WO1996012812A1 - Procede de reduction de la teneur en sucre d'un organisme - Google Patents

Procede de reduction de la teneur en sucre d'un organisme Download PDF

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Publication number
WO1996012812A1
WO1996012812A1 PCT/EP1995/002197 EP9502197W WO9612812A1 WO 1996012812 A1 WO1996012812 A1 WO 1996012812A1 EP 9502197 W EP9502197 W EP 9502197W WO 9612812 A1 WO9612812 A1 WO 9612812A1
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WIPO (PCT)
Prior art keywords
amylase
potato
tubers
organism
glucose
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PCT/EP1995/002197
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English (en)
Inventor
Jette Dina Kreiberg
Tove Martel Ida Elsa Christensen
John Erik Nielsen
Bjarne Munk Hansen
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Danisco A/S
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Publication date
Application filed by Danisco A/S filed Critical Danisco A/S
Priority to EP95923253A priority Critical patent/EP0787192A1/fr
Priority to JP8513606A priority patent/JPH10507365A/ja
Priority to AU27882/95A priority patent/AU2788295A/en
Priority to GB9521449A priority patent/GB2294266B/en
Publication of WO1996012812A1 publication Critical patent/WO1996012812A1/fr
Priority to MXPA/A/1997/002912A priority patent/MXPA97002912A/xx

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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2422—Alpha-amylase (3.2.1.1.) from plant source

Definitions

  • the present invention relates to a method of reducing the level of sugar in an organism. More in particular, the present invention relates to a plant or a part thereof (e.g. fruit, seed, tuber, shoot etc.) or a product thereof, having a reduced level of sugar, and the use of that plant or a part thereof or a product thereof as a foodstuff. Further in particular, the present invention relates to reducing the levels of the reducing sugar glucose in an organism, such as a plant - preferably a potato. In some instances it is desirable to reduce the level of sugar in an organism, in particular a plant.
  • a plant or a part thereof e.g. fruit, seed, tuber, shoot etc.
  • a product thereof having a reduced level of sugar
  • the present invention relates to reducing the levels of the reducing sugar glucose in an organism, such as a plant - preferably a potato. In some instances it is desirable to reduce the level of sugar in an organism, in particular a plant.
  • sugar in particular glucose
  • a foodstuff such as a plant
  • One way to achieve this would be to extract the sugars before forming the foodstuff. However, this process is very laborious and time consumming. It is also very expensive.
  • the presence of reducing sugars in a foodstuff can cause discolouration (such as blackening e.g. by the Maillard reaction (1)) of the foodstuff when it is subjected to high temperatures - such as through a baking or a frying process.
  • discolouration such as blackening e.g. by the Maillard reaction (1)
  • the reducing sugar forms residues with protein material in the foodstuff giving materials that are often black in colour.
  • the higher the levels of reducing sugars so there is an increase in the formation of blackened residues.
  • this is a major problem in the potato industry wherein it is undesirable to have potatoes that produce, for example, blackened pomme frites, chips, crisps or the like by such a reaction.
  • non- and phosphorylated reducing sugars e.g. glucose and fructose
  • a sucrose molecule comprises one glucose molecule and one fructose molecule
  • the general view is that the build-up of reducing sugars during storage of potato tubers, especially cold stored tubers (e.g. storage at 4°C), is due to a splitting of the sucrose molecule (3, 4, 5, 6). This indicates that sucrose is first accumulated and then split into glucose and fructose - more specificly the phosphorylated sugars - during cold storage as shown by Isherwood (1973) (4).
  • a level as high as 5 mg/g tuber can be acceptable, but not if the tubers contain reducing sugars above that level (8, 9).
  • Two varieties which are typically used by the chip/crisp industry are Saturna and Record.
  • a variety like e.g. Bintje is a well-known table potato which can be used for making chips or crisps very early in the season, but soon its build-up of glucose and fructose is too high.
  • a variety completely unsuited for chip/crisp making is Dianella, an old starch variety, its content of reducing sugar is much too high to be used in crisp/chip making.
  • the chip or crisp industry is therefore very dependent on specific varieties which accumulate relatively low levels of reducing sugars compared with other varieties.
  • Another important factor in the determination of any variety's content of reducing sugars is the maturity of the harvested crop before it is stored. This is greatly influenced by the actual growing season, e.g. a cold, rainy season will produce a somewhat immature crop, which will accumulate higher and more fluctuating levels of reducing sugars than a fully mature crop (8, 2, 1, 10).
  • a survey over 3 years of 100 crop sites in the United Kingdom involving monitoring at least 10 parameters (such as temperature, rainfall, soil cultivation etc.) was performed by Kirkman, Young et al (1991) (11).
  • sprouts have to be cut off before slicing of the tubers.
  • the sprouts grow quickly at the warm storage temperature (above 7°C) and, in doing so, use the starch in the tuber leading to losses in production. If they are left unattended then the whole crop is unsuitable for chip or crisp production (12).
  • Starch is an insoluble polysaccharide carbohydrate. It is one of the principal energy reserves of plants. It is often found in colourless plastids (amyloplasts), in storage tissue and in the stroma of chloroplasts in many plants. It comprises two main components: amylose and amylopectin. Both components consist of straight chains of ⁇ (1,4)-linked glycosyl residues but in addition the latter component includes ⁇ (1,6) branches (13).
  • the enzyme ⁇ -amylase is one of the many enzymes involved in the degradation of starch.
  • ⁇ -amylase is best known from cereals where it splits the ⁇ -1-4 glucosidic bindings in the amylose and amylopectin molecules thereby liberating maltose, maltotriose and ⁇ -dextrins (molecules containing the ⁇ -1-6 glucosidic bindings of the amylopectin branches (15, 16).
  • ⁇ -amylase, ⁇ -glucosidase, debranching enzyme, disproportionating enzyme take over producing finally glucose and glucose -1 -phosphate (16).
  • Glucose is quickly phosphorylated by hexokinase to glucose-6-phosphate which is also the product of phosphoglucomutases action on glucose- 1-phosphate.
  • Glucose -6-phosphate is converted by phosphohexose isomerase to fructose-6-phosphate.
  • Glucose-1-phosphate reacts with UTP by the action of UDPG-pyrophosphorylase creating UDPG. This reacts with either fructose or fructose-6-phosphate by the action of either sucrose synthase or sucrose-P-synthase thereby creating sucrose-6-phosphate.
  • a phosphatase cleaves the sucrose-6-phosphate liberating sucrose, then invertase can split sucrose into glucose and fructose.
  • starch phosphorylase which degrades starch primarily by a phosphorylytic reaction (as opposed to a hydrolytic reaction by amylases) (17, 18); phosphofructophosphorylase (PFP) - which is reported to be cold labile thereby blocking the entry of the hexoses into the glycolytic cycle and leading to an accumulation of hexoses (8); phosphofructokinase (PFK) - which is also reported to be cold labile thereby blocking the entry of the glycolytic cycle (8, 19, 20); and invertase - where the levels are higher in cold stored tubers compared with tubers kept at warmer temperatures (21).
  • the first approach was that of Rita Zrenner et al (27), and related to using a 35S promoter to express antisense vacuolar invertase in potatoes.
  • the workers found that in potatoes containing the antisense invertase the amounts of starch, glucose, fructose and sucrose in tubers stored at room temperature showed no difference compared to the control (i.e. non-transformed potato).
  • Tubers which were cold stored showed a strong inhibition of the invertase enzyme (up to 75%) contrary to nontransformed control stored tubers.
  • the expression of the vacuolar invertase mRNA level
  • the third approach was that of Duvening et al (28), Virgin et al (29), Lorberth et al (30), Abel et al (31), Blundy et al (32), Donath and Druger (33), Geuveia (34, 35), Krause et al (36), Rouwendal et al (38), Burrell and Mooney (37) which related to the transformation of potatoes with gene sequences encoding various carbon metabolism enzymes from plants as well as E. coli enzymes. In each of these instances there was either no effect on the sugar and starch quantities, or the levels of starch were lower whereas the level of sugar had considerably increased.
  • this approach does not solve the problem of reducing the levels of reducing sugars in potatoes, let alone the problem of reducing the levels of reducing sugars under cold conditions.
  • this approach does clearly highlight the difficulties faced by the food industry to reduce the levels of reducing sugars - such as glucose and fructose - in the potatoes.
  • a method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in an organism comprising at least partially inhibiting the activity of ⁇ -amylase in the organism.
  • a method of selectively reducing the level of glucose, relative to the level of other reducing sugar(s), in an organism comprising at least partially inhibiting the activity of ⁇ -amylase in the organism, wherein the organism is one that is capable of enzymatically degrading amylopectin or amylose.
  • a cell, tissue, organ or transgenic organism capable of enzymatically degrading amylopectin or amylose comprising an exogenous nucleotide sequence wherein a transcript from the expression thereof at least partially inhibits the activity of ⁇ -amylase in the cell, tissue, organ or transgenic organism.
  • a construct, a transformation vector or an expression vector comprising a promoter and an exogenous nucleotide sequence according to the present invention.
  • a foodstuff prepared from an organism according to the present invention.
  • a sixth aspect of the present invention there is provided baked or fried potato prepared from an organism according to the present invention.
  • a seventh aspect of the present invention there is provided the use of antisense ⁇ -amylase to selectively reduce the level of glucose, relative to the level of other reducing sugar(s), in an organism capable of enzymatically degrading amylopectin or amylose.
  • the activity of the ⁇ -amylase is inhibited by expression in the organism of an exogenous nucleotide sequence coding for a first transcript capable of binding to a second transcript of a nucleotide sequence coding for ⁇ -amylase thereby preventing the translation of the second transcript.
  • the exogenous nucleotide sequence has a sequence that is at least partially anti-sense to the sequence shown as SEQ. ID. NO:1.
  • the exogenous nucleotide sequence has a sequence shown as SEQ. ID. NO:2, or is a variant, homologue or fragment thereof wherein the variant, homologue or fragment thereof can inhibit the activity of ⁇ -amylase (e.g. prevent translation of the second transcript).
  • the exogenous nucleotide sequence is expressed under the control of a 35S promoter.
  • the organism is a plant.
  • the organism is a potato.
  • the foodstuff is a crisp, chip or pomme frite.
  • ⁇ -amylase activity increases in cold conditions.
  • ⁇ -amylase is one of the enzymes involved in the degradation of starch.
  • a potato ⁇ -amylase is the subject of EP-B- 0470145.
  • an anti-sense ⁇ -amylase was placed under the control of a 35S promoter and transformed into a potato plant.
  • the present invention shows that expression of anti-sense ⁇ -amylase led to a significant reduction in the levels of just the reducing sugar glucose - particularly under warm conditions - in the potato over a long period of time.
  • the present invention shows that at least partial inhibition of the endogenous ⁇ -amylase enzyme in stored potato tubers both at warm and cold storage temperatures specifically affects glucose levels in tubers.
  • inventions include the expression of the exogenous nucleotide sequence according to the present invention under the control of a tissue specific promoter, a temperature inducible promoter or a native ⁇ -amylase promoter - including combinations thereof.
  • the present invention also includes constructs comprising the same.
  • FIG. 15 An example of a preferred temperature inducible promoter is the promoter as shown in Figure 15.
  • This promoter which comprises the sequence shown as SEQ. ID. NO:3, is a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoRI fragment isolated from Solanum tuberosum, or a variant, homologue or fragment thereof.
  • tissue specific promoter is the promoter sequence shown as SEQ. I.D. No. 3. This promoter is a tuber specific, cold-inducible promoter.
  • Further embodiments of the present invention include the presence of at least one inhibitor of at least one other of the enzymes involved in the degradation of starch.
  • the expression of the exogenous nucleotide sequence according to the present invention may be in conjunction with the expression of an exogeneous nucleotide sequence that at least partially inhibits the activity of vacuolar invertase - such as anti-sense invertase.
  • selectively reducing relative to other reducing sugar(s) means that the levels of glucose are specifically reduced more so than the levels of other reducing sugars, in particular fructose. In a preferred embodiment the term means that the levels of glucose are lowered substantially more than the levels of fructose.
  • reducing sugar is used in its normal sense in the art - i.e. a sugar capable of acting as a reducing agent in solution as indicated by a positive Benedict's test and ability to decolourise potassium permanganate solution. Most monosaccharides are reducing sugars, as are most disaccharides except sucrose. Typically reducing sugars include glucose and fructose.
  • ⁇ -amylase means that preferably there is at least about 15% inhibition. More preferably there is at least about 50% inhibition. Even more preferably there is at least about 70% inhibition - or even higher (e.g. 95%).
  • inhibiting the activity of ⁇ -amylase includes the organism's ⁇ -amylase per se being inhibited. It also includes reducing the level of replication of the ⁇ -amylase gene. It also includes reducing the level of transcription of the ⁇ -amylase gene. It also includes reducing the level of translation of the transcript of the ⁇ -amylase gene. Preferably, it means the latter.
  • At least partially anti-sense to the sequence shown as SEQ. I.D. No. 1 means that the nucleotide sequence must express a transcript that is at least anti-sense to some of the nucleotide sequence of SEQ. I.D. No. 1 but wherein the nucleotide sequence must be able to inhibit at least partially the activity of ⁇ -amylase.
  • the nucleotide sequence has a sequence that is at least substantially the same as that shown as SEQ. I.D. No. 2 - i.e. is the same as SEQ. I.D. No: 2 or is a variant, homologue or fragment thereof.
  • the nucleotide sequence is that shown as SEQ. I.D. No. 2.
  • the term "construct” - which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes the exogenous nucleotide sequence directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron. intermediate the promoter and the GOI.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred.
  • Various markers exist which may be used in, for example, plants - such as mannose.
  • Other examples of markers include those that provide for antibiotic resistance - such as resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a plant cell.
  • nucleotide' includes genomic DNA, cDNA and synthetic DNA.
  • exogenous means that the nucleotide sequence is not natural to the organism of the present invention.
  • nucleotide sequence coding for anti-sense ⁇ -amylase is not natural to potato.
  • 'organism' in relation to the present invention includes any organism capable of enzymatically degrading amylopectin or amylose. Typical examples of such organisms include plants, algae, fungi and bacteria, as well as cell lines thereof.
  • the term means a plant or cell thereof, preferably a dicot, more preferably a potato.
  • transgenic organism' in relation to the present invention means an organism comprising either an expressable construct according to the present invention or a product of such a construct.
  • the transgenic organism can comprise an exogenous nucleotide sequence (as herein described) under the control of a suitable exogenous promoter, such as the 35S promoter or the cold-inducible promoter (as described herein); or an exogenous nucleotide sequence under the control of a native promoter, such as the native ⁇ -amylase promoter.
  • variant include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the ability to express a transcript that can prevent the translation of the afore-mentioned second transcript, or to act as a promoter in an expression system (such as the transformed cell or transgenic organism according to the present invention), respectively.
  • homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence has the ability to express a transcript that can prevent the translation of the afore-mentioned second transcript, or to act as a promoter, respectively.
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology, more preferably at least 95%, more preferably at least 98% homology.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • Several techniques exist for inserting the genetic information the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
  • the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of a plant such as a plant of the family Solanaceae, in particular of the genus Solanum, especially Solanum tuberosum.
  • the vector system may comprise one vector, but comprises preferably two vectors; in the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
  • One extensively employed system for transformation of plant cells with a given promoter or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • a non-limiting example of such a Ti plasmid is pGV3850.
  • the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect a plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the nucleotide sequence or construct may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used.
  • a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A.
  • Agrobacterium tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
  • Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. See also Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
  • cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 332, pUC series, M13 mp series, pACYC 184 etc.
  • the construct or nucleotide sequence can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is used for the transformation in E. coli.
  • the E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered.
  • a sequence analysis As a method of analysis there is generally used a sequence analysis, a restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid. After each introduction method of the desired nucleotide sequence or construct in the plants further DNA sequences may be necessary. If for example for the transformation, the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells is being intensively studied and is well described in EP 120 516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46 and An et al., EMBO J. (1985) 4:277-284.
  • Direct infection of plant tissues by Agrobacterium is another simple technique which may be employed.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium, e.g. in a solution.
  • the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • a highly preferred embodiment of the present invention therefore relates to a method of lowering the level of a specific reducing sugar (i.e. glucose) in an organism capable of enzymatically degrading amylopectin or amylose by at least partially inhibiting the activity of ⁇ -amylase in the organism.
  • a specific reducing sugar i.e. glucose
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • This sample is an E. Coli bacterial (DH5 ⁇ -) stock containing plasmid pJK4 (described later).
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • DH5 ⁇ -gPAmy 351 (Deposit No. NCIMB 40682).
  • This sample is an E. Coli bacterial stock containing the plasmid pBluescript containing the EcoRI 5.5 genomic DNA fragment isolated from potato (Solanum tuberosum).
  • the EcoRI 5.5 fragment contains the promoter region and part of the 5' untranslated sequence of the structural gene of a potato ⁇ -amylase gene.
  • the plasmid was formed by inserting the EcoRI 5.5 kb potato fragment into the polylinker of the vector pBS (Short et al [1988] Nuc. Acid. Res. 16:7583-7600).
  • the promoter may be isolated from the plasmid by enzyme digestion with EcoRI and then extracted by typical separation techniques (e.g. gels).
  • Figure 1 is a map of each of pIV21 and pEPL;
  • Figure 2 is the nucleotide sequence of the AmyZ4 clone;
  • Figure 3 is a restriction map of the pBKS- plasmid;
  • Figure 4 is a restriction map of gPAmy 351 clone.
  • Figure 5 is the anti-sense nucleotide sequence of the AmyZ4 clone
  • Figure 6 is a restriction map of pJK5
  • Figure 7 is a restriction map of pJK4
  • Figure 8 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 4oC (cold conditions) and 12°C (warm conditions);
  • Figure 9 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12°C (warm conditions);
  • Figure 10 presents graphs of ⁇ -amylase activity of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12°C
  • Figure 11 presents graphs of beta-amylase activity of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12° C (warm conditions);
  • Figure 12 presents graphs of glucose and fructose levels of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12° C (warm conditions);
  • Figure 13 presents graphs of ⁇ -amylase activity of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12°C (warm conditions);
  • Figure 14 presents graphs of beta-amylase activity of a transgenic potato according to the present invention and a control potato at 4°C (cold conditions) and 12oC (warm conditions);
  • Figure 15 is the sequence of the promoter in the gPAmy 351 clone (see above).
  • Figure 4 is a pictorial representation of plasmid gPAmy351.
  • the highlighted portion is a EcoRI - SalI fragment isolated from potato (Solanum tuberosum).
  • the EcoRI - Sail fragment contains the EcoRI 5.5 kb fragment (called subclone Eco 5.5) - which is indicated by the line shown at the bottom of the drawing.
  • the EcoRI 5.5 kb fragment contains the promoter region and part of the 5' untranslated .sequence of the structural gene of a potato ⁇ -amylase.
  • Trie following restriction enzyme sites are shown in Figure 4: E: EcoRI, Ha: HaeIII, Sp: Sspl, H: HindIII, P: Pvul, S: Sall.
  • Tuber of Solanum tuberosum L. ssp. tuberosum cvs Dianella, Satuma, Bintje and Record were harvested from a field located near Holeby in the southern part of Denmark. All four varieties are well established. Dianella is used for starch production, Bintje is predomi ⁇ nantly a table potato, and Satuma and Record are used by the chip/crisp industry.
  • the "Ceralpha” kit and the "Betamyl” kit were obtained from Megazyme (Aust) Pty.Ltd., 6 Altona Place, North Rocks, NSW 2151, Australia.
  • the sugar determination kit (Sucrose/D-Glucose/D-Fructose) was obtained from Boehringer Mannheim. All other chemicals were obtained from Sigma, Merck, Ferak, Difco Lab. or BDH Chemicals Ltd.
  • Transgenic as well as control lines of cv Dianella, cv Satuma, cv Bintje and cv Record were micropropagated in vitro under sterile conditions.
  • Independent transgenic lines transformed with either pJK4 or pJK5 and non-transformed control lines were maintained sterile in agar in a growth chamber.
  • Single node (containing a leaf) cuttings from the steril plants were planted in 5.5 cm pots and placed under cover in a greenhouse. The cover was gradually removed (over 20 days) to harden the plants.
  • the in vitro micropropagated potato plants were planted in a field by hand when they were approx. 10-12 cm tall. The field was managed using ordinary agronomic handling for potato fields.
  • Each line (whether it was transgenic or a non- transgenic control line) was placed in two or four repeats. At harvest all tubers from every line in the two or four repeats were collected, mixed and randomly divided into two portions. One was placed at 4°C (cold storage conditions) and the other at 12°C (warm storage conditions) in the dark.
  • the homogenate was filtered through miracloth and the filtrate centrifuged at 10 krpm for
  • the extracts were divided into two tubes. One for enzyme analyses, which was stored at - 20°C immediately and the other for sugar analysis was boiled for 10 min. and then stored at -20°C.
  • the substrate contains blocked p-nitro- phenyl maltoheptaoside (BPNPG7), glucoamylase and ⁇ -glucosidase in the Ceralpha kit and p-nitrophenyl maltopentaoside (PNPG5) and ⁇ -glucosidase in the Betamyl kit.
  • 0.1 ml substrate was added to 0.35 ml 50 mM Sodium-citrate pH 6.5 and 0.05 ml potato extract and incubated at 30°C for 30 min. At time 0, 10, 20 and 30 min., 0.1 ml was removed and mixed with 0.15 ml stop solution to terminate the reaction. Stop solution was 0.5 M glycin, 2 mM EDTA adjusted to pH 10.0 with NaOH.
  • the activity was expressed as OD 405 h/mg extracted protein. Each sample was determined in duplicate. In the ⁇ -amylase assay, the amount of p-nitrophenol released was directly proportional to the incubation time. This was also the case with the ß-amylase assay after the first 10 min. incubation.
  • Amylose hydrolysis was followed by I 2 /KI staining method. 250 ⁇ l 1,2 % potato amylose in 0.1 M Na-acetat pH 7 was incubated with 50 ⁇ l 4o mg/ml BSA and 100 ⁇ l extract. The enzymatic reaction was performed at 40°C. The reaction was stopped by adding 20 ⁇ l 3 M HCl to 50 ⁇ l enzym reaction sample. 1 ml I 2 /KI (0.026 g/ml I 2 + 0.26 g/ml KI diluted 1: 1000) was added and the amount of amylose was measured spectrophotomerically at 620 nm. Protein concentration was determined according to Bradford (50) using the Bio-Rad protein assay kit (Ca, USA) with ⁇ -globulin as standard.
  • 100 g peeled potatoes were homogenised with 100 ml of buffer (50 mM acetate pH 5.5, 20 mM DTT) and 10 g Dowex in a Warring Blender. After homogenisation the extract was filtered through miracloth and the filtrate was centrifuged at 14.000 rpm for 10 min. at 4°C.
  • the supernatant was precipitated with 20% (NH 4 ) 2 SO 4 by slowly stirring for 30 min.
  • the precipitated proteins were collected by centrifugation and discarded.
  • the supernatant was then applied on a Blue Sepharose column.
  • the affinity column material Blue Sepharose CL-6B (Pharmacia) is the dye Cibacron Blue F3G-A covalently attached to the cross-linked agarose gel Sepharose CL-6B.
  • a 32 ml column was used, equillibrated with 50 mM Tris pH 6.4. The flow was 30 ml/h. 40 ml supernatant sample was applied to the column. Unbound proteins were eluted with 64 ml 50 Tris pH 6.4. The proteins that bound to the affinity material were then eluted with a increasing gradient from 0 - 0,5 M NaCl in total 400 ml of the buffer. Fractions of 8 ml were collected. All operations were performed at 4°C. The fractions were analysed for ⁇ -amylase, ⁇ -amylase and protein.
  • the fractions containing ⁇ -amylase activity were pooled and concentrated appr. 5 times by pressure dialysis using Amicon (membrane YM-10) to 6 ml. This fraction was desalted on a PD-10 gelfiltration column (Pharmacia) equillibrated with 50 mM Tris, 0.1 M NaCl pH 7.
  • the enzymatic reaction mixture contained 200 ⁇ l substrate, 200 ⁇ l 50 mM Na-acetate pH 6.5 and 100 ⁇ l enzyme preparation. The mixture was incubated at 37°C for 20 hr. The reaction was stopped by boiling for 5 min. 20 ⁇ l sample was then analysed on Dionex HPLC. The analysis was performed on a Dionex 4500i with a pulsed electrochemical detector used in pulse amperometric detection mode. A CarboPac PA1 column (4 ⁇ 250 mm) was used. The samples were eluted with the following gradient:
  • Plasmid DNA transformed into E.coli cells of JM109 or DH5 ⁇ was isolated as described in EP-B-0470145. Plasmid DNA transformed into LBA4404 was isolated as follows:
  • the plasmid preparation was as described in EP-B-0470145.
  • small scale preparation of plasmid DNA was performed as follows.
  • Bacterial strains harbouring the plasmids were grown overnight in 2 ml L-Broth (LB) medium with ampicillin added (35 ⁇ g/ml). The operations were performed in 1.5 ml
  • the cells from the overnight culture were harvested by centrifugation for 2 min., washed with 1 ml 10 mM Tris-HCl (pH 8.5), 50 mM EDTA and centrifuged for 2 min. The pellet was suspended in 150 ⁇ l of 15% sucrose, 50 mM Tris-HCl (pH 8.5), 50 mM EDTA by vortexing. 50 ⁇ l of 4 mg/ml lysozyme was added and the mixture was incubated for 30 min. at room temperature and 30 min. on ice. 400 ⁇ l ice cold H2O was added and the mixture was kept on ice for 5 min, incubated at 70-72°C for 15 min. and centrifuged for 15 min. To the supernatant was added 75 ⁇ l 5.0 M Na-perchlorate and
  • the pellet was suspended in 300 ⁇ l 0.3 M Na-acetate and 2-3 vol. cold ethanol was added. Precipitation was accomplished by storing at either 5 min. at -80°C or O/N at -20°C, centrifuging for 5 min., drying by vacuum for 2 min. and redissolving the pellet in 20 ⁇ l H 2 O. The yield was 5-10 ⁇ g plasmid DNA.
  • the banded plasmid DNA was withdrawn from the tubes using a syringe and the ethidium bromide was extracted with CsCl-saturated isopropanol 7-8 times.
  • the CsCl was removed by dialysis in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA for 48 hours with three changes of buffer.
  • the DNA was precipitated by adjusting to 0.3 M Na-acetate and adding 2-3 vol.cold ethanol.
  • the small scale plasmid preparation from E. coli was usually followed by a LiCl precipitation to remove RNA from the DNA solution.
  • the small scale prepared plasmid DNA was dissolved in 100 ⁇ l destilled water. 1 vol of 5M LiCl was added and the mixture incubated at -20°C for 30 min followed by centrifugation at 12,000 rpm. for 15 min, 4°C. The supernatant was transferred to a new eppendorf tube and 2 vol TE buffer or water was added. Precipitation with 2.5 vol of 96% ethanol was accomplished by storing either 10 min. at -80°C, or O/N at -20°C. The DNA was precipitated by centrifuging for 15 min. 12,000 rpm ,at 4°C, drying by vacuum for 2 min and redissolving in 18 ⁇ l of TE or water.
  • Genomic potato DNA was isolated according to Dellaporta et al (1983) and also the work reported in ref. 51. Construction of pEPL and pIV21 (DW2t)
  • pEPL is constructed from pCaMVCN (44, 45) in which the CAT gene is removed by a PstI digestion.
  • a small linker (linker: Pstl-BamHI-BaII-PstI) is inserted into this plasmid PstI site, giving the plasmid called pLise(pL).
  • pL is digested with Hindi and BgIII and the resultant fragment containing the 35S promoter and the NOS terminator is cloned into another pL plasmid digested with EcoRV and BgIII. Both EcoRV and Hindi are blunt ended sites.
  • the resulting construct is called pEnhanccd-Lise (pEL).
  • pEL differs essentially from pCaMVCN in that it contains a variant 35S promoter with a tandem duplication of the 250 bp of the upstream sequence of the promoter.
  • the variant 35S promoter has a transcriptional activity approximately ten times higher than the natural 35S promoter (53).
  • pEL is digested with PstI and BgIII, thereby removing the NOS terminator, and a CaMV terminator (DW2t) is inserted instead. Finally, a linker (PstI-BamHI-SmaI-SacI-SaII-SphI) is inserted into the PstI site situated between the enhanced E35S promoter and the CaMV terminator.
  • This plasmid is called pEPL (see Figure 1).
  • pIV21 is constructed from pHC79 (47) in which a PvuII and BglII digest removed the major part of sequences which are not pBR322 (46) sequences.
  • a PvuII-BglII fragment containing streptomycin resistant (SpRSm R , R702) was ligated in and the resulting plasmid is called pIV1.
  • a HindIII plus EcoRI digest of pIV1 removed the rest of the non-pBR322 sequences.
  • a pUC19 (40) EcoRI/HindIII polylinker was inserted between the HincII/EcoRI sites in pIV1, and this plasmid is called pIV10.
  • the 800bp 35S promoter from pBI121 digested with BamHI and HindIII was cloned into the xbaI-HindIII sites in pIV10 thereby creating the plasmid pIV21.
  • Genomic potato DNA or plasmid DNA was digested with appropriate restriction enzymes and used for Southern transfer (Southern) using Hybond N or N+ membranes (Amersham International) and hybridized according to supplier's instructions (55).
  • the LBA 4404 strain was kept at YMB plates (pH 7.0) containing 100 mg/ml of rifampicin (Sigma) and 500 mg/ml of streptomycin (Sigma). 2.5 ml of LB medium (pH 7.4) was inoculated with the bacteria. The suspension was left growing for 24 hours at 28°C in an incubation shaker at 300-340 rpm. The suspension was then diluted 1:9 with LB and incubated for another 2-3 hours at 28°C and 300-340 rpm. When OD was 0.5-1, 25 ml aliquots of the cells were harvested in 50 ml tubes in a cooling centrifuge at 10.000 rpm, 5 min, 4°C.
  • the tubes were placed on ice and the pellet resuspended in 0.5 ml of 20 mM CaCl 2 . 0.1 ml aliquots of the resuspended cells were quickly frozen in 1 ml cryotubes in liquid nitrogen and stored at -80°C.
  • the plates were incubated for 48 hours at 28°C or until the colonies had a suitable size. This was the first round of selection. Only bacteria transformed with a plasmid containing the NPT II gene conferring kanamycin resistance is able to survive on the kanamycin plate. For the second round of selection six of the obtained colonies were transferred to a YMB plate containing 100 mg/l of rifampicin, 500 mg/l of streptomycin and 50 mg/l of kanamycin. LBA 4404 is resistant to rifampicin and streptomycin and the plasmid confers resistance to kanamycin. The plates were incubated at 28°C until the colonies reached a suitable size (approx. 4-5 days).
  • the colonies were tested for their plasmid content. Plasmid preparations of the colonies were generated and the DNA was digested with appropriate restriction enzymes and run on a 1 % agarose gel to ensure that the plasmid and the inserted fragment had the right size. The digested DNA was blotted onto a Hybond N+ membrane and hybridised with an appropriate radioactively labelled probe (a fragment of the plasmid DNA or insert).
  • a culture of the transformed LBA 4404 bacteria were made by inoculating 2 ml of YMB (pH 7.0) with the bacteria and incubating at 28°C for 24 hours. The suspension was diluted 1:10 and incubated for another 18 hours. The bacteria was centrifuged at 10.000 rph, 4°C for 10 min. and the pellet rinsed twice with 2.5 ml of 2 mM magnesium sulfate, before resuspension in liquid MBa to an OD 660 nm of 0.5.
  • the potato plant material used for transformation was maintained in vitro at MBa medium added 2 ⁇ M. STS (57, 58). By multiplication top shoots as well as nodes were applied, if the leaves were big they were removed. 5 shoots per container with 80 ml medium was left growing at 25°C and 30-35 days after subcultivation the nodes could be used for transformation.
  • the stems of micropropagated plants were cut just above and beneath the node so that only the internodes are used, these may possibly be divided so that the explants are approx. 4 mm long.
  • the explants were floated in the bacterial suspension for 30 min. and blotted dry on a filter paper and transferred to co-cultivation plates (MBa co).
  • the explants were covered with filter paper moistened in liquid MBa, and the plates were covered with cloth for 3 days and left at 25°C. The explants were then washed in liquid MBa containing 800 mg/l. 2 explants per ml were shaken for 18 hours, then blotted dry and transferred to selection medium.
  • the selection medium was solid MBb added 50 mg kanamycin, 800 mg carbenicillin (Duchefa), 0.1 mg GA 3 (Gibberellic Acid,Sigma) and 1 mg t-Zeatin per litre. The carbenicillin was added to kill any remaining Agrobacteria.
  • the selection medium was subcultivated every 3 weeks. Regeneration of whole potato plants
  • a 5 ⁇ M stock of STS was made from 0.19 g of Na.S 2 O 3 -5H 2 O and 10. 19 mg of AgNO 3 dissolved in 7 ml of water and sterilised by filtration.
  • the plantlets were rinsed in lukewarm water to remove residues of media and planted in small pots with TKS 2 instant sphagnum (Flora Gard, Germany). The plantlets were kept moist during the planting and watered after. The pots were placed in a "tent" of plastic with 100 % humidity and 21-23°C, until the plantlets were rooted in the soil. Then the tent was removed and the plants watered regularly. After 4 weeks of growth the plants were potted into large pots (diameter 27 cm) and transferred to a growth chamber with 16 hours day 22°C and 8 hour night 15°C. When the plants had wiltered down, the tubers were harvested. Histochemical localisation of beta-glucuronidase (GUS) activity
  • X-gluc 5-bromo-4-chloro-3-indolyl- ⁇ -glucoronidc
  • X-gluc 5-bromo-4-chloro-3-indolyl- ⁇ -glucoronidc
  • the sections were incubated in X-gluc for 2-12 hours at 37°C. Care was taken to prevent evaporation.
  • the X-gluc was removed and 96 % ethanol was added to the tissue sections to extract chlorophyll and other pigments. Incubation in ethanol was overnight at 5°C and the following day the tissue was transferred to a 2 % sucrose solution and after approx. 1 hour examined in a dissection scope.
  • JMP R version 2 from SAS Institute.
  • JMP is a statistical visualization software for the Apple R Macintosh 11 .
  • pAmyZ4 encodes a 407 amino acid long potato ⁇ -amylase precursor and in addition contains 149 bp 5' and 201 bp 3' untranslated sequences (see figure 2 for the potato AmyZ4 sense sequences) positioned in the EcoRI site of the plasmid pBSK-'s polylinker. See map of pBKS- in figure 3. This cDNA clone is used to prepare two anti-sense clones as follows.
  • An ⁇ -amylase antisense construction was made by digesting the pAMYZ4 cDNA clone with SaCI and EcoRV. This 1640 bp fragment was then subcloned into the SmaI and SacI digested pIV21 (DW2t) plasmid creating the pIVZ4 Sac-Eco plasmid. This places the ⁇ - amylase encoding sequences in the antisense direction downstream of the 35S promoter and upstream of the DW2t terminator. (See figure 5 for the antisense sequence). A partial HindIII digest of pIVZ4Sac-Eco liberates a fragment of approx.
  • This plasmid is called pEPLZ4Sac-Eco and a partial HindIII fragment containing the E35S promoter, the antisense potato sequence and the DW2t terminator was further subcloned into a HindIII digested pBI121, thereby creating the binary plasmid pJK4, see figure 7.
  • DNA of the constructs pJK4 and pJK5 was purified from E.coli JM109 cells (plasmid isolation, see Materials and Methods) and the specific direction of the insert in pJK4 was verified by sequencing using a primer which primes 51 bp before position -90 in the 35S sequence. Transformation and production of potato plants
  • a leaf from each produced plantlets was GUS-tested (see Materials and Methods) and if it was positive then the plant was transferred into a pot and grown in a growth chamber at 22°C 16 h day and 15°C 8 h night. Tubers from pot-grown plants were likewise GUS-tested to verify the transgenic status, and then genomic DNA was isolated as described in Materials and Methods.
  • Table 1 shows the distribution of integrated copies of 40 independent transgenic potato plants. Genomic DNA was isolated from each plants and digested with HindIII. This cleaves at one end of the GUS gene in all the constructions leaving the GUS gene intact and the left border of the T-DNA at the other end (see e.g. figure 6) of the GUS gene.
  • Tables 2 and 3 show the number of independent transgenic lines chosen for the field test 1993 and 1994, respectively.
  • the produced constructs pJK4 and pJK5 do not have any undesirable effects on already well-known existing potato varieties. This is important for an exploitation of the present invention.
  • the transgenic potato plants behave and resemble the specific variety that was originally transformed (cvs Satuma, Record, Dianella or Bintje, see Materials and Methods), and thus were phenotypically true to type.
  • the sugar analysis was performed as described in Materials and Methods. Each single tuber was analysed for its content of the disaccharide sucrose (composed of one glucose linked to one fructose molecule), glucose and fructose.
  • the kit used for measuring these sugars measures the total amount in the tuber extract.
  • Two lines (a transgenic Record and a transgenic Bintje) showed significantly lower levels of reducing sugars compared with the corresponding in vitro micropropagated, non-transformed control lines. Then a possible effect of antisensing the tuber ⁇ -amylase can be seen in 2 out of 28 independent transgenic potato lines.
  • the individual reducing sugar levels (glucose and fructose) of respectively the transgenic Bintje line K125-2.1, transformed with pJK5 (see Materials and Methods) and the nontransformed Bintje control line, after respectively 4°C and 12°C storage are shown in figures 8A and 8B. It is clear from the figures that the sampled tubers from both K125-2.1 and Bintje control accumulate the reducing sugars after storage at 4°C compared with tubers sampled after 12°C storage. Five tubers from each line and storage temperature were sampled on the indicated dates and individually analysed as explained in Materials and Methods. The average reducing sugar content of the sampled five tubers is shown in figure 8. Variation in the sugar content was much larger from tuber to tuber than e.g.
  • the tubers from both lines stored at 12°C are usable for chips/crisps.
  • the upper limit content of reducing sugars in potato tubers used for crisping (chip/crisp making) is shown in figures 8A and 8B by a dashed line (2.5 mg/g tuber fresh weight, 8).
  • the potato variety Bintje is often used as a table potato and in some cases for French fries production. Only very early in the new potato season can Bintje be used for chip/crisp production, this is normally long before the month of October, at that time chips/crisp produced from cv Bintje will be dark brown and taste badly. The reason for this is clearly seen in figure 8B where the tubers from the control Bintje line contain much too high levels of glucose and fructose.
  • the mean ⁇ standard error is indicated and one, two or three stars indicate the level of significance.
  • the amount of reducing sugars in tubers from K125-2.1 is significantly (P>0.001) lower than both 4°C and 12°C storage compared with tubers from control
  • Bintje at the same storage temperature ⁇ -amylase activities in tubers of K125-2.1 and control Bintje after storage at 4°C and 12°C ⁇ -amylase activity was measured in the tuber extracts as explained in Materials and Methods. The enzyme activity was measured in the extracts produced from tubers from which also the sugar content (see above) was measured. The sampling period and dates therefore correspond with the sugar dates (figures 8 and 9). In figure 10 is shown the ⁇ -amylase activity expressed as OD 405nm /h/mg protein and each point represents the average of in most cases five individually analysed tubers. ⁇ -amylase activity (using the megazyme kit) has previously been measured in stored potato tubers by Cochrane et al.
  • Figure 10 and Table 5 show that tubers (on the average) from K125-2.1 have significanyly lower ⁇ -amylase activity compared with the control line Bintje after both 4°C storage (P> 0.001) and 12°C storage (P> 0.01).
  • Figure 10A and table 5 line ⁇ -amylase "4°C storage” show the results with the 4°C stored tubers and Figure 10B and table 5 with the 4°C stored tubers and Figure 10B and Table 5 line ⁇ -amylase "12°C storage”.
  • K125-2.1 . tubers not only have a significantly lower reducing sugar level than the control Bintje line, but also significantly lower ⁇ -amylase activity compared with the control line, whether the tubers are stored at 4°C or 12°C.
  • ß-amylase activities in tubers of K125-2.1 and control Bintje after storage at 4°C and 12°C ß-amylase was measured in the same tuber extracts as ⁇ -amylase. The exact method for determination of ß-amylase is explained in Materials and Methods. As with the sugar measurement there are larger variations seen between the tubers so the average of five (in most cases) individually analysed tubers is shown per point in Figure 11A and B.
  • Figure 11A the ß-amylase activity results of the sampled 4°C stored tubers, obtained from K125-2.1 and control Bintje line.
  • a statistical analysis obtained from the results shows that there is no significant difference in the ß-amylase activity from K125-2.1 or control Bintje tubers (see table 5 ß-amylase line "4°C storage”.
  • Figure 11B and table 5 ß-amylase line "12°C storage” there is no significant difference in the tubers sampled from 12°C storage, ß-amylase activity between K125-2.1 and control Bintje.
  • the reduction in ⁇ -amylase activity seen in the transgenic Bintje line K125-2.1 compared with the non-transformed control Bintje is the result of specific inhibition by the ⁇ -amylase antisense construct (pJK5) and not the result of any coarse control mechanism since only the activity of ⁇ -amylase is affected while ß-amylase stays the same as the control line.
  • Figure 13B shows the level of fructose in both K125-2.1 and control Bintje tubers (sampled at the indicated dates, in most cases the average of 5 individually analysed tubers are shown). It is evident from the figure that there is no significant difference seen between K125.2.1 and control Bintje tubers whether these are sampled from 4°C or 12°C storage (see also Table 3 line “Fructose”). On the contrary, there is a large difference seen in the specific glucose levels of K125-2.1 and control Bintje tubers as shown in Figure 12A.
  • K125-2.1 has a significantly lower glucose level (P> 0.001) compared with control Bintje amounting to a reduction (on the average) of 70% compared with the control. Again the reduction seen previously in the reducing sugar level in K125-2.1's tubers stored at 4°C compared with control Bintje is exclusively in the glucose level (see Figure 12A).
  • potatoes were extracted and the enzymes were partially separated by affinity chromatography (Blue Sepharose CL-6B).
  • affinity chromatography Blue Sepharose CL-6B
  • the proteins binding to the affinity column was further separated by gelfiltration chromatography, resulting in 9 peaks; peak 1 to peak 9.
  • the purification steps are described in detail in Materials and Methods.
  • peak 3 and peak 7 contain ⁇ -amylase activity whereas peak 2 mainly contains ⁇ -amylase activity. These three peaks also degrade amylose, where the highest amylytic activity is found in peak 3 and peak 7. Analysis of the end products on Dionex HPLC showed that peak 7 produces glucose and maltose from hydrolysis of amylose. Similar results are also seen for peak 3, however, the increase of glucose and maltose was much lower than for peak 7. These endproducts were not detected for the remaining peak fractions.
  • the amylolytic activity isolated in peak 7 only accumulates the endproducts, glucose and maltose, with amylose as substrate but not with soluble starch or maltopentaose used as substrates. Peak 3 on the contrary degrades maltopentaose into maltose, maltotriose and maltohexaose. No glucose was detected.
  • the enzyme activity separated in peak 7, ⁇ -amylase produces glucose and maltose from amylose.
  • the reducing sugar level in potato tubers stored at cold temperatures is up to 10 times higher than the sugar level seen in tubers stored at warm temperatures (e.g. 12°C, see e.g. Figures 8A and B). Therefore, by reducing the glucose level as much as 70% in warm stored tubers (and possibly even more with a stronger antisense effect), one can convert many already known varieties into either a chip and/or a crisp variety if they are kept at warm storage. This is beneficial to the industry since many other characteristics are important in selecting a good variety besides its sugar content (e.g. resistance against diseases, yield, eye depth, skin colour form of tuber etc.), and these do not always go together with low reducing sugar content.
  • tubers according to the present invention can be stored cold (below 7°C) and still not accumulate reducing sugars compared with warm stored tubers. This is very beneficial. ⁇ - and ⁇ -amylase activities are higher in cold stored tubers
  • a potato variety which produces tubers that do not accumulate reducing sugars after cold (below 7°C) storage or at least do not have reducing sugar content above the limit for chipping or crisping can be achieved as follows:
  • a cold inductive promoter as e.g. gPAmy351 (see Figure 4) or (65)
  • the antisense ⁇ -amylase sequence e.g. as used in pJK4, pJK5
  • an enhancer in front is transformed into potato varieties like e.g. Satuma or Record (low sugar varieties) or other varieties, as explained in Materials and Methods.
  • Another embodiment of the present invention
  • the two antisense sequences can either be placed one in direct connection with the other under the control of one promoter (or they could have their own promoter and terminator). This could be for example the E35S promoter (as e.g. seen in pJK4) or the 35S promoter (as seen in pJK5) or alternatively a cold inductive promoter as explained above, e.g. gPAmy 351 eventually with enhancer - since antisensing the vacuolar invertase is only effective in cold stored tubers as reported (27).
  • This dual construct would (partly or nearly completely) keep glucose from being released from the degradation of the starch grain by the action of ⁇ -amylase. It would also keep at least partially the glucose and fructose in the form of sucrose molecules thereby removing at least some fructose and perhaps nearly the rest of the glucose. In this way a substantial amount of the glucose and fructose otherwise present in non-transformed control tubers will be absent from these transgenic tubers. Since the amount of glucose and fructose is up till 10 times higher in cold stored tubers at least 90-95% has to be removed by the dual construction in order for the tubers to be stored at cold temperatures.
  • Yet another embodiment of the present invention is a combination of the sequence encoding for either E.coli ADP-glucose pyrophosphorylase (WO91/19806) or the barley ADP-glucose pyrophosphorylase or potatoes own ADP glucose pyrophosphorylase (EP- A-0455316) in a sense orientation in combination with the antisense ⁇ -amylase sequence.
  • the transformed potato varieties could, as described before, be fried after cold and warm storage.
  • the antisense ⁇ -amylase could be beneficially to combine with the PFK gene (sense or antisense), or the PFP(sense or antisense) gene or any other genes participating in either the starch synthesis or starch degradation.
  • anti-sense ⁇ -amylase and at least any one of: branching enzyme (sense or antisense), or sucrose synthase (sense or antisense), or sucrose- P-synthase (sense or antisense), or UDP-glucosepyrophosphorylase (sense or antisense), or ADP-glucose phyrophosphorylase (sense or antisense), or starch phosphorylase (sense or antisense), or beta-amylase (sense or antisense), or ⁇ -glycosidase
  • sense or antisense or phosphohexose isomerase (sense or antisense), or hexokinase (sense or antisense), or fructose-6 phosphate-2 kinase (sense or antisense), or fructose-2,6-bisphosphatase (sense or antisense), or other invcrtases (sense or antisense).
  • Other types of combinations could be the antisense potato ⁇ -amylase with either another plant gene (monocot as well as dicot e.g. ADP-glucose phyrophosphorylase(e.g. from barley) or a bacterial or fungi gene like e.g. the pullanase from bacteria, or other suitable bacterial enzymes.
  • a preferred combination is anti-sense potato ⁇ -amylase with ADP-glucose phyrophosphorylase.
  • Plants 158: 428-436 Steup, M., Robevrek, H. and Mclkonian, M. (1983). Plants 158: 428-436.

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Abstract

On décrit un procédé de réduction de la teneur en sucre d'un sucre réducteur spécifique dans un organisme pouvant dégrader de manière enzymatique l'amylopectine ou l'amylose. Un procédé préféré consiste à inhiber dans cet organisme, au moins partiellement, l'activité de l'α-amylase.
PCT/EP1995/002197 1994-10-21 1995-06-06 Procede de reduction de la teneur en sucre d'un organisme WO1996012812A1 (fr)

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EP95923253A EP0787192A1 (fr) 1994-10-21 1995-06-06 Procede de reduction de la teneur en sucre d'un organisme
JP8513606A JPH10507365A (ja) 1994-10-21 1995-06-06 生物中の糖のレベルを低減させる方法
AU27882/95A AU2788295A (en) 1994-10-21 1995-06-06 A method of reducing the level of sugar in an organism
GB9521449A GB2294266B (en) 1994-10-21 1995-10-19 A method of reducing the level of glucose in an organism
MXPA/A/1997/002912A MXPA97002912A (en) 1994-10-21 1997-04-18 A method of reduction of the sugar level in an organi

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GB9421287.5 1994-10-21
GB9421287A GB9421287D0 (en) 1994-10-21 1994-10-21 A method of reducing the level of sugar in an organism

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004722A1 (fr) * 1996-07-30 1998-02-05 Universität Heidelberg Inhibiteur d'invertase
WO1998040503A1 (fr) * 1997-03-10 1998-09-17 Planttec Biotechnologie Gmbh Molecules d'acide nucleique codant la phosphorylase d'amidon provenant du maïs
WO1998045459A1 (fr) * 1997-04-09 1998-10-15 E.I. Du Pont De Nemours And Company 4-alpha-glucanotransferases intervenant dans les vegetaux
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
WO1999036551A1 (fr) * 1998-01-15 1999-07-22 E.I. Du Pont De Nemours And Company Homologues de phosphoglucomutase vegetale
KR101516421B1 (ko) * 2013-08-22 2015-05-26 김시언 감자의 포도당 함량 속성 측정 방법

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE439048T1 (de) * 2002-11-08 2009-08-15 Bayer Cropscience Ag Prozess zur verminderung des acrylamidgehaltes von hitzebehandelten lebensmitteln

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WO1990012876A1 (fr) * 1989-04-24 1990-11-01 Aktieselskabet De Danske Spritfabrikker (Danisco A/S) GENES DE L'α-AMYLASE DE LA POMME DE TERRE
EP0438904A1 (fr) * 1989-12-21 1991-07-31 Advanced Technologies (Cambridge) Limited Modification du métabolisme végétal
DE4213444A1 (de) * 1992-04-18 1993-10-28 Inst Genbiologische Forschung Verfahren zur Herstellung von Kartoffelpflanzen, deren Knollensprossung unterdrückt ist
WO1994028149A1 (fr) * 1993-05-28 1994-12-08 Monsanto Company Procede d'amelioration de la qualite de pommes de terre stockees

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IE913215A1 (en) * 1990-09-13 1992-02-25 Gist Brocades Nv Transgenic plants having a modified carbohydrate content
GB9117159D0 (en) * 1991-08-08 1991-09-25 Cambridge Advanced Tech Modification of sucrose accumulation

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WO1990012876A1 (fr) * 1989-04-24 1990-11-01 Aktieselskabet De Danske Spritfabrikker (Danisco A/S) GENES DE L'α-AMYLASE DE LA POMME DE TERRE
EP0438904A1 (fr) * 1989-12-21 1991-07-31 Advanced Technologies (Cambridge) Limited Modification du métabolisme végétal
DE4213444A1 (de) * 1992-04-18 1993-10-28 Inst Genbiologische Forschung Verfahren zur Herstellung von Kartoffelpflanzen, deren Knollensprossung unterdrückt ist
WO1994028149A1 (fr) * 1993-05-28 1994-12-08 Monsanto Company Procede d'amelioration de la qualite de pommes de terre stockees

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Title
CLAUSEN, I. G., ET AL.: "SUPPRESSION BY ANTISENSE MRNA OF SYNTHESIS FROM HIGHLY EXPRESSED GENES IN ASPERGILLUS ORYZAE AND ASPERGILLUS NIGER", J. CELL. BIOCHEM. SUPPL., vol. 15D, pages 24 *
TSUTSUMI, N., ET AL.: "SUPPRESSION OF ALPHA-AMYLASE GENE EXPRESSION BY ANTISENSE OLIGONUCLEOTIDE IN CULTEURED BARLEY ALEURONE LAYERS", JAPANESE JOURNAL OF BREEDING, vol. 67, pages 147 - 154 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004722A1 (fr) * 1996-07-30 1998-02-05 Universität Heidelberg Inhibiteur d'invertase
WO1998040503A1 (fr) * 1997-03-10 1998-09-17 Planttec Biotechnologie Gmbh Molecules d'acide nucleique codant la phosphorylase d'amidon provenant du maïs
US6353154B1 (en) 1997-03-10 2002-03-05 Planttec Biotechnologie Gmbh Forschung & Entwicklung Nucleic acid molecules encoding starch phosphorylase from maize
US6686514B2 (en) 1997-03-10 2004-02-03 Planttec Biotechnologie Gmbh Forshung & Entwicklung Nucleic acid molecules encoding starch phosphorylase from maize
WO1998045459A1 (fr) * 1997-04-09 1998-10-15 E.I. Du Pont De Nemours And Company 4-alpha-glucanotransferases intervenant dans les vegetaux
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
US6559364B1 (en) 1997-10-30 2003-05-06 Mogen International N.V. Pre- and postharvest inhibition of remobilisation of storage compounds
WO1999036551A1 (fr) * 1998-01-15 1999-07-22 E.I. Du Pont De Nemours And Company Homologues de phosphoglucomutase vegetale
KR101516421B1 (ko) * 2013-08-22 2015-05-26 김시언 감자의 포도당 함량 속성 측정 방법

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MX9702912A (es) 1997-07-31
CA2202895A1 (fr) 1996-05-02
GB2294266B (en) 1997-06-11
GB2294266A (en) 1996-04-24
GB9421287D0 (en) 1994-12-07
AU2788295A (en) 1996-05-15
EP0787192A1 (fr) 1997-08-06
GB9521449D0 (en) 1995-12-20
JPH10507365A (ja) 1998-07-21

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