SUSTAINED AUTOTROPHIC PRODUCTION OF PLANT SECONDARY METABOLITES

Technical Field

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.

Background Art

The immortalization of animal cells to sustain the production of a desired product, generally an immunoglobulin, 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. In the process of integration, 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. In recent years, the Ti plasmid and T-DNA have been manipulated independently of the bacterium to transform plant cells. These vectors are envisioned as a vehicle for introducing foreign genes. (See, for example, Hoekema, A. et al, Nature (1983) 303:179-180. )

Thus, 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. As with animal cells, 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. Using defined media, 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.

As described above the plant transformation process most studied oσcurs when plant cells are infected with soil bacteria, most particularly Agrobacterium tumefaciens. Transformations of plant cells by viruses, and by other bacteria are known but much less studied. When genes encoding phytohormone synthesis are expressed, continuous growth, i.e., immortality, of the plant cell culture results in the absence of exogenously supplied phytohormones.

As with animal cells, plant cells, actually plant cell protoplasts created by enzymatically digesting away the plant cell wall, can be fused. The field has recently been reviewed. (See, for example, Pelletier, G. and Chupeau, Y., Physiol Vegetale (1984) : 311-333 ) . Several studies suggest that genes expressed in each par¬ ent before fusion remain active in the hybrid created by the fusion process, both parental sets of genes continuing to be expressed over many passages or even following re¬ generation of whole plants from the hybrid.

A number of plant cell fusions have been reported. Puite, K.J., et al, Plant Cell Reports (1985) 4_^:274-276 has described the electrofusion of two different nitrate reductase-deficient mutants, which, after fusion, were able to complement each other to obtain a nitrate reductase-positive phenotype. Fusions have also been ef¬ fected between normal, streptomycin-resistant, N. tabacum cells and an Agrobacterium-transformed streptomycin- sensitive N. tabacum cell (Wullems, G.J. et al, Theor Appl Genet (1980) 56:203-208) . This fusion produced hybrids which could grow in the presence of streptomycin and the absence of phytohormones. Cells of two species of tobacco have been fused to obtain cells which produced peroxidase enzymes characteristic of both parent species (Alfonso, C.L. et al, Biotechnology (1985) 2:811-817).

Other manipulations of plant cells have also been performed successfully. For example, 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 C0₂ is the sole carbon source.

The desirability of using plant cell lines to produce their valuable secondary metabolites, such as, for example, pharmaceuticals, pigments, flavorings and fragrances, anti-viral and anti-neoplastic agents, protein inhibitors, a range of anti-insect agents, antigens, and enzymes, and so forth, has been recognized but not very successfully achieved. The field has been recently reviewed by Misawa, M. in Adv i Biochem Eng/Biotech, Vol. 31, (1985) A. Fiechter (Ed.) Berlin: Springer-Verlag, pp. 59-88.

Yamada, Y. in Cell Culture and Somatic Cell Genetics of Plants (1984) JL:629-635 (Academic Press) reviews technigues for selecting cell lines which produce secondary metabolites and cites a series of papers describing isolation of cell lines which produce pigments, vitamins, alkaloids and steroids. According to Yamada's evidence, even descendants of a single cell guickly become heterogeneous in their ability to produce the secondary metabolite. Furthermore, in order to maintain the cultures, complex nutrient media need be used, and cytokinins need to be supplied to perpetuate growth. In addition, all the cultures cited are organotrophic, rely¬ ing on exogenously supplied sucrose as the sole source of energy and carbon for the cultured cells. None of the cultures were photoautotrophic, using the energy of sunlight and the process of photosynthesis for energy and carbon dioxide as sole carbon source.

There are similarities between the process of i munoglobulin production in animals and the process of secondary metabolite production in plants. Both processes occur in specialized, terminally differentiated cells, which are themselves located in specialized tissues or organs. In plants, the process of secondary metabolite synthesis is often constrained to a specific time or developmental stage in the life of the plant. However, im unoglobulins produced by animal cells are proteins or the primary gene product; plant secondary metabolites are produced by the concentrated action of the enzymes which are the primary gene products. Although secondary metabolite production by plant tissues brought into culture is often lost, it is clear from the work of

Banthorpe, D. et al, Photochemistry (1983) 22:2727-2728, that secondary metabolite-producing cells retain the enzymes necessary to synthesize these secondary metabolites from precursors when maintained in culture.

In the Banthorpe paper, it was shown that although callus from Rosa damascena did not accumulate monoterpenes in culture, extracts of these calluses were able to convert isopentylpyrophosphate (IPP) into monoterpenes, and mevalonate into IPP.

There are a number of secondary plant metabolites which are of great economic and humanitarian value which cannot be produced synthetically or which are produced synthetically only at great expense, or not at all, must be isolated from whole plants or parts thereof, a process which is thereby rendered labor intensive, seasonal, which must needs be often located outside the

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. Exemplary among these are the 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.

Other examples include pigments and a variety of pharmaceuticals, e.g., reserpine, digitoxin, codeine, used in treatment of heart disease and management of pain. Establishment of cell lines which produce these complex components or mixtures, if established in culture so as to produce the desired products from simple starting materi¬

10 als, ideally only CO- and mineral salts, would be a great economic resource. The present invention addresses this goal.

Disclosure of the Invention -^■-* 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

20 are capable of producing the desired secondary metabolites.

Some general background may be relevant. In the single-celled photosynthetic bacteria and eucaryotic algae from which higher plants have evolved, all biochemical

25 pathways, even developmentally expressed pathways, are contained within one cell. As higher plants have evolved from these single-celled progenitors, the separation and compartmentalization of physiological and biochemical functions into discrete tissues and organs has occurred;

30 as plants moved from aguatic to terrestrial environments, the functions of photosynthesis and nutrient acguisition were separated into roots and shoots. Similarly, the process of sexual reproduction was separated into a compartment derived from the shoot (flowers) and the func¬

35 tions of energy metabolism and carbon fixation were dis¬ sected from this reproductive function (in leaves) . Continuing evolution has added variation upon variation to this basic motif. Just as physiological functions have been compartmentalized into distinct organs and tissues, so biochemical functions have been compartmentalized. Biochemical pathways which were once contained within one cell have been segregated and separated into spatially, and sometimes developmentally, distinct cytological compartments. Particular organs also have specialized tissues such as the lactifers and nectaries of flowers, glands on leaves, and nodules on root hairs. 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 fundamental biochemical and cytological features, specific to each plant and the plant-specific secondary metabolites it produces, determines the form the invention will assume in each individual instance.

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.

In one aspect 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.

In another aspect, 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. In the dioma, 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. In further aspects, the invention is directed to a method to produce secondary metabolite using the resulting dioma and to methods to produce the dioma.

In still another aspect, 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. Regardless of whether binary dioma, trioma, or tetraoma forms are used, 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.

In the "binary dioma" the culture comprises two independent cell fusions or diomas. In one fusion, 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. In the second fusion, 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. In further aspects, the invention is directed to producing secondary metabolites using the binary dioma and to methods for producing the binary dioma.

In still another aspect, 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.

In yet another aspect, 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. Iri addition, the inven¬ tion is directed to methods of producing secondary

10 metabolites by culturing said tetraoma, and to methods of preparing said tetraoma.

In a further aspect, 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

20 continuous, sustained growth as a single cell teratoma. This transformed cell is fused with the secondary metabolite synthesizing cell to give a chemoorganotrophic dioma, binary dioma, trioma, or tetraoma. Furthermore, the invention is directed to a method of producing second¬

25 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.

30 Although the foregoing emphasizes the supply of precursor for secondary metabolite by the partner(s) fused to the secondary metabolite-producing cell, it is recognized that other functions may also be served by this partner, in addition to or instead of the supply of pre¬

35 cursor. For example, 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. -*' In still another aspect, 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. 0

Brief Description of the Drawings

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.

Modes of Carrying Out the Invention

The principal object of the invention is the efficient and practical production of 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. However, 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.)

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.

When photoautotrophic, 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.

In general, then, 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- .

In the "monomas" of the invention, 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.

In the diomas of the invention, one of the partners must be transformed to have perfect partner characteristics. Ordinarily, 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, however, occurs where photosynthesis, precursor synthesis, and secondary metabolite synthesis are conducted by three different cells. As set forth above, all three cells may be fused into a trioma, wherein at least one of the three has been transformed to perfect partner characteristics. However, 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. Thus, in one embodiment, 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. (Indeed, 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. In ideal cases, the system is photoautotrophic so that the sole carbon source is carbon dioxide and the energy source is light. In I these instances, the medium in which the system is cultured consists essentially of essential minerals and salts.

Depending on the nature of the secondary metabolite, the desired product of the culture is harvested and purified using standard techniques such as chromatography, size separation, or other standard separa¬ tion methods.

10 An overview of the steps in obtaining the secondary metabolite producing system of the invention is as follows:

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

20 ability to grow in the absence of plant hormones. These resulting plant cell photoautotrophic teratomas are thus capable of fixing carbon dioxide and maintaining themselves perpetually in culture without the addition of hormones. This first parental cell line will confer im- ~"^J mortality and efficient substrate use on its fusion products. If the cell line also produces secondary metabolite, it is the "monoma" form of the system.

However, more typically, a second step is to provide at least a second parent—a secondary metabolite-

30 producing cell. 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.

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.

If required, a third parent to form a component of a trioma is a cell capable of providing the precursor. In the case of rose oil, it is .believed that 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.

To complete this trioma, 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 π 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. This can be accomplished by chromogenic sprays, migration in a variety of separatory systems (thin-layer Si-gels, HPLC, etc.), mass spectroscopy, UV absorption, fluorescence emission, etc. If the two- dimensional absorbent is spatially registered with the culture monolayer, then cells producing the secondary metabolite can be located and isolated from a population of nulls.

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. Using the snap-ring culture dish, it is possible to change the under-medium 10 composition that feeds the immobilized monolayer of plant cells. 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. Using 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.

A specific application of the invention is directed to production of pigments called anthocyanins by colored flowers of Petunia hybrida. 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.

All plant phenolic secondary metabolites arise from the primary metabolite phenylalanine (II), an amino acid produced from Calvin Cycle intermediates via the shikimic acid pathway. Phenylalanine is converted to cinnamic acid (III), and this is converted to chalcone (VI) by addition of three 2-carbon moieties from malonyl CoA (VII). Chalcone is then converted to cyanidin or pelargonidin, the selective hydroxylations occurring at the C-15 level. Precursor studies suggest that cinnamate is the sole precursor, and that it is supplied to the flowers from a separate compartment in the plant, and that this precursor is converted to pigments in the flower.

The source for all plant material referred to in the illustration of petunia pigments is greenhouse-grown plants of P. hybrida of appropriate pigmentation phenotype. Horticultural methods are standard. All media formulations referred to in relation to plant tissue culture are standard compositions which may be found in D.A. Evans et al (Eds.) "Handbook of Plant Tissue Culture" New York: Macmillan, 1983.

Example 1

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. 425-230. 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. JO

In the specific case of the pigment-producing petunia dioma, 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. Using the Omaha snap ring culture dish of the invention 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.

The perfect partner and secondary metabolite producer are fused using standard techniques. Early work on plant cell fusion was reviewed by F. Constabel In vitro (1976) J-2_:743-748; more recently electrically induced plant cell fusion has been Biochim Biophys Acta (1982) 694:227-277. Specific methods for Petunia spp cell fu¬ sions are described by J.B. Power et al Nature (1976) 263:500-502.

The resulting effect of P. hybrida pigment synthesis from CO- screening is accomplished using filter paper-bonded extractants as described for the Omaha blot, manually under the microscope, or by automated methods such as fluorescence activated sorting as described by C.L. Afonso et al Bio/technology (1985) _3_^:811-816-