stably integrated into the plant genome.
  • functions of a virulence region contained on the same plasmid are also employed. The integrated portion of the Ti plasmid permits the production of the cytokinins reguired for continuous growth.
  • transformation process is known in plants.
  • the extent of the correspondence between the transforma ⁇ tion process in plants and the transformation process in animals is impossible to assess in the light of current information.
  • plant cells can be cultured apart from the intact organism. Though complex enough, the nutrient reguirements for culturing plant cells are far less complicated than those for animal cells.
  • explants from many tissues and organs, including developmentally regulated tissues and organs have been brought into tissue culture. Es ⁇ sential ingredients in all plant tissue culture media, phytohormones—growth regulators—supplied exogenously "poise” the plant cell's metabolic processes so the growth ex planta is possible.
  • Husemann W. in Cell Culture and Somatic Cell Genetics of Plants (1984) _1:182-191 (Academic Press) describes the establishment of i photoautotrophic cell lines in culture.
  • the Husemann dis ⁇ closure describes a gradual changeover in the culture medium from sucrose as a carbon source to photoautotrophy where sunlight is the sole energy source and C0 2 is the sole carbon source.
  • United States e.g., plants such as the opium poppy,
  • Papaver somniferum cannot legally be grown in the U.S.), and which is intrinsically a low yield process since the whole plant must be grown in order to be able to isolate secondary metabolite from only a part.
  • chemotherapeutic agent vincristine found in periwinkle
  • flavinoids such as those of Petroselinum hortense or essential oils such as rose oil or other es- sences which cannot be duplicated synthetically due to their complex nature.
  • pigments and a variety of pharmaceuticals e.g., reserpine, digitoxin, codeine, used in treatment of heart disease and management of pain.
  • the invention is directed to efficient cell cultures of plant cells which produce valuable secondary metabolites from simple starting materials.
  • the invention provides plant hybridomas which maintain themselves in culture using carbon dioxide as a carbon source and which
  • This basic primary metabolism is accessed by a multiplicity of bio ⁇ chemical pathways, many of which are compartmentalized in discrete tissues, so that specific biochemicals produced in one locus in the plant are precursors for biochemical pathways which are situated in separate, cytologically distinct loci elsewhere in the plant.
  • the invention provides, through a combination of plant cell transformation and plant cell fusion, in one biological entity or culture, a synthetic process which normally occurs in separated compartments within the intact plant. Secondary metabolite synthesis is integrally coupled to photosynthesis and to carbon fixation, wherein CO- is the sole carbon source and photons are the sole energy source.
  • the cultured immortal cells provided may be of several forms, including monomas, diomas, binary diomas, triomas, and tetraomas.
  • the invention relates to "monomas", applicable to the case in which the secondary metabolite is produced in cells of the intact plant which cells also provide the functions of photosynthesis, synthesis of the reguisite precursors for the secondary metabolite, as well as conversion of the precursors to the secondary metabolite.
  • Monomers are obtained by trans ⁇ formation of this cell to a phenotype of photoautotrophy, phytohormone independence, growth as a single cell teratoma, and continuous production of the secondary metabolite of interest.
  • the invention is also directed to methods of producing secondary metabolites by culturing the resulting monoma, and to methods of preparing the monoma.
  • the invention is directed to a "dioma", a fused cell line produced by fusion of two parental cell lines.
  • the dioma is applicable to the case where the functions of photosynthesis and precursor synthesis are contained in the same compartment within the intact plant, e.g., cells of the mesophyll layer of the leaf, and the function of secondary metabolite synthesis is in a separate compartment in the intact plant, e.g., in a specialized tissue such as the upper epidermis of the petal within a differentiated organ such as the flower.
  • the perfect fusion partner is created by transformation of the photosynthesizing, precursor synthesizing cell, using for example, Ti plasmid to create a phenotype of photoautotrophy, phytohormone independent growth, and growth as a single cell teratoma.
  • the dioma is then created by fusion of the perfect partner with the secondary metabolite synthesizing cells.
  • the invention is directed to a method to produce secondary metabolite using the resulting dioma and to methods to produce the dioma.
  • the invention is directed to binary diomas, triomas and tetraomas. These forms are appropriate to the case where in the intact plant the function of photosynthesis and primary metabolite synthesis is contained in one compartment, conversion of photosynthate and primary metabolite into precursor for the secondary metabolite of interest is contained in a second compartment, and synthesis of secondary metabolite from precursors is contained in a third compartment.
  • the perfect fusion partner is created by transformation of one of the cells, e.g., the photosynthesizing and primary metabolite produc ⁇ ing cell to create the phenotype of photoautotrophy, phytohormone independent growth, and continuous, sustained growth as a single cell teratoma.
  • the culture comprises two independent cell fusions or diomas.
  • the cell synthesizing precursor for secondary metabolite is fused with a perfect partner, thus providing for continuous, sustained, photoautotrophic, phytohormone in ⁇ dependent production of precursor.
  • the cell synthesizing secondary metabolite is also fused with a perfect partner.
  • a binary dioma results when the two types of fused cells, the one producing precursor, the other converting said precursor to secondary metabolite of interest, are cultured.
  • the two fused cell types of the binary dioma may be co-cultured, cultured as an aggregate of the two cell types, or cultured in two stages, for example seguentially wherein the precursor producing dioma would be grown to late log phase initially, followed by addition of the secondary metabolite synthesizing dioma.
  • the invention is directed to producing secondary metabolites using the binary dioma and to methods for producing the binary dioma.
  • the invention is directed to a "trioma" which is the fusion product of three parents: the "perfect" photoautotrophic cell line transformed to produce its own hormones; a secondary metabolite-producing cell; and a cell producing precursors for the secondary metabolite.
  • the invention is also directed to methods of producing secondary metabolites by culturing the resulting trioma, and to methods of prepar ⁇ ing the trioma.
  • the invention is directed to a "tetraoma" in which the dioma synthesizing precursor is fused to the dioma that converts said precursor to secondary metabolite of interest.
  • the inven ⁇ tion is directed to methods of producing secondary
  • the invention is directed to instances in which photoautotrophy is incompatible with synthesis of the secondary metabolite of interest, or -- • - where photoautotrophy is unachievable in the perfect fu ⁇ sion partner, so that transformation, whether by Ti plasmid or by other means, is used to convert the precursor-producing or primary metabolite-producing parental cell to phytohormone independent growth and to
  • ary metabolite of interest through chemoorganotrophic culture on exogenously supplied carbon and energy source, e.g., sucrose, and to methods of producing the chemoorganotrophic monoma, dioma, binary dioma, trioma, or tetraoma.
  • carbon and energy source e.g., sucrose
  • the partner may provide substances which control expression such as inducer or repressor and/ II or may provide specialized energy sources such as 4-C units which may be reguired by the secondary metabolite- synthesizing cell.
  • the invention is directed to a specifically designed apparatus to permit the convenient screening of cell lines for production of secondary metabolites in sandwiched culture media includ ⁇ ing a bonded phase extraction filter.
  • Figure 1 shows a diagram and exploded view of a sandwich assay culture system and a snap-ring holder to contain it.
  • Figure 2 shows the structures of Petunia hybrida secondary metabolites.
  • Secondary metabolite refers to substances made from precursors which substances are not directly involved in the energy metabolism or morphological structure of the plant, wherein the precursors may or may not be thus involved.
  • Secondary metabolites include but are not limited to alkaloids; allergens; amino acids and proteins; antileukemic and antitumor agents; antiviral agents; antimicrobial agents; benzo-compounds including benzoic acid derivatives, benzopyrones, and benzoguinones; carbohydrates including simple sugars and polysaccharides; cardiac glycosides and other cardioactive substances; enzymes and enzyme inhibitors; ethylene; foods, flavors and sweeteners; fragrances and perfumes; furano-compounds; plant growth regulators; insect growth inhibitors; immunochemicals; latex; lipids; miscellaneous medicinals; monofluorocarbon compounds; nucleic acids and derivatives; oils including commercial and volatile oils; organic acids; phenolics; pigments including anthraguinones, flavanoids and chalcones, tannins; steroids, sterols, saponins, and sapogenins; terpenes and terpenoids; plant virus inhibitors; and vitamins.
  • the cells producing the secondary metabolites are grown in culture without an external supply of phytohormones and using an efficient energy and carbon source.
  • At least one cell in the fusions or the monomas of the invention must be a "perfect" partner—i.e., a teratoma which is capable of phytohormone-independent growth.
  • the "perfect” partner may also be a photoautotroph or, less preferably, a chemoorganotroph.
  • its essential characteristics are that it is im ⁇ mortalized so as to grow in the absence of phytohormones in perpetuity.
  • the functions served by this perfect partner may include, in addition to or instead of the sup ⁇ ply of precursor for secondary metabolite, other regula ⁇ tory and/or metabolic functions such as providing inducers or repressors or energy sources.
  • the perfect partner In order to achieve these characteristics, the perfect partner must be transformed. Transformation may be effected using Agrobacterium tumefaciens spheroplasts, naked Ti plasmid DNA isolated from A. tumefaciens, spheroplasts or whole bacteria of related Agrobacterium species, naked DNA isolated therefrom, attenuated, modified, or altered DNA vectors derived therefrom, binary vectors related thereto, or viral DNA, or microbial DNA or RNA transforming nucleic acid.
  • the perfect partner will contribute to the secondary mutabolite producing system, the properties of immortality, of phytohormone-independent growth, multiplication as single cell teratoma and, desir ⁇ ably photoautotrophy.
  • a number of independently isolated clones of transformed perfect partners obtained have the phenotype described; these may differ in genotype, especially by virtue of having different numbers of inser ⁇ tions of transforming DNA in the genome or of having dif ⁇ ferent loci in which the transforming DNA is inserted in the genome.
  • the perfect partner sup ⁇ plies the full range of primary metabolites necessary for growth and multiplication of the secondary metabolite producing system including but not limited to amino acids, vitamins, intermediates of essential biochemical pathways, nucleotides, fatty acids, and photosynthate.
  • the secondary metabolite producing system is capable of indefinite growth in the absence of phytohormones, and forms the secondary metabolite using inexpensive energy and carbon sources, preferably sunlight and CO- .
  • the "perfect” partner itself produces the secondary metabolite, and is preferably photoautotrophic. At a minimum, it must produce the secondary metabolite from a cheap and ef ⁇ ficient carbon and energy source such as sucrose. Photoautotrophy is, however, preferred. The ability of such cells to produce secondary metabolites from a particular source is innate; the methods of the invention confer the characteristics of the perfect partner on the metabolite producing cell.
  • one of the partners must be transformed to have perfect partner characteristics.
  • a cell derived from a por ⁇ tion of the plant which is photoautotrophic is transformed to obtain the characteristics of the perfect partner and the secondary metabolite producing cell is fused in its native state; however, the converse approach can also be employed, wherein the secondary metabolite producing cell is transformed and the .photoautotroph used in its native state in the fusion.
  • Diomas are practical where m photoautotrophy and precursor synthesis occur in the same cell or where the secondary metabolite is obtained from precursors that are the direct product of photosynthesis or synthesis energized by chemoautotrophy.
  • the most typical situation occurs where photosynthesis, precursor synthesis, and secondary metabolite synthesis are conducted by three different cells.
  • all three cells may be fused into a trioma, wherein at least one of the three has been transformed to perfect partner characteristics.
  • the secondary metabolite producing system can also be constructed as a binary dioma which consists of a pair of diomas, which are cultured in relationship to each other, either co-cultured, or in tandem. At least one of the cells fused in each dioma must have perfect partner characteristics.
  • one of the diomas of the binary pair is a fusion between a photoautotroph and precursor producing cell, one of which has perfect partner characteristics and the second dioma is a fusion between the secondary metabolite producing cell and a second cell producing precursor or a photoautotroph, one of which has perfect partner characteristics.
  • the system can be constructed as a dioma/monoma binary pair wherein the monoma is also transformed to the desired growth characteristics.
  • Tetraomas may also be formed by fusion of the two diomas described above.
  • the resulting secondary metabolite producing systems are then cultured using batch culture, continuous culture, immobilized cell culture or any other ways gener ⁇ ally used for culturing cells of higher organisms.
  • a number of fermentors and reactors are available to provide the suitable culture conditions.
  • the system is photoautotrophic so that the sole carbon source is carbon dioxide and the energy source is light.
  • the medium in which the system is cultured consists essentially of essential minerals and salts.
  • the desired product of the culture is harvested and purified using standard techniques such as chromatography, size separation, or other standard separa ⁇ tion methods.
  • the first step is to produce a transformed, preferably, photoautotrophic cell line, preferably from - ⁇ -' the plant type from which the secondary metabolite- producing cell will also be chosen, but not necessarily so.
  • Mesophyll leaf cells are transformed using standard technigues with the Ti plasmid or variant thereof and selected using standard procedures for photoautotrophy and
  • a second step is to provide at least a second parent—a secondary metabolite-
  • the source of this cell will depend on the secondary metabolite desired; in the rose oil- producing case, illustrated below, it can be shown that the essential monoterpene alcohol components of the oil are made in the cells of the upper epidermis of rose pet ⁇ 5 als.
  • These secondary metabolite-producing cells are cultured and selected for ability to produce the desired products by providing substrate, in this case isopentylpyrophosphate (IPP) using selection methods similar to those described by Yamada and improved by the layered culture technique described herein.
  • IPP isopentylpyrophosphate
  • the diomas of the invention are then obtained by fusion of the foregoing two parental cells using standard fusion techniques such as electrofusion or treatment with polyethylene glycol.
  • the fused cells are selected by ability to grow photoautotrophically, and screened for their ability to produce secondary when thus grown.
  • the production can be in suspension culture or immobilized culture.
  • a third parent to form a component of a trioma is a cell capable of providing the precursor.
  • the cells underlying the lower epidermis of the petals provide the less volatile components of the rose oil as well as the precursors for the production of the monoterpenes. Therefore, in the rose oil illustration, the appropriate third parent is derived from cultures of the lower epidermal cells of the petals.
  • the dioma produced above is fused with this third parent cell culture to obtain the trioma cell line selected for photoautotrophic growth and screened for production of the desired product in the absence of precursor.
  • This trioma then can be cultured to produce the desired secondary metabolite using carbon dioxide as a sole carbon source.
  • the specifically designed apparatus of the invention is useful to screen a population of cells to identify and isolate those individual cells within a larger population of nulls which produce the secondary metabolite or alternatively produce the appropriate pre ⁇ cursors.
  • the apparatus employs a stack or sandwich of ⁇ discrete layers of cultured cells and includes a bonded phase extraction filter which binds the secondary metabolite, creating an "Omaha blot".
  • the first reguirement in screening for plant secondary metabolites is to distribute and immobilize the growing cells in a monolayer. Thus immobilized on nutri ⁇ ent medium, the cells may grow and metabolize, while their spatial relationships to one another are preserved for the life of the experiment.
  • the second requirement is an absorbent which can tightly bond, and thus immobilize, the secondary metabolite.
  • the absorbent must be bondable to an inert, two dimensional support, e.g., filter paper.
  • the extraction must work supravitally, so that target cells are not destroyed in the recognition process.
  • the third reguirement is a detection method for the secondary metabolite.
  • a method is described by Knoop, B. and Beiderbeck, R. Z. Naturforsch (1985) 40C, 297 for manipulating plant cell cultures which have been im ⁇ mobilized in agarose monolayers poured on cellophane previously autoclaved and saturated with nutrient medium.
  • the apparatus of the invention is an adaptation of the Knoop method to the problem of plant secondary metabolite production.
  • Figure 1 shows an exploded view of the sandwich assay culture system and of the snap-ring culture dish, the "Omaha dish", to contain it.
  • the apparatus further provides for feeder layer experiments in which two or more agarose-immobilized, cel ⁇ lophane supported plant cell monolayers are brought into apposition with each other.
  • the method and apparatus provides for serial transfer of the same monolayer to dif ⁇ ferently composed nutrient media underlayers.
  • dif ⁇ ferent bonded absorbents the same monolayer of plant cells can be screened for multiple chemical classes of secondary metabolites.
  • the absorbents can be silanes carrying a variety of side chains, each conveying a different bonding specificity, bonded to a filter paper matrix, or, for example, beta cyclo-dextrins.
  • Anthocyanins are in the general class of plant phenolics, possessing one or more aromatic rings bearing one or more hydroxyl substituents. In the flower these compounds occur as glycosides in which a hydroxyl of the aglycone pigment is conjugated to glucose or other sugar.
  • the aglycone por ⁇ tion is a 15-carbon flavonid of the general structure: C fi -C--C fi . Structures cited in the following text are given in Figure 2.
  • Cyanidin (I) is a bright violet anthocyanin pigment produced by . hybrida flowers. Pelargonidin, which is bright carmine, differs in having one fewer hydroxyl substituent than cyanidin.
  • a Dioma Production System from Petunia Petunia mesophyll cells (green) are isolated from leaves and protoplasts prepared using standard methods (see E.C. Cocking Ann Rev Plant Phys (1972) 23:29- 50 for a review) .
  • the isolated mesophyll cell protoplasts are transformed by Agrobacterium tumefaciens bacteria or spheroplasts, or by Ti plasmid to convert the cells to phytohormone independent growth. Transformation of P. hybrida and selection for phytohormone independent growth has been described by M.R. Davey et al in "Advances in Protoplast Research", (1980) L. Ferenczy and G.L. Farkas (Eds.), Pergammon Press, pp.
  • Transformants are sub-selected for photoautotrophy by methods described by W. Hus ' emann in Cell Culture and Somatic Cell Genetics of Plants (1984), Vol. I, I.K. Vasil (Ed.) New York: Academic Press, pp. 182-191. Photoautotrophic transformants are then screened for growth as a single cell teratoma. The resulting set of clonally propagated petunia perfect partners for protoplast fusion have identical phenotypes, but are genotypically heterogeneous, differing in the number and location of T-DNA insertions.
  • the foregoing perfect partner will confer photoautotrophy and phytohormone-independent growth upon the dioma as well as growth as a single cell teratoma, as well as a supply of all primary metabolites and photosynthate necessary for the dioma, and the cinnamic acid precursor of anthocyanin synthesis.
  • the secondary metabolite-producing cell is isolated using the above methods from flower petals.
  • the ability of the isolated petal cells to use exogenously supplied sucrose or cinnamic acid as precursors for anthocyanin pigments is determined. It is also determined using the Omaha culture dish, whether the petunia perfect partner clones can supply the precursor.

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Abstract

On a mis au point un procédé permettant de produire à faible coût des métabolites secondaires en culture cellulaire de plantes. On produit des hybridomes cellulaires de plantes caractérisées par une croissance photoautotrophe perpétuelle et une production métabolite secondaire par fusion de cellules parentales de plantes appropriées, et on les choisit en fonction des propriétés recherchées.
PCT/US1989/002800 1988-06-29 1989-06-28 Production autotrophe soutenue de metabolites secondaires de plantes Ceased WO1990000193A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1997021801A3 (fr) * 1995-12-12 1997-08-14 Xechem Inc Procede de production et d'analyse destine a la production de substances phytochimiques par des cultures de cellules vegetales
WO2015095625A1 (fr) * 2013-12-19 2015-06-25 Rhizoflora, Inc. Composition d'activateur de plantes

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Title
CHEMICAL ABSTRACTS, Volume 103, No. 25, issued 23 Dec. 1985, (Columbus, Ohio, USA), RODDICK et al., "Steroidal glycoalkaloid content of potato, tomato and their somatic hybrids", see page 562, column 2, the abstract No. 211382j, THEORETICAL AND APPLIED GENETICS, 70(6) 655-60 (Eng). *
CHEMICAL ABSTRACTS, Volume 106, No. 11, issued 16 March 1987, (Columbus, Ohio, USA), MERILLON et al., "Alkaloid accumulation in Catharanthus roseus cell lines subcultured with or without phytohormones", see page 332, column 2, the abstract No. 81608m, C.R. ACAD. SCI. SER., 3 1986, 303(16) 689-92 (Fr). *
CHEMICAL ABSTRACTS, Volume 84, No. 11, issued 15 March 1987, (Columbus, Ohio, USA), KHO et al., "Anthocyanin synthesis in a white flowering mutant of Petunia Hybrida by a complementation technique", see page 223, column 1, the abstract No. 71536z, PLANTA, 127(3) 271-9 (Eng). *
CONSTABEL, "Protoplast Technology Applied to Metabolite Production", In Giles ed., INTERNATIONAL REVIEW OF CYTOLOGY, PLANT, PROTOPLASTS, published 1983, by Academic Press (New York, USA), see pages 209-217. *
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PLANT CELL REPORTS, Volume 5, issued March 1986, (New York, USA), HAMILL et al., "Secondary product formation by culture of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes", see pages 111-114. *
WHITAKER et al., In HANDBOOK OF PLANT CELL CULTURE: TECHNIQUE AND APPLICATION, published 1986, by Macmillan Publishing Company (New York, USA), EVANS et al. eds., see pages 264-286. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997021801A3 (fr) * 1995-12-12 1997-08-14 Xechem Inc Procede de production et d'analyse destine a la production de substances phytochimiques par des cultures de cellules vegetales
WO2015095625A1 (fr) * 2013-12-19 2015-06-25 Rhizoflora, Inc. Composition d'activateur de plantes
US10287609B2 (en) 2013-12-19 2019-05-14 Rhizoflora Inc. Plant activator composition
US10612046B2 (en) 2013-12-19 2020-04-07 Rhizoflora Inc. Plant activator composition

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