MXPA01001148A - Plants which synthesize a modified starch, methods for producing the plants, their use, and the modified starch - Google Patents

Abstract

The invention relates to recombinant nucleic acid molecules containing two or more nucleotide sequences which code enzymes involved in the metabolism of starch. The invention also relates to methods for producing transgenic plant cells and plants which synthesize a starch that is modified with regard to the phosphate content and side-chain structure thereof. In addition, the invention relates to vectors and host cells which contain the inventive nucleic acid molecules, to the plant cells and plants produced using the inventive methods, to the starch which is synthesized by the inventive plant cells and plants, and to methods for producing this starch.

Description

PLANTS THAT SYNTHESIZE A MODIFIED STARCH, METHODS TO PRODUCE THESE PLANTS. YOUR USE, AND MODIFIED STARCH FIELD OF THE INVENTION The present invention relates to recombinant nucleic acid molecules that contain two or more nucleotide sequences that encode enzymes involved in starch metabolism; to procedures for generating transgenic cells and plants that synthesize modified starch with respect to its phosphate content and its side chain structure. In addition, the present invention relates to vectors and host cells that contain the nucleic acid molecules according to the invention; to cells and plants that originate from the methods according to the invention; to the starch synthesized by the cells and plants according to the invention; and to processes for the preparation of this starch.

BACKGROUND OF THE INVENTION Taking into account the growing importance of plant constituents as renewable resources, biotechnological research attempts to adapt raw materials to the demands of the processing industry. In this way, to make possible the use of renewable resources in as many fields of application as possible, it is necessary to make available a wide variety of materials. In addition to oils, fats and proteins, the 5 polysaccharides are also important renewable resources of plants. Not only cellulose takes a primordial position in polysaccharides, but also starch, which is one of the most important storage substances in higher plants. In addition to corn, rice and wheat, the potato also plays an important role in particular in the production of starch. 10 The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it is a very complex mixture of different forms of molecules that differ with respect to their degree of polymerization and the occurrence of ramifications in the glucose chains. Therefore, starch is not a raw material uniform. In particular, a difference is made between amylose starch, an essentially unbranched polymer of glucose molecules with -1,4-glycosidic bonds, and amylopectin starch which in turn constitutes a complex mixture of branched glucose chains differently. Other branches are generated by the presence of -1, 6-glycosidic bonds additional. In typical plants used for the production of starch, such as for example corn or potato, the synthesized starch consists of about 25% amylose starch and about 75% amylopectin starch. ^^ ¡g ffigj «te ^ ¿^ ^^^^ gjil | j¡g ^^ -? gttea ^^^ ^^ | The molecular structure of the starch, determined mainly by the degree of branching, the amylose / amylopectin ratio, the average length and distribution of the side chains, and the presence of phosphate groups, are of paramount importance for the important functional properties in starch and its aqueous solutions, respectively. Important functional properties that must be mentioned are, for example, solubility, degradation behavior, film-forming properties, viscosity, color stability, gelation properties and binding and adhesion properties. For several applications, The granule size of the starch may also be important. Also, for certain applications, the production of high amylose starches is of particular interest. In addition, a modified starch contained in the cells of a plant can advantageously alter the behavior of said cells under certain conditions. For example, it is feasible to reduce degradation of starch during the storage of starch-containing organs such as, for example, seeds or tubers, before processing, for example before starch extraction. It is also of interest to produce modified starches that make cells or plant organs that contain such starch, more suitable for processing, for example in the preparation of food products such as popcorn or corn flakes, or slices, chips or potato powder. Of particular interest is the improvement of starches with respect to decreased cold sweetening, ie, decreased release of reducing sugars (in particular glucose) by prolonged storage at low temperatures. It is common to store potatoes especially at temperatures of 4 to 8 ° C to reduce the degradation of starch during storage. The sugars released during this process, in particular glucose, result in undesirable browning reactions during the production of slices or potato chips (what are referred to as the Maillard reactions). The starch that can be isolated from plants is often adapted for particular industrial purposes with the help of chemical modifications that require, as a rule, time and money. For the Therefore, it seems convenient to find possibilities to generate plants that synthesize a starch whose properties already meet the specific demands of the processing industry and thus combine the economic and ecological advantages. One possibility of producing such plants, in addition to measures of reproduction, is the direct genetic alteration of the starch metabolism of starch-producing plants, by means of genetic engineering methods. However, a prerequisite for the same is the identification and characterization of the enzymes involved in the modification of starch synthesis and in the degradation of starch. (starch metabolism), and the isolation of the corresponding DNA sequences encoding these enzymes. The biochemical pathways leading to the synthesis of starch are essentially known. In plant cells, the synthesis of starch It takes place in the plastids. In photosynthetically active tissues, these plastids are chloroplasts; In photosynthetically inactive starch storage tissues, they are the amyloplasts. Important enzymes involved in the synthesis of starch are, for example, branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases, debranching enzymes, disproportionate enzymes, plastid starch phosphorylases, and R1 enzymes ( R1 proteins).

BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide different or alternative genetic engineering methods for modifying starch metabolism in starch-synthesizing plants (e.g., rye, barley, oats, corn, wheat, sorghum and millet, sago, rice, peas, large-leaved pea, yucca, potato, tomato, oilseed rape, soybeans, hemp, flax, sunflower, cowpea, mung beans, beans, plantain or arrowroot), or suitable nucleic acid molecules, by means of which cells can be transformed plants to allow the synthesis of starch varieties advantageously altered. Said altered starch varieties exhibit, for example, modifications with respect to their degree of branching, the amylose / amylopectin ratio, the phosphate content, the granule size of ^ A ^^ & * ^^^^^^^^ g ^ starch and / or the average length and distribution of the side chains (ie, the structure of the side chain). A further object of the present invention is to provide methods that allow the generation of transgenic plants that synthesize a variety of altered (modified) starch. Surprisingly, the transgenic plants or cells that have been transformed with the nucleic acid molecules of the invention, or to be used according to the invention, synthesize a starch that is altered in the particular manner that is described in the invention with regarding its physicochemical properties and / or the structure of its side chain. In contrast, known starches that are synthesized by transgenic plants do not exhibit the alterations according to the invention. In accordance with the invention, these objects are achieved using the forms specified in the claims.

DETAILED DESCRIPTION OF THE INVENTION Therefore, the present invention relates to a recombinant nucleic acid molecule (nucleotide sequence) comprising: (a) at least one nucleotide sequence (polynucleotide or nucleic acid molecule) that encodes a protein with the function of a soluble starch III synthetase, or fragments of said nucleotide sequence; and (b) one or more nucleotide sequences encoding a protein selected from group A, which consists of proteins that function as branching enzymes (BE I, lia and llb), ADP glucose pyrophosphorylases, starch synthetases linked to granule, soluble starch synthetases I, II, or other, debranching enzymes, disproportionate enzymes, plastid starch phosphorylases, R1 enzymes, amylases and glycosidases; or fragments thereof - preferably synthetases of soluble starch II, soluble starch synthetases I and / or branching enzymes, and fragments thereof; and nucleic acid molecules that hybridize to one of said nucleotide sequences or fragments thereof, preferably a deoxyribonucleic acid molecule or a ribonucleic acid molecule, especially a cDNA molecule. Especially preferred is a nucleic acid molecule that hybridizes specifically with one of said nucleotide sequences or fragments thereof. Nucleotide sequences which are suitable according to the invention, and which encode a protein having the function of a soluble starch synthetase III, are described, for example, in EP-A-0779363. For the purposes of this invention, it is understood that the term "nucleotide sequence encoding a protein having the function of a soluble starch synthetase III" means in particular sequences whose coding region has a length of 3000-4500 bp, preferably 3200-4250 bp, especially 3400-4000 bp, and whose homology with the entire coding region of a nucleic acid encoding a protein having the function of a starch synthetase amounts to at least 70%, preferably at least 80%, especially at least 90%, and most preferably at least 95%. Nucleotide sequences which are suitable in accordance with the invention, and which encode a group A protein, are for example soluble starch synthetases (type I, II or other) or granule-linked starch synthetase isoforms (e.g. , Hergersberg, 1988, thesis for PhD, University of Cologne, Abel, 1995, thesis for PhD, FU Berlin, Abel et al, 1996, Plant Journal 10 (6): 981-991, Visser and others, 1989, Plant Sci. , 64: 185-192; van der Leij et al., 1991, Mol. Gen. Genet. 228: 240-248; EP-A-0779363; WO 92/11376; WO 96/15248, in which SSSB is a soluble starch synthetase I and GBSSII is a soluble starch II synthetase; WO 97/26362; WO 97/44472; WO 97/45545; Delrue et al., 1992, J. Bacteriol. 174: 3612-3620; Baba et al., 1993, Plant Physiol. 103: 565-573; Dry and others, 1992, The Plant Journal 2, 2: 193-202, or in the EMBL X74160 database entries; X58453; X88789); Branching enzyme sophomores (branching enzymes I, Na, llb), debranching enzyme isoforms (debranching enzymes, isoamylases, pullulanases) or disproportionation enzyme sophorms, which are described for example in WO 92/14827; WO 95/07335; WO 95/09922; WO 96/19581; WO 97/22703; WO 97/32985; WO 97/42328; Takaha et al., 1993, J. Biol. Chem., 268: 1391-1396, or in the EMBL X83969 database entry, and those for ADP glucose pyrophosphorylases, plastid starch phosphorylase isoforms and R1 enzymes ¿^ ^^^ Ü * l > i ^^^? fefeeS < ifc | (R1 proteins), which are described, for example, in EP-A-0368506; EP-A-0455316; WO 94/28146; DE 19653176.4; WO 97/11188; Brisson et al., 1989, The Plant Cell 1: 559-566; Buchner et al., 1996, Planta 199: 64-73; Camirand et al., 1989, Plant Physiol. 89 (Suppl 4) 61; Bhatt & Knowler, J. 5 Exp. Botany 41 (suppl.) 5-7; Lin et al., 1991, Plant Physiol. 95: 1250-1253; Sonnewald et al., 1995, Plant Mol. Biol. 27: 567-576; DDBJ No. D23280; Lorberth et al., 1998, Nature Biotechnology, 16: 473-477, and also for amylases and glucosidases. The nucleotide sequences that are used properly In accordance with the invention, they are of prokaryotic or eukaryotic origin, preferably of bacterial, fungal or vegetable origin. For the purposes of the present invention, the term "fragment" denotes portions of the nucleic acid molecules according to the invention, or that are used according to the invention, which have a length of at least 15 bp, preferably at least 150 bp, especially preferably at least 500 bp, but whose length is generally not greater than 5000 bp, preferably 2500 bp. For the purposes of the present invention, "hybridization" denotes a hybridization under conventional hybridization conditions, preferably under severe conditions such as those described by Sambrook et al., "Molecular Cloning, A Laboratory Manual" [Molecular Cloning, Laboratory Manual] 2nd. Edition (1989) Cold Spring Harbor Laboratory Press, Cold ^^^^^^ ^^^^^^ rfsstó ^^^^^^^ Spring Harbor, New York). In particular, the term "fragment" comprises biologically active molecules. It is especially preferred that a "specific hybridization" take place under the following very severe hybridization conditions: Hybridization buffer: SSC 2x; solution Denhardt 10x (Ficoll 400 + PEG + BSA, ratio 1: 1: 1); SDS 0.1%, EDTA 5 mM; 50 mM Na2HPO4; Herring sperm DNA 250 g / ml; TRNA 50 g / ml; or 0.25 M sodium phosphate buffer solution pH 7.2; 1 mM EDTA; SDS 7%; at 10 Hybridization temperature T = 55 at 68 ° C, Wash buffer: 0.2x SSC; SDS 0.1% and Washing temperature: T = 40 to 68 ° C. Molecules that hybridize with the nucleic acid molecules according to the invention, or that are used in accordance with The invention also encompasses fragments, derivatives and allelic variants of the nucleic acid molecules according to the invention or which are used according to the invention. It is understood that "fragments" mean not only portions of the nucleic acid molecules that are long enough to encode a functionally active portion of the proteins that 2nd are described. Within the context of the present invention, the term "derivative" means that the sequences of these molecules differ from the nucleic acid sequences according to the invention, or that they are used according to the invention, in one or more positions, and exhibit a high ^ k5 ^^ á * i j! ^ 'degree of homology with these sequences. Homology means a sequential identity of at least 60%, preferably more than 70%, and especially more than 85%, in particular more than 90%, and most preferably more than 95%. Deviations from the nucleic acid molecules according to the invention, or which are used according to the invention, can be originated by means of one or more deletions, substitutions, insertions (addition) or recombinations. In addition, homology means that there is a functional and / or structural equivalence between the nucleic acid molecules in question and the proteins encoded by them. The nucleic acid molecules which are homologous to the molecules according to the invention, or which are used according to the invention, and which constitute derivatives of these molecules, are as a rule variations of these molecules which constitute modifications which exert a biological function the same, virtually identical or similar. They can be variations of natural occurrence, for example sequences of other plant species, or mutations, it being possible that these mutations occur naturally or have been introduced by directed mutagenesis. The variations can also be synthetic sequences. Allelic variants may be naturally occurring variants or synthetic variants or variants generated by recombinant DNA technology. The nucleic acid molecules according to the invention, or used according to the invention, can be DNA molecules, in particular cDNA molecules or, if appropriate, the combination of genomic molecules. In addition, the nucleic acid molecules according to the invention, or which are used according to the invention, can be RNA molecules. The nucleic acid molecules according to the invention, or which are used according to the invention, or fragments thereof, can be obtained, for example, from natural sources, or they can be generated by means of recombinant technology or can be generate by synthesis. To express the nucleic acid molecules according to the invention, or used according to the invention, in sense or antisense orientation in plant cells, they bind to DNA regulatory elements that ensure transcription in plant cells. These include, in particular, promoters. In general, any promoter that is active in plant cells is suitable for expression. The promoter can be chosen in such a way that the expression is constitutive or is only in a particular tissue, at a particular time point of the development of the plant, or at a point of time determined by external factors that may be, for example, inducible chemically or biologically. With respect to the transformed plant, the promoter can be homologous or heterologous, as can the nucleotide sequence. Examples of suitable promoters are the 35S RNA promoter of cauliflower mosaic virus, and the corn ubiquitin promoter, for constitutive expression; the B33 patatin promoter (Rocha-Sosa et al., 1989 EMBO J. 8: 23-29) for tuber-specific expression in potatoes, or a promoter that ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., 1987, Proc. Nati, Acad. Sci., USA 84: 7943-7947, Stockhaus et al., 1989, EMBO J. 8: 2445-2451); the Ca / b promoter (for example, U.S. Patent Nos. 5656496, 5639952, Bansal et al., 1992, Proc. Nati. Acad. Sci. USA, 89: 3654-3658), and the Rubsico SSU promoter (see, for example, for example, U.S. Patent Nos. 5034322 and 4962028), or a specific endosperm expression of the glutelin promoter (Leisy et al., Plant Mol. Biol. 14 81990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366; Yoshihara et al., FEBS Lett, 383 (1996) 213-218), the shrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380), the wheat HMG promoter, the USP promoter, the phaseolin promoter, or promoters of maize zein genes (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93). A terminator sequence terminating the nucleic acid molecule according to the invention can serve to correctly end the transcription, and to help transcribe a tail of poly-A, which is considered to have the function of stabilizing the transcripts. These elements have been described in the literature (see Gielen et al., 1989, EMBO J. 8: 23-29) and as a rule, they are interchangeable as desired. The nucleic acid molecules according to the invention, or used according to the invention, can be used to generate transgenic cells and plants that show increase and / or reduction of the activity of the soluble starch synthetase III and at least a additional enzyme of starch metabolism. For this purpose, the nucleic acid molecules according to the invention, or used according to the invention, are introduced into suitable vectors, provided with the nucleic acid regulatory sequences that are necessary for efficient transcription in plant cells, and enter the plant cells. On the one hand, there is the possibility of using the nucleic acid molecules according to the invention, or used according to the invention, to inhibit the synthesis of endogenous soluble starch III synthesis and / or at least one additional protein of group A in the cells. This can be achieved with the help of antisense constructs, in vivo mutagenesis, a cosuppression effect that occurs, or with the help of properly constructed ribozymes. On the other hand, the nucleic acid molecules according to the invention, or used according to the invention, can be used to express the soluble starch synthetase III and / or at least one additional group A protein, in cells of transgenic plants, and thus lead to a greater activity, in the cells, of the enzymes that have been expressed in each case. In addition, there is the possibility of using the molecules according to the invention, or that are used according to the invention, to inhibit the synthesis of the endogenous soluble starch III synthetase and the overexpression of at least one additional protein of group A in the cells. Finally, the nucleic acid molecules according to the invention, or used according to the invention, can also be used h ¿^^^? %to? > s ^ e * - ^ ^ i X ...- to express the soluble starch synthetase III and to inhibit at least one additional group A protein in cells of transgenic plants. The last two mentioned embodiments of the invention thus lead, in the cells, to a simultaneous inhibition and increase in the activity of the enzymes that are inhibited or expressed, respectively. The invention further relates to a vector comprising a nucleic acid molecule according to the invention. The term "vector" encompasses plasmids, cosmids, viruses, bacteriophages and other vectors conventionally used in genetic engineering, which contain the nucleic acid molecules according to the invention and which are suitable for transforming cells. Preferably, such vectors are suitable for transforming plant cells. Preferably, they allow the integration of the nucleic acid molecules according to the invention, if appropriate together with flanking regulatory regions, into the genome of the plant cell. Examples are binary vectors such as pBinAR or pBinB33, which can be used in gene transfer mediated by agrobacteria. In a preferred embodiment, the vector according to the invention is distinguished by the fact that the nucleotide sequence encoding a protein having the function of a soluble starch synthetase III or fragments thereof, is present in sense orientation or antisense.

In a further preferred embodiment, the vector according to the invention is distinguished by the fact that the nucleotide sequence encoding one or more proteins selected from group A or fragments thereof is present in sense or antisense orientation. In another preferred embodiment, the vector according to the invention is distinguished by the fact that the nucleotide sequence encoding a plurality of proteins selected from group A or fragments thereof, is partially in sense orientation and partially in orientation of antisense Preferably, the vector according to the invention comprises one or more regulatory elements that ensure the transcription and synthesis of an RNA in a prokaryotic or eukaryotic cell. In addition, by means of the usual techniques of Molecular Biology (see for example Sambrook and others, 1989, "Molecular Cloning, A Laboratory Manual" [Molecular Cloning, Laboratory Manual] 2nd. Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), it is possible to introduce several mutations into the DNA sequences according to the invention, or to be used according to the invention, which leads to the synthesis of proteins with biological properties that can be altered. On the one hand, it is possible to generate deletion mutants in which sequences are generated, by progressive deletions from the 5 'end or from the 3' end of the coding DNA sequences, which lead to the synthesis of analogously truncated proteins.

For example, such deletions at the 5 'end of the DNA sequence allow directed production of enzymes which, due to the removal of the relevant transit or signal sequences, are no longer located in their original compartment (homolog), but in the cytosol, or which, due to the addition of other signal sequences, are located in one or more other compartments (heterologous) (eg plasto, vacuole, mitochondria, apoplast). On the other hand, it is also possible to introduce point mutations at positions where an altered amino acid sequence affects, for example, the activity of the enzyme or the regulation of the enzyme. In this way, it is possible to generate for example mutants that have an altered value of KM or Kcat, or that are no longer subject to the regulatory mechanisms normally present in the cell, for example by allosteric regulation or covalent modification. For manipulation of genetic engineering in prokaryotic cells, the DNA sequences according to the invention, or used according to the invention, or fragments of these sequences, can be introduced into plasmids that allow mutagenesis or a sequence altered by the recombination of DNA sequences. Base exchanges can be made or natural or synthetic sequences can be added, with the help of normal methods of Molecular Biology (see Sambrook et al., 1989, in the aforementioned place). To link the DNA portions with one another, adapters or linkers can be fixed in the portions. In addition, manipulations that provide adequate restriction cleavage sites or that remove excessive DNA or restriction cleavage sites that are no longer needed may be employed. Where insertions, deletions or substitutions are required, in vitro mutagenesis, initiator repair, restriction or ligation can be employed. The analytical methods that are generally carried out are sequence analysis, restriction analysis and, if appropriate, other methods of Biochemistry and Molecular Biology. The invention also relates to a transgenic host cell with altered metabolism of starch, in particular to prokaryotic or eukaryotic cells, preferably bacterial or plant cells (monocotyledonous or dicotyledonous) (for example E. coli, Agrobacterium, Solananceae, Poideae, rye, barley , oats, corn, wheat, sorghum and millet, sago, rice, peas, large-seeded peas, cassava, potatoes, tomatoes, oilseed rape, soybeans, hemp, flax, sunflower, cowpeas, mung beans, beans, plantains or arrowroot) containing one or more nucleic acid molecules according to the invention or one or more vectors according to the invention, or derived from said cell. Another subject of the invention is a transgenic host cell with altered starch metabolism, in particular prokaryotic or eukaryotic cells, preferably bacterial or plant cells (e.g. E. coli, Agrobacterium, Solananceae, Poideae, rye, barley, oats, corn, wheat) , sorghum and millet, sago, rice, peas, large-leaved pea, cassava, potato, tomato, oilseed rape, soybeans, hemp, flax, sunflower, cowpea, beans Jindm? TiA mung, bean, banana or arrowroot), which contain (a) at least one nucleotide sequence encoding a protein having the function of a soluble starch synthetase III or fragments thereof, and (b) a or more nucleotide sequences encoding a protein selected from group A or fragments thereof, or nucleotide sequences which hybridize with these nucleic acid molecules, or which derive from said cell. The host cells according to the invention can be generated not only by using the nucleic acid molecules according to the invention, but also by means of successive transformation (for example, the so-called "super-transformation"), using a plurality of portions of the nucleotide sequence according to the invention, or a plurality of vectors comprising portions of the nucleotide sequence according to the invention, which encode a protein having the function of a soluble starch synthetase III or fragments thereof and, in addition, one or more proteins selected from the Group A, consisting of branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases I, II or other debranching enzymes, disproportionate enzymes, plasmoxylated starch phosphorylases, amylases, glycosidases, R1 enzymes, fragments of the same - preferably synthetases of soluble starch II, soluble starch synthetases I and / or branching enzymes and also fragments thereof - and nucleic acid molecules that hybridize with one of said nucleotide sequences or fragments thereof, in a plurality of steps of cell transformation. Here, the cell is transformed (a) with at least one nucleic acid molecule encoding a protein having the function of a soluble starch synthetase III, its fragment, or with a vector comprising said nucleic acid molecule, and (b) is transformed simultaneously or in succession with one or more nucleic acid molecules that encode a protein that is selected from group A, consisting of branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases I , II or other debranching enzymes, disproportionate enzymes, amylases, glycosidases, plasto starch phosphorylases and R1 enzymes, or fragments thereof - preferably soluble starch synthetases II, soluble starch synthetases I and / or branching enzymes and their fragments - , and nucleic acid molecules that hybridize with one of said nucleotide sequences or fragments thereof, or one or more vectors that co They comprise one or more of said nucleic acid molecules in any desired sequence. A further subject of the invention is a process for the production of a host cell, a bacterial cell, a plant cell or a transgenic plant, which synthesize a modified starch, where they are integrated into the genome of a cell (a) at minus one nucleic acid molecule encoding a protein having the function of a soluble starch synthetase III or fragments thereof, and (b) one or more nucleic acid molecules encoding a protein selected from group A, consisting of branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases I, II or others, debranching enzymes, disproportioning enzymes, amylases, glycosidases, plasmid starch phosphorylases and R1 enzymes, or fragments thereof - preferably soluble starch synthetases II, soluble starch synthetases I and / or branching enzymes and fragments thereof, and nucleic acid hybridizing with one of said nucleotide sequences or fragments thereof; and optionally a complete plant of the transgenic plant cell is regenerated. A further embodiment of the present invention relates to a method for generating a host cell, a bacterial cell, a plant cell or a transgenic plant that synthesizes a modified starch, comprising integrating one or more nucleic acid molecules according to the invention. , or one or more vectors according to the invention, in the genome of a cell, and optionally regenerating a whole plant of the transgenic plant cell. By providing the nucleic acid molecules according to the invention, it becomes possible to take part in the metabolism of the starch of the plants, with the help of genetic engineering methods, and to alter it in such a way as to obtain as a result the synthesis of a starch. modified which is altered with respect to the starch synthesized in wild-type plants for example in terms of structure, water content, protein content, lipid content, fiber content, ash / phosphate content, amylose / amylopectin ratio, distribution of molecular mass, degree of branching, granule size, granule shape and crystallization, or in their physicochemical properties such as flowability and absorption behavior, gelation temperature, viscosity, thickener capacity, solubility, gel structure, transparency, thermal stability , stability against shear stress, stability against acids, tendency to suffer degradation dation, gelation, freeze-thaw stability, complex formation, iodine binding, film formation, adhesion power, stability against enzyme, susceptibility to digestion and reactivity. There is also the possibility of increasing production in plants properly constructed by genetic engineering, increasing the activity of proteins that are involved in the metabolism of starch, for example by overexpressing suitable nucleic acid molecules, or by providing mutants that are no longer subject to regulatory mechanisms of the cell, and / or that exhibit different temperature dependencies with respect to its activity. A particularly pronounced increase in production may be the result of increased activity of one or more proteins that are involved in the metabolism of starch in specific cells of the starch storage tissues of the transformed plants, such as, for example, in the tuber in the case of potatoes, or in the endosperm in the case of corn or wheat. The economic importance and the advantages of these possibilities of taking part in the metabolism of the starch, are enormous. .tfmVm &fStmmi When the nucleic acid molecules according to the invention are expressed in plants, or which are used according to the invention, it is possible in principle that the synthesized protein is located in any desired cell compartment of the plant . To achieve localization 5 in a particular compartment (cytosol, vacuole, apoplast, plastids, mitochondria), if necessary, the transit or signal sequence that ensures the location must be suppressed (or removed) and, if necessary, the The rest of the coding region must be linked to DNA sequences that ensure localization in the particular compartment. These sequences are known (see, for example, Braun et al., EMBO J: 11 (1992), 3219-3227; Wolter et al., Proc. Nati, Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al. Plant J. (1991), 95-106). The localization in certain parts of plants or tissues can be ensured, for example, by specific promoters that are well known to the person skilled in the art. The generation of plant cells with reduced activity of a protein involved in starch metabolism can be achieved, for example, by expressing an appropriate antisense RNA, a sense RNA to achieve a cosuppression effect, by mutagenesis in vivo, or expressing a properly constructed ribozim that cuts specifically transcripts that encode one of the proteins involved in the metabolism of starch, using a nucleic acid molecule according to the invention; preferably expressing an antisense transcript.

For this purpose, it is possible to use, on the one hand, a DNA molecule that encompasses the entire sequence encoding a protein involved in the metabolism of the starch, including any flanking sequences that may be present, and also DNA molecules that only comprise 5 portions of the coding sequence, these portions having a minimum length of 15 bp, preferably at least 100-500 bp, in particular more than 500 bp. As a rule, DNA molecules are used that are shorter than 5000 bp, preferably shorter than 2500 bp. It is also possible to use DNA sequences that exhibit a high degree of homology with the sequences of the DNA molecules according to the invention, but they are not completely identical to them. The homology minimum must be greater than about 65%. In particular, it is preferred to use sequences with a homology of about 75 to 85%, particularly about 85 to 95%. The expression of ribozymes to reduce the activity of specific proteins in cells is known in the art and is described for example in EP-B1-0 321 201. The expression of ribozymes in plant cells was described, for example, by Feyter et al. (Mol. Gen. Genet, 250 (1996), 329-338) The proteins involved in the metabolism of starch can also be reduced in plant cells according to the invention by means of the so-called "mutagenesis in". vivo ", where a hybrid RNA-DNA-oligonucleotide is introduced into cells s,. ~ . -. , tntMa i »-, and« -ase, sas ut} . ("quimeroplasto") to transform them (Kipp PB and others, Poster Session in the 5th International Congress of Plant Molecular Biology, September 21-27, 1997, Singapore, RA Dixon and CJ Arntzen, Report of the Meeting on Metabolic Engineering in Transgenic Plants, Keystone Symposium, Copper Mountain, Colorado, USA, TIBTECH 15 (1997), 441-447, International Patent Application WO 95/15972, Kren et al, Hepatology 25 (1997), 1462-1468, Cole-Strauss and others, Science 273 81996), 1386-1389). A portion of the DNA component of the RNA-DNA-oligonucleotide used for this purpose is homologous to a nucleic acid sequence of an endogenous protein, but exhibits a mutation with respect to the nucleic acid sequence of the endogenous protein, or contains a heterologous region included by the homologous regions. The base pairing between the homologous regions of the RNA-DNA-oligonucleotide and the endogenous nucleic acid molecule, followed by homologous recombination, allows the mutation or heterologous region contained in the DNA component of the RNA-DNA-oligonucleotide to be transferred to the genome of a plant cell. This leads to reduced activity of the protein involved in starch metabolism. Alternatively, enzymatic activities involved in starch metabolism of plant cells can be reduced by means of a cosuppression effect. This method is described, for example, by Jorgensen (Trends Biotechnol., 8 (1990), 340-344), Niebel et al. (Curr. Top, Microbiol.Immunol., 197 81995), 91-103), Flavell, et al. (Curr. Top Microbiol Immunol. & -? # c "&" - 197 (1995), 43-46), Palaqui and Vaucheret (Plant Mol. Biol. 29 81995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet 243 (1994), 613-621). To inhibit the synthesis in transformed plants of a plurality of enzymes involved in the biosynthesis of starch, it is possible to use DNA molecules for transformation, which contain simultaneously, in antisense orientation and under the control of a suitable promoter, a plurality of regions. which encode the relevant enzymes. Each sequence may be under the control of its promoter or, alternatively, the sequences may be transcribed by a binding promoter as a fusion, so that the synthesis of the proteins in question is inhibited at approximately the same level or at a different level. With respect to the length of the individual coding regions that are used in such a construction, what has already been mentioned above for the generation of antisense constructions is also applied here. In principle, there is no upper limit for the number of antisense fragments transcribed starting from a promoter in such a DNA molecule. However, as a rule, the resulting transcript should not exceed a length of 25 kb, preferably 15 kb. Therefore, the nucleic acid molecules according to the invention, or that are used according to the invention, make it possible to transform plant cells and simultaneously inhibit the synthesis of a plurality of enzymes.

In addition, it is possible to introduce the nucleic acid molecules according to the invention, or that are used according to the invention, into traditional mutants that are deficient or defective with respect to one or more genes of starch biosynthesis (Shannon and Garwood , 1984, in Whistler, BeMiller and Paschall, "Starch: Chemistry and Technology" [Starch: Chemistry and Technology], Academic Press, London, 2nd Edition, 25-86). These defects may be related, for example, to the following proteins: cell-bound starch synthetase (GBSSI) and soluble starch synthetases (SS I, II, III and others), branching enzymes (BE I, lia and 11 Ib) , debranching enzymes (R enzymes, isoamylases, pullulanases), disproportionate enzymes and plasto starch phosphorylases. In this manner, the present invention also relates to transgenic plant cells which are obtained by means of a process according to the invention, which have been transformed with a vector or nucleic acid molecule according to the invention, or which are used according to the invention, and to plant cells or transgenic plants derived from said transformed cells. The cells according to the invention contain one or more nucleic acid molecules according to the invention, or which are used according to the invention, these being preferably linked with one or more DNA regulatory elements (eg promoter, enhancer, terminator) that ensure transcription in plant cells, in particular a promoter. The cells according to the invention can be distinguished from naturally occurring plant cells, inter alia, by the fact that they contain a nucleic acid molecule according to the invention, which does not occur naturally in these cells; or by the fact that said molecule exists integrated in a site in the genome of the cell in which it would not appear otherwise, that is, in a different genomic medium. In addition, the transgenic plant cells according to the invention can be distinguished from plant cells of natural occurrence, by the fact that they contain at least one copy of a nucleic acid molecule according to the invention, stably integrated in its genome, if appropriate, in addition to copies of such a molecule that occur naturally in the cells or nucleic acid molecules to be used in accordance with the invention. If the molecule or nucleic acid molecules introduced into the cell (s), are additional copies of those already occurring naturally in the cells, then the plant cells according to the invention can be distinguished from naturally occurring plant cells, in particular by the fact that this additional copy, or these additional copies, they are located in genome sites where they would not occur naturally, or do not occur naturally. This can be reviewed, for example, with the help of a Southern blot analysis. Preferred plant cells according to the invention are those in which the enzymatic activity of the individual enzymes involved in the metabolism of the starch is increased and / or reduced in each case by at least 10%, preferably by at least 30%. %, and especially at least 50%. Furthermore, preferably, the plant cells according to the invention can be distinguished from natural plant cells by at least one of the following characteristics: if the nucleic acid molecule that has been introduced is heterologous with respect to the plant cell, the cells Transgenic plants display the transcripts of the nucleic acid molecules according to the invention that have been introduced. These can be detected, for example, by Northern blot analysis. For example, plant cells according to the invention contain one or more proteins encoded by a nucleic acid molecule according to the invention, or used according to the invention, which has been introduced. This can be detected, for example, by immunological methods, in particular by means of a Western blot analysis. If the introduced nucleic acid molecule according to the invention, or that is used according to the invention, is homologous with respect to the plant cell, the cells according to the invention can be distinguished from natural cells, for example, based on the additional expression of the nucleic acid molecules according to the invention, or which are used according to the invention. For example, transgenic plant cells contain more or less transcripts of the nucleic acid molecules according to the invention, or that are used according to the invention. This can be detected, for example, by analysis ^^ - ftj Northern blot. "More" or "less" in this context means preferably at least 10% more or less, preferably at least 20% more or less, and especially at least 50% more or less transcribed than the corresponding non-transformed cells. In addition, the cells preferably exhibit a corresponding increase or decrease in the protein content encoded by the nucleic acid molecules that have been introduced (at least 10%, 20% or 50%). The transgenic plant cells can be regenerated in whole plants by means of techniques known to those skilled in the art. The plants obtained by regenerating the transgenic plant cells according to the invention, and the methods for generating transgenic plants by regenerating whole plants of the plant cells according to the invention, are also subject of the present invention. Another subject of the invention are the plants that contain the transgenic plant cells according to the invention. In principle, the transgenic plants can be plants of any species, that is, not only monocotyledonous plants, but also dicotyledonous plants. Preferably, the plants are useful plants and starch storage plants such as, for example, cereal species (rye, barley, oats, corn, wheat, sorghum and millet, etc.), sago, rice, peas, large-seeded pea , yucca, potato, tomato, oilseed rape, soybean, hemp, flax, sunflower, cowpea, mungbean, or arrowroot.

The invention also relates to the propagation material of the plants according to the invention, for example, fruits, seeds, tubers, rhizomes, shoots, cuts, calluses, protoplasts, cell cultures, and the like. The alteration of the enzymatic activities of the enzymes involved in starch metabolism results in a starch of altered structure in the plants generated by the methods according to the invention. A large number of cloning vectors is available for prepare the introduction of foreign genes in higher plants, vectors that contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, the pUC series, the M13mp series, pACYC184, and the like. The desired sequences can be introduced into the vector at a cutting site of adequate restriction. The obtained plasmid is used to transform E. coli cells. The transformed E. coli cells are grown in a suitable medium, and then harvested and lysed. The plasmid is recovered. The analytical methods to characterize the plasmid DNA obtained are generally restriction analysis, gel electrophoresis and other methods of Biochemistry and Molecular Biology (Sambrook et al., In the aforementioned place). After each manipulation, the plasmid DNA can be cut and the DNA fragments obtained can be ligated with other DNA sequences. > S? Fo? I &? T ila.-.

Each plasmid DNA sequence can be cloned in the same plasmid or in other plasmids. A large number of techniques are available to introduce DNA into a host plant cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformants, protoplast fusion by means of polyethylene glycol (PEG), injection, DNA electroporation, the introduction of DNA by the biolistic method, and other possibilities ("Gene Transfer to Plants", pages 24-29, eds: Potrykus I. and Spangenberg G., Springer Verlag Berlin Heidelberg 1995). The injection and electroporation of DNA in plant cells does not require particular aspects of the plasmids or the DNA used. Simple plasmids such as for example pUC derivatives can be used. However, if you are going to regenerate whole plants from these transformed cells, the presence of a selectable marker gene is required. Depending on the method of introduction of the desired genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmids are used to transform the plant cell, at least the right margin, but frequently both the right and left margins of the T-DNA of the Ti and Ri plasmids, must be linked to the genes that are they will introduce as a flanking region. < . **. * # & * .. * If an agrobacteria is used for transformation, the DNA to be introduced must be cloned into specific plasmids, either in an intermediate vector or in a binary vector. The intermediate vectors can be integrated into the agrobacterial Ti or Ri plasmids by homologous recombination, due to sequences that are homologous to sequences in the T-DNA. The first also contains the vir region, which is required for the transfer of T-DNA. Intermediate vectors can not be replicated in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). The vectors binaries are capable of replicating in E. coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker that is framed by the left and right margin regions of the T DNA. They can be directly transformed into the agrobacteria (Holsters et al. (1978) Mol. Gen. Genet. 163: 181- 187). The agrobacteria that acts as the cell The host must contain a plasmid carrying a vir region. The vir region is required to transfer the T-DNA to the plant cell. Additional DNA T may be present. The transformed agrobacteria is used to transform plant cells. The use of T-DNA to transform plant cells has been investigated extensively and described in EP 120516; Hoekema, in "The Binary Plant Vector System", Offserdrukkeerij Kanters B.V., Alblasserdam (1985) chapter V; Fraley et al., Crit. Rev. Plant Sci. 4: 1-46, and An et al. (1985) EMBO J. 4: 277-287.

- Sas * ^ it ~ To transfer the DNA to the plant cell, vegetable explants can be co-cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes. It is then possible to regenerate whole plants of the infected plant material (for example, leaf section, stem sections, roots, but also protoplasts or plant cells grown in suspension cultures) in a suitable medium which may contain, for example, antibiotics or biocides to select the transformed cells. The resulting plants can then be examined for the presence of the introduced DNA. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (see, for example, Willmitzer L., 1993, "Transgenic plants" in "Biotechnology, a Multi-Volume Comprehensive Treatise" [Biotechnology]. , Comprehensive treatise on volumes] (HJ Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge). Although the transformation of dicotyledonous plants by means of vector systems of Ti plasmid with the help of Agrobacterium tumefaciens is well established, a more recent work suggests that monocotyledonous plants are also really accessible for transformation by means of vectors based on agrobacteria (Chan and others, Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282). Alternative systems for the transformation of monocotyledonous plants are the transformation by means of the biolistic method, protoplast transformation, the electroporation of partially permeabilized cells, and the introduction of DNA by means of glass fibers. Specifically different methods for the transformation of corn have been described in the literature (see for example WO 95/06128, EP 0 513 849, EP 0 465 875). EP 292 435 discloses a method by means of which fertile plants can be obtained starting from a granular corn husk, friable, free of mucus. In this context, Shillito et al. (Bio / Technology 7 (1989) 581) have observed that the regenerative capacity of fertile plants requires the production of suspended callus cultures, of which can make a protoplast culture in division with the ability to regenerate plants. Prioli and Sóndahl (Bio / Technology 7 (1989), 589) also describes the regeneration and obtaining of fertile maize plants from maize protoplasts. Once the introduced DNA is integrated into the genome of the The plant cell is generally stable therein and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker that mediates the resistance of the transformed plant cells to a biocide or antibiotic such as kanamycin, G 418, bleomycin, hygromycin, phosphinotycin, and the like. Therefore, the marker The chosen individual must allow the selection of the transformed cells on the cells lacking the introduced DNA. Within the plant, the transformed cells develop in the usual way (see also McCormick et al. (1986) Plant Cell Reports 5: 81-84). The resulting plants can develop normally and hybridize with plants that have the same transformed germplasm or other germplasm. The resulting hybrids have the corresponding phenotypic characteristics. 5 Two or more generations must be developed to ensure that the phenotypic characteristic is retained and inherited in a stable manner. Also, seeds or the like must be harvested to ensure that the phenotype in question or other characteristics have been retained. Due to the expression of a nucleic acid molecule of In accordance with the invention, the transgenic cells and plants according to the invention synthesize a starch whose properties, for example physicochemical properties, are altered in comparison with the starch synthesized in wild-type plants. A further subject of the invention is the starch that is obtained from a cell and plant of the invention, from its propagation material, or in accordance with a method of the invention. Another subject of the invention is a method of producing starch in a manner known per se, in which the host cells, plant cells, plants, and parts of plants according to the invention, or their propagation material, are processed or integrate in the method. In a preferred embodiment, the starch according to the invention is distinguished in that its phosphate content is reduced by at least 30%, preferably at least 50%, especially at least 70%, and i-MJsife e ??? m * > • «^ s? ISJtt & tffet'g ^ .l áa-isfe * ^, á m HSkatieti * -.-? > 'very preferably at least 90%, compared to a starch obtainable from a non-transformed (ie, wild-type) cell or plant, and because its content of glucan (see fraction 3 in example 13), after treatment with isoamylase in the exclusion volume of a column HPLC system composed of 2 TSK-gel 2000SW columns connected in series and a TSK-gel 3000SW column in 10 mM sodium acetate, pH 3.0 (at a flow rate of 0.35 ml / min as shown in example 13), increases at least 50%, preferably at least 150%, especially at least 300%, and most preferably at least 500%. In a further embodiment, the starch according to the invention is distinguished in that its phosphate content increases by at least 10%, preferably at least 30%, and most preferably at least 50%, compared to a starch obtainable from a cell or plant not transformed (ie, wild-type), and because its content of glucan (see fraction 3 of Example 13), after treatment with amylase in the volume of exclusion of a HPLC system of column composed of TSK-gel 2000SW columns connected in series and a TSK-gel 3000SW column in 10 mM sodium acetate, pH 3.0 (at a flow rate of 0.35 ml / min as shown in example 13), increases at least 50% , preferably at least 150%, especially at least 300%, and most preferably at least 500%. Methods for extracting starch from plants or from starch storage organs of plants are known to those skilled in the art. For example, Eckhoff et al. Describe processes for extracting starch from corn kernels (Cereal Chem. 73 (1996) 54-57). As a rule, the extraction of maize starch on an industrial scale is carried out by means of wet milling. In addition, processes for extracting starch from a variety of starch storage plants are described, for example, in "Starch Chemistry and Technology" (Editors: Whistler, BeMiller and Paschall (1994)). 2nd Edition, Academic Press Inc. London Ltd; ISBN 0-12-746270-8; see, for example, chapter XII, pages 412-468, production of corn starches and sorghum: Watson; Chapter XIII, pages 469-479, production of tapioca starches, arrowroot and sago: Corbishley and Miller; Chapter XIV, pages 479-490, potato starch: production and uses: Mitch; chapter XV, pages 491 to 506, wheat starch: production, modification and uses: Knight and Oson; and chapter XVI, pages 507 to 528: production and uses of rice starch: Rohmer and KIem). The devices normally used to extract starch from plant material are separators, decanters, hydrocyclones, spray dryers and fluidized bed dryers. A further embodiment of the present invention also includes the use of the starch according to the invention in the industry, preferably for the production of food products, packaging materials or disposable products. The starch according to the invention can be modified chemically and / or physically by means of methods known to those skilled in the art, and is suitable in its modified or unmodified form for a variety of applications in the food or non-food sector. In principle, the possible uses of starch in accordance with the invention can be divided into two important sectors. One sector covers the products of starch hydrolysis, mainly glucose and glucose units, which are obtained by chemical or enzymatic means. They are used as starting material for other chemical modifications and processes such as fermentation. What may be important here is simplicity and design The cost of a hydrolysis process such as is generally carried out essentially enzymatically using amyloglucosidase. A financial savings would be feasible using less enzyme. This could be done by altering the structure of the starch, for example by increasing the surface area of the granule, improving its susceptibility to degradation by reducing the degree of branching, or of a steric structure that limits the access of the enzymes used. The other sector in which the starch according to the invention can be used as a native starch due to its polymeric structure, can be divided into two additional fields of application: 1. The food industry Starch is a traditional additive for a large number of food products in which its function is essentially to agglutinate aqueous additives or cause an increase in viscosity or increase the gelation. The important characteristics are the flow characteristics, the sorption characteristics, the swelling temperature, the gelatinization temperature, the viscosity, the thickening power, the solubility of the starch, the transparency, the structure of the gel, the thermal stability, the stability against shear stress, stability against acids, the tendency to undergo degradation, film forming capacity, freeze-thaw stability, susceptibility to digestion, and the ability to form complexes, for example, with ions inorganic or organic 2. The non-food industry In this important sector, starch is used as an auxiliary for various preparation processes or as a product additive. When starch is used as an auxiliary, mention should be made in particular of the paper and cardboard industry. Starch acts mainly for retarding purposes (retaining solids), agglutinating filler particles and fines, as a hardener and for dehydration. In addition, the advantageous properties of the starch with respect to rigidity, hardness, sound, touch, luster, softness, bond strength and surface treatment. 2. 1 Paper and cardboard industry Within the papermaking process, four fields must be distinguished, that is, surface treatment, coating, kneading and sprinkling. With 80% of the consumption, the starch for surface treatment usually represents the highest amount of starch, 8% is used as a coating starch, 7% as a kneading starch and 5% as a starch for aspersion. The demands on starch with respect to surface treatment are essentially high whiteness, an adapted viscosity, very stable viscosity, good film formation and little dusting. When used for coating, solid content, adequate viscosity, high agglutination capacity and high affinity of pigments play an important role. When used as a kneading additive, a fast, uniform and lossless distribution, high mechanical strength and complete retention on paper cloth is important. If the starch is used in the spraying sector, a suitable solids content, high viscosity and a high agglutination capacity are again of importance. 2. 2 The adhesives industry An important field of application of starches is in the adhesives industry, where potential uses can be divided into four subsections: use as a pure starch paste, use in starch pastes that they have been treated with special chemical agents, the use of starch as an additive for synthetic resins and polymer dispersions, and the use of starches as extenders for synthetic adhesives. 90% of the starch-based adhesives are used in the sectors of * More corrugated cardboard, production of bags and paper bags, production of mixed materials for paper and aluminum, production of cardboard boxes and gummy adhesives for wraps, stamps and the like. 2. 3 Textile industry and textile care products industry An important field of application for starches as auxiliaries and additives is the production of textile products and products for the care of textile products. The following four fields of application should be distinguished within the textile industry: the use of starch as a sizing agent, that is, as an auxiliary to soften and reinforce the burning behavior as a protection of the tensile forces applied during weaving, and increase the resistance to abrasion during weaving; starch as a textile finishing agent, in particular after quality reducing pretreatments such as bleaching, dyeing and the like; starch as a thickener in the preparation of dye pastes to prevent dye exudation; and starch as an additive for warp agents for sewing threads. 2.4 Building materials industry The fourth field of application is the use of starches as additives in building materials. An example is the production of gypsum boards, where the starch that is mixed with the gypsum suspension is gelatinized with water, and diffuses to the surface of the mortar core and joins the cardboard with the core. Other fields of application are the mixture for plaster and mineral fibers. In the case of ready mix concrete, starch products are used to retard hardening. 2. 5 Soil stabilization A limited market for starch products is the production of soil stabilizers, which are used to temporarily protect soil particles from water when it is artificially disturbed. According to current knowledge, the combination products of starch and polymer emulsions match the previously used products with respect to their erosion and scale-reducing effect, but they are notably less expensive. 2. 6 Use in crop and fertilizer protection products One field of application for using starch is in crop protection products to alter the specific properties of those products. In this way, the starches are used to improve the wetting capacity of crop protection products and fertilizers, for the dosed supply of active ingredients, to convert liquid active ingredients, volatile active ingredients and / or active ingredients of malodor, into conformable, stable, microcrystalline substances, to mix incompatible compounds and to prolong the duration of action by reducing their decomposition. 2. 7 Pharmaceutical, medicinal and cosmetic industry 5 Another field of application is the sector of the pharmaceutical, medicinal and cosmetic industry. In the pharmaceutical industry, starches are used as binders for tablets or to dilute the binder in capsules. In addition, starches are used as tablet disintegrants, since they absorb fluids after they have been swallows, swelling in a short period to such an extent that the active ingredient is released. Medicinal lubricating powders and powder for wounds are made based on starch for quality reasons. In the cosmetic sector, starches are used, for example, as vehicles for powder additives such as fragrances and salicylic acid. A relatively applicable field of the starch is that of toothpastes. 2. 8 Addition of coal starch and carbon agglomerates A field of application for starch is as an additive for coal and coal agglomerates. With the addition of starch, the coal can agglomerate or form into agglomerates, in terms of high quality, thus preventing the early decomposition of the agglomerates. In the case of charcoal for steak, the addition of starch reaches an amount between 4 and 6%; in the case of heated coal, between 0.1 and 0.5%. In addition, These starches are becoming important as binders, since the emission of harmful substances can be markedly reduced when starches are added to charcoal and coal agglomerates. 2.9 Preparation of smoothed ore and coal sediment In addition, starch can be used as a flocculant in the preparation of smoothed ore and coal sediment. 2. 10 Casting Auxiliary 10 An additional field of application is as an additive for foundry auxiliaries. Several casting methods require cores made with sand treated with binders. The binder used predominantly until now is bentonite, which is treated with modified starches, in most cases inflatable starches. Purpose to add the starch is to increase the fluidity and improve the agglutination power. In addition, the inflatable starches can meet other demands of production engineering, such as being dispersible in cold water, rehydratable and easily miscible with sand, and having high water binding capacity. 20 2.11 Use in the rubber industry Starch is used in the rubber industry to improve technical and visual quality. The reasons are the improvement of the luster of the x ^^ t í¿ '^^ ^^ - surface, improved management and appearance; for this purpose, the starch is dispersed on the sticky rubberized surfaces of the rubber materials before cold curing; and also the improvement of the printing capacity in the rubber. 2. 12 Production of leather substitutes Modified starches can also be sold for the production of leather substitutes. 2. 13 Starch in synthetic polymers In the polymer sector, the following fields of application can be contemplated: the use of starch degradation products in the processes (the starch is only a filler, there is no direct link between the synthetic polymer and the starch) or, alternatively, the use of starch degradation products in the production of the polymers (the starch and the polymer form a stable bond). The use of starch as a pure filler is not competitive compared to other substances such as talc. However, this is different when the specific properties of the starch impact the spectrum of characteristics of the final products, significantly altering said spectrum. An example of this is the use of starch products in the treatment of thermoplastics such as polyethylene. Here, the starch and the synthetic polymer are combined by coexpression at a ratio of 1.1 to produce a master batch from which various products are made together with granulated polyethylene, using conventional treatment techniques. By using starch in polyethylene films, a greater permeability of substances can be achieved in the case of hollow bodies, a better water vapor permeability, a better antistatic behavior, a better anti-block behavior and a better printing capacity with aqueous inks . The current disadvantages refer to insufficient transparency, reduced tensile strength and reduced elasticity. Another possibility is the use of starch in polyurethane foams. By adapting the starch derivatives and by optimizing the process engineering, it is possible to control the reaction between synthetic polymers and the hydroxyl groups of the starches in a targeted manner. This results in polyurethane films having the following spectrum of properties, due to the use of starch: a reduced coefficient of heat expansion, a reduced shrinkage behavior, improved depression-tension behavior, an increase in water vapor permeability without altering the incorporation of water, a reduced flammability, and a reduced final tensile strength, without drop formation of the combustible parts, halogen free and reduced aging. The disadvantages that still there are a reduced printing capacity and a reduced impact resistance. Product development is currently no longer restricted to movies. Solid polymer products can also be prepared such as pots, slabs and plates, with a starch content of more than 50%. In addition, starch / polymer mixtures are considered advantageous since their biodegradability is much higher. Starch graft polymers have become increasingly important due to their extremely high water binding capacity. They are products with a skeleton of starch and a side chain of a synthetic monomer, grafted following the principle of chain mechanism of free radicals. The currently available starch graft polymers are distinguished by a better water binding and retention capacity of up to 1000 g of water per g of starch combined with high viscosity. The fields of application for these superabsorbents have been widespread in recent years and are, in the hygiene sector, the products of diapers and pads, and in the agricultural sector, for example seed coatings. What is decisive for the application of novel starches constructed by genetic engineering, is on the one hand the structure, water content, protein content, lipid content, fiber content, ash / phosphate content, amylose / amylopectin ratio, distribution of molecular mass, degree of branching, size and shape of granule, and crystallization; and on the other hand, also the characteristics that affect the following characteristics: flow and sorption behavior, gelatinization temperature, viscosity, thickening power, solubility, gel structure, transparency, heat stability, shear stability, stability to acids , tendency to undergo degradation, gel formation, freeze-thaw stability, complex formation, iodine binding, film formation, adhesive power, stability to enzymes, digestion capacity and reactivity. The production of modified starches by means of genetic engineering can, on the one hand, alter, for example, the properties of the starch derived from the plant, in such a way that no further modification is required by means of chemical or physical alterations. On the other hand, starches that have been altered by genetic engineering can be subjected to to additional chemical modification, which leads to quality improvements for some of the fields of application mentioned above. These chemical modifications are known in principle. In particular, they are modifications by treatment with heat and pressure, treatment with organic or inorganic acids, enzymatic treatment, oxidations or esterifications leading, for example, to the formation of phosphate starches, nitrate starches, sulfate starches, xanthan starches, acetate starches and citrate starches. In addition, monohydric or polyhydric alcohols can be used in the presence of strong acids to prepare starch ethers, resulting in alkyl ethers, o-allylethers, starchy hydroxyalkylether and O-carboxylmethyl ethers, starch ethers containing N, S-containing starch ethers, cross-linked starches or starch graft polymers.

One use of the starches according to the invention is the industrial application, preferably for food products or for the preparation of packaging materials and disposable articles. The following examples serve to illustrate the invention and do not constitute a restriction in any way.

Abbreviations: BE branching enzyme bp base pairs 10 GBSS granulated beta-linked starch synthase IPTG isopropyl- -D-thiogalactopyranoside soluble starch synthetase PMSF phenylmethylsulfonyl fluoride Means and solutions used in the examples: SSC 20x 175.3 g NaCI 88.2 g sodium citrate up to 1000 ml with double distilled water pH 7.0 with 10 N NaOH Shock absorber A 50 mM Tris-HCl pH 8.0 DTT 2.5 mM EDTA 2 mM PMSF 0.4 mM glycerol 10% sodium dithionite 0.1% Shock absorber B Tris-HCl 50 mM pH 7.6 DTT 2.5 mM EDTA 2 mM Shock absorber C sodium citrate 0.5 M pH 7.6 Tris-HCl 50 mM pH 7.6 DTT 2.5 mM EDTA 2 mM TBS 10x Tris-HCl 0.2 M pH 7.5 NaCl 5.0 M TBST IOx TBS 10x Tween 20, 0.1% (v / v) Elution buffer Tris 25 mM pH 8.3 glycine 250 mM Dialysis buffer Tris-HCl 50 mM pH 7.0 NaCl 50 mM 2 mM EDTA-14.7 mM mercaptoethanol 0.5 mM PMSF Protein buffer 50 mM sodium phosphate buffer pH 7.2% 10 mM EDTA 0.5 mM PMSF-14.7 mM mercaptoethanol DESCRIPTION OF THE FIGURES Figure 1 represents a schematic profile of RVA temperature (viscosity and temperature versus time [min]), this with the viscometer parameters T = gelatinization temperature, temperature at the beginning of gelatinization; Max specifies the maximum viscosity; Min specifies the minimum viscosity; End specifies the viscosity at the end of the measurement; Set is the difference (D) between Min and End (backspace). Figure 2 shows the side chain distribution of the determined amylopectin samples, on the right by means of HPAEC-PAC (voltage [mV] against Time [min]) and on the left determined by gel penetration chromatography (stream [nC] ] against Time [min]). A = control (wild type No. 1); B = (asSSIl, No. 7); C = (asSSIII, No. 8); D = (asSSIl asSSIII, No. 13); E = (asSSIl asSSIII, No. 14). The numbers given in square brackets in the description of the figures, refer to the numbers of the starch samples that are described in tables 1 and 2. The following methods were used in the examples: yes? t & mjM iSÉ .A * »~» ~ - »¿* > 1. Cloning method The vector pBluescript II SK (Stratagene) was used for cloning in E. coli. For the transformation of plants, constructs of 5 genes were cloned into the binary vector pBinAR Hyg (Hófgen &Willmitzer, 1990, Plant Sci. 66: 221-230) and pBinB33-Hyg. 2. Bacterial strains and plasmids E. coli strain DH5a (Bethesda Research 10 Laboratories, Gaithersburg, E.U.A.) was used for the Bluescrpt pBluescript II KS vector (Stratagene) and for the pBINAR Hyg and pBinB33 Hyg constructs. E. coli strain XL1-Blue was used for excision in vivo. pBinAR 15 Plasmid pBinAR is a derivative of the binary vector plasmid pBin19 (Bevan, 1984), which was constructed as follows: A fragment of 529 bp was isolated spanning nucleotides 6909-7437 of the 35S promoter of cauliflower mosaic virus from plasmid pDH51, as an EcoRI / Kpnl fragment (Pietrzak et al., 1986); HE ligated between the EcoRI and Kpnl cleavage sites of the polylinker pUC18, and was named plasmid pUC18-35S. With the aid of the restriction endonucleases Hindlll and Pvull, a 192 bp fragment was isolated from plasmid pAGV40 (Herrera-Estrella et al., 1983), which covers DNA from the Ti plasmid j < -fe »jm4A &amp? bñt0BS * tt¿S? & K & amp; t > '"J * pTiACHd (Gielen et al., 1984, nucleotides 11749-11939). After having provided the Pvull cut site with Sphl linkers, the fragment was ligated between the Sphl and Hindlll cut sites of pUC18-35S, and this was called pA7 plasmid. In addition, all the polylinker comprising the 35S promoter and the oes terminator was cut with EcoRI and HindIII and ligated into the pBin19 cut appropriately. This produced the plant expression vector pBinAR (Hófgen and Wíllmitzer, 1990). pBinB33 The promoter of the B33 gene of patatin Solanum tuberosum (RóchaSosa et al., 1989) was ligated, as a Dral fragment (nucleotides -1512- + 14) into the vector pUC19 cut with Sstl, which had been shaved at its ends with the T4 DNA polymerase help. This yielded the plasmid pUC19-B33. The B33 promoter was cut from this plasmid with EcoRI and Smal and ligated into the pBinAR vector cut suitably. This produced the plant expression vector pBinB33. pBinAr-Hyg Starting from plasmid pA7 (see description of pBinAR vector), the EcoRI-HindIII fragment comprising the 35S promoter, the oes terminator and the polylinker portion between the 35S promoter and the oes terminator, was introduced into the pBin-Hyg plasmid cut properly. pBinB33-Hyg Starting from the plasmid pBinB33, the EcoRI-HindIII fragment comprising the B33 promoter, part of the polyslinker and the terminator oes, is cut and ligated into the pBin-Hyg vector cut properly. This produced the plant expression vector pBinB33-Hyg. 3. Transformation of Agrobacterium tumefaciens The DNA was transferred by direct transformation following the method of Hoffgen and Willmitzer (1988, Nucleic Acids Res. 16: 9877). The plasmid DNA of the transformed agrobacteria was isolated following the method of Birnboim and Doly (1979, Nucleic Acids Res. 7: 1513-1523), subjected to suitable restriction cut, and then analyzed by gel electrophoresis. 4. Transformation of potatoes The plasmids were transformed into potato plants (Solanum tuberosum L. cv. Desiree, Vereinigte Saatzuchten eG, Ebstorf) with the help of the C58C1 strain of Agrobacterium tumefaciens (Díetze et al. (1995) in "Gene Transfer to Plants "[Transfer of genes to plants], pages 24-29, editors: Potrykus I. and Spangenberg G., Springer Verlag, Deblaere et al., 1985, Nucí Acids Res. 13: 4777-4788). Ten small leaves of a sterile potato culture that had been cut with a scalpel were placed in 10 ml of an MS medium (Murashige and Skoog (1962) Physiol. Plant 15: 473) supplemented with 2% sucrose and containing 50 ml of a culture of Agrobacterium tumefaciens developed during the night under selection conditions. After gently shaking the culture for 3-5 minutes, it was incubated two more days in the dark. For callus induction, the leaves were then placed on MS medium supplemented with 1.6% glucose, naphtylacetic acid 5 mg / l, benzyllaminopurine 0.2 mg / l, claforan 250 mg / l, kanamycin 50 mg / l and Bactoagar 0.80%. After incubating the leaves for one week at 25 ° C and 3000 Lux, they were placed in MS medium supplemented with 1.6% glucose, zeatin ribose 1.4 mg / l, naphthylacetic acid 20 mg / l, gibberellic acid 20 mg / l, claforan 250 mg / l, kanamycin 50 mg / l and Bactoagar 0.80%, to induce rods.

. Plant cultivation regime Potato plants were stored in a greenhouse under the following regime: Light period 16 hours at 25,000 Lux and 22 ° C Dark period 8 hours at 15 ° C Atmospheric humidity 60% 6. Radioactive labeling of the DNA fragments The DNA fragments were radiolabelled with the help of a "DNA Random Primer Labeling" labeling equipment from Boehringer Mannheim (Germany) following the manufacturer's instructions. 7. Determination of the activity of starch synthetase The determination of starch synthetase activity was done by determining the incorporation of 14C-glucose of ADP [14C-glucose] into an insoluble methanol / KCl product as described by Denver and Smith, 1992, Plant 186: 609 -617. 5 8. Detection of soluble starch synthase in the native gel To detect the activity of soluble starch synthetases by non-denaturing gel electrophoresis, tissue samples of potato tubers were hydrolyzed in 50 mM Tris-HCl pH 7.6, 2 mM DTT, EDTA 2.5 mM, 0 glycerol 10% and 0.4 mM PMSF. Electrophoresis was carried out in a MíniProtean II chamber (BioRAD). The monomer concentration in the 1.5 mm thick gels was 7.5% (w / v), and 25 mM Tris-glycine pH 8.4 was used as buffer for the gel and buffer for the run. Identical amounts of protein extract were applied, and separation was carried out for 2 5 hours at 10 mA per gel. Gels with activity were subsequently incubated in 50 mM Tricine-NaOH pH 8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP-glucose, 0.1% amylopectin (w / v) and 0.5 M sodium citrate. The formed glucans were stained with Lugol's solution. 0 9. Starch analysis The starch formed by the transgenic potato plants was characterized by the following methods: (a) Determination of the amylose / amylopectin ratio in the starch of potato plants. Starch was isolated from potato plants by means of normal methods, and the amylose: amylopectin ratio was determined by the method described by Hovenkamp-Hermelink et al. (Potato Research 31 (1988) 241-246). (b) Determination of phosphate content in potato starch, some glucose units can be phosphorylated on carbon atoms at positions C2, C3 and C6. To determine the degree of phosphorylation at the C6 position of glucose, 100 mg of starch was hydrolysed for 4 hours at 95 ° C in 1 ml of 0.7 M HCl (Nielsen et al. (1994) Plant Physiol. 105: 1 11-1 17). After neutralization with 0.7 M KOH, 50 ml of the hydrolyzate was subjected to a visual-enzymatic test to determine glucose-6-phosphate. The absorption change of the test batch (imidazole 100 mM / HCl) was monitored; 10 mM MgCl 2; NAD 0.4 mM; 2 units of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; 30 ° C) at 334 nm. Total phosphate was determined as described by Ames, 1996, Methods in Enzymology VIII, 115-118. (c) Analysis of the amylopectin side chains To analyze the distribution and length of the side chains in the starch samples, 1 ml of a 0.1% starch solution was digested with 0.4 U of soamilasa (Megazyme International Ireland Ltd. , Bray, Ireland) s * overnight at 37 ° C in a 100 mM sodium citrate buffer, pH 3.5. The rest of the analysis was carried out as described by Tomlinson et al. (1997), Plant J. 11: 31-47, unless otherwise specified. (d) Determination of granule size The granule size was determined using a "Lumosed" photosedimentometer from Retsch GmbH, Germany. For this purpose, 0.2 g of starch was suspended in approximately 150 ml of water and measured immediately. The program supplied by the manufacturer calculated the average diameter of the starch granules, assuming an average starch density of 1.5 g / l. (e) Gelatinization properties The gelatinization or viscosity properties of the starch were recorded using a Vískograph E viscometer from Brabender OHG, Germany, or using a Rapid Visco Anayzer from Newport Scientific Pty Ltd., Investment Support Group, Warriewood NSW 2102, Australia. When Viskograph E is used, a suspension of 20 g of starch in 450 ml of water is subjected to the following heating program: heat at 3 min from 50 ° C to 96 ° C, keep constant 30 minutes, cool to 3 min up to 30 ° C, and again keep constant for 30 minutes. The temperature profile gave the characteristic properties of gelatinization. When measured using the Rapid Visco Analyzer (RVA), a suspension of 2 g of starch in 25 ml of water was subjected to the following heating program: suspend for 60 seconds at 50 ° C, heat at 12 min at 50 ° C at 95 ° C, keep constant for 2.5 minutes, cool to 12 ° C / min up to 50 ° C, and again keep constant for 2 minutes. The RVA temperature profile gave the viscometric parameters of the test starches for maximum viscosity (Max), final viscosity (End), gelatinization temperature (T), the minimum viscosity (Min) that occurs after the maximum viscosity, and the difference between the minimum and final viscosity (setback, Set) (see table 1 and figure 1). (f) Determination of the strength of the gel To determine the strength of the gel by means of an analyzer Texture Analyzer, 2 g of starch were gelatinized in 25 ml of water (see RVA measurement) and then stored for 24 hours in a sealed container at 25 ° C with the exclusion of air. Samples were mounted under the probe (circular seal) of a TA-XT2 Texture Analyzer (Stable Micro Systems), and gel strength was determined with the following parameter values: Test speed 0.5 mm Penetration depth 7 mm Contact area (of the seal) 1 13 mm2 Pressure / contact area 2 g . Determination of glucose, fructose and sucrose To determine the content of glucose, fructose and sucrose, small portions of potato tuber (approximately 10 mm in diameter) were frozen in liquid nitrogen, and then extracted for 30 minutes at 80 ° C in 0.5 ml of 10 mM HEPES, pH7.5; 80% ethanol (vol / vol). The supernatant, which contains the soluble materials, was removed and the volume determined. The supernatant was used to determine the amount of soluble sugars. The quantitative determination of glucose, fructose and sucrose soluble in a batch with the following composition was carried out: 100. 0 mM midazole / HCI, pH 6.9 1.5 mM MgCl2 0.5 mM NADP + 1.3 mM ATP 10-50 I sample 1.0 unit of yeast glucose-6-phosphate dehydrogenase The batch was incubated for 5 minutes at room temperature. Subsequently, the sugars were determined by photometry by measuring the absorption at 340 nm after the successive addition of: 1. 0 unit of yeast hexokinase (to determine glucose) 1.0 unit of yeast phosphoglucoisomerase (to determine fructose) and 1.0 unit of yeast invertase (to determine sucrose) 11. Determination of water incorporation capacity (WUC) To determine the water incorporation capacity, soluble material was removed from the starch that had been swollen at 70 ° C, by means of centrifugation (10 minutes at 10,000 x g), and then the residue was weighed. The water incorporation capacity of the starch was based on the initial amount of starch corrected by the soluble material. WUC (g / g) = (residue- (initial amount-soluble) / (initial quantity-soluble) EXAMPLES OF USE EXAMPLE 1 Preparation of plasmid p35S SSI-Hyg An Asp718 / Xbal fragment of 1831 bp containing a partial cDNA coding for potato SS I (Abel G. 81995), thesis for PhD, Freie Universitát Berlin), between Asp718 and the Xbal cut site of the vector pBinAR- was introduced. Hyg in antisense orientation with respect to the 35S promoter. ? * gjgfó EXAMPLE 2 Preparation of plasmid p35S-SSI-Kan A 2384 bp EcoRI fragment containing cDNA coding for potato SS I was shaved at its ends (Abel, 1995, in the aforementioned place), and was introduced into the vector pBinAR, which had previously been cut with Smal, in orientation of meaning with respect to the 35S promoter.

EXAMPLE 3 Preparation of plasmid p35S SSII-Kan A 1959 bp Smal / Asp718 fragment containing a partial cDNA encoding SSII from potato was shaved at its ends (Abel, 1995, called GBSS II therein), and introduced into the Smal cut site of the pBinAR vector in antisense orientation with respect to the 35S promoter.

EXAMPLE 4 Preparation of Plasmid pB33-SSII-Hvq 0 A Smal / Sall fragment of 2619 bp containing a CDNA coding for potato SS II (Abel, 1995, in the aforementioned place) in the vector pBinB33-Hyg, which had previously been cut with Smal and Sali, in sense orientation with respect to the B33 promoter. • ^ ^ - ^ & ^ a EXAMPLE 5 Preparation of plasmid p35S SSIII-Hyq An Asp718 / Xbal fragment of 4212 bp containing a cDNA encoding the SS III of potato (Abel et al., 1996, Plant J. 10 (6): 981-991) was inserted between the ASP718 and the Xbal cut site. vector pBinAR-Hyg, in antisense orientation with respect to the 35S promoter.

EXAMPLE 6 Preparation of plasmid p35S-SSIII-Kan An EcoRI fragment of 4191 bp containing a cDNA encoding the SS III of potato was shaved at its ends (Abel et al., 1996, in the aforementioned place), and was introduced into the Smal cut site of the vector pBinAR in orientation of meaning with respect to the 35S promoter.

EXAMPLE 7 Preparation of Plasmid pB33 BE SSIII-Kan A 1650 bp Hindlll fragment containing a partial cDNA encoding potato BE enzyme was shaved at its ends (Kossman et al., 1991, Mol. &Gen. Genetics 230 (1-2): 39-44), and it was introduced into the pBinB33 vector that had previously been cut with Smal, in antisense orientation with respect to the B33 promoter. The resulting plasmid was opened by cutting with BamHI. At the cut site, a 1362 bp BamHI fragment containing a partial cDNA encoding the potato SS III enzyme was introduced (Abel et al., 1996, in the cited place), again in antisense orientation with respect to the promoter. B33.

EXAMPLE 8 Preparation of plasmid p35S SSII-SSIII-Kan An EcoRV / Hincll fragment of 1546 bp, which contained a partial cDNA coding for the SS II of potato (Abel, 1995, in the aforementioned place), was cloned into the vector pBluescript II KS, which had been cut with EcoRV / Híncll, was then excised by a Asp718 / BamHI digestion and introduced in antisense orientation with respect to the 35S promoter in the vector POINTS that had been digested in the same way. Then, a 1356 bp BamHI fragment containing a partial cDNA encoding potato SS III (Abel et al., 1996, in the aforementioned place) was introduced at the BamHI cleavage site of the vector pBinAR-SSI1, again in the orientation of antisense with respect to the 35S promoter.

EXAMPLE 9 Preparation of Plasmid pB33 SSl SSIII-Kan A 2384 bp EcoRI fragment containing a cDNA coding for SS I from potato was shaved at its ends (Abel, 1995, in the cited place), and was cloned into the Smal cut site of the pBinB33 vector in antisense orientation. with respect to the B33 promoter. A 1362 bp BamHI fragment containing a partial cDNA coding for potato SS III (Abel et al., 1996, in the above-mentioned place) was introduced at the BamHI cleavage site of the resulting vector, again in antisense orientation with with respect to the B33 promoter.

EXAMPLE 10 Preparation of plasmid p35S SSII-Hyq 15 A 1959 bp Smal / Asp718 fragment containing a partial cDNA coding for SS II (Abel, 1995, in the aforementioned place) was shaved at its ends and introduced into the Smal cut site of the pBinAR-Hyg vector in antisense orientation with respect to the 35S promoter. ~ w && > s & * Si- -a ^ ** s ^ m§ & amp; amp; ~ '~ EXAMPLE 10B Preparation of Plasmid pB33 R1-Hyq A 1.9 kB R1 fragment of S. tuberosum was obtained (WO 97/11188) by digestion with Asp718 of the pBluescript vector. The fragment was cloned into the Asp718 cut site behind the B33 promoter in antisense orientation of vector pB33-Binar-Hyg. This vector contains a resistance to hygromycin.

EXAMPLE 11 Introduction of plasmids in the qenoma of potato cells The plasmids given in Examples 1 to 10 were transferred to agrobacteria, individually and / or in succession, with the aid of which, the potato cells were transformed as described above. Subsequently, whole plants of the transformed plant cells were regenerated. Transgenic plants of the genotype asSSI-asSSII-asSSIII were generated by transformation with the plasmid p35S SSI-Hyg described in Example 1, and subsequent transformation with the plasmid p35S SSII-SSIII-Kan described in example 8. Transgenic plants of the genotype were generated. genotype asSSII-asSSI- asSSIII by transformation with the plasmid p35S SSII-Hyg described in ^^ m ^ ^ ^^ ^^^^^ a ^^ i example 10, and subsequent transformation with the plasmid pB33 SSl SSIII-Kan described in example 9. As a result of the transformation, the transgenic potato plants synthesized varieties of altered starch.

EXAMPLE 12 Physicochemical characterization of modified starches The starch formed by the transgenic plants generated according to example 11, differs from the starch synthesized by wild-type (potato) plants for example, with respect to their phosphate or amylose content and the viscosities and gelatinization properties, which were determined by RVA Table 1 shows the results of the physicochemical characterization of the modified starches. In the antisense constructs, the enzymatic activities of the suppressed soluble starch synthetases were reduced by up to 85% with respect to the untransformed control plants.

^^^ ^^ ^^ ¿^^ PICTURE, 1 Properties of modified starches TABLE 1 (continued) Keys: SS1 = isoform I of starch synthetase; SSII = sophorm II of starch synthetase; SSIII = isoform III of starch synthetase; BE = branching enzyme; as = antisense; oe = overexpressed (sense); cos = cosuprimido (sense); data of the Rapid Visco Analyzer (RVA): max designates the maximum viscosity; min the minimum viscosity; end the viscosity at the end of the measurement; set is the difference (D) between min and end (recoil) and T is the temperature of gelatinization. The percentages are based on the wild type (= 100%). & & js ¿j¡sß ~ ** 3t * m * ~. .. "^ ^ - Ba-aii EXAMPLE 13 Characterization of the side chains of modified starches The glucan chains were separated after removing the amylose by means of thymol precipitation (Tomlinson et al., In the cited place), using a high performance anion exchange chromatography system with an amperometric detector (HPEAC-PAD, Dionex) . The samples (10 mg / ml amylopectin) were dissolved in 40% DMSO and 1/10 part by volume of 100 mM sodium acetate pH 3.5, and 0.4 U of soamilase (Megazyme) were added. After incubation, 10 μl of the sample was applied to the column system and eluted as described by Tomlinson and others (in the cited place). Figure 2 shows the results of the HPEAC-PAD analysis regarding the length and distribution of the side chains of the starch samples Nos. 1, 7, 8, 13 and 14 (see tables 1 and 2). Another HPLC system to detect the side chain distribution, consisted of 3 columns connected in series (2 TSK-Gel 2000SW and one TSK-Gel 3000SW, TosoHaas, Stuttgart, Germany) as described by Hizukuri ((1986) Carbohydr, Res. 147: 342-347). 100 I of the prepared sample was applied to the column system. The eluent used was 10 mM sodium acetate, pH 3.0, at a flow rate of 0.35 ml / min. The glucans were detected by means of a refractive index detector (Gynkotek), and the chain lengths of linear eluted glycans were t ^ - _ .Arflir .m. t '«K? J determined by means of mass spectroscopy and iodometry (Hizukuri (1986), in the cited place). Figure 2 shows the results of the gel-chromatographic analysis of HPLC, with respect to length and distribution of the side chains of starch samples Nos. 1, 7, 8, 13 and 14 (see tables 1 and 2) . Table 2 shows the percentages of several side chain fractions of the starches that were analyzed. Fraction 1 represents the percentage of the A and B1 chains (Hízukuri (1986), in the aforementioned place); fraction 2 represents the percentage of chains B2, B3 and B4 (Hizukuri (1986), in the aforementioned place); and fraction 3 shows the percentage of the high molecular weight glucan molecules eluting in the elution volume. TABLE 2 Distribution of amylopectin side chains of modified starch Go ¿¿S -.Sfefl.

Claims (30)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for the preparation of a transgenic host cell, characterized in that they are integrated into the genome of a cell simultaneously or sequentially, (a) at least one nucleotide sequence that encodes a protein with the function of a starch synthetase soluble III, or fragments of said nucleotide sequence, and (b) one or more nucleotide sequences that encode a protein selected from group A, consisting of proteins that function as branching enzymes, ADP glucose pyrophosphorylases, bound starch synthetases to granule, soluble starch synthetases, debranching enzymes, disproportionate enzymes, plastid starch phosphorylases, R1 enzymes, amylases and glycosidases, or fragments thereof.

2. The method according to claim 1, wherein the cell is a bacterial or plant cell.

3. A transgenic host cell, comprising (a) at least one nucleotide sequence encoding a protein having the function of a soluble starch synthetase III, or fragments of said nucleotide sequence, and (b) a more nucleotide sequences encoding a protein selected from group A, consisting of proteins that have the function of branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases, debranching enzymes, disproportionate enzymes, starch phosphorylases of plastids, R1 enzymes, amylases and glucosidases, or fragments thereof.

4. The host cell according to claim 3, further characterized in that it is a bacterial or plant cell.

5. A recombinant nucleic acid molecule, comprising (a) at least one nucleotide sequence encoding a protein having the function of a soluble starch synthetase III, or fragments of said nucleotide sequence, and (b) one or more nucleotide sequences encoding a protein selected from group A, consisting of proteins having the function of branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases, debranching enzymes, disproportioning enzymes, phosphorylases of plast starch, R1 enzymes, amylases and glucosidases, or fragments thereof.

6. The nucleic acid molecule according to claim 5, further characterized in that it is a molecule of deoxyribonucleic acid.

7. The nucleic acid molecule according to claim 6, further characterized in that it is a cDNA molecule.

8. The nucleic acid molecule according to claim 5, further characterized in that it is a ribonucleic acid molecule.

9. - A nucleic acid molecule that hybridizes specifically with a nucleic acid molecule as claimed in one or more of claims 5 to 8.

10. A vector comprising a nucleic acid molecule 5 as claimed in one or more of claims 5-9.

11. A vector comprising a nucleic acid molecule as claimed in one or more of claims 5-9, characterized in that the nucleotide sequence encoding a protein having the function of a soluble starch synthase, or fragments of the 10 itself, it is present in sense or antisense orientation.

12. A vector comprising a nucleic acid molecule as claimed in one or more of claims 5-9, characterized in that the nucleotide sequence encoding one or more proteins selected from group A, or fragments thereof , it is present 15 in sense orientation or antisense.

13. A vector comprising a nucleic acid molecule as claimed in one or more of claims 5-9, characterized in that the nucleotide sequence encoding one or more proteins selected from group A, or fragments thereof , is partially present in sense orientation and partially in antisense orientation.

14. A vector comprising a nucleic acid molecule as claimed in one or more of claims 5-9, which is linked to one or more regulatory elements that ensure the transcription and synthesis of an RNA in a prokaryotic or eukaryotic cell.

15. The method according to one or more of claims 1 and 2, further characterized in that one or more nucleic acid molecules as claimed in one or more of the claims are integrated into the genome of a cell. -9, or one or more vectors as claimed in one or more of claims 10-14.

16. The host cell according to one or more of claims 3 and 4, further characterized in that it contains one or more nucleic acid molecules as claimed in one or more of claims 5-9, or one or more vectors as claimed in one or more of claims 10-14.

17. A method of generating a transgenic plant that synthesizes a modified starch, characterized in that a whole plant of a plant cell is regenerated as claimed in one or more of claims 3, 4 and 16.

18.- One plant comprising one or more cells as claimed in one or more of claims 3, 4 and 16.

19. The plant according to claim 18, further characterized in that it is a starch storage plant.

20. The plant according to claim 19, further characterized in that it is a utility plant.

21. - The plant according to one or more of claims 18-20, further characterized because it is a wheat, corn, potato or rice plant.

22. The propagation material of a plant as claimed in one or more of claims 18-21.

23. A process for the preparation of starch by means of a method known per se, characterized in that the cells claimed in one or more of claims 3, 4 and 16, the plants claimed in the claim, are integrated into the process. one or more of claims 18-21, or the propagation material claimed in claim 22.

24.- A starch that can be obtained from a cell as claimed in one or more of claims 3, 4. and 16, a plant as claimed in one or more of claims 18-21, or of the propagation material claimed in claim 22.

25.- The starch according to claim 24, further characterized by having , compared to a starch obtained from a non-transformed plant or cell, a phosphate content reduced by at least 30%, and a glucan content after treatment with isoamylase in the elution volume of a colu system HPLC mna, composed of 2 columns TSK-Gel 2000SW and one column TSK-Gel 3000SW connected in series in 10 mM sodium acetate pH 3.0, which is increased by at least 50%.

26. - The starch according to claim 24, further characterized in that it has, compared to a starch obtained from a non-transformed plant or cell, a phosphate content increased by at least 10%, and a content of glucan after treatment with isoamylase in the elution volume of an HPLC column system, composed of 2 columns TSK-Gel 2000SW and one column TSK-Gel 3000SW connected in series in 10 mM sodium acetate pH 3.0, which is increased at least in fifty%.

27. The use of a starch as claimed in one or more of claims 24-26, in the industry, preferably for the production of food products, packaging materials or disposable articles.

28. The use of (a) at least one nucleotide sequence that encodes a protein that has the function of a soluble starch synthase III, or fragments of said nucleotide sequence, and (b) one or more nucleotide sequences encoding a protein selected from group A, consisting of proteins that function as branching enzymes, ADP glucose pyrophosphorylases, synthetases of granule-bound starch, soluble starch synthetases I, II or others, debranching enzymes, disproportionate enzymes, phosphorylases of plastid starch, R1 enzymes, amylases and glycosidases, or fragments thereof; for the production of transgenic cells, preferably bacterial or plant cells. • i * v

29. - The use of one or more nucleic acid molecules as claimed in one or more of claims 5-9, or of one or more vectors as claimed in one or more of claims 10-14, for the generation of transgenic cells, preferably bacterial or plant cells.

30. The use of the cells claimed in one or more of claims 3, 4 and 16, of the plants claimed in one or more of claims 18-21, or of the propagation material claimed in Claim 22, for the production of starch.