[go: up one dir, main page]

WO2004058962A1 - Procede de production de plantes contenant des amidons a teneur en phosphate accrue - Google Patents

Procede de production de plantes contenant des amidons a teneur en phosphate accrue Download PDF

Info

Publication number
WO2004058962A1
WO2004058962A1 PCT/EP2003/014960 EP0314960W WO2004058962A1 WO 2004058962 A1 WO2004058962 A1 WO 2004058962A1 EP 0314960 W EP0314960 W EP 0314960W WO 2004058962 A1 WO2004058962 A1 WO 2004058962A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
starch
phosphoglucomutase
expression
reduction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2003/014960
Other languages
English (en)
Inventor
Jens Pilling
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience AG
Original Assignee
Bayer CropScience AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer CropScience AG filed Critical Bayer CropScience AG
Priority to AU2003290126A priority Critical patent/AU2003290126A1/en
Publication of WO2004058962A1 publication Critical patent/WO2004058962A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)

Definitions

  • the present invention relates to a method for increasing the phosphate content in plant starches from genetically modified plants.
  • the genetic modification consists in particular in the introduction of one or more foreign nucleic acid molecules whose presence, or expression, leads to a reduction of the activity of at least one phosphoglucomutase (PGM) in comparison with corresponding wild-type plant cells which has (have) not been genetically modified.
  • PGM phosphoglucomutase
  • the polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it constitutes a highly complex mixture of different forms of molecules which differ with regard to their degree of polymerization and the occurrence of branches of the glucose chains. Starch therefore does not constitute a uniform raw material. Starch consists of two chemically different components, amylose and amylopectin. In typical plants used for starch production, such as, for example, maize, wheat or potato, amylose starch amounts to approximately 20% - 30% in the starch which has been synthesized and amylopectin starch to approximately 70% - 80%.
  • the functional properties of starch are affected greatly not only by the amylose/amylopectin ratio and the phosphate content, but also by the molecular weight, the pattern of the side-chain distribution, the ionic content, the lipid and protein content, the mean size of the starch grains, the starch grain morphology and the like.
  • Important functional properties which may be mentioned in this context are, for example, solubility, the retrogradation behaviour, the water-binding capacity, the film-forming properties, the viscosity, the gelatinization properties, the freeze-thaw stability, the stability to acids, the gel strength and the like.
  • the size of the starch grain too may be of importance for various applications.
  • Imported sucrose is degraded in the cytosol by sucrose synthase to give UDP-glucose and fructose.
  • the resulting UDP-glucose is subsequently converted into glucose-1 -phosphate by UDP-glucose phyrophosphorylase (UGPase).
  • UDP-glucose phyrophosphorylase UDP-glucose phyrophosphorylase
  • the second product of the sucrose synthase reaction namely fructose, is phosphorylated by fructokinase to give fructose-6-phosphate.
  • Fructose-6-phosphate is converted into glucose-6-phosphate by phosphoglucose isomerase and subsequently into glucose-1 -phosphate by phosphoglucomutase.
  • glucose-6-phosphate is the main precursor which is taken up by the amyloplast for starch synthesis (Tauberger et. al. 2000, Plant Journal 32(1), 43-53).
  • Glucose-6-phosphate is converted in the amyloplast by the plastid phosphoglucomutase to give glucose-1 -phosphate, which is converted by ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) to give ADP-glucose (M ⁇ ller- Rober et al. 1992).
  • This nucleotide sugar is the direct precursor for the starch synthases (SS; EC 2.4.1.21), which form -1 ,4-glycosidic bonds between the glucose units to give amylose.
  • the branching enzymes (EC 2.4.1.24) are responsible for producing amylopectin; following hydrolytic cleavage of the ⁇ 1 ,4-glycosidic glucose chains, they generate ⁇ -1 ,6- giycosidic branching (Tauberger 1999, PhD thesis).
  • plants with a reduced phosphoglucomutase activity reveal an increased glucose-6-phosphate content, but also fructose-6-phosphate and glucose-1 -phosphate content (Tauberger 1999, loc. cit.). A trend to a reduced starch content and an increased sucrose and glucose accumulation is also observed.
  • WO 98-01574 describes pea plants in which the activity of the plastid phosphoglucomutase has been reduced owing to the mutation of an Rug3 enzyme and which have an increased sucrose content and a longer harvest window.
  • WO 01-75128 too relates to the production of legumes with a reduction of the activity of the plastid phosphoglucomutase.
  • the aim is an increased protein content combined with a prolonged grain-filling period.
  • EP 1174510 describes plastid PGM sequences from maize, rice, soybeans and bullrush and the methodology for generating transgenic maize and soybeans with a modified plastid phosoglucomutase content.
  • a method for increasing the phosphate content in a plant starch by reducing the activity of at least one phosphoglucomutase in the plant (cell) used for starch production has not been described as yet.
  • the phosphate content can be modified in principle both by recombinant approaches and by the subsequent chemical phosphorylation of native starches (see, for example in: Starch Chemistry and Technology. Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall. Academic Press, New York, 1988, 349-364).
  • chemical modifications are, as a rule, expensive and time-consuming and lead to starches whose physico- chemical properties may differ from in-vivo modified starches.
  • the starches produced by the method according to the invention furthermore differ from chemically phosphorylated starches by a modified phosphorylation pattern and, after gelatinization of the starches and gelling, by modified gel strength characteristics.
  • Novel functionalities are in great demand in the starch industry. Physical and/or chemical modifications of starch are required for a variety of applications. Starch suspensions with an increased phosphate content offer improved clarity and an improved low-temperature stability. Starches with an increased phosphate content permit the development of advantageous products mainly in papermaking, ready meals, dressings, dietetic products, in the baking industry and the like.
  • the present invention thus relates to a method for increasing the phosphate content in starches from genetically modified plant cells in comparison with corresponding unmodified wild-type plant cells, wherein a plant cell is genetically modified, with the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified.
  • a plant may be regenerated from, or using, the cells thus generated, using methods with which the skilled worker is familiar, and this plant can be used, if appropriate, for generating further plants.
  • the genetic modification can take the form of any genetic modification which leads to a reduction of the activity of one or more phosphoglucomutases which are endogenous to the plant cell in comparison with corresponding plant cells, of wild-type plants, which have not been genetically modified.
  • the term "genetically modified” means that the genetic information of the plant cell is modified, the activity of at least one phosphoglucomutase being reduced.
  • genetically modified plant cells according to the invention show a reduction of the expression of one or more phosphoglucomutase genes which are endogenous to the plant cell in comparison with corresponding plant cells, of wild-type plants, which have not been genetically modified.
  • “genetically modified” is also understood as meaning a plant in which a reduction of the activity of at least one phosphoglucomutase has been brought about by means of a mutation.
  • the mutants may take the form of either spontaneously occurring mutants or of mutants which have been generated by the targeted application of mutagens.
  • wild-type plants or wild-type plant cells refers to those plants, or plant cells, which act as starting material in the method according to the invention, i.e. before the reduction according to the invention of the activity or expression of at least one phosphoglucomutase.
  • they take the form of plants or plant cells which are not genetically modified with regard to the activity or expression of phosphoglucomutase.
  • genetically modified plants or plant cells which are genetically modified otherwise than regarding the activity or expression of phosphoglucomutase may also constitute the starting material according to the invention.
  • the term "reduction of the activity” refers to a reduction of the enzymatic activity of a phosphoglucomutase protein in the cells and/or a reduction of the expression of endogenous genes encoding proteins of a phosphoglucomutase, and/or a reduction of the amount of phosphoglucomutase protein in the cells.
  • the genetic modification which has been carried out may also lead to the expression of inactive PGM protein in the plant cell. Details on the methodology of the activity determination are found under "General Methods".
  • phosphoglucomutases are understood as meaning enzymes (E.C. 5.4.2.2.) which catalyze the conversion between glucose-1 -phosphate and glucose-6-phosphate (ap Rees and Morrell 1990, Am. Potato J. 67: 835-847). Proteins which have been identified as plastid phosphoglucomutases have already been isolated from spinach (M ⁇ hlbach and Schnarrenberger, 1978, Planta 141 : 65-70) and from pea (Salvucci et al., 1990, Plant Physiol. 93: 105-109). At least two PGM isoforms, a plastid form and a cytosolic form, exist in potato.
  • the cytosolic PGM also referred to as PGM I, is indispensible for the sugar and starch metabolism and catalyzes the conversion reaction between glucose-1 -phosphate and glucose-6-phosphate.
  • the potato cytosolic phosphoglucomutase (PGM I) is a protein of approx.
  • the cytosolic PGM ensures the supply of glucose-6-phosphate from glucose-1 -phosphate in the cytosol, firstly for transport into the amyloplasts and secondly also for glycolysis (energy supply).
  • the concentrations of the soluble sugars sucrose and glucose remain unchanged.
  • the genetic modification can be brought about by introducing one or more foreign nucleic acid molecules into a plant cell.
  • the present invention thus relates to a method for increasing the phosphate content in starches of genetically modified plants by introducing one or more foreign nucleic acid molecules into a plant cell.
  • the presence and/or expression of these foreign nucleic acid molecules leads to the reduction of the activity of at least one phosphoglucomutase in comparison with corresponding unmodified plant cells from wild-type plants.
  • a plant can be regenerated from or using the genetically modified cells thus prepared, using customary methods, and, if appropriate, further plants can be generated from the above plant.
  • the reduction of the expression can be determined for example by measuring the amount of phosphoglucomutase-protein-encoding transcripts, for example by Northern Blot analsysis (Tauberger et al., 2000, The Plant Journal, 23(1), 43-53), quantitative RT-PCR or what are known as microarrays.
  • "Reduced” in this context means a reduced amount of transcripts in comparison with corresponding cells which have not been genetically modified or wild-type cells, preferably by at least 50%, in particular by at least 70%, preference by at least 85% and especially preferably by at least 95%.
  • the reduction of the amount of phosphoglucomutase proteins can be determined for example by immunological methods such as Western Blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay). These methods are known to the skilled worker and described in, for example, Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual; Cold Spring Harbour Laboratory Press.
  • Reduced means, in this context, a reduced amount of phosphoglucomutase protein in comparison with corresponding cells which have not been genetically modified or wild-type cells, preferably by at least 50%, in particular by at least 70%, by preference by at least 85% and especially preferably by at least 95%.
  • foreign nucleic acid molecule or “foreign nucleic acid molecules” are understood as meaning, in the context of the present invention, such a molecule which either does not occur naturally in corresponding plant cells or which does not occur naturally in the plant cells in the specific spatial arrangement or which is localized at the location in the genome of the plant cell where it does not occur naturally.
  • the foreign nucleic acid molecule is preferably a recombinant molecule which consists of various elements whose combination or specific spatial arrangement does not occur naturally in plant cells.
  • the activity of at least one plastid phosphoglucomutase may be reduced by the method according to the invention in a preferred embodiment.
  • a plastid phosphoglucomutase is understood as meaning, in this context, an enzyme which catalyzes the conversion reaction between glucose-1 - phosphate and glucose-6-phosphate in the plastid.
  • the plastid PGM plays an important role in the transport of glucose-6-phosphate into the amyloplasts and the supply of glucose-1 -phosphate.
  • it is another functionally important enzyme for the regulation of starch biosynthesis in storage tissue (Fl ⁇ gge and Weber, 1994, Planta 194: 181- 185; Fl ⁇ gge 1985, J. Experim. Botany 46: 1317-1323; Schott et al., 1995, Planta 196: 647-652; Kammerer et al, 1998, Plant Cell 10: 105-117).
  • cytosolic PGM may likewise take place within the scope of the present invention.
  • the genetic modifications required may be carried out simultaneously or in succession.
  • the increase in the phosphate content of the starch which is brought about by the method according to the invention leads in particular to an increased phosphate content in the C-6 position of the starch produced thus in comparison with starch from corresponding wild-type plant cells which have not been genetically modified.
  • the term “phosphate content” of the starch refers to the content of phosphate bonded covalently in the form of starch phosphate monoesters.
  • the term “increased phosphate content” means that the total phosphate content of covalently bonded phosphate and/or the phosphate content in the C-6 position of the starch synthesized in the plant cells according to the invention is increased in comparison with starch from plant cells of corresponding wild-type plants, preferably by at least 40%, by preference by at least 60%, especially preferably by at least 80%.
  • the term "phosphate content in the C6 position” is understood as meaning the content of phosphate groups which are bound at the carbon atom position "6" of the glucose monomers of the starch.
  • the positions C2, C3 and C6 of the glucose units in the starch may be phosphorylated in vivo.
  • the determination of the phosphate content in the C6 position can be performed via a glucose-6- phosphate determination using a visual-enzymatic assay (Nielsen et al., Plant Physiol. 105, (1994), 111-117).
  • the foreign nucleic acid molecule(s) introduced into the plant cell is, or are, selected from the group consisting of a) DNA molecules which encode at least one antisense RNA which brings about a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); b) DNA molecules which, via a cosuppression effect, lead to a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); c) DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of at least one endogenous gene which encodes PGM protein(s); d) nucleic acid molecules introduced by means of in-vivo mutagenesis, which lead to a mutation or an insertion of a heterologous sequence in at least one endogenous gene encoding PGM protein(s), the mutation or insertion bringing about a reduction of the expression of said at least one gene or the synthesis of inactive PGM proteins; e) DNA molecules which simultaneously encode at
  • An example of a tool which may be used for inhibiting gene expression by means of antisense or cosuppression technology is a DNA molecule which encompasses all of the sequence encoding a phosphoglucomutase protein, including any flanking sequences, or else DNA molecules which only encompass parts of the coding sequence which, however, must be long enough in order to bring about an antisense effect, or cosuppression effect, in the cells.
  • Sequences which are generally suitable for effective antisense or cosuppression inhibition have a minimum length of 15 bp, preferably a length of 100-500 bp; in particular sequences with a length of over 500 bp.
  • DNA sequences which have a high degree of homology with the sequences which occur endogenously in the plant cell and which encode phosphoglucomutase proteins.
  • the minimum degree of homology should exceed approx. 65%.
  • sequences with at least 90%, in particular between 95% and 100% homology is to be preferred.
  • introns i.e. of noncoding regions of genes which encode phosphoglucomutase proteins.
  • the reduction of the phosphoglucomutase activity in the plant cells may also be achieved by what is known as "in-vivo mutagenesis", where an RNA-DNA hybrid oligonucleotide ("chimeroplast”) is introduced into cells by transforming cells (Kipp, P.B. et al., Poster Session at the " 5 th International Congress of Plant Molecular Biology, 21st-27th September 1997, Singapore; R. A. Dixon and C.J.
  • a part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous phosphoglucomutase gene, but, in comparison with the nucleic acid sequence of an endogenous phosphoglucomutase gene, contains a mutation or a heterologous region which is surrounded by the homologous regions.
  • the mutation or heterologous region which is present in the DNA component of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell by base pairing of the homologous regions of the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule, followed by homologous recombination. This leads to a reduction of the activity of one or more phosphoglucomutase proteins.
  • mutagenesis is to be understood as meaning any type of mutation such as, for example, deletion, point mutation (nucleotide substitution), insertion, inversion, gene conversion or chromosome translocation.
  • the mutation may be generated by the use of chemical agents or high-energy radiation (for example x-rays, neutron, gamma or UV radiation).
  • chemical agents or high-energy radiation for example x-rays, neutron, gamma or UV radiation.
  • mutants in plant species which reproduce predominantly vegetatively has been described for example for potatoes which produce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoretical and Applied Genetics 75, 217-221) and for mint with an increased oil yield or modified oil quality (Dwivedi et al., 2000, Journal of Medicinal and Aromatic Plant Sciences 22, 460-463). All of these methods are suitable in principle for generating the plant cells according to the invention and the starch produced by them.
  • the identification of mutations in the genes in question may be effected with the aid of methods known to the skilled worker.
  • Methods which may be employed in particular in this context are analyses based on hybridizations with probes (Southern blot), the amplification by means of polymerase chain reaction (PCI), the sequencing of relevant genomic sequences and the search for individual nucleotide substitutions.
  • An example of a method for identifying mutations with the aid of hybridization patterns is the search for restriction fragment length polymorphisms (RFLP) (Nam et al., 1989, The Plant Cell 1 , 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750).
  • RFLP restriction fragment length polymorphisms
  • PCR-based method is the analysis of amplified fragment length polymorphisms (AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem et al., 2001 , Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259, 150-160).
  • AFLP amplified fragment length polymorphisms
  • amplified fragments which have been cleaved with restriction endonucleases may also be used for identifying mutations (Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753).
  • Methods for determining SNPs are, inter alia, by Qi et al. (2001 , Nucleic Acids Research 29 (22), 116) Drenkard et al. (2000, Plant Physiology 124, 1483-1492) and Cho et al.
  • TILLING targeting induced local lesions in genomes
  • the plant cells according to the invention may also be generated with the aid of homologous transposons, i.e. transposons which are naturally present in the plant cells in question.
  • homologous transposons i.e. transposons which are naturally present in the plant cells in question.
  • Ramachandran and Sundaresan 2001 , Plant Physiology and Biochemistry 39: 234-252
  • the possibility of generating mutants with the aid of retrotransposons and methods for identifying mutants have been described by Kumar and Hirochika (2001 , Trends in Plant Science 6 (3), 127-134).
  • heterologous transposons in different species has been described both for dicotyledonous plants and for monocotyledonous plants: for example for rice (Greco et al., 2001 , Plant Physiology 125, 1175-1177; Liu et al., 1999, Molecular and General Genetics 262, 413- 420; Hiroyuki et al., 1999, The Plant Journal 19 (5), 605-613; Jeon and Gynheung, 2001 , Plant Science 161 , 211-219), barley (2000, Koprek et al., The Plant Journal 24 (2), 253-263) Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717, Schmidt and Willmitzer, 1989, Molecular and General Genetics 220, 17-24; Altmann et al., 1992, Theoretical and Applied Genetics 84, 371-383; Tissier et al., 1999, The Plant Cell 11 , 1841-1852), tomato (Beco
  • the plant cells and plants according to the invention and the starch produced by them may be generated or produced with the aid of both homologous and heterologous transposons, the use of homologous transposons also being understood as meaning those which already naturally exist in the plant genome.
  • the T-DNA insertion mutagenesis is based on the ability of certain segments (T-DNA) of Ti plasmids from Agrobacterium to integrate into the genome of plant cells.
  • the site of integration into the plant chromosome is not fixed, but may take place at any given site. If the T-DNA integrates into a segment of the chromosome which constitutes a gene function, the result may be a modified gene expression and thus modified activity of a protein encoded by the gene in question.
  • the integration of a T-DNA into the coding region of a protein frequently leads to a situation where the protein in question can no longer be synthesized by the cell in question in active form, or not at all.
  • T-DNA insertions for generating mutants has been described for example for Arabidopsis thaiiana (Krysan et al., 1999, The Plant Cell 11 , 2283-2290; Atipiroz- Leehan and Feldmann, 1997, Trends in Genetics 13 (4), 152-156; Parinov and Sundaresan, 2000, Current Opinion in Biotechnology 11 , 157-161) and rice (Jeon and An, 2001 , Plant Science 161 , 211-219; Jeon et al., 2000, The Plant Journal 22 (6), 561-570).
  • the reduction of the phosphoglucomutase activity in plant cells may also be brought about by the simultaneous expression by sense and antisense molecules of the target gene to be repressed in each case, preferably the phosphoglucomutase gene.
  • chimeric constructs which contain "inverted repeats" of the target gene, or parts of the target gene, in question.
  • the chimeric constructs encode sense and antisense RNA molecules of the target gene in question.
  • Sense and antisense RNA are synthesized simultaneously in planta as one RNA molecule, it being possible for sense and antisense RNA to be separated from each other by means of a spacer and to form a double-stranded RNA molecule.
  • Sense and antisense sequences of the target gene, or target genes may also be expressed separately from one another by means of the same or different promoters (Nap, J-P et al, 6 th International Congress of Plant Molecular Biology, Quebec, 18th-24th
  • RNA molecules of promoter DNA molecules in plants in trans may lead to methylation, and transc ptional inactivation, of homologous copies of these promoters, which shall hereinbelow be referred to as target promoters (Mette et al., EMBO J. 19, (2000), 5194-5201).
  • target promoter via inactivating the target promoter, to reduce the gene expression of a specific target gene (for example phosphoglucomutase gene) which is naturally under the control of this target promoter, i.e. the DNA molecules which comprise the target promoters of the genes to be repressed (target genes) are in this case - in contrast to the original function of promoters in plants - used as DNA molecules which can be transcribed themselves and not as control elements for the expression of genes or cDNAs.
  • a specific target gene for example phosphoglucomutase gene
  • Constructs which are preferably used for generating the double-stranded target promoter RNA molecules in planta, where they may be present as RNA hairpin molecules are those which contain "inverted repeats" of the target promoter DNA molecules, the target promoter DNA molecules being under the control of a promoter which governs the gene expression of said target promoter DNA molecules. These constructs are subsequently introduced into the genome of plants. The expression of the "inverted repeats" of said target promoter DNA molecules results in the formation of double-stranded target promoter RNA molecules in planta (Mette et al., EMBO J. 19, (2000), 5194-5201). The target promoter may thus be inactivated.
  • PGM proteins can be achieved by expressing nonfunctional derivatives, in particular trans- dominant mutants, of such proteins and/or by expressing antagonists/inhibitors of such proteins.
  • Antagonists/inhibitors of such proteins encompass for example antibodies, antibody fragments or molecules with similar binding properties.
  • a cytoplasmic scFv antibody was employed 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).
  • nucleic acids which reduce the activity of a target gene are, for example, the cauliflower mosaic virus 35S RNA promoter and the maize ubiquitine promoter for constitutive expression, the Patatin gene promoter B33 (Rocha-Sosa et al., EMBO J.
  • the MCPI promoter of the potato metallocarbopeptidase inhibitor gene (Hungarian patent application HU9801674) or the potato GBSSI promoter (international patent application WO 92/11376) for a 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.
  • the Shrunken-1 promoter (Werr et al., EMBO J. 4, (1985), 1373-1380), the wheat HMG promoter, the USP promoter, the phaseolin promoter, or promoters of maize zein genes (Pedersen et al., Cell 29, (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).
  • the expression of the foreign nucleic acid molecule(s) is particularly advantageous in those plant organs which store starch.
  • Such organs are, for example, the tuber of the potato plant or the grains, or the endosperm, of maize, wheat or rice plants. It is therefore preferred to use promoters which mediate the expression in these organs.
  • promoters which are only activated at a point in time which is determined by external influences (see, for example, WO 93/07279). Promoters of heat-shock proteins, which permit simple induction, may be of particular interest in this context.
  • seed-specific promoters such as, for example, the Vicia faba USP promoter, which ensures seed-specific expression in Vicia faba and other plants, may be (Fiedler et al., Plant Mol. Biol. 22, (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225, (1991), 459-467). Others which may be employed are fruit-specific promoters, such as, for example, described in WO91/01373.
  • a termination sequence which serves for the correct termination of the transcription process and the addition of a poly-A tail to the transcript, which is assumed to have a function in stabilizing the transcripts, may be present.
  • Such elements have been described in the literature (cf., for example, Gielen et al., EMBO J. (1989), 8:23-29) and can be exchanged as desired.
  • the genetically modified plant cells generated by the method according to the invention synthesize a modified starch whose physico-chemical properties, in particular the phosphate content, the viscosity behavior and/or the gel strength, is modified in comparison with starch synthesized in wild-type plants so that it is better suited to specific uses.
  • the starch of the plant cells generated by the present method has an increased phosphate content in comparison with starch from plant cells of corresponding wild-type plants, so that this starch is better suited to specific uses.
  • gel strength means the resistance of a starch gel to deformation under specific conditions.
  • Gel strength can be measured for example by measuring the force required when a standardized measuring rod is immersed into a starch gel, or the force required for pulling a standardized measuring rod out of a starch gel is estimated.
  • the gel strength shall be determined with the aid of a Texture Analyzer under the conditions described in the methodological part.
  • amylose content is determined by the method of Hovenkamp-Hermelink et al. (Potato Research (1988), 31 :241-246) which has been described below for potato starch. This method can also be applied to isolated starches of other plant species. Methods for isolating starches are known to the skilled worker.
  • the present invention relates to the use of one or more nucleic acid molecules for increasing the phosphate content in plant starch, wherein a plant cell is genetically modified, the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison to corresponding wild-type plant cells (which have not been genetically modified).
  • the at least one nucleic acid molecule used in this context is selected among the groups illustrated hereinabove.
  • the use according to the invention of one or more nucleic acid molecules leads to an increased phosphate content in the C6 position of the starch in comparison with starch of corresponding wild-type plant cells (which have not been genetically modified).
  • the at least one phosphoglucomutase whose expression and/or activity has been reduced may be a plastid phosphoglucomutase.
  • the starch obtained by the method according to the invention preferably exhibits an increased gel strength.
  • the term "increased gel strength” refers to an increase in gel strength preferably by at least 30%, in particular by at least 50%, by preference by at least 70% and especially preferably by at least 100%, by a maximum of not more than 150% or not more than 200% in comparison with the gel strength of starch of corresponding wild-type plant cells (which have not been genetically modified).
  • the modified starch produced by the method according to the invention is not only distinguished by an increased phosphate content in comparison with starch from corresponding wild-type plants, but also by a modified side- chain distribution.
  • the present invention relates to genetically modified plant cells which synthesize a modified starch, the modified starch being characterized in that it has an modified side-chain distribution
  • the amount of amylopectin side chains with a dp of 8 to 15 in the starch produced by the method according to the invention is increased by at least 5%, by preference at least 10%, in particular by at least 20%.
  • the amount of the side chains with a dp of 16-23 in the starch produced by the method according to the invention can preferably be reduced by at least 10%, by preference by at least 15%, in particular by at least 20% in comparison with the amount of the corresponding amylopectin side chains in wild-type plants.
  • the method according to the invention for the production of a starch with increased phosphate content encompasses the extraction of the starch from a plant and/or from starch-storing parts of such a plant and/or from a plant cell of such a plant and/or from propagation material of such a plant, said plant having been generated by the above-described method for increasing the phosphate content by reducing the activity of at least one phosphoglucomutase in genetically modified plant cells.
  • These plant cells may belong to any plant species, i.e. both to monocotyledonous and 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, particularly industrial, purposes.
  • the invention preferably relates to fiber-developing plants (for example flax, hemp, cotton), oil-storing plants (for example oilseed rape, sunflower, soybean), sugar-storing plants (for example sugarbeet, sugarcane, sugar millet) and protein-storing plants (for example legumes).
  • the invention relates to plant cells from starch-storing plants (for example, wheat, barley, oats, rye, potatoes, maize, rice, pea, cassava); potato plant cells are especially preferred.
  • starch-storing plants for example, wheat, barley, oats, rye, potatoes, maize, rice, pea, cassava
  • potato plant cells are especially preferred.
  • a variety of techniques are available for introducing DNA into a plant host cell. These techniques encompass the transformation of plant cells with T- DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, protoplast fusion, injection, the electroporation of DNA, the introduction of DNA by means of the biolistic approach and other possibilities.
  • any promoter which is active in plant cells is suitable for expressing the foreign nucleic acid molecule(s).
  • the promoter may be chosen in such a way that expression in the plants according to the invention takes place constitutively or only in a specific tissue, at a specific point in time of plant development or at a point in time determined by external influences.
  • the promoter may be homologous or heterologous.
  • Figure 1 shows a graphic representation of the change in the amylopectin side chain distribution in comparison with the wild type (WT).
  • Fig. 2 shows a schematic representation of vector pA7
  • Fig. 3 shows a schematic representation of vector JP 6-101
  • Fig. 4 shows a schematic representation of vector pBinAr-Met
  • Fig. 5 shows a schematic representation of vector JP 7-116
  • 35S cauliflower mosaic virus promoter
  • OCS polyadenylation sequence (terminator), Agrobacterium tumefaciens octopine synthase gene
  • Dhfr dehydrofolate reductase (resistance to methodrexate)
  • Seq ID 1 Nucleic acid sequence amplified from the published sequence of the plastid phosphoglucomutase (AC: AJ240053) with the primers JP-1a (5'-TTT gTC gAC ATC CAC ACg AgT TTC AAT TCC-3') and JP-1b (5'-ggg CAA CgC gAT CTA gAg AAC C-3') by RT-PCT technology as a 1.2 kb DNA pPGM2 fragment from total RNA from potato tubers;
  • Seq ID 2 Amino acid sequence of the plastid phosphoglucomutase from potato of Seq ID 1 above.
  • Starch was isolated from potato plants by standard methods, and the amylose/amylopectin ratio was determined by the method described by
  • the positions C2, C3 and C6 of the glucose units may be phosphorylated.
  • 50 mg of starch are hydrolyzed for 4 hours at 95°C in 500 ⁇ l of 0.7 M HCI.
  • the mixtures are subsequently centrifuged for 10 minutes at 15500 g and the supematants removed.
  • 7 ⁇ l of the supematants are mixed with 193 ⁇ l of imidazole buffer (100 mM imidazole, pH 7.4; 5 mM MgCI 2 , 1 mM EDTA and 0.4 mM NAD).
  • imidazole buffer 100 mM imidazole, pH 7.4; 5 mM MgCI 2 , 1 mM EDTA and 0.4 mM NAD. The measurement was carried out in a photometer at
  • the total phosphate content was determined by the method of Ames (Methods in Enzymology VIII, (1966), 115-118).
  • Approximately 50 mg starch are treated with 30 ⁇ l of ethanolic magnesium nitrate solution and ashed for 3 hours at 500°C in the muffle furnace. The residue is treated with 300 ⁇ l of 0.5 M hydrochloric acid and incubated for 30 min at 60°C. An aliquot portion is subsequently made up to 300 ⁇ l with 0.5 M hydrochloric acid, and this is added to a mixture of 100 ⁇ l of 10% strength ascorbic acid and 600 ⁇ l of 0.42% strength ammonium molybdate in 2 M sulfuric acid and incubated for 20 min at 45°C. A photometric determination at 820 nm is now carried out, using a phosphate calibration series as the standard.
  • Step 1 First, the starch suspension was stirred for 10 minutes at 960 rpm and subsequently heated, initially for one minute, at 50°C at a measuring rate of 160 rpm.
  • Step 2 The temperature is increased from 50°C to 95°C at a heating rate of 12°C per minute.
  • Step 3 The temperature is kept at 95°C for 2.5 minutes
  • Step 4 The starch suspension is cooled from 95°C to 50 C C in steps of 12°C per minute
  • Step 5 The temperature is held at 50°C for 2 minutes. After the program has finished, the stirrer is removed and the beaker covered. The gelatinized starch is now available for texture analysis after 24 hours.
  • the profile of the RVA analysis contains characteristic values which are shown for comparing different measurements and substances.
  • the following terms are to be understood as follows in connection with the present invention:
  • the maximum viscosity is understood as meaning the highest viscosity value measured in cP (centipoise) which is obtained in step 2 or 3 of the temperature profile.
  • the minimum viscosity is understood as meaning the lowest viscosity value, measured in cP, which is observed in the temperature profile after the maximum viscosity. This normally takes place in step 3 of the temperature profile.
  • the final viscosity is understood as meaning the viscosity value, measured in cP, which is observed at the end of the measurement.
  • the peak temperature is understood as meaning the temperature in the temperature profile at which the viscosity first increases by 25 cP over a period of 20 sec.
  • the supernatant is then treated with 3 volumes of ethanol, and the amylopectin which precipitates is separated by centrifugation for 5 minutes at 2000 g (RT).
  • the pellet (amylopectin) is then washed with ethanol and dried using acetone.
  • a 1% strength solution is prepared by adding DMSO to the pellet, of which 200 ⁇ l are treated with 345 ⁇ l of water, 10 ⁇ l of 0.5 M sodium acetate (pH 3.5) and
  • the relative amount of short side chains in the total of all side chains is determined via the determination of the percentage of a particular side chain in the total of all side chains.
  • the total of all side chains is determined via the determination of the total area under the peaks which represent the degrees of polymerization 6 to 23 in the HPCL chromatogram.
  • the percentage of a specific side chain in the total of all side chains is determined via the determination of the ratio of the area under the peak which represents this side chain in the HPLC chromatogram to the total area.
  • the protein was extracted following a modified method of Geigenberger and Stitt 1993 (Planta 189: 329-339).
  • Leaf material was extracted following a modified method of Geigenberger and Stitt 1993 (Planta 189: 329-339).
  • the phosphoglucomutase activity was determined following the method of Galloway and Dugger (1994, Physiol. Plantarum 92: 479-486). The activity assay was carried out in a buffer consisting of 80 mM Hepes-
  • the T-DNA of the plasmid (JP7-116) was transferred into potato plants (var. Desiree) with the aid of agrobacteria as described by Rocha- Sosa et al. (EMBO J., (1989), 8:23-29).
  • the primers JP-1a (5'-TTT gTC gAC ATC CAC ACg AgT TTC AAT TCC-3') and JP-1 b (5'-ggg CAA CgC gAT CTA gAg AAC C-3') were designed using the published sequence of the plastid phosphoglucomutase (AC: AJ240053), and a 1.2 kb DNA pPGM2 fragment from the total RNA from potato tubers was amplified by means of RT-PCT technique (Stratagene ProSTARTM HF single-tube RT-PCR system). The resulting DNA fragment (see SEQ ID No.
  • the vector pBinAR-Met was used for the subsequent cloning.
  • the vector pBinAR-Met originates from plasmid pGPTV-DHFR (Becker et al. 1992, Plant Mol. Biol. 20: 1195-1197), which constitutes a derivative of the vector pBin19.
  • pBinAR-Met contains the dhfr gene, which confers resistance to methotrexate, and instead of the 3' end of the nopalin synthase gene it contains the 3' end of gene 7 of the T-DNA of the Ti plasmid pTiACH ⁇ (Nukleotide 2106-2316; Gielen et al., 1984, EMBO J 3: 835-846).
  • the EcoRI-/-//77dlll fragment comprising the 35S promoter, the ocs terminator and the interposed portion of the polylinker was ligated into the suitably cleaved plasmid pGPTV-DHFR.
  • the resulting vector was referred to as pBinAR-Met (Fig. 4).
  • the plasmid pBinAR is a derivative of the vector plasmid pBin19 (Bevan 1984, Nucl Acids Res 12: 8711-8721 ; Frisch et al., 1995, Plant. Mol. Biol. 27:405-409; GenBank U09365) and was constructed as follows: a 529 bp fragment, which encompasses the nucleotides 6909-7437 of the cauliflower mosaic virus 35S RNA promoter, was isolated from the plasmid pDH51 (Pietrzak et al. 1986, Nucl. Acids Res.
  • a 192 bp fragment which encompasses the polyadenylation signal (3' end) of the octopine synthase gene (gene 3) of the T-DNA of the Ti plasmid pTiACH ⁇ (Gielen et al., 1984, loc. cit.) (nucleotides 11749-11939) was isolated from the plasmid pAGV40 (Herrera-Estrella et al. 1983, Nature 303:209-213) with the aid of the restriction endonucleases Hind ⁇ and PvuW.
  • the entire polylinker comprising the 35S promoter and the ocs terminator was excised from pA7 using EcoRI and Hind ⁇ and ligated into the suitably cleaved pBin19. This gave rise to the plant expression vector pBinAR (H ⁇ fgen and Willmitzer 1990, Plant Science 66:221-230).
  • the 35S promoter OCS cassette was replaced by the EcoRI-Hindlll cassette from vector JP 6-101 , which cassette consisted of the 35S promoter, pPGM2 and OCS terminator, via the EcoRI-Hindlll cleavage sites of the vector.
  • the resulting vector was termed JP 7-116 (Fig.5 ).
  • This wash procedure is repeated 3 more times, so that the starch is resuspended five times in total in fresh tap water. Thereafter, the starches are dried at 37°C to a water content of 12-17% and homogenized in a mortar. The starches are now available for analyses.
  • Example 1 The starch of various independent lines originating from the transformation described in Example 1 was isolated from potato tubers. The physico- chemical characteristics of this starch were subsequently analyzed. The results of the characterization of the modified starches are shown in Table 1 (Tab. 1) for a selection of certain plant lines which act as an example.
  • the analysis of the amylopectin side-chain distribution was carried out as described above.
  • the table contains an overview over the individual peak areas of the HPAEC chromatogram in relation to the total peak areas of wild-type plants (Desiree) and of aPGM2 (250JP 005), aPGM3 (250JP 026) and aPGM4 (250JP 060) plants (potato plants with a reduced activity of an aphosphoglucomutasell protein).
  • the number of glucose monomers in the individual side chains is shown as dp.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne un procédé permettant d'accroître la teneur en phosphate d'amidons à partir de cellules végétales génétiquement modifiées en comparaison avec les cellules végétales de type sauvage non modifiées correspondantes, selon lequel une cellule végétale est génétiquement modifiée, cette modification génétique entraînant une réduction de l'activité d'au moins une phosphoglucomutase en comparaison avec les cellules végétales de type sauvage qui n'ont pas été génétiquement modifiées. L'invention concerne en outre l'utilisation d'une ou de plusieurs molécules d'acide nucléique, dont la présence et/ou l'expression entraîne une réduction de l'activité d'au moins une phosphoglucomutase en comparaison avec les cellules végétales de type sauvage correspondantes qui n'ont pas été génétiquement modifiées, afin d'accroître la teneur en phosphate dans de l'amidon.
PCT/EP2003/014960 2002-12-30 2003-12-29 Procede de production de plantes contenant des amidons a teneur en phosphate accrue Ceased WO2004058962A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003290126A AU2003290126A1 (en) 2002-12-30 2003-12-29 Method for producing plants containing starches with an increased phosphate content

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02029067 2002-12-30
EP02029067.2 2002-12-30

Publications (1)

Publication Number Publication Date
WO2004058962A1 true WO2004058962A1 (fr) 2004-07-15

Family

ID=32668751

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/014960 Ceased WO2004058962A1 (fr) 2002-12-30 2003-12-29 Procede de production de plantes contenant des amidons a teneur en phosphate accrue

Country Status (2)

Country Link
AU (1) AU2003290126A1 (fr)
WO (1) WO2004058962A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3039140A4 (fr) * 2013-08-29 2017-01-25 Sveriges Stärkelseproducenters Förening Upa Plante transgénique

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1001029A1 (fr) * 1996-07-09 2000-05-17 Unilever Plc Procede pour augmenter la teneur en sucrose des plantes
WO2001012782A2 (fr) * 1999-08-12 2001-02-22 Aventis Cropscience Gmbh Cellules vegetales et plantes transgeniques a activite modifiee des proteines gbssi et be
WO2002034923A2 (fr) * 2000-10-23 2002-05-02 Bayer Cropscience Gmbh Cellules vegetales et plantes de monocotyledone permettant de synthetiser de l'amidon modifie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1001029A1 (fr) * 1996-07-09 2000-05-17 Unilever Plc Procede pour augmenter la teneur en sucrose des plantes
WO2001012782A2 (fr) * 1999-08-12 2001-02-22 Aventis Cropscience Gmbh Cellules vegetales et plantes transgeniques a activite modifiee des proteines gbssi et be
WO2002034923A2 (fr) * 2000-10-23 2002-05-02 Bayer Cropscience Gmbh Cellules vegetales et plantes de monocotyledone permettant de synthetiser de l'amidon modifie

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FERNIE ALISDAIR R ET AL: "Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism.", PLANTA (BERLIN), vol. 214, no. 4, February 2002 (2002-02-01), pages 510 - 520, XP002240369, ISSN: 0032-0935 *
FERNIE ALISDAIR R ET AL: "Potato plants exhibiting combined antisense repression of cytosolic and plastidial isoforms of phosphoglucomutase surprisingly approximate wild type with respect to the rate of starch synthesis.", PLANT PHYSIOLOGY AND BIOCHEMISTRY (PARIS), vol. 40, no. 11, 20 November 2002 (2002-11-20), pages 921 - 927, XP002240371, ISSN: 0981-9428 *
LYTOVCHENKO ANNA ET AL: "Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase.", PLANTA (BERLIN), vol. 215, no. 5, September 2002 (2002-09-01), pages 802 - 811, XP002240370, ISSN: 0032-0935 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3039140A4 (fr) * 2013-08-29 2017-01-25 Sveriges Stärkelseproducenters Förening Upa Plante transgénique

Also Published As

Publication number Publication date
AU2003290126A1 (en) 2004-07-22

Similar Documents

Publication Publication Date Title
US8017832B2 (en) Plant cells and plants which synthesize a starch with an increased final viscosity
CA2603919C (fr) Amidon a haute teneur en phosphate
EP1725666B1 (fr) Plantes presentant une activite reduite de l'enzyme de phosphorylation de l'amidon phosphoglucane dikinase
US7112718B2 (en) Transgenic plants synthesizing high amylose starch
EP1869089B1 (fr) Amidon de pomme de terre glutineux phosphoryle
US20060253929A1 (en) Plants with reduced activity of a class 3 branching enzyme
WO2004058962A1 (fr) Procede de production de plantes contenant des amidons a teneur en phosphate accrue
AU2002316971C1 (en) Transgenic plants synthesising high amylose starch
AU2002316971A1 (en) Transgenic plants synthesising high amylose starch

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP