FIELD OF THE INVENTION

This invention relates to plant genetic engineering and more particularly to the expression of foreign proteins in plants and specifically to recombinant gene constructs used to introduce foreign proteins into plants and to transgenic plants and their seeds which express a human, animal or fish protein. BACKGROUND OF THE INVENTION

Collagen, in the form of insoluble fibers or fibrous aggregates, is the major proteinaceous constituent of most vertebrates and many invertebrates. In mammals, collagen is the most abundant body protein forming the major protein constituent of connective tissue, skin, tendon, cartilage and bone.

One type of collagen, collagen I, is the major component of gelatin, a protein useful in food production as a jellying agent, stabilizer, emulsifier, thickener, foaming agent, water binder, crystal growth modifier, glaze, adhesive, binder and fining agent. When blended into milk, gelatin adds a consistency and desirable "mouth feel" to the milk. Gelatin used in the food industry is typically obtained by isolating collagenous material from animal skins and bones or from fish by means of an extensive extraction process. This typically requires several days of treating the hides and bones with lime and/or acids and includes several washing steps.

There is a growing demand for gelatin suitable for the needs ofthe kosher, Muslim and Seventh Day Adventist consumer. These consumers have dietary restrictions against consumption of pork and pork products. Most commercially available gelatin is produced from pork. In the past, only small quantities of properly certified kosher or non-pork gelatin has been produced from limited supplies of hides and bones derived from kosher animals or from skins of kosher fish. The price of kosher gelatin ranges from 6 to 15 times the price of standard bovine or porcine gelatin. The preparation of kosher gelatin is expensive and the limitations of raw material make its production in large quantities on a regular basis extremely difficult.

There is, therefore, a need for a method of producing a ready supply of kosher gelatin which is inexpensive to prepare.

Gelatin is one ofthe most versatile natural products known. It is a material with a wide range of physical and biochemical properties which are responsible for the numerous and varied applications in which gelatin plays an important part. Gelatin is useful in the photographic and leather industries and for pharmaceutical and medical uses, printing applications, coated abrasives and adhesive uses, among other things. Possibly the most important single property of gelatin is its gel-forming characteristic. Many ofthe other physical properties of gelatin, such as its protective-colloidal, setting, swelling and film- forming properties are useful in the photographic process, paper-making and textile industry.

There is therefore a need for large quantities of gelatin which have the biochemical and biophysical properties required for use in the photographic, pharmaceutical/medical, leather, glue, printing and other industries.

Industrial uses of corn are mainly from corn starch in the wet-milling industry and corn flour in the dry-milling industry. The industrial uses for corn starch and corn flour are based on functional properties, such as viscosity, film formation, adhesive properties, and ability to suspend particles. Corn starch is an example of a class of food gums known as seed gum. Besides the usefulness of corn starch as a food thickener, both corn starch and corn flour have applications in the paper and textile industries, as well as in connection with adhesives, building materials, foundry binders, laundry starches, explosives, oil-well muds, and other mining applications.

Collagen is useful in medical, reconstructive, therapeutic and cosmetic applications in humans. For instance, collagen injection therapy is used to treat urinary incontinence (Appell, R.A., Urologic Clinics of North American 21 :177-182 (1994)); photodamaged skin (Goldhar et al., Canadian Family Physician 39:352-356, 359-363 (1993)); acne scars (Matsuoka. L.Y., Clinics in Plastic Surgery 20:35-41 (1993); Low et al., Journal of Dermatologic Surgery & Oncology 18:981-986 (1992)); peridontal tissue regeneration (Dowell et al., British Dental Journal 171:125-127 (1991)) and as a drug delivery system to target drugs for the eye (Friedberg et al, Ophthalmology 98:725-732 (1991)). Collagen for these purposes is typically obtained from tissues of farm animals such as cows or pigs. As such collagen is essentially a foreign protein, immunogenic responses may present problems. To some extent, these problems may be minimized by treating the animal-derived collagen to decrease immunogenicity. However, there remains a need for a ready supply of human collagen for pharmacological, therapeutic and cosmetic applications.

In our co-pending application. Serial No. 08/445,254 filed June 2, 1995, is described a method for producing in milk of a female, non-human mammal a compound endogenous to the mammal but not naturally secreted in its milk and of generating an isogenic mammal which has been genetically altered to produce in its milk an endogenous compound which is normally secreted by other tissues in the mammal. The compound is preferably procollagen or collagen or fragments thereof, which may easily be converted to gelatin. The compound may be recovered from the collected milk or used directly in food production. The disadvantage is that the gelatin obtained from the collagen produced in the milk is dairy, which, for the kosher consumer, limits its use to dairy products only. There is a need for a non-mammalian and non-dairy source of gelatin for the vegetarian market. Furthermore, a special extraction process which is not typically part of milk processing is required to recover collagen or gelatin from the milk of isogenic mammals. Additionally, significant time is required to produce a herd of isogenic milk producing mammals.

Plant genetic engineering techniques are utilized to obtain plants having improved characteristics or traits and to incorporate heterologous genes from a source other than the transformed plant into plant cells to obtain desired traits or to produce useful polypeptides in the plant cells. Thus, transgenic plants have been developed to produce crops of increased value.

Transgenic plants have been developed which produce foreign biological proteins. The 5' regulatory region and putative signal sequence of a rice alpha amylase gene was fused to a bacterial gene encoding β-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of alpha amylase was suppressed by the presence of sucrose in the medium and induced by its absence. Ming- Tsair Chan et al.. J. Biol. Chem. 269:17635-17641 (1994). Turpen et al., Bio/Technology 13:53-57 (1995), disclose malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. The authors engineered hybrid virions of tobacco mosaic virus that contain malaria-parasite immunodominant sequences recognized by protective monoclonal antibodies against these parasites and that yet retain their infectivity.

Herbers et al. in Bio/Technology 13:63-66 (1995) disclose expression at high levels of a thermostable, microbial xylanase from Clostridium thermocellum in the apoplast of transgenic tobacco. The expressed xylanase was easily purified.

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. The transgenic plants produced HBsAg which is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast. Mason et al. in Proc. Nat. Acad. Sci., USA, 89:11745-11749 (1992). The immunological response elicited in vivo by using recombinant HBsAg purified from transgenic tobacco leaves was qualitatively similar to that obtained by immunizing mice with yeast-derived recombinant HBsAg (commercial vaccine). Both B- and T-cell epitopes are preserved when the antigen is expressed in transgenic tobacco. Thanavala et al. in Proc. Nat. Acad. Sci. USA, 92:3358-3361 (1995).

Mori et al. in FEBS 13403, vol. 336, no. 1, 171-174 (1993), disclose construction of transgenic tobacco plants expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative carrying the human gamma interferon gene.

The genes encoding the heavy and light chains of a mouse monoclonal antibody (mAb Guy's 13) have been cloned and expressed in tobacco plants, Nicotiana tabacum. Transgenic plants were regenerated that secrete full-length Guy's 13 antibody. Antigen binding studies confirmed the fidelity of assembly and demonstrated that the antibody is fully functional. Furthermore, the plant antibodies retained the ability to aggregate streptococci, which confirms that the bivalent antigen-binding capacity ofthe full length antibodies is intact. The results demonstrate that IgA as well as IgG class antibodies may be assembled correctly in tobacco plants and suggest that transgenic plants may be suitable for high-level expression of more complex genetically engineered immunoglobulin molecules. Ma, et al. in Eur. J. Immunol. 24:131-138 (1994).

Plant genetic engineering is employed to obtain plants having improved characteristics or traits, such as virus resistance, insect resistance, herbicide resistance, enhanced stability, improved plant taste or nutritional value, altering, starch, oil and protein profile, yield or quality. For instance, work is being done on tomatoes that may be vine- ripened and shipped without bruising, and to provide tomatoes which are better tasting, have improved color and higher vitamin content and which contain more solids. Other projects are directed to generate tomatoes with improved viscosity, i.e., thickness and texture, which means fewer tomatoes are required to generate the same amount of catsup.

Pectin, used to make jelly thicken or gel, occurs naturally in many fruits and vegetables, giving them their firmness. The pectin in ripening tomatoes is degraded by the

enzyme polygalacturonase (PG). As pectin is destroyed, the cell walls of tomatoes break down and soften, making them difficult to successfully ship to market. Reducing the amount of PG in tomatoes slows cell wall breakdown and results in a fruit which remains firm for a longer time at ambient temperature. Calgene, Inc. of Davis, California developed a tomato which incorporates a gene that essentially attaches itself to the PG gene and inactivates it. Such transgenic plants produce drastically reduced levels of PG. This slows the natural softening process that accompanies ripening and allows the Flavr Savr™ tomato to spend more time on the vine than other tomatoes. This results in a more flavorful tomato which is firm enough to be shipped.

U.S. Patent No. 5,202,422 to Hiatt et al. issued April 13, 1993 discloses a method for producing a glycopolypeptide multimer by introducing first and second mammalian genes encoding the constituent parts ofthe multimer into first and second respective members of a plant species, generating a progeny from the first and second plant species members, and isolating the glycopolypeptide multimer from the progeny plant.

U.S. Patent No. 5,188,958 to Moloney et al. issued February 23, 1993 discloses transgenic Brassica species cells (such as rapeseed and rutabaga) produced by transformation of cell cultures with foreign DNA which when expressed will alter the phenotype ofthe transgenic cells using a manipulated Agrobacterium transformation system followed by regeneration of plants from transformed cells. An example of such foreign DNA is the gene for kanamycin resistance. The cells and the plants produced thereby are capable of expressing the foreign gene.

U.S. Patent No. 5,384,253 to Krzyzek et al. issued January 24, 1995 discloses a method to increase the susceptibility of cultured Zea mays cells to stable transformation with recombinant DNA via electroporation so that the cells retain their ability to regenerate fertile, transgenic Zea mays plants containing the introduced DNA which may be inherited by progeny ofthe transformed plant.

D'Halluin et al., Transgenic Maize Plants by Tissue Electroporation, The Plant Cell, 4:1495-1505 (1992) describe the production of normal, fertile transgenic monocots by creating transformation-competent cells from immature zygotes and type I callus by gentle partial hydrolysis of certain cell wall components, such as pectin. This results in enhanced permeability ofthe cell wall to exogenous DNA while not destroying cell viability.

One problem with using transgenic plants to express foreign genes is that plants may not always properly fold or process mammalian proteins and may lack post-translational enzymes that are mammalian specific so as to provide the desired degree of biological or physiological activity in the resulting protem. OBJECTS AND SUMMARY OF THE INVENTION

It is an object ofthe invention to provide a method of producing a ready supply of gelatin from a specific non-mammalian source.

It is an object ofthe invention to provide a method of producing a collagen compound in plants.

It is another object ofthe invention to provide a method of inexpensively producing high yields of kosher gelatin in a short period of time.

It is a further object ofthe invention to provide a method of producing an inexpensive, ready supply of human collagen and procollagen for medical and cosmetic applications.

Another object ofthe invention is to provide a method of producing a ready supply of gelatin by producing a collagen compound in plants by genetic manipulation.

It is another object ofthe invention to provide a method of producing large quantities of gelatin having the biochemical and biophysical properties suitable for use in the food, photographic, pharmaceutical/medical, paper, leather and glue industries.

It is yet another object ofthe invention to provide a method of producing large quantities of non-Bloom gelatin suitable for microencapsulation and photographic applications.

It is also an object ofthe invention to provide a method of producing gelatin from plants from which food gums are extracted to achieve an enhanced gelatin effect.

A still further object ofthe invention to provide a gene construct for producing a collagen compound in plants.

Another object of this invention is the development of transgenic plants capable of reproduction which produce a collagen compound.

Still another object ofthe present invention is the production of a low cost collagen compound in plants.

Yet another object ofthe invention is to provide a method of easily and inexpensively producing human collagen for therapeutic and cosmetic uses.

An additional object ofthe invention is the development of transgenic corn which produces a collagen compound.

These and other objects ofthe invention are accomplished by providing a method of producing transgenic plants capable of expressing a collagen compound. The method comprises introducing a gene construct into cells of a plant. The gene construct comprises a DNA sequence coding for the collagen compound operably linked upstream (5') to a plant promoter sequence capable of initiating and directing transcription in plant cells ofthe DNA sequence. The gene construct is integrated into the plant genome such that, upon transformation, the plant cells express the collagen compound. Transgenic plants are regenerated from the transformed plant cells and grown for a time and under conditions sufficient to permit expression ofthe collagen compound. The collagen compound is then recovered from the transgenic plants.

The collagen compound is preferably procollagen, collagen I through collagen XIX or fragments thereof. The collagen may be of human, animal or fish origin, and preferably of bovine origin. Specifically, the collagen compound may comprise collagen I alpha 1, collagen I alpha 2 and other recombinant collagen products, such as collagen II through collagen XIX, inclusive, and fragments thereof. The procollagen forms of bovine collagen I through XIX and fragments thereof may also be produced by the methods of this invention.

Additionally, in an embodiment ofthe invention, a gene construct comprises at least a DNA sequence coding for the collagen compound operably linked upstream (5') to a promoter sequence capable of initiating and directing transcription in plant cells ofthe DNA sequence. When such a gene construct is incorporated into a plant genome, the DNA sequence coding for the collagen compound is expressed in the plant's cells.

In further embodiments ofthe invention, there is provided a collagen compound- producing plant cell produced by the method described above and a collagen-compound producing plant cell descended from the above plant cell. In yet another embodiment ofthe invention, there is provided collagen produced from plants and seeds, gelatin produced from plants and seeds and a dairy or vegetarian food product which includes gelatin produced from plants.

In other aspects, the invention is directed to plant cells transformed with the gene constructs described above and to plants regenerated from or containing these transformed plant cells. In further aspects, the invention is directed to methods to produce plants and

seeds which produce a collagen compound which method comprises cultivation ofthe transgenic plants ofthe invention followed by recovery ofthe collagen compound as collagen or gelatin.

The invention contemplates genetically altering plants, preferably com plants, to program them to synthesize any one or more ofthe collagen proteins I through XIX, procollagen or any collagen fragments of human, animal or fish origin. The collagen producing com plants are used as an inexpensive method to generate plant derived gelatin. In addition, gelatin from such plants is valuable in the manufacturing of gelatin coated capsules for prescription and over-the-counter pharmaceuticals. Com is the preferred plant as com starch is an essential filler substance used in the pharmaceutical industry in making pills and capsules. Thus, the transgenic com plants ofthe invention are a source of both com starch and gelatin, the major ingredients for gelatin coated capsules. The vegetarian collagen compound ofthe invention has utility in food production and in pharmacology, medicine, agriculture and the cosmetics industry. An additional advantage of using com as the transgenic plant is the recovery of collagen or gelatin without added cost as technology is already well developed to extract protein from com, for instance, by a wet milling process. For non-pharmaceutical uses, the recovered collagen or gelatin is not required to be of high purity.

The collagen compounds that may be produced by the methods of this invention include, for instance, collagen I and fragments thereof and specifically, collagen I alpha 1 and collagen I alpha 2 and other recombinant collagen products, such as collagen II through collagen XIX, inclusive, and fragments thereof. The procollagen forms of collagen I through XIX and fragments thereof may also be produced by the methods of this invention. The collagen compound may be of human, animal or fish origin. In its broadest applications, the present invention is not limited to any particular species of mammals or fish. For instance, mammals such as swine, goat, sheep, oxen and cattle may be used and cold and warm water fish may be used. Advantageously, kosher mammals and fish are used. The preferred fish are carp (Cyprinus Carpio), tilapia (Tilapia Nilotica and the like) and Nile Perch (Lates Niloticus). In these fish, gelatin having a Bloom strength in the same range as hide gelatins (about 250-300 Bloom) may be derived but their melting points are in the range of approximately three degrees to six degrees centigrade lower than in bone gelatin. Standard

Knox ® 300 Bloom gelatin (derived from bovine bone) has a melting point of 29°C, while fish gelatins have a melting point in the range of 23 - 26°C.

It is contemplated that the method ofthe invention may be used to tailor-make collagen or gelatin that meets the needs of a specific industry or application. For instance, where gelatin with a high Bloom strength and low melting point is required for a certain application, it is possible to prepare a transgenic plant which expresses collagen from which such a gelatin having the desired characteristics may be recovered by providing an appropriate gene constmct comprising a DNA sequence coding for a suitable collagen compound.

These and other features, objects and advantages ofthe invention will be apparent from the following description ofthe preferred embodiment, taken in conjunction with the accompanying drawings and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by way of example and not limitation in the Figures ofthe accompanying drawings in which like references denote like or corresponding parts, and in which:

Fig. 1 shows a gene constmct according to the invention; and

Fig. 2 shows an altemative gene constmct according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides a method of producing transgenic plants capable of expressing a collagen compound comprising introducing a gene constmct into cells of a plant. The gene constmct comprises a DNA sequence coding for the collagen compound operably linked upstream (5') to a promoter sequence capable of initiating and directing transcription in plant cells ofthe DNA sequence. The gene constmct is integrated into the plant genome such that upon transformation the plant cells express the collagen compound. Transgenic plants are regenerated from the plant cells which have been transformed and grown for a time and under conditions sufficient to permit expression ofthe collagen compound.

The present invention is capable of providing an unlimited supply of kosher source gelatin, with the long term costs being less than that of standard gelatin. By providing transgenic collagen-compound producing plants, there will never be a shortage of raw material. The present invention contemplates producing transgenic com cells via

electroporation which are capable of regeneration to yield fertile, transgenic com plants. The transgenic com plants produce a collagen compound, specifically procollagen or any member ofthe collagen family I through II or fragments thereof. Ordinarily, com does not produce collagen in any form.

As a result of integration ofthe gene constmct in the plant genome, the gene constmct will be present in all or substantially all ofthe cells ofthe plant tissue after transformation and regeneration. Altematively, expression ofthe collagen compound gene may be targeted to particular plant tissue or particular stages in the development ofthe plant, by the use of tissue or organ specific, or developmental stage specific promoters which can be expressed in the plant cell of choice.

Since collagen I is the major component of gelatin, gene constmcts are introduced into a plant, preferably com, which instructs the plant to produce a collagen compound in its cells. The collagen compound is extracted from com as collagen or gelatin using known wet milling technology. As the destabilization temperature of collagen I is usually above 41° C, a 2-4 minute heat treatment ofthe collagen compound at or above this temperature and subsequent cooling to room temperature or below, i.e., approximately 25° C or below, will convert the collagen compound to gelatin. Altematively, heat treatment for a longer period at lower temperatures and subsequent cooling to room temperature or below will convert the collagen compound to gelatin. While procollagen or collagen does not automatically have any gelling characteristics, measured by Bloom strength, the method ofthe invention contemplates obtaining gelatin preferably with a Bloom strength in the range of 0 to 350 Bloom and preferably in the range of 90-300 Bloom, i.e., from non-Bloom gelatin to full- strength Bloom gelatin, and possessing the biochemical and biophysical properties necessary to meet the particular applications and needs ofthe food production, medical, therapeutic, cosmetic, photographic, glue, leather and other industries.

There is also a need for non-Bloom gelatin for applications such as microencapsulation and photography. Microencapsulation involves safely enclosing materials in microcapsules for later use for such purposes as holding an agent for controlled initiation of a chemical reaction, for controlling the release rate or masking the taste of pharmaceuticals, or for timely release of a flavor or odor. Non-Bloom gelatin is typically obtained from fish collagen and is stable, i.e., it does not gel, at room temperature. The preferred fish collagen for this purpose is obtained from cod, haddock and pollock, but may

be obtained from other fish as well. Accordingly, the method ofthe invention contemplates obtaining non-Bloom gelatin for use in microencapsulation and photography applications. For applications calling for a higher Bloom gelatin having a lower melting point than standard gelatin, it is contemplated that a DNA sequence coding for a collagen compound obtained from tilapia fish (Tilapia Nilotica and the like), carp (Cyprinus Carpio ofthe Cyprinidae family) and Nile Perch (Lates Niloticus), among others, is suitable. Such gelatin is useful in producing soft gelatin capsules. Definitions

The terms plant, selectable marker genes and reporter genes, transcription termination sequences, transformation and operably linked have the following meanings in this application and claims:

As used here, the term plant includes plant cells in planta and plant cells and plant protoplasts in culture, plant cell tissue cultures from which com plants can be regenerated, aggregations of plant cells such as is present as a disorganized mass in a callus, plant clumps, and plant cells that are intact in plants or parts of plants, such as embryos, pollen, flowers, kernels, ears, cobs, leaves, shoots, seeds, fruit, husks, stalks, roots, root tips, anthers, silk and the like.

By "selectable marker genes and reporter genes" is meant a DNA sequence coding for a phenotypical trait by means of which transformed cells may be selected from untransformed cells.

By "transcription termination sequences" is meant any nucleic acid sequence which determines the position ofthe 3' end of a transcript. Transcription termination sequences include polyadenylation sites. By "transformation" is meant the act of causing a cell to contain a DNA sequence which did not originate in that cell. By "operably linked" is meant the chemical fusion of two fragments of DNA in a proper suit ion and reading frame to be transcribed into functional RNA.

The preferred type of com species is obtained from the American Type Culture Collection (ATCC), Rockville, Md. 20852 U.S.A. For example, a tissue culture of regenerable cells of a plant of inbred com line (for example, public inbred lines H99, Pa91) that is highly regenerable from calli, as described in Hodges et al., Bio./Technol. 4:219-223 (1986), is preferred. Applications

Normally, extraction of gelatin from skins and bones is an expensive process and contributes at least 20% ofthe cost of producing gelatin. As contemplated herein, it is not necessary to isolate, extract or purify the collagen compound from the com plant in an additional, separate process; the wet-milling process which is typically used to extract com starch and com gluten may be used to extract the collagen compound. The resulting gelatin may be used in the production of cultured dairy products such as yogurt, ice cream, pudding, sour cream and other milk-based products. This has the advantage of utilizing com for both com starch and gelatin. The resultant gelatin is usable in the food industry with the added benefit of a low-cost, vegetarian (non-dairy) kosher product. Com is the preferred plant as it has the advantages of low raw material cost (in the range of $2.00 to $2.50 per bushel or 56 pounds of dry kernels) and an existing wet milling extraction process.

The resultant gelatin may also be used in such applications as photography, adhesives. pharmaceutical and medical uses, paper and textile making and other uses by conventional methods. The recovered collagen may be used in medical and therapeutic procedures and for cosmetics applications.

There are several advantages to using transgenic plants as bioreactors over bacteria, yeast or isogenic cattle. While the estimated cost of generating transgenic plants may be high initially, the yield and overall cost ofthe resultant collagen and gelatin will be very low. Collagen or gelatin may be extracted from transgenic com plants using existing wet-milling technology. In contrast, while collagen or gelatin produced in milk of isogenic cattle may be used directly in the production of dairy foods, other uses for the collagen or gelatin requires extraction ofthe collagen or gelatin from the milk. This extraction process is an additional, separate step which is not part of milk processing as currently practiced. In practice, bacteria and yeast bioreactors can be very expensive to produce foreign proteins.

The use of transgenic plants has other applications as well. With appropriate gene constructs the expression ofthe inserted genes in transgenic plants may be controlled in a tissue-specific and in a differentiation-specific manner. Plants may be altered not only to produce a collagen compound but also to obtain an "enhanced gelatin effect" when the collagen compound is produced in a plant which is also the source of a food gum such as com starch, guar gum. locust bean gum, etc. By "enhanced gelatin effect" is meant that there are additional, noticeable effects on gelling properties as a result ofthe synergistic interaction of the collagen compound from which gelatin is derived and the food gum which is produced by

the same plant. Although the primary effect is that of gelling, it is understood that this can affect the overall properties and cause general improvement in the resulting gel. The collagen and food gum may either interact during growth ofthe plant or altematively, where the collagen and the food gum are present in different cellular compartments ofthe plant, the enhanced effect is initiated during processing ofthe crop, when collagen/gelatin and the food gum are brought together. Technologies associated with milling of food gum producing crops and starch producing crops are adapted to harvest collagen, gelatin or a gelatin-food gum combination.

Examples of suitable monocotyledonous plants into which the gene constmct may be inserted include com (Zea mays Linnaeus), rice, wheat, barley, oats, millet, sorghum amaranth, onion, asparagus and sugar cane. Suitable dicotyledonous plants include alfalfa, soybean, cotton, clover, sugarbeet, sunflower, carrot, celery, cabbage, cabbage, broccoli, brussel sprouts, radish, rapeseed, cucumber, pepper, canola, bean, lettuce, cauliflower, spinach, artichoke, pea, okra, squash, kale, collard greens, potato, tobacco, tomato, tea and coffee and the like.

Other suitable plants foi use in this invention are plants from which food gums are derived. Food gums or hydrocolloids are water-soluble or -dispersible polysaccharides (glycans) and their derivatives and gelatin (which is a protein rather than a polysaccharide). Gums are useful due to their capacity to thicken or gel aqueous systems at low concentration. Gums are utilized as binders, bodying agents, bulking agents, crystallization inhibitors, clarifying agents, cloud agents, emulsifying agents, emulsion stabilizers, encapsulating agents, film formers, flocculating agents, foam stabilizers, gelling materials. mold release agents, protective colloids, suspending agents, suspension stabilizers, swelling agents, syneresis inhibitors, texturing agents and whipping agents, in coatings and for water absoφtion and binding.

Polysaccharide food gums include seed gums such as com starch, guar gum and locust bean gum; tuber and root gums such as potato starch, tapioca starch and konjac mannan. Gums such as algins, carrageenans and agar are seaweed extracts.

Other suitable plants useful in the invention are ofthe family Leguminosae and include the guar plant, Cyanoposis tetragonolobus, the source of guar gum, and the locust or carob tree, Ceratonia siliqua, from which locust bean gum is derived. Also suitable are seaweeds including Irish moss (Chondrus crispus) from which the polysaccharide gum

carrageenan is extracted, and giant kelp, Macrocytis pyrifera, which is the principal source of commercial alginate. Other related brown seaweeds which are also suitable are rockweed (Ascophyllum nodosum) and the several Laminaria species, from which alginates are recovered. Many seaweeds belonging to the Gracillaria, Gelidium, and Acanthopeltis groups yield agar on extraction and these plants are also suitable in the method ofthe invention.

Food chemists have found that combining two food gums, such as K-carrageenan and locust bean gum, yields a desirable effect on the food being prepared. A more elastic gel with markedly greater gel strength, viscosity, gel elasticity and less syneresis is produced. The combination of K-carrageenan with konjac mannan also produces desirable results. At high concentrations, carrageenan increases the gel strength of guaran, but at low concentrations it produces only an increase in viscosity. The combination of two food gums has never before been accomplished in plants.

It is advantageous to insert the gene constmct ofthe invention in plants which produce food gums to achieve an enhanced gelatin effect due to the interaction ofthe food gum and the collagen compound. It should be understood that the present invention is not limited to the plants described herein and other plants may be similarly employed. Collagen-Gelatin Transition

The typical vertebrate collagen molecule is a rigid rod about 3000 A long and 15 A in diameter, with a particle weight of about 3 x 10 ⁵ g/mole. It is composed of three polypeptide chains of approximately equal length, with an average weight per chain of about 1 x 10 ⁵ g/mole. The triple helix of collagen I is a heterotriplex containing the products of two different collagen-encoding genes and is designated [α](I)] _{2 2} (I). Collagen I triplexes contain two protein chains encoded by the CollAl gene and one protein chain encoded by the CollA2 gene. When heated above a critical temperature or as a consequence of changes in a solvent environment that lower the critical temperature below the experimental temperature, the rigid three-stranded collagen molecule collapses to a mixture of random-coil components collectively termed gelatin. This treatment results in the separation of non-covalently cross¬ linked molecules into three single-chain random-coil units, termed the α-components.

In mammals, fibroblasts typically synthesize and secrete collagen. The following co- translational and post-translational modifications occur when collagen is produced in fibroblasts: cleavage of "signal" peptides at the N-termini ofthe chains, hydroxylation ofthe Y-position proline and lysine residues, hydroxylation of a few X-position proline residues,

addition of galactose or addition of galactose and then glucose, to some ofthe hydroxylysine residues, addition of a mannose-rich oligosaccharide to the C propeptides, association ofthe C-terminal propeptides, and finally formation of both intrachain and interchain disulfide bonds in the propeptides. After secretion of procollagen from fibroblasts, the N propeptides are cleaved by a procollagen N proteinase and the C propeptides are cleaved by a separate procollagen C proteinase. The collagen then self-assembles into fibrils. Lysyl oxidase converts some lysine and hydroxylysine residues to aldehyde derivatives that form cross-links with similar residues in adjacent molecules as disclosed by Prockop, DJ et al., New England J. Med. 311:376-386 (1984).

Most proteins require extensive post-translational modification in order to exhibit full biological activity. In a preferred embodiment, post-translational modifications and biological activity ofthe compound is not a concern. The posttranslational events ofthe protein of interest, collagen, may be important but not critical to its capacity to gel. As an objective ofthe invention is the production of gelatin which has a random coil configuration, the presence of appropriate enzymes to ensure proper assembly ofthe collagen into triplexes is immaterial. So long as the collagen compound produced in the plant has the ability to gel when recovered, the stmcture, nature and biological activity ofthe collagen compound is not critical. Only a limited region ofthe collagen protein is required for the resulting gelatin to display a gelling effect. G. Stainsby in The Science and Technology of Gelatin, edited by A.G. Ward and A. Courts, Academic Press 1977, pg. 196-199. Accordingly, the collagen compound may be a fragment of a protein, such as a fragment of procollagen or collagen. Specifically, the collagen compound may comprise the COLIA1 chain, the COLIA2 chain, both the COLI A 1 and COLIA2 chains or appropriate portions thereof. In one embodiment, the collagen compound may be collagen I alpha 1 which has the ability to gel as a homotrimer. Of course, gelatin derived from classical bovine collagen I which is comprised of two chains of COLI Al and one chain of COLI A2 will exhibit a higher Bloom strength than other gelatins, so that classical bovine collagen I may be the preferred collagen compound for some applications. Gene constructs

The invention also contemplates a gene constmct which comprises a DNA sequence coding for the collagen compound operably linked upstream (5') to a promoter sequence capable of initiating and directing transcription in plant cells of said DNA sequence. The

gene constmct is integrated into the plant genome such that upon transformation the plant cells express the collagen compound. Transgenic plants are regenerated from the plant cells which have been transformed and grown for a time and under conditions sufficient to permit expression ofthe collagen compound.

The invention is also directed to seeds obtained by growing the transgenic collagen compound-producing plants, collagen produced from plants, gelatin produced from plants and food products which include gelatin produced from plants.

The proper regulatory signals must be present in the proper location with respect to the gene for the newly inserted collagen gene to express the protein for which it codes in the plant cell. These regulatory signals include a promoter sequence that directs the cellular machinery to produce RNA and a polyadenylation sequence which is a non-translated region that terminates transcription in plant cells and causes the addition of polyadenylate nucleotides to the 3' end ofthe RNA to stabilize the RNA in the cytoplasm for subsequent translation ofthe RNA to produce protein.

The promoter may be any constitutive, inducible, tissue or organ specific, or developmental stage specific promoter which can be expressed in the plant cell of choice. Constitutive promoters, such as the 35S promoter of cauliflower mosaic vims, which direct RNA production in many or all tissues of a plant and during most stages of development when integrated into the genome of transgenic plants are preferred. In particular, the 35S promoter from the cauliflower mosaic vims (CaMV35S), which has been shown to be the strongest constitutive promoter known in plants and which confers expression in both dicots and monocots, is preferred. Odell et al.. Nature, 313:810-812 (1985); Jensen et al., Nature, 321:669-674 (1986); Jefferson et al., EMBO J., 6:3901-3907 (1987); Kay et al., Science, 236:1299-1302 (1987); Sanders, et al., Nucleic Acids Research, 4:1543-1558 (1987). In particular, a chimeric promoter with doubled CaMV enhancer elements, referred to herein as a CaMV35S doubled enhancer promoter, is preferred.

It is critical that the gene constmct consisting of at least a promoter and the DNA sequence coding for the collagen gene integrates into the genome ofthe com so that it is stably inherited by progeny ofthe transgenic com. Stable transformation comprises the chromosomal integration, including incorporation into plastid chromosomes, ofthe introduced DNA so that the integrated gene sequences are passed on to progeny ofthe transformed plant. Stably transformed cells must also be capable of regenerating fertile,

transgenic plants. Transient transformation, in contrast, does not generate transgenic plants as the introduced DNA is eventually lost.

Electroporation is the presently preferred method of introducing foreign DNA into com. Fromm et al. Nature. 319:791-793 (1986); Jones et al., Plant Mol. Biol., 13, 501 (1989); Yang et al.. Plant Cell Reports, 7, 421 (1988); D'Halluin et al., The Plant Cell R:1495-1505 (1992) and Krzyzek et al., U.S. Patent No. 5,384,253.

Thus, the invention contemplates cloning the appropriate bovine collagen genes from genomic DNA or from messenger RNA as cDNA. A cDNA constmct will not contain all the introns while the genomic DNA will. Preferably, bovine collagen type I (either the αl or α2 chains, or both chains or fragments thereof) is cloned. Appropriate DNA sequences containing the bovine collagen compound gene or fragments thereof linked to a plant specific promoter such as the CaMV35S promoter are constmcted and used to transform com (maize). Constructs are made using specific restriction enzymes which recognize certain sequences of bases on the DNA strand and cut where those sequences appear. Ligases are enzymes which rejoin DNA segments. The cut DNA is ligated (joined) to a DNA vector which allows the gene of interest to become incorporated into the plant genome. The vector may contain viral and/or plasmid features. Heterozygous transgenic plants that synthesize the collagen compound are generated. Transformation occurs when the gene carried by the vector is incorporated into the DNA ofthe plant where it initiates production ofthe desired collagen compound. Using backcrossing techniques well known to those of ordinary skill in the art, homozygous maize for commercial planting is then generated. Transformed plants are grown and either pollinated with the same transformed strain or different strains. The resulting progeny expressing the collagen compound is then identified. Two or more generations may be grown to insure that the collagen compound is stably maintained and inherited and then seeds are harvested for use to provide plants expressing the desired collagen compound. Similar procedures are followed to obtain collagen compound of human or fish origin. Cloning The Bovine Collagen I Genes Overview

The entire complement of genes in an organism that encodes proteins needed for development and life is called the genome. In mammals, the genes are organized on chromosomes with each chromosome being a single long molecule of double stranded DNA. The DNA in the chromosomes functions as a blueprint for proteins. Many genes are encoded

as a single unintermpted sequence of nucleotides. Some genes are arranged in segments with coding regions (exons) intermpted by non-coding regions (introns). When a particular protein is needed, the code to synthesize the protein is transferred from the DNA into a molecule of messenger RNA (mRNA) by a process called transcription. The 5' end ofthe mRNA encodes the protein's amino terminus (beginning with the first exon) and the 3' end encodes the carboxyl terminus. Nucleotides preceding the first exon are referred to as being "upstream" from the gene and nucleotides after the last exon are "downstream" from the gene. RNA transcription is initiated at a specific upstream DNA sequence called a promoter. Transcription rate may be accelerated by the presence of sequences in the DNA called enhancer elements and these may be upstream or downstream ofthe promoter. After transcription, the mRNA may be further processed by splicing before leaving the nucleus. The completed mRNA enters the cytoplasm where it encounters ribosomes which translate the codes contained in the mRNA into the appropriate protein.

Where not specified, recombinant DNA procedures follow the methods of Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), incoφorated herein by reference. Restriction enzymes and T4 ligase are obtained from commercial sources, such as New England Biolabs of Beverly, Massachusetts, and used according to the manufacturer's recommendations . Cloning the human collagen gene

The gene encoding the alpha I polypeptide chain of human type I collagen (COL1A1) has been cloned as disclosed by Barsh, G.S. et al, Joumal of Biological Chemistry 259(23): 14906- 14913 (1984). incoφorated herein by reference. It spans a large area (18,000 base pairs) and is composed of multiple exons. In addition to the upstream promoter, the COL1A1 gene transcription may also be regulated by sequences within the first intron (noncoding region between the first and second exons) as disclosed by Bomstein, P., Proc. Natl. Acad. Sci, USA 84:8869-8873 (1987). Cloning the genomic bovine collagen I alpha I gene and bovine collagen I alpha II gene

A procedure similar to that for cloning human collagen type I alpha 1 is used for cloning bovine collagen. Specifically, a bovine genomic library is created by isolating DNA from bovine fibroblasts, cutting it with appropriate restriction enzymes to generate large fragments of DNA and cloning them into a cosmid vector, using standard techniques such as

disclosed in Sambrook and Maniatis, Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (1989). Large segments of DNA (30-42 kilobases) are cloned into a single cosmid. The resulting library is screened with cDNA probes made by restriction digestion ofthe human collagen I alpha 1 cDNA clone in the conventional way disclosed by Tromp, G., Biochemical Joumal 253:919-922 (1988), incoφorated herein by reference, to identify and isolate the bovine collagen I alpha I gene. The cDNA is preferred for screening instead ofthe genomic human clone because the cDNA contains only exons. The exons of collagen are highly conserved between species whereas introns may not be highly conserved as disclosed by Bernard, M.P., Biochemistry, 22(22):5213-5223 (1983). The cDNA probes allow detection ofthe exons within the bovine genomic fragments contained in the bovine genomic library. The library is screened with a probe which encodes the 5' region ofthe human collagen gene and subsequently positive clones are then screened with human probes encoding the far 3' region ofthe collagen gene. The double screening may result in a single clone containing the entire bovine COLlAl gene. If a full length clone is not contained within a single cosmid, the library may be rescreened with the 3' probe and positive clones analyzed for sequences which overlap the clones "pulled" from the library with the 5' probe. Restriction enzyme analysis of each resulting clone enables creation of a hybrid clone encoding the entire gene by splicing into the overlapping intron regions. Furthermore, clones which hybridize with the 5' probe but not the 3' probe may be used to identify the bovine collagen promoter and enhancer elements. These techniques result in (a) a full length genomic clone encoding the bovine COLlAl gene and (b) a clone containing the collagen transcriptional promoter and enhancer elements. These recombinant constmcts are then used to create clones to be used for gene constmcts to generate transgenic com. The same strategy is followed to clone the bovine collagen type I alpha 2 gene. The cloned human COL1A2 cDNA is used in the conventional manner disclosed by Kuivaniemi, et. al., Biochemical Joumal 525:633-640 (1988), incoφorated herein by reference, to create 5" and 3' probes for the isolation of a bovine collagen type I alpha 2 full length genomic clone and regulatory elements.

Cloning the bovine cDNA encoding the bovine type I alpha 1 collagen polypeptide and bovine type I alpha 2 collagen polypeptide cDNA libraries are created from mRNA transcripts and do not contain the intronic sequences ofthe genomic gene. Many genomic genes are silent in differentiated or

specialized cells. The active genes are transcribed and proteins synthesized from the messages. Therefore, tissue specific or differentiation specific clones can be generated from the mRNAs being synthesized at the time of RNA extraction. RNA is extracted from specialized bovine cells which synthesize collagen type I and double strand cDNA is synthesized using standard techniques to create a DNA bovine library encoding for bovine type I alpha 1 collagen. The average size mRNA in a cell is usually around 2 kilobases. However, the human collagen type I (and other collagen) message is quite large (4-5 kilobases) and bovine collagen type I is similar in size. While most plasmid vectors used in cloning are able to handle inserts of this size, it may be advantageous to clone into lambda phage vectors which can accept inserts as large as 10 kilobases without replication problems. Plasmids containing smaller inserts will outgrow those containing large inserts and may make screening more difficult. It may be necessary to enrich for large mRNAs by size fractionation over sucrose density gradients. After enrichment the resulting RNAs are reverse transcribed into cDNA and ligated into the vector of choice. This results in a library enriched for the bovine collagen I transcript. The library is screened with the human collagen alpha 1 cDNA probe as discussed above with respect to a genomic library. Positive clones are sequenced to determine whether they are full length. If no full length clones are obtained, clones which contain areas of overlap may be spliced together to create a full length bovine cDNA. This results in a clone encoding bovine type I alpha 1 collagen. This cDNA clone encodes for a procollagen protein. This clone may be used to create gene constmcts by ligating it with the appropriate promoter and enhancers for generating transgenic com.

The collagen type I alpha 2 gene is isolated in a similar manner using the human collagen type I alpha 2 cDNA as a probe as discussed above with respect to a genomic library to screen the cDNA bovine collagen type I alpha 2 library. In all other respects, the process for collagen type I alpha 2 is the same as for collagen type I alpha 1. Cloning any collagen compound gene

Similar processes, as outlined above, may be followed for cloning any other collagen compound gene such as collagen II through collagen XIX and fragments thereof, of human, animal or fish origin. The resulting clones are used for DNA constmcts to generate transgenic com. It is understood that new probes for use in cloning collagen and new methods of cloning different types of collagen may be developed for use in the invention. Creation of clones for generating transgenic plants

Recombinant collagen gene constmcts are used to generate transgenic plants which are able to produce procollagen, the collagen I alpha 1 and/or collagen I alpha 2 chains or fragments thereof in their cells. Either a constitutive promoter, such as the CaMV35S promoter, or a desired tissue specific or differentiation specific promoter is then ligated to the gene encoding the desired collagen compound using standard techniques now common in the art. A transcription termination sequence is ligated downstream ofthe stmctural gene to provide for efficient termination. Specific Examples of Gene Constructs

Two different types of gene constmcts shown in Figs. 1 and 2 and described below are preferred. The preferred gene constmcts are integrative (integrate into host DNA). Constructs using the cDNA bovine clone

The cloned bovine cDNA prepared as described above is put under the regulatory control ofthe cauliflower mosaic vims 35S promoter utilizing conventional genetic manipulation techniques. Constructs using the cDNA human, animal or fish clone

The cloned human, animal or fish cDNA prepared as described above is put under the regulatory control ofthe cauliflower mosaic vims 35S promoter utilizing conventional genetic manipulation techniques Generating transformed plant cells

The preferred gene constmcts for producing procollagen or collagen to be used to produce gelatin are as follows:

Referring to Fig. 1, the preferred gene constmct comprises a CaMV35S doubled enhancer promoter fused to the collagen compound gene, which may be the cloned bovine collagen type I alpha 1 gene or the collagen type I alpha 2 gene or the procollagen genes or any member ofthe collagen compound gene family as defined above 5' to the start codon for protein translation. A transcription termination sequence, including a polyadenylation signal, from the nopaline synthase or octopine synthase genes or from CaMV is fused to the 3' end of the collagen compound gene to ensure cessation of translation. After transcription termination, polyadenylic acid "tails" are added to the 3' end of mRNA precursor for message stability. This results in a first gene fragment. A second gene fragment carrying the neomycin resistance gene under the regulatory control of a second CaMV35S promoter and a transcription termination sequence are fused to the 3' end ofthe first gene fragment. The

resulting gene construct is cloned into a bacteria vector in toto in order to grow the construct for DNA purification. The gene constmct is then introduced into a plant for integration into the host chromosome as discussed below.

Other elements such as leader sequences, introns, enhancers, polyadenylation sequences and the like may optionally be included in the foreign DNA to improve expression or functioning ofthe introduced DNA in the plant by affecting transcription, stability ofthe mRNA, etc. For example, insertion ofthe maize alcohol dehydrogenase 1 intron 1 (Adh-1) (Callis et al., Genes and Develop., 1, 1183 (1987)) or the rice actin 1 intron 1 (Act-1) sequences (McElroy et al., Mol. Gen. Genet. 231:150-160 (1991)), between a promoter and a coding sequence in a particular recombinant DNA constmct leads to a 10-65 fold increase in production of a reporter enzyme in transgenic maize plants. (Callis et al., supra). However, even without inclusion of an intron or enhancer element in the gene constmct. sufficient expression for a selectable marker to facilitate identification and selection of transformed cells may often be obtained. Klein et al., Plant Physiol., 91, 440 (1989). Gene constmcts containing the Adh-1 or Act-1 enhancer elements contained within the introns are preferred to increase expression ofthe collagen compound gene in maize cells. Referring to Fig. 2, the rice actin 1 intron 1 or com alcohol dehydrogenase intron 1 is inserted between the CaMV35S doubled enhancer promoter and the collagen compound gene. The Act 1 intron 1 gene is obtained by digestion of plasmid pBCG-A4 with Xbal and Ncol as disclosed in McElroy et al., Mol. Gen. Genet. 231:150-160 (1991). The Adh 1 intron 1 sequence is obtained by restriction digest of plasmid pCI,GusN with Xbal and Smal as disclosed by McElroy et al., Mol. Gen. Genet. 231 :150-160 (1991). Constructs using the cDNA bovine clone

This constmct encodes the collagen alpha 1 and collagen alpha 2 procollagen genes. In one embodiment, the cDNAs encoding procollagen are cut with appropriate restriction enzymes to remove the nucleic acids residues which encode procollagen amino and carboxyl terminal amino acids. This latter constmct is referred to as bovine collagen cDNA. These terminal amino acids are needed for correct association of two collagen type I alpha 1 and one collagen type I alpha 2 procollagen chains to form the procollagen triple helix. The terminal amino acids are cleaved when the procollagen molecule is secreted from cells and this cleaved molecule is referred to as collagen. In this embodiment ofthe invention, it may not

be necessary for procollagen to be secreted by the plant cells as long as the protein may be stored without degradation.

The CaMV35S promoter used herein is described in Benfey and Chua, Science 250:959-966 (1990). Analysis ofthe CaMV 35S promoter indicates two domains, domain A from nucleotide -90 to +8. and domain B from nucleotide -343 to -90, and five subdomains, Bl through B5. whose end points are indicated below, located upstream ofthe Tata region (- 46 to +8) ofthe CaMV35S promoter.

B5 B4 B3 B2 Bl Al TaTa

-343 -301 -208 -155 -105 -90 -46 +8

The promoter sequences used are derived from plasmid pKM794 described in Omirulleh et al., Plant Molecular Biology, 21:415-428 (1993) as attributed to Drs. E. Fejes and F. Nagy (unpublished). Plasmid pKM794 contains a CaMV doubled enhancer promoter sequence fused to the GUS gene with NOS terminator. The doubled enhancer promoter constmct of plasmid pKM794 contains a tandem repeat of domains B3 to Al (nucleotides - 208 to -46) inserted upstream ofthe promoter domains Al and Tata (nucleotides -90 to +8) of the CaMV35S promoter. This entire doubled enhancer/promoter unit is excised from plasmid pKM794 by cleaving with Hind III Bam HI.

Octopine Ti plasmids carry an ocs gene which encodes octopine synthase (lysopine dehydrogenase). Koncz et al., EMBO J. 2:1597-1603 (1983) provides a functional analysis of ocs. Nopaline Ti plasmids encode the nopaline synthase gene (nos) (sequenced by Depicker et al.. J. Mol. App. Gen. 1 :561-573 (1982). A functional analysis of nos is provided by Shaw et al., Nucl. Acids Res. 12:7831-7846 (1984).

The CaMV35S doubled enhancer promoter sequence is fused to the bovine collagen/procollagen type I alpha I or bovine collagen/procollagen type I alpha 2 or human collagen/procollagen clone using established molecular biology techniques, following Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

A transcription termination sequence including a polyadenylation signal is fused to the 3' end ofthe CaMV35S doubled enhancer promoter sequence-collagen compound gene. The transcription termination sequence may be obtained from the same source as the promoter (i.e.. from CaMV) or may be obtained from a different source. Omirulleh et al.,

Plant Mol. Bio. 21:415-428 (1993) discloses a 260 base pair sequence containing a 3' transcription terminator sequence ofthe nopaline synthase gene in plasmid pKM794. This 260 base pair sequence is derived from restriction digest of plasmid pKM794 with Sacl and EcoRI and is ligated to the 3' end ofthe doubled enhancer promoter sequence-collagen compound gene using techniques as disclosed by Maniatis. Altematively, restriction digest of plasmid pNA2G disclosed by Omirulleh et al., Plant Mol. Bio. 21:415-428 (1993) with EcoRI and PstI releases the CaMV35S terminator/polyadenylation fragment, which is then ligated to the 3' end ofthe doubled enhancer promoter sequence-collagen compound gene as discussed above. Another vector constmct, pDE108. disclosed in D'Halluin, The Plant Cell 4:1495-1505 (1992), uses an octopine synthase termination/polyadenylation nucleotide sequence which is released from pDE108 by cleavage with Ncol and Hind III and subsequently ligated to the 3' end ofthe doubled enhancer promoter sequence-collagen compound gene.

The foreign DNA coding for a collagen compound to be introduced into the plant preferably contains a selectable marker or a reporter gene, or both, effective in plant cells to aid in isolation of transformed cells and to promote identification and selection of transformed cells. The selectable marker may altematively be carried on a separate piece of DNA and used in a co-transformation procedure. The selectable marker and/or reporter genes are flanked with appropriate regulatory sequences to enable expression in plants. Selectable markers useful in the invention include genes encoding antibiotic and herbicide resistance such as are well known in the art. A preferred dmg resistance marker is the gene whose expression results in neomycin resistance. After transforming the plant cells. those cells having the gene constmct will be identified by their ability to grow on a medium containing the particular antibiotic.

Reporter genes encode easily assayable marker proteins whose expression is manifested by some easily detectable property, e.g., change in phenotype or enzyme activity. Reporter genes are not present in or expressed by the recipient cell or tissue. They are used to determine whether a specific foreign DNA sequence can transform a plant cell. Preferred reporter genes include the luciferase genes from firefly Photinus pyr amis. After the foreign DNA is introduced into the recipient cells, cells are assayed for expression ofthe reporter gene.

The first gene fragment comprising the doubled enhancer promoter sequence-collagen compound gene-transcription termination sequence is fused to an EcoRI/Hindlll fragment of plasmid pDE108 containing the entire sequence ofthe CaMV promoter - neomycin resistance gene - octopine synthase gene terminator region (3' ocs) described by D'Halluin et al., The Plant Cell 4: 1495-1505 (1992). This entire new constmct is cloned into a bacterial vector such as pBluescript (Stratagene, Califomia) at an appropriate restriction site(s) for growth and isolation ofthe DNA. Before transformation of plant cells, the gene constmct is excised from the bacterial vector with appropriate restriction enzymes and purified by agarose gel electrophoresis to remove any bacterial vector DNA. The resulting gene constmct will be used for transformation. Corn Tissue Culture

Unless otherwise specified, com tissue culture procedures are as described in Green and Rhodes, "Plant Regeneration in Tissue Culture of Maize", Maize for Biological Research (Plant Molecular Biology Association, Charlottesville, Va. 1982, at 367-372) and in Duncan, et al., "The Production of Callus Capable of Plant Regeneration From Immature Embryos of Numerous Zea Mays Genotypes", 165 Planta 322-332 (1985). Methods of preparing and maintaining calli from maize tissue are described in R. Phillips et al., Com and Com Improvement, Agronomy Society of America (3d ed., 1988) at pages 345-387. Transformation of maize cells

Methods to introduce the collagen compound gene into plants include the direct transfer of DNA by microinjection. electroporation, DNA entry aided by polyethylene glycol. injection of plant tissue with Agrobacterium tumefaciens engineered to carry foreign DNA into plants, liposome fusion, use of a particle gun to inject DNA-coated microprojectiles and the like. There are advantages and disadvantages to each of these methods. A particular method of introducing the gene constmct into a particular plant species may not be the most effective for another plant species. For instance, Agrobacterium mediated transformation has been used successfully to transform dicotoleydonous plants but its utility for monocot transformation is limited. Electroporation is the presently preferred method for transformation of com so that the foreign DNA is stably integrated in the com plant genome and inherited by progeny ofthe transformed com plants. D'Halluin et al., The Plant Cell, 4:1495-1505 (1992). U.S. Patent No. 5,384,253 to Krzyzek et al, incoφorated herein by reference, discloses a preferred method of transforming com via electroporation.

To successfully produce fertile transgenic plants by electroporation, target cells are treated to render them competent for uptake of foreign DNA without significantly reducing viability. These "transformation-competent" cells incoφorate the foreign DNA at appropriately high frequencies to stably transform a sufficient number of cells. The transformed cells are capable of maintaining cell division and regenerative capacity throughout the selection processes necessary to confirm and identify stably transformed cells. The transformed regenerated plants are capable of passing on the introduced DNA to progeny so that the progeny express the introduced DNA.

Three types of cells are preferred for use in transformation and subsequent production of transgenic maize: (a) immature zygotes isolated from ears, (b) type I callus produced by culture of immature zygotes, and (c) suspension cultures of type II callus generated from the type I callus cultures. Preparation of callus and suspension cell cultures

Regenerable maize suspension cell cultures may be derived from a number of plant tissues. Preferably, the cell cultures are derived from calli generated from immature maize embryos which are removed from the kernels of an ear when the embryos are about 1-3 mm in length (about 9-14 days after pollination). Embryos are aseptically isolated and placed on nutrient agar initiation/maintenance media with the embryo root/shoot axis down (scutellum up). The initiation maintenance media (i.e., F medium, obtained from Gibco, Grand Island, New York) consists of N6 basal media (Chih-ching, Proceedings of Symposium on Plant Tissue Culture. May 25-30, 1978, Science Press, Peking, pp. 43-50) with 2% (w/v) sucrose, 1.5 mg/liter 2,4-dichlorophenoxyacetic acid, 6 mM proline, 200 mg/l casein hydrolysate and 0.25% Gelrite (Kelco, Inc., San Diego). Callus tissue (type I) appears and grows from the scutellum after several days to a few weeks. The callus tissue from the scutellum is evaluated for friable consistency and the presence of well-defined somatic embryos which would indicate that the cultures are regenerable under proper conditions. Tissue is of "friable consistency" when it is easily dispersed without causing injury to the cells. Tissue meeting this definition is transferred to fresh media and subcultured on a routine basis about every two weeks.

After about 4-6 months, the established callus cultures is referred to as type II callus and is transferred to liquid growth media. Methods for producing regenerable suspension cell cultures are described by C. E. Green et al. in Maize for Biological Research, Plant Molec.

25

Biol. Assoc. (1982) at pages 367-372; R. Phillips et al., Com and Com Improvement. Agronomy Soc. Amer., (3d ed., 1988) at pages 345-381; and I. Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vol. I, Laboratory Procedures and Their Applications, Academic Press (1984) at pages 152-158. The liquid growth media for suspension cell cultures is typically of similar formulation to F media with ABA (abscisic acid) (lO.sup -7 M) added to augment regenerative capacity and enhance culture vitality. The callus is preferably not sieved prior to introduction into the liquid growth media. The cultures in liquid media are subcultured as appropriate to maintain active growth. Preparation of Transformation-Competent Cells

"Transformation-competent cells". defined as cells having an increased ability to take up, express and integrate foreign DNA by electroporation as compared to untreated cells, are prepared by partially enzymatically degrading the cell walls ofthe cells by the controlled exposure of cells to one or more pectin-degrading enzymes. Hydrolysis ofthe pectin component ofthe cell wall is believed to increase the cell's permeability to foreign DNA while preserving the viability ofthe cell. In contrast, previous production of transgenic dicots has been accomplished by completely enzymatically degrading the cell walls of type II callus thereby creating a type of competent cells termed protoplasts. However, electroporation of monocot protoplasts (i.e., maize) results in extremely low transformation rates and often phenotypically abnormal plants (i.e., reduced size, seed number and viability). Krzyzek et al.. supra; D'Halluin et al., The Plant Cell 4:1495-1505 (1992).

Transformation competent cells prepared as disclosed in Krzyzek et al., U.S. Patent No. 5,384,253, are moφhologically and physiologically different from protoplasts in a number of ways including the shape and conformation. Transformation-competent cells retain the out-of-round shape of callus cells and consist of stable multicell clumps in culture while protoplasts are spherical and unless they reversibly agglutinate, do not clump. As portions ofthe cell wall are still intact, transformation-competent cells are stainable with Tinapol BPOT (Ciba-Geigy), a cellulose-specific stain. No staining is observed with protoplasts as the cells wall is completely degraded.

Polysaccharidase enzymes such as one or more pectin degrading enzymes are used to break down part ofthe maize cell walls. The term "pectin degrading enzyme" includes enzymes that catalyze the breakdown of pectin and pectin subunits. Examples of pectin- degrading enzymes include endopectin lyase, pectin lyase, pectolyase,

endopolygalacturonase, and polygalacturonase, as well as pectinase. Other enzymes such as xylanase, cellulase, hemicellulase, driselase, transeliminase, or macerozyme, may also be used in combination with pectin-degrading enzymes. Pectolyase (Sigma Chem. Co., St. Louis, Mo.), a combination of endopectin lyase and endopolygalacturonase, is preferably used to prepare transformation-competent cells. It is also available from Seishin Pharmaceutical Co. (Tokyo, Japan) as "Pectolyase Y-23".

Enzymatic treatment is carried out for a time sufficient to partially break down the maize cell walls without adversely affecting the viability, mitotic activity or regenerative capacity ofthe cells as disclosed by Krzyzek et al., supra. For a dilute solution of enzyme(s) (0.1-1%) digestion time ranges from about 0.75 to 3.0 hours, preferably from about 1.5-2.0 hours for a packed volume of about 1-2 ml of maize cells/5 ml enzyme buffer at about room temperature. Optionally, the cultured maize cells are declumped by sieving or filtering to increase the cell wall surface area exposed to enzymatic action. Following partial degradation ofthe cells walls, the cells are washed with buffer in an amount sufficient to substantially remove residual enzymes. Electroporation of Suspension Cultures

The gene constmct ofthe invention is introduced into cultures of transformation- competent maize cells prepared as described above by electroporation to obtain transformed cells which are regenerable into fertile transgenic maize plants. Potter et al., PNAS USA, 81, 7161 (1984) describes a suitable electroporation apparatus for use in the invention. Such devices typically consist of an electronic device including a capacitor. The capacitor is charged and incoφorated into a circuit in series with the cells to be electroporated in an electroporation buffer. The capacitor is then discharged so deliver a current pulse to the cells. The waveform, pulse length and number of pulses delivered and pulse field conditions may be varied to optimize cell viability and levels of expression ofthe transformed cells.

Electroporation of transformation-competent cells is preferably performed within about 45 minutes of their preparation, although transformation-competent cells left for as long as 3 hours before electroporation may be transformed at acceptable frequencies. Electroporation is performed in an electroporation chamber in the presence of electroporation buffer at room temperature (20° - 30° C). K.J. Paite, Plant Cell Reports, 4, 274 (1985). The formulation, osmolarity, pH, concentration and ionic composition and other parameters ofthe electroporation buffer are adjusted to optimize cell viability and transformation.

The electroporation buffer must be compatible with, and must not be toxic or otherwise adversely affect the cells to be transformed. The electoporation buffer will generally contain the gene constmct, a buffering agent to maintain the pH ofthe electroporation buffer between about 7 and 7.7, preferably at about 7.5, and an osmoticum. Suitable buffering agents include HEPES, (N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid]) and sodium phosphate, and mixtures thereof. The osmoticum in the electroporation buffer is a compound(s) that helps maintain an osmotic balance between the extracellular medium and the interior ofthe transformation-competent maize cells, so cell viability is preserved. Preferred osmotica are sugars such as fructose, sucrose and the like and polyols, preferably C ₂ - C ₆ polyols such as mannitol, sorbitol, glycerol and the like. The optimal concentration ofthe osmoticum is approximately 0.3-0.5M. The use of mannitol was found to lead to a consistently higher rate of transformation than using either sorbitol or sucrose.

The gene constmct present in the electroporation buffer may be in supercoiled or linear form. In preferred embodiments, linear double-stranded DNA, e.g., from recombinant plasmids, is employed. Although concentrations as low as about 1 μg/ml per 1.0 ml of electroporation buffer may be used, it is preferred that the DNA gene constmct be at a concentration of about 100 μg/ml or greater. Selection of Transformants

The cells are placed on a recovery medium following electroporation and penetration ofthe gene constmct into at least some ofthe transformation-competent cellls. The recovery medium is preferably a solid callus maintenance medium containing mannitol. The mannitol is preferably removed after one week and recovery on maintenance media is continued for an additional week before selection begins. The extent ofthe recovery period varies depending on the selection agent used, and/or the number of cells electroporated. The recovery period affords cells an opportunity to recover from electroporation and permits the cells to proliferate and stabilize so that adequate numbers of cells may be generated to facilitate selection and subsequent regeneration.

Identification and selection of those cells which both contain the gene constmct and which are capable of regeneration to form plants is begun between 1 day and 4 weeks, preferably at about 1.5 - 2.5 weeks following recovery. There are two methods for selecting the transformed cells. The first comprises assaying transformed cells within calli or plants

regenerated therefrom for the expression of reporter genes or assessing the phenotypic effects ofthe gene constmct, if any. The second, preferred method comprises identifying the transformed cells by growing the cells in the presence of an antibiotic, i.e., kanamycin, which is toxic for non-transformed cells, but allows growth of those cells transformed with and expressing the introduced gene constmct including the selectable marker gene (i.e., neomycin resistance). Selection conditions are chosen to optimize growth and accumulation ofthe transformed cells while simultaneously inhibiting growth ofthe non-transformed cells. The selection agent's effect on cell viability, regeneration capacity and fertility is monitored and determined by experimentally establishing the concentration range which inhibits growth. This may be done by plotting a growth inhibition curve for the given selective agent and tissue being transformed.

A selection regimen may include sequential changes in the concentration ofthe agent and multiple selection cycles. Concentration and cycle length vary depending on the selection agent used. For instance, transformed suspension cultures may be selected by growing the cultures in the presence of kanamycin 200 mg/L for a period of 3-6 weeks followed by selection on 60 mg/l for 3 - 6 weeks.

Transformation of putative transformants is confirmed by phenotypic and/or genotypic analysis. For example, for collagen encoded by the gene constmct ofthe invention, an immunologic specific assay using anti-collagen antibodies may be used. The presence ofthe gene constmct may also be confirmed by conventional means, i.e., by Southern blot or by polymerase chain reaction (PCR). As the gene constmct includes a plant expressible marker encoding the neomycin resistance gene, the plant cells are selected by culturing the plant cells in the present of neomycin. Plant Regeneration and Seed Production

Transformed cell lines are regenerated into transgenic whole plants in a conventional manner well known in the art and the fertility ofthe resultant plants is determined. Transformed cell lines which test positive by genotypic and/or phenotypic analysis are placed on a media which promotes tissue differentiation and plant regeneration. Regeneration procedures typically include reducing the level of auxin and adding sucrose which discontinues proliferation of a callus and promotes somatic embryo development or other tissue differentiation. An example of a regeneration procedure is given in C. E. Green et al., Maize for Biological Research, Plant Molec. Biol. Assoc, Charlottesville. Va., pps. 367-372

(1982). The regenerated plants may be transferred to standard soil conditions and cultivated in a conventional manner. After the gene constmct is stably incoφorated into regenerated transgenic plants, it can be transferred to other plants by appropriate sexual crosses and selfs as described by M. Neuffer, ibid, at pages 19-30. Any of a number of standard breeding techniques may be used depending on the species to be crossed. The plants are grown and harvested using conventional procedures. Analysis of Rl progeny

The initial plants generated from the transformed callus are termed the RO generation or RO plants. The term Rl progeny or the Rl generation refers to the seeds produced by various sexual crosses ofthe RO generation plants. The progeny derived from the germination of Rl seeds are also referred to as the Rl generation.

Various tissues ofthe Rl generation are analyzed to confirm the successful transmission and inheritance ofthe gene constmct in the sexual crosses described above. The analysis is performed as described above for analysis ofthe electroporated callus for evidence of transformation, except that plants and plant parts rather than callus are being analyzed. Production of Commercial Hybrid Seed

It is advantageous to incoφorate the gene constmct ofthe invention into many varieties of hybrids which differ in maturity, yield, pest resistance, and other agronomic traits. Hybrids adapted to growth in one part ofthe com belt may not be adapted to growth in other parts ofthe com belt due to variations in such traits as maturity, disease, and insect resistance. It is therefore advantageous to integrate the gene constmct into many parental hybrid lines to increase the hybrid combinations that may be produced containing the gene constmct. This is accomplished by breeding programs in which backcrossing is performed by crossing the initial fertile, transgenic plant to an elite inbred line and then crossing the progeny back to the elite parent using conventional methods. Elite lines are pure breeding inbreds having commercially valuable traits as a result of classical breeding methods. Some ofthe progeny from this cross will carry the gene constmct and some will not. The plants carrying the gene constmct are then crossed again to the elite parent resulting in progeny which segregate once more. Repeated crosses are performed until the original elite line is converted to a genetically engineered line containing the gene constmct and all other important traits present in the elite parent. A separate backcrossing program is employed for every elite line to be converted to a genetically engineered elite line. Using conventional breeding techniques, it is possible for

both parents of a hybrid seed com to be homozygous for the gene constmct. Com breeding techniques required to transfer genes from one line or variety to another are well-known.

Altematively, the gene constmct may be delivered by electroporation into immature zygotic embryos preconditioned with a mild enzymatic treatment of immature embryos or into type I callus preconditioned with cutting and preplasmolysis of type I callus. D'Halluin et al., The Plant Cell 4:1495-1505 (1992). The next section describes an electroporation treatment of immature zygotic embryos, followed by a description of electroporation of type I callus, following the procedure of D'Halluin et al., supra. Electroporation, Selection and Regeneration of Immature Zygotic Embryos

Freshly excised immature embryos of H99 or Pa91 are treated for 1-3 minutes with an enzyme solution containing CPW salts (Frearson et al, Dev. Bio. 33, 130-137 (1973)) supplemented with 10% mannitol, 5 mM Mes, pH 5.6 and 0.3% macerozyme (Kinki Yakult, Nishinomiya, Japan) or other combinations of pectin degrading enzymes. The embryos are then rinsed in appropriate buffers to remove residual enzymes. The transformation- competent cells are transferred to a microcuvette containing 200μL maize EPM electroporation buffer (80 mM KCl, 5 mM CaCl ₂ , 10 mM Hepes and 0.425 M mannitol, pH 7.2) and 10- 20 μg/ml gene constmct and incubated for 1 hour. The cuvettes are then incubated in an ice bath for 10 min. and then transferred to an electroporation chamber. Several pulse protocols may be tested to determine the optimal conditions. For instance, electroporation may be carried out by discharging one pulse with a field strength of 375 V/cm from a 900 μF capacitor as described by D'Halluin et al., The Plant Cell 4:14951505 (1992). The pulse strength, capacitance, and electroporation apparatus are as described by Dekeyeser et al., Plant Cell 2:591-602 (1990). After electroporation, the embryos are transferred to recovery media (F medium or F medium supplemented with 0.2M mannitol) or directly to selection media (F medium supplemented with 0.2M mannitol and 200 μg/ml kanamycin) and cultured in the dark at 23 °C. Those transferred to recovery media are cultured 10 days then transferred to selection media. They are maintained on selection media with mannitol for 14 days then transferred to selection media without mannitol, but still containing kanamycin 200 μg/ml. The cells are then subcultured in 3 week intervals for 6-8 weeks. For regeneration, the developing embryogenic tissue is isolated and transferred to MS medium (Murashige et al., Physiol. Plant 15:473-497 (1962)) supplemented with 5 mg/L 6-benzyl- aminopurine for line H99 and 5 mg/L zeatin for line Pa91 and cultured at 23 °C for 10 to 14

days. The embryogenic tissue is then transferred to MS medium without hormones and 6% sucrose. Developing shoots are transferred to half-strength MS medium with 1.5% sucrose for further development into plantlets. The plantlets are transferred to soil and grown to maturity in the greenhouse. Electroporation, Selection and Regeneration of Type I Callus

Developing type I callus of Pa91 is cultured for about 2 months on Mah 1 VII substrate (N6 medium (Chu et al., Sci. Sin. Peking 18, 659-668 (1975)), supplemented with 100 mg/L casein hydrolysate, 6 mM 1-proline, 0.5 g/L 2-(N-moφholino)ethanesulfonic acid (Mes), 1 mg/L 2,4-D, and 2% sucrose solidified with 1.6 g/L Phytage (Sigma), and supplemented with 0.75 g/L MgCl ₂ , pH 5.8) subcultured every 14 to 20 days. Embryogenic tissue is dissected from the developing type I callus and preconditioned by cutting in about 1.5 mm thick pieces in EPM buffer without KCl. and preplasmolysis for about 3 hours in this buffer. The 150 mg of callus fragments are then transferred to cuvettes containing 200μL of EPM supplemented with 80 mM KCl. Electroporation, selection and regeneration protocols are as described above for immature zygotic embryos. Confirmation of Transformation of Callus

To show that callus lines grown on kanamycin selection medium have acquired the collagen compound gene, DNA is isolated from pieces of each kanamycin resistant callus line and from unselected control callus. Dellaporta et al.. Plant Mol. Biol. Rep. 1, 19-21 (1983). Southern blot is performed on restriction enzyme digested DNA according to standard protocols as disclosed by Maniatis. however using the radiolabelled bovine collagen as a probe. Polymerase chain reaction is performed on DNA using primers to amplify selected sections ofthe bovine collagen gene. Standard protocols are employed as disclosed by the Perkin Elmer PCR manual. This confirms integration ofthe bovine collagen compound gene into the maize genome.

Immunological assays or in situ hybridizations are performed on tissue from plants showing integration. Frozen sections are prepared and stained with antibodies directed against type I collagen. Extraction of the Collagen Compound

Progeny containing the desired collagen compound is identified by electrophoresis or Elisa technology. After cultivation, the transgenic plant is harvested to recover the produced

collagen compound or gelatin using conventional methods. This harvesting step may consist of harvesting the entire plant, or only part ofthe plant commercially harvested.

There are two major milling processes for com. Dry milling of com separates the germ from the endosperm. The endosperm is recovered in the form of coarse grits, com meal and com flakes, or it may be passed through fine rollers and reduced to com flour.

The collagen compound may be extracted from the com as collagen or gelatin by means of an existing com wet-milling process. In the wet-milling process, after the com kemels are cleaned to remove coarse material such as dust, chaff, cobs, stones and insects, the com is steeped in large tanks of warm water generally containing acid and sulfur dioxide (a sulf rous acid solution) to soften the com and render the starch granules separable from the protein matrix that envelopes them. If the temperature is sufficiently high, the collagen compound will be converted to gelatin during this step and will be extracted as gelatin by separation in the steep water or upon completion ofthe remaining steps described below. About 7% ofthe kernel's dry substance is leached out during this step, forming protein-rich steep water used as a feed ingredient. The softened kemels are coarsely ground to release the germs. The course grind produces a pulpy material containing germ, fiber (hull), starch, protein and collagen compound which is passed through a hydrocyclone separator where the germ is recovered. Because of their high oil content, the germs are lighter than the starch, protein, and fiber fractions and can easily be separated. The hulls and endosperm, the heavier particles, are discharged from the bottom ofthe hydrocyclone tube for further processing and the lighter germs are drawn off the top ofthe vortex. The germs are washed free of remaining starch, dried and the com oil is removed by expelling or solvent extraction. The spent germs are used as a feed ingredient.

The slurry now contains the fiber (hulls) and the protein, starch and collagen compound fractions ofthe endosperm. The starch-protein-fiber-collagen compound slurry is subjected to an intense milling to release additional starch from the fiber. The fiber is then wet-screened from the starch-protein slurry, washed free of starch, and dried to form the major component of com gluten feed. The best fiber can be additionally purified to become com bran. The starch-protein-collagen compound slurry is separated into its component parts by passing it through combinations of high speed centrifuges and hydrocyclones to separate the heavier starch and collagen compound from the lighter protein. The protein fraction is filtered and dried in rotary or flash driers to yield com gluten, which is rich in the com

protein known as zein. The starch slurry is dewatered and dried to produced co starch, which may be used as such or further converted into com symp by the hydrolytic action of acid or starch-splitting enzymes. The collagen compound fraction is separated by high speed centrifuge, dried and used in cosmetic, medical, reconstmctive, therapeutic and industrial applications. Altematively, the collagen compound may be recovered as a gelatin-com starch combination in the same fraction using high speed centrifugation.

The presence of residual collagen in the com plant after collagen/gelatin extraction does not affect its use in animal feed. In addition to being biocompatible, collagen is an ubiquitous protein already naturally present in animals.

While a number of embodiments ofthe present invention have been shown and described, it will be obvious to one skilled in the art that many changes and modifications may be made thereto without departing from the spirit and scope ofthe invention. It will be appreciated that the specific gene constmct is not critical to the invention, and various other gene constmcts that are the most suitable to particular hosts may be used.