# Patent

## Title
Transgenic plants synthesizing high amylose starch

## Abstract

Transgenic plant cells and plants are described which synthesize a starch which is modified in comparison with corresponding wild type plant cells and plants. The plant cells and plants described show a reduced activity of R1, BEI and BEII proteins. Furthermore, modified starches as well as methods for their production are described.

## Background
FIELD OF THE INVENTION
The present invention relates to genetically modified plant cells and plants wherein the genetic modification leads to the reduction of the activity of R1 and BEI and BEII proteins in comparison with corresponding plant cells of wild type plants that have not been genetically modified. Furthermore, the present invention relates to means and methods for the production thereof. Plant cells and plants of that type synthesise a modified starch characterised in that it has an amylose content of at least 75% and—in comparison with starch of corresponding wild type plants which have not been genetically modified—a reduced phosphate content and/or a modified distribution of the side chains and/or an increased gel strength in the texture analyser and/or a modified starch granule morphology and/or a modified average starch granule size. Thus, the present invention also relates to starch that can be synthesised by the plant cells and plants of the invention as well as methods for the production of this starch.
With regard to the increasing importance of plant ingredients as renewable raw material sources in the past few years, one of the problems of research in the field of biotechnology is to endeavour adjustment of these raw materials to the requirements of the processing industry. For allowing an application of renewable raw materials in as many as fields as possible, it is furthermore necessary to achieve a great variety of substances.
Apart from oils, fats and proteins, polysaccharides represent the essential renewable raw materials from plants. Among the polysaccharides, starch plays a central role beside cellulose. It is one of the most important storage substances in higher plants. For allowing as wide an application of starch as possible, it seems desirable to provide plants which are able to synthesise modified starch that is particularly suitable for different purposes. One possibility of providing such plants is—apart from cultivating—the purposeful genetic modification of the starch metabolism of starch-producing plants by genetic engineering.
The polysaccharide starch is a polymer of chemically uniform basic building blocks—the glucose molecules. It is, however, a very complex mixture of different molecule forms which differ with regard to their polymerisation degree and the occurrence of branchings of the glucose chains. Thus, starch is not a uniform raw material. There are two chemically different components of starch: the amylose and the amylopectin. In plants typically used for the production of starch, such as e.g. maize, wheat or potato, the synthesised starch consists of about 20%–30% of amylose starch and of about 70%–80% of amylopectin starch.
Amylose was considered a linear polymer for a long time, consisting of α-1,4-glycosidically bound α-D-glucose monomers. In recent studies, however, the presence of about 0.1% α-1,6-glycosidic branching points has been proven (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1–10; Takeda et al., Carbohydr. Res. 132, (1984), 83–92).
As a rule, the complete separation of the amylose from the amylopectin is very difficult so that the quality of the amylose strongly depends on the type of the separation method chosen.
There are different methods for the determination of the amylose content. Some of these methods are based on the iodine-binding capacity of the amylose which can be determined potentiometrically (Banks & Greenwood, in W. Banks & C. T. Greenwood, Starch and its components (page 51–66), Edinburgh, Edinburgh University Press), amperometrically (Larson et al., Analytical Chemistry 25(5), (1953), 802–804) or spectrophotometrically (Morrison & Laignelet, J. Cereal Sc. 1, (1983), 9–20). The determination of the amylose content can also be carried out calorimetrically by means of DSC (differential scanning calorimetry) measurements (Kugimiya & Donovan, Journal of Food Science 46, (1981), 765–770; Sievert & Holm, Starch/Stärke 45 (4), (1993), 136–139). Moreover, it is possible to determine the amylose content by using the SEC (size exclusion chromatography) of native or debranched starch. This method was particularly recommended for the determination of the amylose content of genetically modified starches (Gérard et al., Carbohydrate Polymers 44, (2001), 19–27).
The choice of the analysis method used for the determination of the amylose content of a starch has a crucial influence on the size of the amylose figures determined as could be shown by various studies (Shi et al., J. Cereal Science 27, (1998), 289–299; Gérard et al., Carbohydrate Polymers 44, (2001), 19–27).
In contrast to the amylose, the amylopectin is branched to a larger degree and exhibits about 4% branching points which occur due to the presence of additional α-1,6-glycosidic linkings. The amylopectin is a complex mixture of glucose chains branched differently.
A further essential difference between amylose and amylopectin is the molecular weight. While amylose—depending on the origin of the starch—has a molecular weight of 5×105−106 Da, the molecular weight of amylopectin is between 107 and 108 Da. Both macromolecules can be differentiated from each other by their molecular weight and their different physico-chemical properties, which can be made apparent in the simplest way by their different iodine-binding properties.
The functional properties of the starch are strongly influenced—apart from the amylose/amylopectin ratio and the phosphate content—by the molecular weight, the pattern of the side chain distribution, the content of ions, the lipid and protein content, the average starch granule size and the starch granule morphology etc. Important functional properties to be mentioned are, for example, the solubility, the retrogradation behaviour, the water binding capacity, the film formation properties, the viscosity, the pasting properties, the freeze-thaw-stability, the acid stability, the gel strength etc. The starch granule size, too, can be important for different applications.
The ratio of amylopectin and amylose has a strong influence on the physico-chemical properties of the starches and, thus, on the possible applications of these starches. Since methods for the separation of these two components are very time-consuming and costly, such methods are no longer used on a large technical scale (Yound, A. H. in: Starch Chemistry and Technology. Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall. Academic Press, New York, 1984, 249–283). For a plurality of applications it would be desirable to have starches at disposal which still contain only one of the two polymers or at least one of the two starch components in an enriched form.
So far, both mutants and plants produced by genetic engineering have been described which, in comparison with corresponding wild type plants, exhibit a modified amylopectin/amylose ratio.
For example, the so-called “waxy” mutant from maize exhibiting a mutation in the gene encoding the starch granule bound starch synthase I (abbreviated: GBSSI) (Akasuka and Nelson, J. Biol. Chem., 241, (1966), 2280–2285; Shure et al., Cell 35 (1983), 225–233), produces a starch essentially consisting of amylopectin. For potato, genotypes were produced both by means of chemical mutagenesis of a haploid line (Hovenkamp-Hermelink et al., Theor. Appl. Genet., 225, (1987), 217–221) and by means of antisense inhibition of the GBSSI-gene, whose starches essentially consist of amylopectin starch. In comparison with starches of the corresponding wild type plants, such waxy potato starches do not exhibit any differences with regard to phophate content, the morphology of the starch granule or the ion content (Visser et al., Starch/Stärke, 49, (1997), 438–443).
Furthermore, maize mutants are commercially available which exhibit starches with amylose contents of about 50% or about 70% (amylose content determined by potentiometric determination of the iodine-binding capacity) and which are designated Hylon V® or HylonVII® (National Starch and Chemical Company, Bridgewater, N.J., U.S.A.). Moreover, also maize hybrids have been described which synthesise so-called “low amylopectin starch” (LAPS) and exhibit a content of high molecular (“high mol weight”) amylopectin of about 2.5% and an amylose content of about 90% (potentiometric determination of the iodine-binding capacity) (Shi et al., J. Cereal Science 27, (1998), 289–299).
Furthermore, transgenic potato plants have been described which, due to the antisense-inhibition of the branching enzyme I (=BEI) and the branching enzyme II (=BEII) gene, synthesise a potato starch which exhibits an amylose content of up to 75% by colorimetric determination of the amylose content according to the method described by Morrison and Laignelet (J. Cereal Sci. 1, (1983), 9–20) (Schwall et al., Nature Biotechn. 18, (2000), 551–554). These potato starches are characterised by a phosphate content of the starch which is up to 6 times higher compared to corresponding wild type plants. Furthermore, the international patent application WO 97/11188 describes transgenic potato plants which, due to their antisense inhibition of the R1 gene and the BEI gene synthesise a starch with an amylose content of more than 70%, the amylose content having been determined according to the method by Hovenkamp & Hermelink (Potato Research 31, (1988), 241–246).
Transgenic potato plant cells and potato plants synthesising a starch having an amylose content of more than 75% (calorimetric determination of the amylose content according to Hovenkamp & Hermelink (Potato Research 31, (1988), 241–246) and, at the same time, a reduced phosphate content in comparison with corresponding wild type plants have not been described in the state of the art so far. The same applies to the potato starches that can be isolated from these potato plant cells and plants and to methods for the production of such starches. However, the provision of such starches is desirable since their physico-chemical properties can be expected to be advantageously useful for various industrial applications.
Thus, the technical problem underlying the present invention is to provide plant cells and plants synthesising starch which has an amylose content of more than 75% (calorimetric determination of the amylose content according to Hovenkamp & Hermelink (Potato Research 31, (1988), 241–246) and a reduced phosphate content in comparison with the phosphate content of starch from corresponding wild type plant cells and plants that have not been genetically modified, as well as to provide such starch which differs from the starches described in the state of the art in its structural and/or functional properties and is, thus, more suitable for general and/or specific purposes.
This technical problem has been solved by providing the embodiments characterised in the claims.
SUMMARY OF THE INVENTION
Thus, the present invention relates to a transgenic plant cell which is genetically modified, wherein the genetic modification leads to a reduction of the activity of one or more R1 proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEI proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEII proteins occurring endogenously in the plant cell in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified.
The genetic modification may be any genetic modification leading to a reduction of the activity of one or more R1 proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEI proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEII proteins occurring endogenously in the plant cell in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified.
In this context, the term “transgenic” means that the plant cells of the invention differ in their genetic information from corresponding plant cells which are not genetically modified due to a genetic modification, in particular the introduction of one or more foreign nucleic acid molecules.
In this context, the term “genetically modified” means that the plant cell is modified in its genetic information due to the introduction of one or more foreign nucleic acid molecules and that the presence and/or the expression of the foreign nucleic acid molecule/s lead/s to a phenotypic modification. In this context, phenotypic modification preferably relates to a measurable modification of one or more functions of the cells. Genetically modified plants cells of the invention, for example, exhibit a reduction of the expression of one or more R1 genes occurring endogenously in the plant cell and a reduction of the expression of one or more BEI genes occurring endogenously in the plant cell and a reduction of the expression of one or more BEII genes occurring endogenously in the plant cell in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified, and/or a reduction of the activity of one or more R1 proteins occurring endogenously in the plant cell and a reduction of the activity of one or more BEI proteins occurring endogenously in the plant cell and a reduction of the activity of one or more BEII proteins occurring endogenously in the plant cell in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified.
Within the meaning of the present invention, the term “reduction of the activity” means a reduction of the expression of endogenous genes encoding R1, BEI and/or BEII proteins and/or a reduction of the amount of R1, BEI and/or BEII proteins in the cells and/or a reduction of the enzymatic activity of the R1, BEI and/or BEII proteins in the cells.
In the context of the present invention, the term “reduction of expression” refers to a reduction of the amount of transcripts of the respective endogenous gene in a plant cell of the invention as compared to a corresponding wild-type plant cell. The reduction of the expression can, for instance, be determined by measuring the amount of transcripts encoding R1, BEI or BEII proteins, e.g. by means of Northern blot analysis or RT-PCR. In this context, a reduction preferably means a reduction of the amount of transcripts in comparison with corresponding cells that have not been genetically modified by at least 50%, in particular by at least 70%, more preferably by at least 85% and most preferably by at least 95%.
The reduction of the amount of R1, BEI and/or BEII proteins can, for instance, be determined by means of Western blot analysis. In this context, a reduction preferably means a reduction of the amount of R1, BEI and/or BEII proteins in comparison with corresponding cells which have not been genetically modified by at least 50%, in particular by at least 70%, more preferably by at least 85% and most preferably by at least 95%.
Methods for determining the reduction of the enzymatic activity of the R1, BEI and BEII proteins are known to the person skilled in the art and will be described further below for each protein individually. In the context of the present invention, the term “R1 protein” relates to proteins which have been described, for example, in Lorberth et al. (Nature Biotech. 16, (1998), 473–477) and in the international patent applications WO 98/27212, WO 00/77229, WO 00/28052 and which have the following characteristics. Important characteristics of R1 proteins are i) their amino acid sequence (see, for example, GenBank Acc. No. A61831, Y09533); ii) their localisation in the plastides of plant cells; iii) their ability to influence the degree of phosphorylation of starch in plants.
Further, the term “R1 protein” refers to a protein catalysing the phosphorylation of starch in dikinase-type reaction in which three substrates, an a-polyglucan, ATP and H2O are converted into three products, an a-polyglucan-P, AMP and orthophosphate (Ritte et al., PNAS 99(10) (2002), 7166–7171).
The inhibition of the R1 gene encoding an R1 protein from potato in transgenic potato plants, for example, leads to a reduction of the phosphate content of the starch which can be isolated from the potato tuber. Moreover, Lorberth et al. were able to demonstrate that the R1 protein from Solanum tuberosum is able to phosphorylate bacterial glycogen when the corresponding R1 cDNA is expressed in E. coli (Lorberth et al., Nature Biotech. 16, (1998), 473–477).
Ritte et al. (Plant J. 21, (2000), 387–391) were able to show that the R1 protein from Solanum tuberosum in potato plants binds to starch granules in a reversible way, wherein the strength of the binding to the starch granule depends on the metabolic status of the plant. In potato plants, the protein is mainly present in starch granule bound form in leaves that have been kept in the dark. After exposing the leaves to light, however, the protein is mainly present in the soluble form which is not bound to the starch granule.
Furthermore, the inhibition of the expression of the R1 gene from potato in transgenic potato plants or in the tubers thereof leads to a reduction of the so-called “cold-induced-sweetenings” (Lorberth et al., Nature Biotech. 16, (1998), 473–477).
In the context of the present invention, the term “R1 protein” also relates to proteins which exhibit a significant homology (identity) of at least 60%, preferably of at least 80%, more preferably of at least 90% to the amino acid sequence stated under SEQ ID NO: 6 or under the GenBank Acc. No. Y09533 or A61831, and which are able to modify the degree of phosphorylation of polysaccharides such as, for example, starch and/or glycogen. Preferably, the R1 protein originates from potato (GenBank Acc. No. Y09533 or A61831).
Preferably, an R1 protein, as addressed in the embodiments of the present invention, is encoded by a nucleic acid molecule which hybridises, advantageously under stringent conditions, with the nucleic acid molecule having the nucleotide sequences shown under SEQ ID NO: 5 and which encodes a polypeptide having the activity of an R1 protein.
Within the present invention the term “hybridization” means hybridization under conventional hybridization conditions (also referred to as “low stringency conditions”), preferably under stringent conditions (also referred to as “high stringency conditions”), as for instance described in Sambrook and Russell (2001), Molecular Cloning, A Laboratory Manual, CSH Press, Cold Spring Harbour, N.Y., U.S.A. Within an especially preferred meaning the term “hybridization” means that hybridization occurs under the following conditions:

    Hybridization buffer: 2×SSC; 10× Denhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or
    
        0.25 M of sodium phosphate buffer, pH 7.2;
        1 mM EDTA
        7% SDS
    
    
    Hybridization temperature T=60° C.
    Washing buffer: 2×SSC; 0.1% SDS
    Washing temperature T=60° C.


Nucleic acid molecules which hybridize with a nucleic acid molecule having the nucleotide sequence shown under SEQ ID NO: 5 can, in principle, encode a R1 protein from any organism expressing such a protein.
Such hybridizing nucleic acid molecules can for instance be isolated from genomic libraries or cDNA libraries of plants. Alternatively, they can be prepared by genetic engineering or chemical synthesis.
Such nucleic acid molecules may be identified and isolated with the use of a nucleic acid molecule encoding an R1 protein as disclosed herein or parts of such a molecule or reverse complements of such a molecule, for instance by hybridization according to standard methods (see for instance Sambrook and Russell (2001), Molecular Cloning. A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., U.S.A).
Nucleic acid molecules possessing the same or substantially the same nucleotide sequence as indicated in SEQ ID NO: 5 or parts thereof can, for instance, be used as hybridization probes. The fragments used as hybridization probes can also be synthetic fragments which are prepared by usual synthesis techniques, and the sequence of which substantially coincides with that of a nucleic acid molecule specifically described herein.
The hybridizing nucleic acid molecules also comprise fragments, derivatives and allelic variants of the nucleic acid molecule having the nucleotide sequence shown under SEQ ID NO: 5. Herein, fragments are understood to mean parts of the nucleic acid molecules which are long enough to encode an R1 protein. In this connection, the term derivative means that the sequences of these nucleic acid molecules differ from the sequence of an above-described nucleic acid molecule in one or more positions and show a high degree of homology to such a sequence. In this context, homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably of at least 65%, more preferably of at least 70%, even more preferably of at least 80%, in particular of at least 85%, furthermore preferred of at least 90% and particularly preferred of at least 95%. Most preferably homology means a sequence identity of at least n %, wherein n is an integer between 40 and 100, i.e. 40≦n≦100. Deviations from the above-described nucleic acid molecules may have been produced, e.g., by deletion, substitution, insertion and/or recombination.
Preferably, the degree of homology is determined by comparing the respective sequence with the nucleotide sequence of the coding region of SEQ ID No: 5. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the ClustalW program (Thompson et al., Nucleic Acids Research 22 (1994), 4673–4680) distributed by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE) at the European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from several websites including IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and EBI (ftp://ftp.ebi.ac.uk/pub/software/) and all sites with mirrors to the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
When using ClustalW program version 1.8 to determine whether a particular sequence is, for instance, 90% identical to a reference sequence according to the present invention, the settings are set in the following way for DNA sequence alignments:

    KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.


For protein sequence alignments using ClustalW program version 1.8 the settings are the following: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.
Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding nucleic acid molecules or proteins encoded thereby. Nucleic acid molecules which are homologous to one of the above-described molecules and represent derivatives of these molecules are generally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, for instance sequences from other microorganisms, or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The allelic variants may, e.g., be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques.
The proteins encoded by the different variants of the nucleic acid molecule having the nucleotide sequence shown under SEQ ID NO: 5 possess certain characteristics they have in common. These include for instance enzymatic activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc.
In the context of the present invention, the term “R1 gene” relates to a nucleic acid molecule (e.g. cDNA, DNA) encoding an “R1 protein” as described above. Nucleic acid molecules encoding R1 proteins have been described for various plants such as, e.g. maize (WO 98/27212 A1), rice (WO 00/28052 A1) and wheat (WO 00/77229 A1). Preferably, the R1 gene originates from potato, an R1 cDNA from potato with the nucleotide sequence stated under SEQ ID NO: 5 or GenBank Acc. No. Y09533 or A61831 is particularly preferred.
In the context of the present invention, the term “branching enzyme” or “BE protein” (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase, E.C. 2.4.1.18) relates to a protein catalysing a transglycosylation reaction, wherein (α-1,4-linkings of an α-1,4-glucan donor are hydrolysed and the α-1,4-glucan chains released thereby are transferred to an α-1,4-glucan acceptor chain and are thereby converted into α-1,6-linkings. Within the meaning of the present invention, the term “BE gene” is a gene encoding a “BE protein”.
In the context of the present invention, the term “BEI protein” relates to a branching enzyme (=BE) of the isoform I, preferably the BEI protein originates from potato plants.
The designation of the isoforms follows the nomenclature suggested by Smith-White and Preiss (Smith-White and Preiss, Plant Mol. Biol. Rep. 12 (1994), 67–71; Larsson et al., Plant Mol. Biol. 37, (1998), 505–511). This nomenclature assumes that all branching enzymes exhibiting a higher homology (identity) on the amino acid level to the BEI protein from maize having the amino acid sequence shown under SEQ ID NO: 9 (GenBank Acc. No. D11081; Baba et al., Biochem. Biophys. Res. Commun. 181 (1), (1991), 87–94; Kim et al., Gene 216, (1998), 233–243) than to the BEII protein from maize having the amino acid sequence shown under SEQ ID NO: 10 (GenBank Acc. No. AF072725, U65948) are designated branching enzymes of the isoform I or, in short, BEI proteins.
In the context of the present invention, the term “BEI gene” relates to a nucleic acid molecule (e.g. cDNA, DNA) encoding a “BEI protein”, preferably a BEI protein from potato plants. Such nucleic acid molecules have been described for numerous plants, for example for maize (GenBank Acc. No. D11081, AF 072724), rice (GenBank Acc. No. D11082), pea (GenBank Acc. No. X80010) and potato. Different forms of the BEI gene or the BEI protein from potato have been described, for example, by Khoshnoodi et al. (Eur. J. Biochem. 242 (1) (1996), 148–155), GenBank Acc. No. Y08786 and by Kossmann et al. (Mol. Gen. Genet. 230 (1991), 39–44). In potato plants, the BEI gene is expressed mainly in the tubers and hardly in the leaves (Larsson et al., Plant Mol. Biol. 37, (1998), 505–511).
Preferably, a BEI protein, as addressed in the embodiments of the present invention, is encoded by a nucleic acid molecule which hybridises, advantageously under stringent conditions, with the nucleic acid molecule having the nucleotide sequence shown under SEQ ID NO: 7 and which encodes a polypeptide having branching enzyme activity.
The definition for the term “hybridisation” as defined above in connection with R1 proteins applies equally for the definition of nucleic acid molecules hybridising with the nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 7. Preferably, a BEI protein as referred to herein displays a sequence identity of at least 60%, in particular of at least 75%, preferably of at least 85%, more preferably at least 90% and even more preferably at least 95% to the amino acid sequence depicted under SEQ ID NO: 8.
In the context of the present invention, the term “BEII protein” relates to a branching enzyme (=BE) of the isoform II, preferably it originates from potato plants. Within the meaning of the present invention, all enzymes exhibiting a higher homology (identity) on the amino acid level to the BEII protein from maize (GenBank Acc. No. AF072725, U65948) than to the BEI protein from maize (GenBank Acc. No. D 11081, AF 072724) are to be designated BEII protein.
In the context of the present invention, the term “BEII gene” relates to a nucleic acid molecule (e.g. cDNA, DNA) encoding a “BEII protein”, preferably a BEII protein from potato plants. Such nucleic acid molecules have been described for numerous plants, for example, for potato (GenBank Acc. No. AJ00004, AJ011888, AJ011889, AJ011885, AJ011890), maize (AF072725, U65948), barley (AF064561), rice (D16201) and wheat (AF286319). In potato plants, the BEII gene is expressed mainly in the leaves and hardly in the tubers (Larsson et al., Plant Mol. Biol. 37, (1998), 505–511).
Preferably, a BEII protein, as addressed in the embodiments of the present invention, is encoded by a nucleic acid molecule which hybridises, advantageously under stringent conditions, with the nucleic acid molecule having the nucleotide sequence shown under SEQ ID NO: 9 and which encodes a polypeptide having branching enzyme activity.
The definition for the term “hybridisation” as defined above in connection with R1 proteins applies equally for the definition of nucleic acid molecules hybridising with the nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 3. Preferably, a BEII protein as referred to herein displays a sequence identity of at least 60%, in particular of at least 75%, preferably of at least 85%, more preferably at least 90% and even more preferably at least 95% to the amino acid sequence depicted under SEQ ID NO: 4.
In a preferred embodiment of the present invention, the genetic modification of the transgenic plant cell of the invention is the introduction of one or more foreign nucleic acid molecules the presence and/or expression of which leads to a reduction of the activity of R1 and BEI and BEII proteins in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified.
Preferably, this reduction of activity is achieved by inhibiting the expression of the endogenous genes encoding R1 proteins, BEI proteins and BEII proteins.
The production of the plant cells of the invention can be achieved by different methods known to the person skilled in the art, e.g. by methods leading to an inhibition of the expression of endogenous genes encoding an R1, BEI or BEII protein. These include, for example, the expression of a corresponding antisense RNA, the provision of molecules or vectors mediating a co-suppression effect, the expression of a ribozyme constructed accordingly which specifically cleaves transcripts encoding an R1, BEI or BEII protein or the so-called “in-vivo mutagenesis”. Furthermore, the reduction of the R1 and/or the BEI and/or the BEII activity in the plant cells may also be caused by the simultaneous expression of sense and antisense RNA molecules of the target gene to be repressed, preferably of the R1 and/or the BEI and/or the BEII gene, a technique which is commonly referred to as RNA interference (RNAi) (Bosher and Labouesse, Nature Cell Biology 2, (2000), E31–E36; Waterhouse et al., PNAS 95, (1998), 13959–13964). Furthermore, by the use of double-stranded RNA molecules comprising promoter sequences, a transcriptional inactivation of the promoter can be achieved. These and further methods for reducing the activity of proteins will be described in more detail below. All these methods are based on the introduction of one or more foreign nucleic acid molecules into the genome of plant cells.
Within the context of the present invention, the term “foreign nucleic acid molecule” is understood to be a molecule which either does not occur naturally in corresponding plant cells or which does not occur naturally in the plant cells in the concrete spatial order or which is located at a position in the genome of the plant cell at which it does not occur naturally. The foreign nucleic acid molecule preferably is a recombinant molecule which consists of various elements the combination or specific spatial order of which does not occur naturally in plant cells.
The foreign nucleic acid molecule can, for instance, be a so-called “triple construct” which is understood to be a single vector for plant transformation which contains both the genetic information for inhibiting the expression of one or more endogenous R1 genes and for inhibiting the expression of one or more BEI and BEII genes or the presence or expression of which leads to the reduction of the activity of one or more R1, BEI and BEII proteins.
In another embodiment, the foreign nucleic acid molecule may be a so-called “double construct” which is understood to be a vector for plant transformation which contains the genetic information for inhibiting the expression of two of the three target genes (R1, BEI, BEII gene) or the presence or expression of which leads to the reduction of the activity of two of the three target proteins (R1, BEI, BEII proteins). In this embodiment of the invention, the inhibition of the expression of the third target gene and/or the reduction of the activity of the third target protein takes place by means of a separate foreign nucleic acid molecule which contains the corresponding genetic information for exerting this inhibiting effect.
In another embodiment of the invention, it is not a triple construct that is introduced into the genome of the plant cell but several different foreign nucleic acid molecules, one of these foreign nucleic acid molecules being for example a DNA molecule which, for instance, is a co-suppression construct leading to a reduction of the expression of one or more endogenous R1 genes, and a further foreign nucleic acid molecule being a DNA molecule encoding, for example, an antisense RNA leading to a reduction of the expression of one or more endogenous BEI and/or BEII genes. In principle, as regards the construction of the foreign nucleic acid molecules, it is also suitable to use every combination of antisense, co-suppression and ribozyme constructs or in-vivo mutagenesis, which all lead to a simultaneous reduction of the gene expression of endogenous genes encoding one or more R1, BEI and BEII proteins or which lead to a simultaneous reduction of the activity of one or more R1, BEI and BEII proteins.
In this case, the foreign nucleic acid molecules can be introduced simultaneously (“co-transformation”) or consecutively, i.e. one after the other (“super transformation”), into the genome of the plant cell.
In another embodiment of the invention, at least one antisense RNA is expressed for reducing the activity of one or more R1 proteins and/or BEI proteins and/or BEII proteins in plant cells.
For inhibiting the gene expression by means of antisense or co-suppression technology, it is possible to use for instance a DNA molecule which comprises the entire sequence encoding an R1 and/or BEI and/or BEII protein, including flanking sequences that may optionally be present, as well as DNA molecules which only comprise parts of the coding sequence and/or flanking sequences, wherein these parts must be long enough to lead to an antisense effect or a co-suppression effect in the cells. In general, sequences having a minimum length of 15 bp, preferably a length of 100 to 500 bp, in particular sequences having a length of more than 500 bp, are suitable for an efficient antisense or co-suppression inhibition. Usually, DNA molecules which are shorter than 5000 bp, preferably sequences which are shorter than 2500 bp are used.
For antisense or co-suppression approaches, it is also suitable to use DNA sequences which have a high degree of homology to the sequences that occur endogenously in the plant cell and that encode R1, BEI or BEII proteins. The minimum degree of homology should be higher than 65%. It is preferred to use sequences having a homology of at least 90%, in particular between 95 and 100%.
Moreover, also introns, i.e. of non-coding regions of genes encoding R1, BEI and/or BEII proteins, are conceivable for use to achieve an antisense or a co-suppressive effect.
The use of intron sequences for inhibiting the gene expression of genes encoding proteins of the starch biosynthesis has been described in the international patent applications WO 97/04112, WO 97/04113, WO 98/37213, WO 98/37214.
The person skilled in the art knows how to achieve an antisense and a co-suppressive effect. The method of co-suppression inhibition was described, for instance, in Jorgensen (Trends Biotechnol. 8 (1990), 340–344), Niebel et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 91–103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43–46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995, 149–159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311–317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613–621).
The skilled persons are also familiar with the expression of ribozymes for reducing the activity of certain enzymes in cells and is described, for example, in EP-B1 0 321 201. The expression of ribozymes in plant cells has been described, for instance, in Feyter et al. (Mol. Gen. Genet. 250, (1996), 329–338).
Furthermore, the R1 and/or BEI and/or BEII activity in plant cells may also be reduced by the so-called “in-vivo mutagenesis” in which a hybrid RNA-DNA oligonucleotide (“chimeroplast”) is introduced into cells by means of the transformation of cells (Kipp, P. B. et al., Poster at the 5th International Congress of Plant Molecular Biology, 21–27September 1997, Singapore; R. A. Dixon and C. J. Arntzen, Meeting report to “Metabolic Engineering in Transgenic Plants”, Keystone Symposia, Copper Mountain, Colo., U.S.A., TIBTECH 15, (1997), 441–447; international patent application WO 95/15972; Kren et al., Hepatology 25, (1997), 1462–1468; Cole-Strauss et al., Science 273, (1996), 1386–1389).
A part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous R1, BEI and/or BEII gene, it has, however, a mutation in comparison with an endogenous R1, BEI and/or BEII gene or it contains a heterologous region which is embraced by the homologous regions.
It is possible to transfer the mutation or heterologous region contained in the DNA component of the RNA-DNA molecule into the genome of a plant cell by means of base pairing of the homologous regions of the RNA-DNA oligonucleotide and the endogenous nucleic acid molecule, followed by homologous recombination. As a result, the activity of one or more R1, BEI and/or BEII proteins is reduced.
In addition, the reduction of the R1 and/or BEI and/or BEII activity in the plant cells may also be caused by the simultaneous expression of sense and antisense RNA molecules of the target gene that is to be repressed, preferably of the R1 and/or the BEI and/or the BEII gene.
This can be achieved, for instance, by using chimeric constructs which contain inverted repeats of the respective target gene or of parts of the target gene. In this case, the chimeric constructs encode sense and antisense RNA molecules of the respective target gene. Sense and antisense RNA are synthesised simultaneously in planta as one RNA molecule, wherein the sense and the antisense RNA may be separated from each other by a spacer and form a double-stranded RNA-molecule. It was possible to show that the introduction of inverted repeat DNA constructs into the genome of plants is a very efficient method for repressing the genes corresponding to the inverted repeat DNA constructs (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95, (1998), 13959–13964; Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67–82; Singh et al., Biochemical Society Transactions vol. 28, part 6 (2000), 925–927; Liu et al., Biochemical Society Transactions vol. 28, part 6 (2000); 927–929; Smith et al., Nature 407, (2000), 319–320; international patent application WO 99/53050 A1). Sense and antisense sequences of the target gene or of the target genes can also be expressed separately using the same or different promoters (Nap, J.-P. et al., 6th International Congress of Plant Molecular Biology, Quebec, 18–24 June 2000; Poster S7–27, lecture session S7).
Thus, it is also possible to reduce the R1 and/or BEI and/or BEII activity in the plant cells by the production of double-stranded RNA molecules of R1 and/or BEI and/or BEII genes. Preferably, inverted repeats of DNA molecules of R1 and/or BEI and/or BEII genes or cDNAs are introduced into the genome of plants for this purpose, wherein the DNA molecules to be transcribed (R1, BEI or BEII gene or cDNA or fragments of these genes or cDNAs) are under the control of a promoter which controls the expression of said DNA molecules.
Furthermore, it is known that in planta the formation of double-stranded RNA molecules of promoter DNA molecules in plants can lead in trans to a methylation and a transcriptional inactivation of homologous copies of these promoters which are called target promoters in the following (Mette et al., EMBO J. 19, (2000), 5194–5201).
Therefore, it is possible to reduce the gene expression of a certain target gene (e.g. R1, BEI or BEII gene) by means of the inactivation of the target promoter, the target gene being naturally controlled by this target promoter.
This means that, in this case, in contrast to the original function of promoters in plants, the DNA molecules which comprise the target promoters of the genes to be repressed (target genes) are not used as control elements for the expression of genes or cDNAs but as transcribable DNA molecules themselves.
For producing the double-stranded target promoter RNA molecules in planta which may be present there as RNA hairpin molecules, constructs are preferred to be used which contain inverted repeats of the target promoter DNA molecule, the target promoter DNA molecules being under the control of a promoter which controls the gene expression of said target promoter DNA molecules.
Then, these constructs are introduced into the genome of plants. The expression of the inverted repeats of said target promoter DNA molecules leads to the formation of double-stranded target promoter RNA molecules in planta (Mette at el., EMBO J. 19, (2000), 5194–5201). In this way, it is possible to inactivate the target promoter.
Therefore, the R1 and/or BEI and/or BEII activity in the plant cells can also be reduced by generating double-stranded RNA molecules of promoter sequences of R1 and/or BEI and/or BEII genes. For this purpose, inverted repeats of promoter DNA molecules of R1 and/or BEI and/or BEII promoters are preferred to be introduced into the genome of plants, the target promoter DNA molecules to be transcribed (R1, BEI and/or BEII promoter) being under the control of a promoter which controls the expression of said target promoter DNA molecules. The promoter sequences from R1and/or BEI and/or BEII genes necessary for carrying out the present embodiment can be provided by methods known to the skilled person and described in the literature such as in Sambrook and Russell (2001), Molecular Cloning, CSH Press, Cold Spring Harbor, N.Y., U.S.A. The methods may for example include the preparation of a genomic library from the plant in which the activity of R1, BEI and BEII proteins shall be reduced, screening of the library for clones containing the sequence flanking the coding region of the respective gene in 5′-direction by the help of a probe comprising a coding sequence for the R1 or BEI or BEII protein as described above and finally sequencing positive clones by conventional techniques.
Moreover, the skilled person knows that the reduction of activity of one or more R1, BEI and/or BEII proteins can be achieved by means of the expression of non-functional derivatives, in particular trans-dominant mutants of such proteins, and/or by means of the expression of antagonists/inhibitors of such proteins.
Antagonists/inhibitors of such proteins comprise, for instance, antibodies, antibody fragments or molecules having similar binding properties. A cytoplasmatic scFv antibody, for example, was used for modulating the activity of the phytochrome A protein in genetically modified tobacco plants (Owen, Bio/Technology 10 (1992), 790–4; Review: Franken, E., Teuschel, U. and Hain, R., Current Opinion in Biotechnology 8, (1997), 411–416; Whitelam, Trends Plant Sci. 1 (1996), 268–272).
Therefore, a plant cell of the invention is also subject matter of the present invention, wherein said foreign nucleic acid molecule the presence and/or expression of which causes a reduction of R1, BEI and BEII activity in said plant cell is selected from the group consisting of

    a) DNA molecules which encode at least one antisense RNA leading to a reduction of the expression of endogenous genes encoding R1 proteins and/or BEI proteins and/or the BEII proteins, preferably encoding R1, BEI and BEII proteins;
    b) DNA molecules which, through a co-suppression effect, lead to a reduction of the expression of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins;
    c) DNA molecules encoding at least one ribozyme which specifically cleaves transcripts of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins;
    d) nucleic acid molecules which have been introduced by means of in-vivo mutagenesis and which lead to a mutation or an insertion of a heterologous sequence in the genes encoding endogenous R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins, wherein the mutation or insertion leads to a reduction of the expression of genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, or the synthesis of inactive R1 and/or BEI and/or BEII proteins; and
    e) DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, wherein said antisense RNA and said sense RNA form a double-stranded RNA molecule which leads to a reduction of the expression of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins.


In another embodiment, the present invention relates to transgenic plant cells which synthesises a modified starch. The transgenic plant cells of the invention synthesise a modified starch which is modified in its physico-chemical properties, in particular the amylose/amylopectin ratio, the phosphate content, the viscosity behaviour, the size of the starch granules and/or the form of the starch granules in comparison with starch synthesised in wild type plants so that it is more suitable for specific purposes of application.
It was surprisingly found that the composition of the starch is modified in the plant cells of the invention in such a way that it has an amylose content of at least 75% and a reduced phosphate content in comparison with starch from plant cells from corresponding wild type plants, so that said starch is more suitable for specific purposes of application.
A plant cell of the invention which contains modified starch having an amylose content of at least 75% and a reduced phosphate content compared to the starch of corresponding plant cells of wild type plants, the cells not being genetically modified, is also subject matter of the present invention.
In the context of the present invention, the amylose content is determined according to the method by Hovenkamp-Hermelink et al. described below in connection with potato starch (Potato Research 31, (1988), 241–246). This method can also be used for isolated starches of other plant species. The person skilled in the art is familiar with methods for isolating starches.
Within the meaning of the present invention, the term “phosphate content” relates to the content of phosphate bound covalently in form of starch phosphate monoesters.
In the context of the present invention, the expression “reduced phosphate content” means that the overall phosphate content of phosphate covalently bound and/or the phosphate content in the C-6 position of the starch synthesised in the plant cells of the invention is reduced by at least 20%, preferably by at least 50%, more preferably by at least 80% in comparison with starch from plant cells of corresponding wild type plants, the cells not being genetically modified.
Within the meaning of the present invention, the term “phosphate content in the C-6 position” is understood to be the content of phosphate groups which are bound to the carbon atom position “6” of the glucose monomers of the starch. In principle, the positions C2, C3 and C6 of the glucose units may be phosphorylated in the starch in vivo. In connection with the present invention, the phosphate content in the C-6 position (=C6-P content) can be determined through the determination of the glucose-6-phosphate by means of an optic-enzymatic test (Nielsen et al., Plant Physiol. 105, (1994), 111–117) (see below).
In the context of the present invention, the expression “overall phosphate content” of the starch is understood to be the content of phosphate bound covalently in form of starch phosphate monoesters in the C2, C3 and C6 position of the glucose units. According to the invention, the content of phosphorylated non-glucans such as, e.g. phospholipids, is not included in the term “overall phosphate content”. Thus, phosphorylated non-glucans must be separated quantitatively before determining the overall phosphate content. The skilled person knows methods for separating the phosphorylated non-glucans (e.g. phospholipids) from the starch. Methods for determining the overall phosphate content are known to the person skilled in the art and are described below.
In a preferred embodiment of the invention, the plant cells of the invention synthesise a starch which has a phosphate content in the C-6 position of the glucose monomers of up to 15 nmol C6-P mg−1 starch, in particular of up to 10 nmol C6-P mg−1 starch, preferably of up to 7 nmol C6-P mg−1 starch, more preferably of up to 4 nmol C6-P mg−1 starch.
In another embodiment, the present invention therefore relates to plant cells according to the invention which synthesise a modified starch, wherein the modified starch is characterised in that it has a modified distribution of the side chains. It has been shown that the starch modified in the plant cells of the invention is characterised not only by an increased amylose content and a reduced phosphate content compared to the starch of corresponding wild type plants, but also by a modified distribution of the side chains.
In this embodiment, the term “modified distribution of the side chains” is understood to be an increase in the proportion of short side chains having a DP of 26 to 31 by at least 50%, preferably by at least 100%, more preferably by at least 150% and especially preferred by at least 200% in comparison with the proportion of short side chains having a DP of 26 to 31 of amylopectin from wild type plants. Moreover, the term “modified distribution of the side chains” means an increase of the proportion of short side chains having a DP of 26 to 31, wherein the increase of the proportion of short side chains having a DP of 26 to 31 is not higher than 800%, in particular not higher than 500% compared to the proportion of short side chains having a DP of 26 to 31 of amylopectin from wild type plants. The quantity “DP” means the degree of polymerisation.
The proportion of short side chains is determined by the determination of the proportion in percent that a certain side chain has in the overall proportion of all side chains. The overall proportion of all side chains is determined through the determination of the overall height of the peaks which represent the polymerisation degrees of DP 6 to 40 in the HPLC chromatogram. The proportion in percent that a certain side chain has in the overall proportion of all side chains is determined by the determination of the ratio of the height of the peak representing this side chain in the HPLC chromatogram to the overall height. The program Chromeleon 6.20 by Dionex, USA can, for instance, be used for determining the peak areas.
In another preferred embodiment, the present invention relates to plants cells of the invention which synthesise a modified starch which form a gel after pasting in a 60% (w/v) CaCl2 solution, the gel having an increased gel strength compared to the gel from starch of corresponding wild type plant cells that have not been genetically modified.
Within the meaning of the present invention, the term “increased gel strength” means an increase in the gel strength by at least 1000%, in particular by at least 2500%, preferably by at least 5000% and more preferably by at least 10,000%, by 40,000% at the most or by 30,000% at the most in comparison with the gel strength of starch of corresponding wild type plant cells that have not been genetically modified.
In the context of the present invention, the gel strength is to be determined by means of a texture analyzer under the conditions described below. In this case, the pasting of the starch is achieved in an aqueous 60% (w/v) CaCl2 solution since in a purely aqueous system, it is not possible to achieve pasting of the starch at normal pressure.
In a further preferred embodiment, the present invention relates to plant cells of the invention which, in addition to the aforementioned properties, the starch of which has a modified morphology of the starch granules.
In comparison with high-amylose starches which are known so far, in particular with high-amylose potato starches, the starches of the plant cells of the invention are not only modified in the amylose content, the phosphate content, the distribution of the side chains, the viscosity behaviour and the gel formation behaviour, but also in a modified morphology of the starch granules, which renders these starches more suitable for certain purposes of application.
These starches, in particular the potato starches, could, for instance, be used instead of rice starches since, after mechanical fragmentation, the starches of the invention have an average size of the starch granules which is similar to that of rice starches. Compared to rice starches, the starches of the invention, in particular the potato starches, however have the advantage that they can be sedimented to larger units having the form of a bunch of grapes (cf. Example 2) as small starch granules form bunch-of-grapes-like agglomerations, which may be of advantage in the extraction and processing of the starch and by which the costs may be reduced.
Preferably, the morphology of the starch granules contained in the plant cells of the invention is characterised by an agglomeration of small starch granules having the form of a bunch of grapes.
In a preferred embodiment, the starches contained in the plant cells of the invention are characterised in that the average granule size is reduced compared to the average granule size of corresponding cells of wild type plants which are not genetically modified.
In the context of the present invention, the term “average granule size” means the granule size which can be determined using, for instance, a photo sedimentometer of the type “Lumosed FS1” by Retsch GmbH (see below).
In a further preferred embodiment of the invention, a reduced average granule size is a reduction of the average granule size by at least 20%, preferably by at least 40% and more preferably by at least 60%.
In another preferred embodiment, the starches of the plant cells of the invention are characterised by an average granule size of less than 20 μm, in particular of less than 18 μm, preferably of less than 16 μm and more preferably of 10–15 μm.
In another preferred embodiment of the invention, the starches of the plant cells of the invention are characterised in that the proportion of granules having an average granule size of less than 20 μm is at least 70%, preferably at least 75% and more preferably at least 80%.
After mechanical fragmentation of the starch, which may be carried out as described below, the starches of the plant cells of the invention have a proportion of granules having a granule size of less than 20 μm of at least 80%, preferably of at least 90% and more preferably of at least 95%.
A plurality of techniques is available for introducing DNA into a plant host cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation means, the fusion of protoplasts, injection, the electroporation of DNA, the introduction of DNA by means of the biolistic approach, and other possibilities.
The use of the Agrobacteria-mediated transformation of plant cells has been researched into intensively and described sufficiently in EP120516; Hoekema, in: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1–46 and An et al. EMBO J. 4, (1985), 277–287. For the transformation of potato, see, for example, Rocha-Sosa et al. (EMBO J. 8, (1989), 29–33.).
The transformation of monocotyledonous plants by means of Agrobacterium-based vectors has also been described (Chan et al., Plant Mol. Biol. 22, (1993), 491–506; Hiei et al., Plant J. 6, (1994) 271–282; Deng et al., Science in China 33, (1990), 28–34; Wilmink et al., Plant Cell Reports 11, (1992), 76–80; May et al., Bio/Technology 13, (1995), 486–492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550–555; Ritchie et al., Transgenic Res. 2, (1993), 252–265). An alternative system for the transformation of monocotyledonous plants is the transformation by the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37–48; Vasil et al., Bio/Technology 11 (1993), 1553–1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317–325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625–631), protoplast transformation, the electroporation of partially permeabilised cells, and the introduction of DNA by means of glass fibers. The transformation of maize, in particular, has been described repeatedly in the literature (cf., for example, WO 95/06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833–844; Gordon-Kamm et al., Plant Cell 2, (1990), 603–618; Koziel et al., Biotechnology 11 (1993), 194–200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721–726).
The successful transformation of other cereal species has also been described, for example in the case of barley (Wan and Lemaux, see above; Ritala et al., see above; Krens et al., Nature 296, (1982), 72–74) and wheat (Nehra et al., Plant J. 5, (1994), 285–297). For the expression of the foreign nucleic acid molecule (foreign nucleic acid molecules), in principle, any promoter which is active in plant cells can be used. The promoter can be chosen in such a way that expression in the plants according to the invention is constitutive, or only in a particular tissue, at a particular point in time of plant development, or at a point in time determined by external factors. With respect to the plant, the promoter may be homologous or heterologous. Examples of suitable promoters are the promoter of the cauliflower mosaic virus 35S RNA and the ubiquitin promoter from maize for constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23–29), the MCPI promoter of the metallocarboypeptidase inhibitor gene from potato (Hungarian patent application HU9801674) or the GBSSI promoter from potato (international patent application WO 92/11376) for tuber-specific expression in potatoes, or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943–7947; Stockhaus et al., EMBO J. 8 (1989), 2445–2451), the Ca/b promoter (see, for example, U.S. Pat. No. 5,656,496, U.S. Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89, (1992), 3654–3658) and the rubisco SSU promoter (see, for example, U.S. Pat. No. 5,034,322, U.S. Pat. No. 4,962,028), or the glutelin promoter for an endosperm-specific expression (Leisy et al., Plant Mol. Biol. 14 (1990), 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 HMG promoter from wheat, 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). The expression of the foreign nucleic acid molecule (the foreign nucleic acid molecules) is of particular advantage in organs of the plant that store starch. Such organs are, e.g., the tuber of the potato plant or the kernels or the endosperm of maize, wheat or rice plants. Thus, promoters mediating the expression in these organs are preferred to be used.
However, it is also possible to use promoters which are only activated at a point in time which is determined by external factors (see, for example, WO 93/07279). Promoters of heat shock proteins, which permit simple induction, may be of particular interest in this context. Furthermore, seed-specific promoters such as, for example, the Vicia faba USP promoter which ensures seed-specific expression in Vicia faba and other plants (Fiedler et al., Plant Mol. Biol. 22, (1993), 669–679; Bäumlein et al., Mol. Gen. Genet. 225, (1991), 459–467) can be used. Other promoters which can be employed are fruit-specific promoters as described, for example, in WO 91/01373. A termination sequence which serves for the correct termination of the transcription and for adding a poly-A tail to the transcript, which is understood to have a function in stabilising the transcripts, may furthermore be present. Such elements have been described in the literature (cf., for example, Gielen et al., EMBO J. 8 (1989), 23–29) and are freely exchangeable.
The plant cells according to the invention may belong to any plant species, i.e. to monocotyledonous or dicotyledonous plants. They are preferably plant cells from agriculturally useful plants, i.e. plants which are grown by man for the purposes of nutrition or for technical, in particular industrial, purposes. The invention preferably relates to fibre-forming (for example flax, hemp, cotton), oil-storing (for example rape, sunflower, soy bean), sugar-storing (for example sugar beet, sugar cane, sugar millet) and protein-storing plants (for example leguminous plants). In a further preferred embodiment, the invention relates to fodder plants, in particular forage grass and pasture grass (alfalfa, clover, etc.) and vegetable plants (for example tomato, lettuce, chicory).
In another preferred embodiment, the invention relates to plant cells from starch-storing plants (for example wheat, barley, oat, rye, potato, maize, rice, pea, cassava), particularly preferred are plant cells from potato.
The plant cells of the invention can be used for regenerating whole plants.
The plants obtainable by regenerating the transgenic plant cells of the invention are also subject matter of the present invention.
Furthermore, plants which contain the transgenic plant cells of the invention are also subject matter of the invention.
The transgenic plants may, in principle be plants belonging to any plant species, i.e. both monocotyledonous and dicotyledonous plants. They are preferably useful plants, i.e. plants which are grown by man for the purposes of nutrition or for technical, in particular industrial, purposes. The invention preferably relates to plant cells of fibre-forming (for example flax, hemp, cotton), oil-storing (for example rape, sunflower, soy bean), sugar-storing (for example sugar beet, sugar cane, sugar millet) and protein-storing plants (for example leguminous plants). In a further preferred embodiment, the invention relates to fodder plants, in particular forage grass and pasture grass (alfalfa, clover, etc.) and vegetable plants (for example tomato, lettuce, chicory).
In another preferred embodiment, the invention relates to starch-storing plants (for example wheat, barley, oat, rye, potato, maize, rice, pea, cassava), particularly preferred are potato plants.
The present invention also relates to a method for the production of a transgenic plant cell which synthesises a modified starch, wherein a plant cell is genetically modified by introducing one or more foreign nucleic acid molecules, the presence and/or expression of which leads to a reduction of the activity of R1, BEI and BEII proteins compared to corresponding plants cells of wild type plants, the cells not being genetically modified.
In a preferred embodiment of the method of the invention, the modified starch is characterised in that it has an amylose content of at least 75% and a reduced phosphate content in comparison with starch from corresponding wild type plants which are not genetically modified.
The present invention also relates to a method for producing a transgenic plant which synthesises modified starch, wherein

    a) a plant cell is genetically modified by introducing one or more foreign nucleic acid molecules the presence and/or expression of which leads to a reduction of the activity of R1, BEI and BEII proteins compared to corresponding plant cells of wild type plants, the cells not being genetically modified;
    b) a plant is regenerated from the cell produced according to a); and
    c) optionally further plants are produced from the plant produced according to step b).


In a preferred embodiment of the method of the invention, the modified starch is characterised in that it has an amylose content of at least 75% and a reduced phosphate content compared to the starch from corresponding wild type plants which are not genetically modified.
In another embodiment of the method of the invention, the modified starches moreover have a modified distribution of the side chains and/or a modified morphology of the starch granules and/or a reduced average size of the starch granules and/or form a gel after pasting in an aqueous 60% (w/v) CaCl2 solution, the gel having an increased gel strength in comparison with a gel of starch from corresponding wild type plants which are not genetically modified. The same as has already been said above in connection with the plant cells of the invention also applies to the genetic modification introduced according to step a). Regeneration, of plants according to step b) can be made using methods known to the person skilled in the art.
Further plants of the methods of the invention can be produced according to step c) by means of vegetative propagation (for example using cuttings, tubers or by means of callus culture and regeneration of whole plants) or by generative propagation. Generative propagation is preferably done under controlled conditions, i.e. selected plants having specific properties are crossed with each other and propagated. The person skilled in the art obviously knows that, for producing the plant cells and plants of the invention, also transgenic plants can be used in which the activity of one or two of the aforementioned proteins has already been reduced and which, according to the method of the invention, only have to be genetically modified in such a way that the activity of the second or third protein is also reduced.
In addition, the skilled person knows that the aforementioned super-transformation is not necessarily carried out with primary transformants but preferably with pre-selected stable transgenic plants which advantageously have already been tested for, e.g. fertility, stable expression of the foreign gene, hemizygosity and heterozygosity, etc. in corresponding experiments.
The present invention also relates to the plants obtainable by the methods of the invention.
The present invention also relates to propagation material of plants of the invention containing plant cells of the invention as well as of the plants produced according to the methods of the invention. Within the meaning of the present invention, the term “propagation material” comprises parts of the plant which are suitable for producing progeny by the vegetative or generative route. Examples which are suitable for vegetative propagation are cuttings, callus cultures, rhizomes or tubers. Other propagation material encompasses, for example, fruits, seeds, seedlings, protoplasts, cell cultures and the like. The propagation material is preferably seeds.
Furthermore, the present invention relates to the use of one or more foreign nucleic acid molecules encoding proteins having the enzymatic activity of R1, BEI and BEII proteins and to the use of fragments of said foreign nucleic acid molecules for producing plant cells or plants of the invention synthesising a modified starch.
In another embodiment of the invention, the plant cells of the invention synthesise a modified starch due to the use according to the invention of one or more foreign nucleic acid molecules, the modified starch being characterised in that it has an amylose content of at least 75% and/or a reduced phosphate content compared to the starch of corresponding wild type plants which have not been genetically modified and/or a modified distribution of the side chains and/or a modified morphology of the starch granules and/or a reduced average size of the starch granules and/or a modified starch which forms a gel after pasting in an aqueous 60% (w/v) CaCl2 solution, the gel having an increased gel strength in comparison with a gel of starch from corresponding wild type plant cells which are not genetically modified.
In another embodiment, the present invention relates to the use of one or more foreign nucleic acid molecules for producing plants of the invention, wherein the foreign nucleic acid molecule is a molecule, or the foreign nucleic acid molecules are several molecules selected from the group consisting of

    a) DNA molecules which encode at least one antisense RNA leading to a reduction of the expression of endogenous genes encoding the R1 proteins and/or BEI proteins and/or the BEII proteins, preferably encoding R1, BEI and BEII proteins;
    b) DNA molecules which, through a co-suppression effect, lead to the reduction of the expression of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins;
    c) DNA molecules encoding at least one ribozyme which specifically cleaves transcripts of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins;
    d) nucleic acid molecules which have been introduced by means of in-vivo mutagenesis and which lead to a mutation or an insertion of a heterologous sequence in the genes encoding endogenous R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins, wherein the mutation or insertion leads to a reduction of the expression of the genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, or the synthesis of inactive R1 and/or BEI and/or BEII proteins; and
    e) DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, wherein said antisense RNA and said sense RNA form a double-stranded RNA molecule which leads to a reduction of the expression of endogenous genes encoding R1 proteins and/or BEI proteins and/or BEII proteins, preferably encoding R1, BEI and BEII proteins.


As has already been explained before, the foreign nucleic acid molecules can be introduced simultaneously or consecutively, i.e. one after the other, into the genome of the plant cell. The simultaneous introduction of the foreign nucleic acid molecules saves time and costs, i.e. the co-transformation in which, preferably in one transformation experiment according to the aforementioned methods of the invention, foreign nucleic acid molecules are introduced into the plant cell, the presence and optionally the expression of which lead to the reduction of the activity of one or more R1 proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEI proteins occurring endogenously in the plant cell and to the reduction of the activity of one or more BEII proteins occurring endogenously in the plant cell in comparison with corresponding plant cells of wild type plants, the cells not being genetically modified.
Thus, the present invention also relates to compositions containing at least one of the foreign nucleic acid molecules defined according to the invention, these foreign nucleic acid molecules being suitable for producing the transgenic plant cells and/or the transgenic plants of the invention. Preferably, the presence and/or expression of these foreign nucleic acid molecules in plant cells leads to the reduction of the activity of R1 and BEI and BEII proteins compared to corresponding plant cells of wild type plants, the cells not being genetically modified.
In this case, in the composition of the invention, the nucleic acid molecules the presence and/or expression of which in the plant cell and/or the plant leads to the reduction of the activity of R1 and BEI and BEII proteins compared to corresponding plant cells of wild type plants, the cells not being genetically modified, can be contained either separately or together in one recombinant nucleic acid molecule. In the former case, the composition of the invention can, for instance, contain two or more recombinant nucleic acid molecules and/or vectors the joint presence of which in the plant cell leads to said phenotype. In the latter case, a recombinant nucleic acid molecule contains the genetic information leading to the reduction of the activity of R1 and BEI and BEII proteins compared to corresponding plant cells of wild type plants, the cells not being genetically modified.
In such a recombinant molecule, for instance, the above-described foreign nucleic acid molecules the presence and/or expression of which in a plant cell leads to the reduction of the activity of R1 and BEI and BEII proteins compared to corresponding plant cells of wild type plants, the cells not being genetically modified, can be present as one chimeric gene or as separate genes. Examples of such double or multiple constructs have been described numerously in the literature.
The aforementioned recombinant nucleic acid molecules can be present in any host cell.
In another embodiment, the present invention therefore also relates to a host cell, in particular a plant cell, containing a composition of the invention.
The plant cells and plants of the invention synthesise a starch, in particular in their starch-storing organs, which is modified in its physico-chemical properties, in particular the phosphate content and/or the amylose content, preferably the phosphate content and the amylose content, and/or the distribution of the side chains and/or the viscosity behaviour and/or the morphology of the starch granules and/or the average size of the starch granules in comparison with starch synthesised in wild type plants.
Thus, starch which is obtainable from the plant cells, plants and/or propagation material of the invention is also subject matter of the invention.
In a preferred embodiment, the starch of the invention is characterised in that it has an amylose content of at least 75% and a reduced phosphate content in comparison with starch from corresponding wild type plants which are not genetically modified.
The meaning of the term “increased gel strength” has already been defined in connection with the description of the plant cells of the invention.
In comparison with high-amylose starches which are known so far, in particular with high-amylose potato starches, the starches of the invention are not only modified in the amylose content, the phosphate content, the distribution of the side chains, the viscosity behaviour and the gel formation behaviour, but also in a modified morphology of the starch granules, which renders these starches more suitable for certain purposes of application.
The starches of the invention, in particular the potato starches, could, for instance, be used instead of rice starches since, after mechanical fragmentation, the starches of the invention have an average size of the starch granules which is similar to that of rice starches. Compared to rice starches, the starches of the invention, in particular the potato starches, however have the advantage that they can be sedimented to larger units having the form of a bunch of grapes (cf. Example 2) as small starch granules form bunch-of-grapes-like agglomerations, which may be of advantage in the extraction and processing of the starch and by which the costs may be reduced.
Preferably, morphology of the starch granules of the starch of the invention is characterised by an agglomeration of small starch granules having the form of a bunch of grapes.
In another embodiment, the starches of the invention are characterised in that the average granule size is reduced compared to the average granule size of corresponding wild type plants which are not genetically modified.
In the context of the present invention, the term “average granule size” means the granule size which can be determined using, for instance, a photo sedimentometer of the type “Lumosed FS1” by Retsch GmbH (see below).
In another embodiment of the invention, a reduced average granule size is a reduction of the average granule size by at least 20%, preferably by at least 40% and more preferably by at least 60%.
In another embodiment, the starches of the invention are characterised by an average granule size of less than 20 μm, in particular of less than 18 μm, preferably of less than 16 μm and more preferably of 10–15μm.
In another embodiment of the invention, the starches of the invention are characterised in that the proportion of granules having an average granule size of less than 20 μm is at least 70%, preferably at least 75% and more preferably at least 80%.
After mechanical fragmentation of the starch, which may be carried out as described below, the starches of the invention have a proportion of granules having a granule size of less than 20 μm of at least 80%, preferably of at least 90% and more preferably of at least 95%.
In a particularly preferred embodiment, the starch of the invention is a potato starch. Moreover, the present invention relates to a method for producing the starches of the invention comprising the step of extracting the starch from a plant (cell) of the invention and/or from starch-storing parts of such a plant.
Preferably, such a method also comprises the step of harvesting the cultivated plants and/or starch-storing parts of said plants prior to extracting the starch and, particularly preferably, the step of cultivating the plants of the invention prior to the harvesting.
The person skilled in the art knows methods for extracting the starch from plants or from starch-storing parts of plants. Furthermore, methods for extracting the starch from various starch-storing plants have been described, e.g. in “Starch: Chemistry and Technology (editors.: Whistler, BeMiller and Paschall (1994), 2ndedition, Academic Press Inc. London Ltd.; ISBN 0-12-746270-8; cf., e.g. chapter XII, page 412–468: maize and sorghum starches: production; by Watson; Chapter XIII, page 469–479: tapioca, arrowroot and sago starches: production; by Corbishley and Miller; Chapter XIV, page 479–490: potato starch: production and uses; by Mitch; Chapter XV, page 491 to 506: wheat starch: production, modification and uses; by Knight and Oson; and Chapter XVI, page 507 to 528: rice starch: production and uses; by Rohmer and Klem; maize starch: Eckhoff et al., Cereal Chem. 73 (1996) 54–57), the extraction of maize starch on an industrial scale is generally achieved by wet milling. Apparatuses usually used in processes for extracting starch from plant materials are separators, decanters, hydrocyclones, spray dryers and fluidized-bed dryers.
Moreover, starch which is obtainable using the aforementioned method of the invention is also subject matter of the invention.
The starches according to the invention can be modified afterwards by processes known to the skilled person and are suitable, in their unmodified or modified forms, known to the skilled person and are suitable, in their unmodified or modified forms, for a variety of applications in the food or non-food sector.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Schematic representation of the expression vector ME 5/6 as described further below.
FIG. 2: Light-microscopic view of starch granules of wild type potato plants.
FIG. 3: Light-microscopic view of starch granules of 072VL036 potato plants having a reduced gene expression of the R1 and BEI gene.
FIG. 4: Light-microscopic view of starch granules of 203MH010 potato plants according to the invention.



DETAILED DESCRIPTION OF THE INVENTION
The Following Methods were Used in the Examples
Analysis of the Starch
a) Determination of the Amylose/Amylopectin Ratio
Starch was isolated from potato plants according to standard techniques and the ratio of amylose to amylopectin was determined using the method described by Hovenkamp-Hermelink et al. (Potato Research 31, (1988), 241–246).
b) Determination of the Phosphate Content
The positions C2, C3 and C6 of the glucose units may be phosphorylated in the starch. For determining the C6-P content of the starch, 50 mg starch are hydrolysed in 500 μl 0.7 M HCl for 4 hours at 95° C. Then, the mixtures are centrifuged for 10 min at 15,500 g and the supernatants are taken. 7 μl of the supernatants are mixed with 193 μl imidazole buffer (100 mM imidazole, pH 7.4; 5 mM MgCl2, 1 mM EDTA and 0.4 mM NAD). The measuring was carried out in the photometer at 340 nm. After establishing a basic absorption, the enzyme reaction was started by adding 2 u glucose-6 phosphate dehydrogenase (from Leuconostoc mesenteroides, Boehringer Mannheim). The change in the absorption is directly proportional to the concentration of the G-6-P content of the starch.
The overall phosphate content was determined according to the method by Ames (Methods in Enzymology VIII, (1966), 115–118).
30 μl ethanolic magnesium nitrate solution are added to about 50 mg starch and ashed for three hours at 500° C. in a muffle furnace. 300 μl 0.5 M hydrochloric acid are added to the residue and incubated for 30 min at 60° C. Then, an aliquot is filled to 300 μl 0.5 M hydrochloric acid, added to a mixture of 100 μl 10% ascorbic acid and 600 μl 0.42% ammonium molybdate in 2 M sulphuric acid and incubated for 20 min at 45° C.
Then, a photometric measurement is conducted at 820 nm, using a phosphate calibration series as a standard.
c) Determination of the Gel Strength (Texture Analyser)
2 g starch (TS) are dissolved in 25 ml of an aqueous 60% (w/v) CaCl2 solution and pasting is achieved in an RVA apparatus (temperature program: cf. item d) “Determination of the viscosity properties by means of a Rapid Visco Analyser (RVA)”) and then it is stored in a closed container for 24 hours at room temperature. The samples are fixed under a probe (cylindrical stamp with a planar surface) of a texture analyser TA-XT2 by Stable Micro Systems (Surrey, UK) and the gel strength is determined using the following parameters:
















test speed
 0.5 mm/s



depth of penetration
  7 mm



contact area
 113 mm2



pressure
  2 g










d) Determination of the Viscosity Properties by Means of a Rapid Visco Analyser (RVA)

2 g starch (TS) are added to 25 ml H2O and used for the analysis in a Rapid Visco Analyser (Newport Scientific Pty Ltd., Investment Support Group, Warriewod NSW 2102, Australia). The apparatus is used according to the manufacturer's instructions. For determining the viscosity of the aqueous solution of the starch, the starch suspension is first heated to 50° C. for 1 min, then it is heated from 50° C. to 95° C. at a speed of 12° C. per minute. Subsequently, the temperature is maintained at 95° C. for 2.5 min. Then, the solution is cooled down from 95° C. to 50° C. at a speed of 12° C. per minute. The viscosity is determined over the whole time.
e) Determination of Glucose, Fructose and Sucrose
The content of glucose, fructose and sucrose is determined according to the method described by Stitt et al. (Methods in Enzymology 174, (1989), 518–552).
f) Analysis of the Distribution of the Side Chains of the Amylopectin by Means of Ion Exchange Chromatography
For separating amylose from amylopectin, 200 mg starch are dissolved in 50 ml-reaction vessels with 12 ml 90% (v/v) DMSO in H2O. After adding 3 volumes ethanol, the precipitate is separated by a 10 min-centrifugation at about 1800 g at room temperature (RT). The pellet is then washed with 30 ml ethanol, dried and dissolved in 40 ml 1% (w/v) NaCl solution at 75° C. After cooling down the solution to 30° C., about 90 mg thymol are added slowly and this solution is incubated for at least 60 h at 30° C. Then, the solution is centrifuged for 30 min at 2000 g (RT). 3 volumes ethanol are then added to the supernatant and the precipitating amylopectin is separated by means of 5 min-centrifugation at 2000 g (RT). The pellet (amylopectin) is then washed with ethanol and dried using acetone. By adding DMSO to the pellet, a 1%-solution is prepared 200 μl of which are added to 345 μl water, 10 μl 0.5 M sodium acetate (pH 3.5) and 5 μl isoamylase (dilution of 1:10; Megazyme) and incubated for about 16 h at 37° C. An aqueous 1:5 dilution of this digestion is then filtered with an 0.2 μm-filter and 100 μl of the filtrate are analysed by means of ion exchange chromatography (HPAEC-PAD, Dionex). The separation is carried out with a PA-100 column (with a corresponding pre-column), the detection is carried out amperometrically. The elution conditions are as follows:












t (min)
solution A (%)
solution B (%)

















 5
0
100


35
30
70


45
32
68


60
100
0


70
100
0


72
0
100


80
0
100


stop





solution A—0.15 M NaOH


solution B—1 M sodium acetate in 0.15 M NaOH






The relative proportion of short side chains in the overall proportion of all side chains is determined by determining the proportion in percent that a certain side chain has in the overall proportion of all side chains. The overall proportion of all side chains determined through the determination of the overall height of the peaks which represent the polymerisation degrees of DP 6 to 40 in the HPLC chromatogram. The proportion in percent that a certain side chain has in the overall proportion of all side chains is determined by the determination of the ratio of the height of the peak representing this side chain in the HPLC chromatogram to the overall height of all peaks having a DP of 6 to 40. The program Chomeleon 6.20 by Dionex, USA was used for determining the peak heights. The parameters of the evaluation software that were to be adjusted were as follows:













retention
parameter




time (min)
name
parameter value
channels


















0.000
Inhibit Integration
on
All channels


20.000
Lock Baseline
on
All channels


20.600
Inhibit Integration
off
All channels


20.600
Minimum Height
0.001 (Signal)
All channels


45.000
Inhibit Integration
on
All channels









g) Determination of the Granule Size

Starch was extracted from the potato tubers according to standard methods and washed several times with water in a 10 l-bucket (ratio height of the bucket/diameter of the bucket=approx. 1.1). For obtaining the starch samples which were finally subjected to the determination of the granule size, the starches were left to stand for about 4 h after washing to achieve as complete a sedimentation of the starches as possible.
The granule size was then determined by means of a photo sedimentometer of the type “Lumosed FS1” by Retsch GmbH, Germany using the software V.2.3.
The software adjustments were as follows:














data of the substance:
calibration no.
0



density [kg/m3]
1500


sedimentation fluid:
type
water



viscosity [Pa s]
0.001



density [kg/m3]
1000



addition
—



measurement data
5 min



sieve diameter [μm]
250



passage [%]
100



measurement range
4.34–117.39 μm



calibration
N



temperature
20° C.









The distribution of the granule size was determined in an aqueous solution and according to the manufacturer's instructions and based on the literature of e.g. H. Pitsch, Korngröβenbestimmung; LABO-1988/3 Fachzeitschrift für Labortechnik, Darmstadt.
h) Mechanical Fragmentation of the Strach
About 0.5 g of each starch were placed in a coffee mill (manufacturer: Mellert, type: M85, Germany) and ground six times for 30 s each. Between two intervals, the grinding was interrupted for 20 s each. The distribution of the granule size was determined as described in item g).
i) Water Binding Capacity
For determining the water binding capacity (WBC), the supernatant was weighed after separating the soluble portion by centrifugation of the starch swollen at 70° C. The water binding capacity (WBC) of the starch was set in relation to the weighed portion of the starch corrected by the soluble mass.

WBC (g/g)=(residue−(weighed portion−soluble portion))/(weighed portion−soluble portion).

The expression vector ME5/6 (cf. FIG. 1) was used in the Examples:
Preparation of the Expression Vector ME5/6
pGSV71 is a derivative of the plasmid pGSV7 which is derived from the intermediary vector pGSV1. pGSV1 is a derivative of pGSC1700 the construction of which has been described by Cornelissen and Vanderwiele (Nucleic Acids Research 17, (1989), 19–25). pGSV1 was obtained from pGSC1700 by deletion of the carbenicillin resistance gene as well as deletion of the T-DNA sequences of the TL-DNA region of the plasmid pTiB6S3.
pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar et al., Gene 2, (1977), 95–113) as well as the replication origin of the Pseudomonas plasmid pSV1 (Itoh et al., Plasmid 11, (1984), 206). pGSV7 additionally contains the selectable marker gene aadA from the transposon Tn1331 from Klebsiella pneumoniae which confers resistance to the antibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24 (3), (1990), 218–226; Tolmasky and Crosa, Plasmid 29 (1), (1993), 31–40).
The plasmid pGSV71 was obtained by cloning a chimeric bar gene between the border regions of pGSV7. The chimeric bar gene contains the promoter sequence of the cauliflower mosaic virus for initiating the transcription (Odell et al., Nature 313, (1985), 180), the bar gene from Streptomyces hygroscopicus (Thompson et al., EMBO J. 6, (1987), 2519–2523) and the 3′-non-translated region of the nopaline synthase gene of the T-DNA of pTiT37, for terminating the transcription and polyadenylation. The bar gene confers resistance to the herbicide glufosinate ammonium.
In position 198–222, the T-DNA contains the right border sequence of the TL-DNA of the plasmid pTiB6S3 (Gielen et al., EMBO J. 3, (1984), 835–846). There is a polylinker sequence between the nucleotide 223–249. The nucleotides 250–1634 contain the P35S3 promoter region of the cauliflower mosaic virus (Odell et al., cf. above). The coding sequence of the phosphinothricine resistance gene (bar) from Streptomyces hygroscopicus (Thompson et al., 1987, cf. above) is contained between the nucleotides 1635–2186. The two terminal codons at the 5′ end of the bar wild type gene were replaced by the codons ATG and GAC. There is a polylinker sequence between the nucleotides 2187–2205. The 260 bp-TaqI fragment of the non-translated 3′ end of the nopaline synthase gene (3′nos) from the T-DNA of the plasmid pTiT37 (Depicker et al., J. Mol. Appl. Genet. 1, (1982), 561–573 is located between the nucleotides 2206 and 2465. The nucleotides 2466–2519 contain a polylinker sequence. The left border sequence of the TL-DNA from pTiB6S3 (Gielen et al., EMBO J. 3, (1984), 835–846) is located between the nucleotides 2520–2544.
The vector pGSV71 was then cleaved with the enzyme PstI and blunted. The promoter B33 and the ocs cassette were cleaved from the vector pB33-Kan as an EcoRI-HindIII fragment, blunted and inserted into the vector pGSV71 which had been cleaved with PstI and blunted. The vector obtained served as a starting vector for the construction of ME5/6. An oligonucleotide containing the cleavage sites EcoRI, PacI, SpeI, SrI, SpeI, NotI, PacI and EcoRI was inserted into the PstI cleavage site between the B33 promoter and the ocs element of the vector ME4/6 by duplicating the PstI cleavage site. The expression vector obtained was called ME5/6.
Description of the Vector pSK-Pac
pSK-Pac is a derivative of pSK Bluescript (Stratagene, USA) into which PacI cleavage sites flanking the multiple cloning site (MCS) were inserted.
The following Examples illustrate the invention:
EXAMPLE 1
Production of Transgenic Potato Plants having a Reduced Gene Expression of an R1, BEI and BEII Gene
For producing transgenic plants having a reduced activity of a BEI, R1 and BEII protein, first transgenic plants were generated in which the BE1 activity and the amount of protein R1 were reduced. For this purpose, both the T-DNA of the plasmid pB33-aR1-Hyg and the T-DNA of the plasmid pB33-a-BE1-Kan were transferred simultaneously into potato plants using Agrobacteria as described by Rocha-Sosa et al. (EMBO J. 8, (1989), 23–29).
For constructing the plasmid pB33-aR1-Hyg and the plasmid pB33-aBE1-Kan, first the expression vectors pB33-Kan and pb33-Hyg, respectively, were constructed. For this purpose, the promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989, cf. above) was ligated as DraI fragment (nucleotides −1512 to +14) into the vector pUC19 (GenBank Acc. No. M77789) which had been cleaved with SstI, the ends of said vector having been blunted by means of the T4-DNA polymerase. In this way, the plasmid pUC19-B33 was obtained. The B33 promoter was cleaved from this plasmid with EcoRI and SmaI and ligated into the vector pBinAR which had been cleaved correspondingly. In this way, the plant expression vector pB33-Kan was obtained. The plasmid pBinAR is a derivative of the vector plasmid pBin19 (Bevan, Nucl. Acids Research 12, (1984), 8711–8721) and was constructed by Höfgen and Willmitzer (Plant Sci. 66, (1990), 221–230). Starting from plasmid pB33-Kan, the EcoRI-HindIII fragment comprising the B33 promoter, a portion of the polylinker and the ocs terminator from pB33-Kan were cleaved and ligated into the vector pBIB-Hyg (Becker, Nucleic Acids Res. 18 (1), (1990), 203) which had been cleaved correspondingly. As a result, pB33-Hyg was obtained.
Then, an approximately 2000 bp-Asp718 fragment of the plasmid pRL1 which contains the nucleotide sequence of about +2850 to about +4850 of the R1 cDNA from Solanum tuberosum (Lorberth, Charakterisierung von RL1: ein neues Enzym des Stärkemetabolismus. Dissertation Freie Universität Berlin) in antisense orientation into the Asp718 cleavage site of the plasmid described before. The resulting plasmid was called pB33-aR1-Hyg. For constructing the plasmid pB33-aBE1-Kan, analogously to the aforementioned strategy, the promoter region of the patatin-class-I gene B33 from Solanum tuberosum—a SmaI/HindIII fragment which has a length of about 3100 bp and contains a partial cDNA for the BE1 enzyme from potato (Kossmann, Klonierung und funktionelle Analyse von Genen codierend für am Kohlenhydratstoffwechsel der Kartoffel beteiligte Proteine, Dissertation Technische Universität Berlin, (1992))—was first blunted and inserted into the SmaI cleavage site of the vector pBinAR-Hyg (cf. above) in antisense orientation with regard to the B33 promoter.
After the transformation, different lines of transgenic potato plants could be identified by means of Western blot analysis, the tubers of said potatoes having a content of the R1 protein which was reduced significantly. Further analyses showed that isolated starch of the line 36 had the highest amylose content of all transformants examined independently of each other.
Plants of said line were then transformed with the plasmid pGSV71-aBE2-basta as described by Rocha-Sosa et al. (EMBO J. 8 (1989), 23–29).
Plasmid pGSV71-aBE2-basta was constructed by screening according to standard procedures a tuber-specific potato cDNA library with a DNA fragment which had been amplified by RT-PCR (primer 1 (SEQ ID No. 1): 5′-gggggtgttggctttgacta and primer 2 (SEQ ID No. 2) 5′-cccttctcctcctaatccca; Stratagene ProSTARTM HF Single-Tube RT-PCR system) with total-RNA from tubers as template. In this way, a DNA fragment which has a size of about 1250 bp (cf. SEQ ID No. 3) and which was then subcloned as a EcoRV-SmaI fragment into the EcoRV cleavage site of the cloning vector pSK-Pac (cf. above) and finally ligated as PacI fragment into the expression vector ME5/6 (FIG. 1) in antisense orientation. As a result, the plasmid pGSV71-aBE2-basta was obtained.
From plants which were obtained by the transformation with the plasmid pGSV71-aBE2-basta and which showed a reduced R1, BEI and BEII gene expression, said plants being called 203MH plants, tissue samples of tubers of the independent transformants were taken and their amylose content was determined (cf. methods). The starches of the independent lines the tubers of which had the highest amylose content were used for further analysing the starch properties (cf. Example 2).
EXAMPLE 2
Analysis of the Starch of Plants Having a Reduced R1, BEI and BEII Gene Expression
The starch of different independent lines of the transformation 203 MH described in Example 1 was isolated from potato tubers. Then, the physico-chemical properties of this starch were analysed. The results of the characterisation of the modified starches are shown in Table 1 (Tab. 1) for an exemplary selection of certain plant lines.
















TABLE 1







phosphate

RVA
RVA
RVA
RVA
RVA
gel strength,




in C6
amylose
max
min
fin
set
T
60% (w/v) CaCl2


no.
genotype
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
























1
Desiree
100
22
100
100
100
100
100
100



(wild type)












3
203MH010
25
92
no pasting in H2O
approx. 17,000


5
203MH055
33
80

not measured


6
203MH080
31
91

approx. 10,600





Legend:


R1 = R1 enzyme, BEI = branching enzyme I, BEII = branching enzyme II, as = antisense


RVA = Rapid Visco Analyser,


max = maximum viscosity,


min = minimum viscosity


fin = viscosity at the end of the measurement,


set = set back = difference of min und fin


T = pasting temperature


The values in % are related to the wild type (=100%) except for the amylose content.






The distribution of the side chains of the amylopectin was analysed as described above. The following Table (Tab. 2) contains an overview of the proportions of the individual peak heights of the HPAEC chromatograms within the overall peak height of wild type plants (Desiree), of 072VL036 plants (potato plants having a reduced gene expression of the R1 and BEI gene) and of selected lines of the transformations 203MH (cf. Example 1: potato plants having a reduced gene expression of the R1, BEI and BEII gene):









TABLE 2








proportion of the



no. of
individual peak heights within the overall peak


glucose
height of every potato line in %












units
Desi Mix
072VL036
203MH10
203MH61
203MH80















dp6
2.48
1.2
2.6
1.7
2.7


dp7
1.90
1.2
1.6
1.1
1.5


dp8
1.46
1.4
1.0
0.8
1.2


dp9
2.48
2.2
1.3
1.1
1.5


dp10
4.38
3.5
2.1
1.9
2.1


dp11
6.28
4.8
3.1
2.8
3.0


dp12
7.30
5.6
3.6
3.6
3.3


dp13
7.88
6.1
3.6
3.9
3.3


dp14
7.88
6.3
3.9
4.4
3.6


dp15
7.30
6.3
4.2
4.4
3.3


dp16
6.72
6.1
4.2
4.7
3.3


dp17
5.84
5.9
4.2
4.7
3.3


dp18
5.26
5.7
4.4
4.7
3.3


dp19
4.82
5.5
4.4
4.7
3.3


dp20
4.38
5.1
4.4
4.7
3.3


dp21
3.94
4.7
4.4
4.7
3.6


dp22
3.50
4.2
4.2
4.7
3.6


dp23
3.07
3.8
4.2
4.4
3.9


dp24
2.48
3.4
4.4
4.4
4.2


dp25
2.19
3.1
4.2
4.4
4.2


dp26
1.90
2.7
4.2
4.2
4.5


dp27
1.75
2.4
3.9
4.2
4.5


dp28
1.31
2.0
3.6
3.6
4.5


dp29
1.02
1.6
3.4
3.3
4.2


dp30
0.88
1.3
2.9
2.8
3.9


dp31
0.58
1.1
2.6
2.5
3.3


dp32
0.44
0.8
2.1
1.9
2.7


dp33
0.29
0.7
1.8
1.7
2.4


dp34
0.29
0.5
1.8
1.4
2.4


dp35
0.00
0.4
1.6
1.1
1.8


dp36
0.00
0.3
1.0
0.8
1.5


dp37
0.00
0.2
0.8
0.6
0.9


dp38
0.00
0.2
0.5
0.0
0.9


dp39
0.00
0.0
0.0
0.0
0.0


dp40
0.00
0.0
0.0
0.0
0.0


Total
100.00
100.0
100.0
100.0
100.0









If the proportion of peak heights of the individual chain lengths (indicated in DP) in the overall peak height is compared, a considerable shift towards side chains having a DP>26 can be seen with regard to the distribution of the side chains of the amylopectin of the 203MH plants compared to the amylopectin of wild type plants and also to 072VL plants. If the mean is calculated from the relative proportions of the side chains having a DP of 26 to DP 31, the following values are obtained (Tab. 3):













TABLE 3







Desi







Mix
072VL036
203MH10
203MH61
203MH80





















mean of the
1.24
1.85
3.43
3.43
4.15


relative


proportions


of DP


26 to DP 31


change in %
100
149
276
276
335


compared to


Desi Mix


(=100%)









The amylopectin of 203MH plants is characterised by an increased proportion of side chains having a DP of 26 to DP 31 compared to amylopectin of wild type plants and also of 072VL plants.
Furthermore, the morphology of the starch granules was examined:
The surface of the starch granules of wild type plants appears smooth under the light microscope. The form of the granules is round to oval, no “internal structures” being noticeable. Moreover, a uniform distribution of the different granule sizes can be seen (cf. FIG. 2).
Starch granules of 072VL036 plants (FIG. 3) have a very heterogeneous appearance. Only some granules appear smooth, others have grooves, some show “bunch-of-grapes-like agglomerations”. Other granules have cross-recess-like structures. The spectrum of granule sizes is broad, smaller granules making up a greater proportion than is the case with starch granules of wild type plants.
The morphology of the granules of the line 203MH010 (FIG. 4), too, is heterogeneous, though less apparent than in 072VL036 plants. The surface of almost all granules has grooves, most of the granules show bunch-of-grapes-like agglomerations. Sometimes, particles can be seen which look like fragments of these agglomerated structures. The size distribution is relatively broad, smaller granules dominate though.
Furthermore, the granule size was determined using a photo sedimentometer of the type “Lumosed” by Retsch GmbH, Germany.
The average granule size of both untreated starch samples and samples which, prior to the granule size determination, were subjected to an overall 3-minute mechanical fragmentation was measured (for conduction see above) (Tab. 4).
In addition, the proportion of starch granules having a size of <20 μm was determined (Tab. 5)







TABLE 4







average granule size [μm]













mechan.



sample
untreated
fragmentation















Wt
23.86
22.27



072VL036
16.88
16.78



203MH010
14.78
11.31



203MH066
14.59
11.82



203MH080
14.31
12.14

















TABLE 5







Proportion of granules < 20 μm [%]













mechan.



sample
untreated
fragmentation















Wt
51.7
49.3



072VL036
69.7
69.9



203MH010
85.8
92.9



203MH066
83.7
90.6



203MH080
88.4
91.1










The results show that both the average granule size and the proportion in percent of starch granules <20 μm of the starches of the invention differ significantly from wild type starches as well as from starches derived from 072VL036 plants.
After mechanical fragmentation of the starches, these differences are even more significant than without mechanical treatment.

## Claims

1. A transgenic plant cell which has been genetically modified, the plant cell comprising:
a) in an antisense orientation relative to a promoter, a nucleic acid sequence of at least 500 nucleotides that hybridizes under high stringency conditions to a gene encoding an endogenous starch phosphorylase (R1) protein and that leads to the reduction, compared to the non-genetically modified plant cell, of the expression of one or more endogenous starch phosphorylase (R1) proteins;
b) in an antisense orientation relative to a promoter, a nucleic acid sequence of at least 500 nucleotides that hybridizes under high stringency conditions to a gene encoding an endogenous branching enzyme I (BEI) protein and that leads to the reduction, compared to the non-genetically modified plant cell, of the expression of one or more endogenous branching enzyme I (BEI) proteins; and
c) in an antisense orientation relative to a promoter, a nucleic acid sequence of at least 500 nucleotides that hybridizes under high stringency conditions to a gene encoding an endogenous branching enzyme II (BEII) protein and that leads to the reduction, compared to the non-genetically modified plant cell, of the expression of one or more endogenous branching enzyme II (BEII) proteins.

2. A transgenic plant cell comprising at least one nucleic acid molecule comprising:
a) a nucleic acid sequence comprising at least 500 nucleotides of a starch phosphorylase (R1) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a starch phosphorylase (R1) protein compared to expression in the non-transgenic plant cell;
b) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme I (BEI) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a branching enzyme I (BEI) protein compared to expression in the non-transgenic plant cell; and
c) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme II (BEII) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a branching enzyme II (BEII) protein compared to expression in the non-transgenic plant cell.

3. The transgenic plant cell according to claim 1 or 2 which synthesizes a modified starch.

4. The transgenic plant cell according to claim 3, wherein the modified starch has an amylose content of at least 75% and a reduced phosphate content compared to starch from a non-transgenic plant cell.

5. The transgenic plant cell according to claim 3, wherein the modified starch has a modified distribution of the side chains.

6. A transgenic plant comprising the transgenic plant cell according to claim 1 or 2.

7. A method for producing a transgenic plant cell that synthesizes a modified starch, comprising the step of introducing into a plant cell at least one nucleic acid molecule, wherein said at least one nucleic acid molecule comprises:
a) a nucleic acid sequence comprising at least 500 nucleotides of a starch phosphorylase (R1) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a starch phosphorylase (R1) protein compared to expression in the non-transgenic plant cell;
b) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme I (BEI) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a branching enzyme I (BEI) protein compared to expression in the non-transgenic plant cell; and
c) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme II (BEII) gene operably linked to a promoter in an antisense orientation leading to reduced expression of an endogenous gene encoding a branching enzyme II (BEII) protein compared to expression in the non-transgenic plant cell;
thereby producing the transgenic plant cell that synthesizes a modified starch.

8. The method of claim 7, wherein the modified starch has an amylose content of at least 75% and a reduced phosphate content in comparison with starch from a non-transgenic plant cell.

9. A method for producing a transgenic plant that synthesizes a modified starch comprising the step of
regenerating a plant from a plant cell produced according to the method of claim 7,
thereby producing the transgenic plant that synthesizes a modified starch.

10. The method according to claim 9, wherein the modified starch has an amylose content of at least 75% and a reduced phosphate content in comparison with starch from a non-transgenic plant.

11. A transgenic plant obtained by the method according to claim 9 or 10.

12. The transgenic plant according to claim 6 which is a starch-storing plant.

13. The transgenic plant according to claim 12 which is a potato plant.

14. Propagation material of the plant according to claim 6, wherein the propagation material comprises the transgenic plant cell.

15. A nucleic acid molecule comprising:
a) a nucleic acid sequence comprising at least 500 nucleotides of a starch phosphorylase (R1) gene operably linked to a promoter in an antisense orientation, which when expressed in a plant cell leads to reduced expression of an endogenous gene encoding a starch phosphorylase (R1) protein compared to a the non-transformed plant cell;
b) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme I (BEI) gene operably linked to a promoter in an antisense orientation, which when expressed in a plant cell leads to reduced expression of an endogenous gene encoding a branching enzyme I (BEI) protein compared to the non-transformed plant cell; and
c) a nucleic acid sequence comprising at least 500 nucleotides of a branching enzyme II (BEII) gene operably linked to a promoter in an antisense orientation, which when expressed in a plant cell leads to reduced expression of an endogenous gene encoding a branching enzyme II (BEII) protein compared to the non-transformed plant cell.

16. A host cell comprising the nucleic acid molecule according to claim 15.

17. The host cell according to claim 16, wherein the cell is a plant cell.

18. The transgenic plant according to claim 11 which is a starch-storing plant.

19. The transgenic plant according to claim 18 which is a potato plant.

20. Propagation material of the plant according to claim 11, wherein the propagation material comprises the transgenic plant cell.