WO1998035051A1 - Pommes de terre transgeniques avec reduction des niveaux d'activite de la tubercule-phosphorylase de type l ou h alpha-glucan et avec reduction de l'adoucissement au froid - Google Patents

Pommes de terre transgeniques avec reduction des niveaux d'activite de la tubercule-phosphorylase de type l ou h alpha-glucan et avec reduction de l'adoucissement au froid Download PDF

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WO1998035051A1
WO1998035051A1 PCT/CA1998/000055 CA9800055W WO9835051A1 WO 1998035051 A1 WO1998035051 A1 WO 1998035051A1 CA 9800055 W CA9800055 W CA 9800055W WO 9835051 A1 WO9835051 A1 WO 9835051A1
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plant
tubers
glu
leu
potato
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PCT/CA1998/000055
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English (en)
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Lawrence Michael Kawchuk
John David Armstrong
Dermot Roborg Lynch
Norman Richard Knowles
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Her Majesty The Queen In Right Of Canada As Represented By The Department Of Agriculture And Agri-Food Canada
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Priority claimed from US08/868,786 external-priority patent/US5998701A/en
Application filed by Her Majesty The Queen In Right Of Canada As Represented By The Department Of Agriculture And Agri-Food Canada filed Critical Her Majesty The Queen In Right Of Canada As Represented By The Department Of Agriculture And Agri-Food Canada
Priority to BR9807214-5A priority Critical patent/BR9807214A/pt
Priority to AU58493/98A priority patent/AU724942B2/en
Priority to NZ336766A priority patent/NZ336766A/xx
Priority to CA002275885A priority patent/CA2275885C/fr
Priority to EP98901894A priority patent/EP1009839A1/fr
Priority to JP53344998A priority patent/JP2001511007A/ja
Priority to PL98334962A priority patent/PL334962A1/xx
Priority to HU0000542A priority patent/HUP0000542A3/hu
Publication of WO1998035051A1 publication Critical patent/WO1998035051A1/fr

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • 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

Definitions

  • the invention relates to the inhibition of the accumulation of sugars in potatoes by reducing the level of ⁇ glucan L-type tuber phosphorylase or glucan H-type tuber phosphorylase enzyme activity in the potato plant.
  • the sugars that accumulate are predominantly glucose, fructose, and sucrose. It is primarily the glucose and fructose (reducing sugars) that react with free amino groups when heated during the various cooking processes such as frying via the Maillard reaction, resulting in the formation of brown pigments (Burton, 1989, Shallenberger et al., 1959). Sucrose produces a black colouration when fried due to caramehzation and charring.
  • the ideal reducing sugar content is generally accepted to be 0.1% of tuber fresh weight with 0.33% as the upper limit and higher levels of reducing sugars are sufficient to cause the formation of brown and black pigments that results in an unacceptable fried product (Davies and Viola, 1992).
  • PFK phosphofructokinase
  • ADPGPP ADPglucose pyrophosphorylase
  • starch The degradation of starch is believed to involve several enzymes including -amylase (endoamylase), ⁇ -amylase (exoamylase), amyloglucosidase, and -glucan phosphorylase (starch phosphorylase).
  • -amylase endoamylase
  • ⁇ -amylase exoamylase
  • amyloglucosidase amyloglucosidase
  • starch phosphorylase starch phosphorylase
  • the tuber L-type c l,4 glucan phosphorylase (EC 2.4.1.1) isozyme (GLTP) (Nakano and Fukui, 1986) has a low affinity for highly branched glucans, such as glycogen, and is localized in amyoplasts.
  • the monomer consists of 916 amino acids and sequence comparisons with phosphorylases from rabbit muscle and Escherichia coli revealed a high level of homology, 51% and 40% amino acids, respectively.
  • the nucleotide sequence of the GLTP gene and the amino acid sequence of the GLTP enzyme are shown in SEQ LD NO: 1 and SEQ ID NO: 2, respectively.
  • the H-type tuber -glucan phosphorylase isozyme H (GHTP) (Mori et al., 1991) has a high affinity for glycogen and is localized in the cytoplasm.
  • the gene encodes for 838 amino acids and shows 63% sequence homology with the tuber L-type phosphorylase but lacks the 78-residue insertion and 50-residue amino-terminal extension found in the L- type polypeptide.
  • the nucleotide sequence of the GHTP gene and the amino acid sequence of the GHTP enzyme are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • a third isozyme has been reported (Sonnewald et al., 1995) that consists of 974 amino acids and is highly homologous to the tuber L-type phosphorylase with 81 % identity over most of the polypeptide. However, the regions containing the transit peptide and insertion sequence are highly diverse.
  • This isozyme is referred to as the leaf L-type phosphorylase since the mRNA accumulates equally in leaf and tuber, whereas the mRNA of the tuber L-type phosphorylase accumulates strongly in potato tubers and only weakly in leaf tissues.
  • the tuber L-type phosphorylase is mainly present in the tubers and the leaf L-type phosphorylase is more abundunt in the leaves (Sonnewald et al., 1995).
  • the nucleotide sequence of the leaf L-type phosphorylase gene and the amino acid sequence of the leaf L-type phosphorylase enzyme are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the role of the various starch degrading enzymes is not clear, however, and considerable debate has occurred over conflicting results. For example, reduced expression of the leaf L-type phosphorylase (Sonnewald et al., 1995) had no significant influence on starch accumulation. Sonnewald et al.
  • the inventors have found that surprisingly, reduction of the level of ⁇ glucan L-type tuber phosphorylase (GLTP) or glucan H-type tuber phosphorylase (GHTP) enzyme activity within the potato tuber results in a substantial reduction in the accumulation of sugars in the tuber during propagation and storage, relative to wildtype potatoes, particularly at storage temperatures below 10°C, and specifically at 4°C. It is remarkable that, given the complexity of carbohydrate metabolism in the tuber, reduction in the activity of a single enzyme is effective in reducing sugar accumulation in the tuber. The inventors' discovery is even more surprising in light of the previously discussed work of Sonnewald et al.
  • Tubers in which cold-induced sweetening is inhibited or reduced may be stored at cooler temperatures without producing high levels of reducing sugars in the tuber which cause unacceptable darkening of fried potato products.
  • Cold storage of tubers storage results in longer storage life, prolonged dormancy by limiting respiration and delaying sprouting, and lower incidence of disease.
  • Reduction in GLTP or GHTP activity in potato plants and tubers can be accomplished by any of a number of known methods, including, without limitation, antisense inhibition of GLTP or GHTP mRNA, co-suppression, site-directed mutagenesis of wildtype GLTP or GHTP genes, chemical or protein inhibition, or plant breeding programs.
  • the invention provides modified potato plants having a reduced level of ⁇ glucan L-type tuber phosphorylase (GLTP) or glucan H-type tuber phosphorylase (GHTP) activity in tubers produced by the plants, relative to that of tubers produced by an unmodified potato plant.
  • GLTP ⁇ glucan L-type tuber phosphorylase
  • GHTP glucan H-type tuber phosphorylase
  • the invention provides a potato plant transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene.
  • the aforementioned DNA sequence may be inserted in the expression cassette in either a sense or antisense orientation.
  • Potato plants of the present invention could have reduced activity levels of either one of GLTP or GHTP independently, or could have reduced activity levels of both GLTP and GHTP.
  • the inventors have found that reduction of activity levels of GLTP or GHTP enzymes in potato plants results in potato tubers in which sugar accumulation, particularly over long storage periods at temperatures below 10°C, is reduced.
  • the invention further extends to methods for reducing sugar production in tubers produced by a potato plant comprising reducing the level of activity of GLTP or GHTP in the potato plant.
  • such methods involve introducing into the potato plant an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene.
  • the DNA sequence may be inserted in the expression cassette in either a sense or antisense orientation.
  • a direct measure of improved cold-storage characteristics is a reduction in the level of GLTP or GHTP enzyme activity detected in potatoes after harvest and cold-storage.
  • Transformed potato varieties have been developed wherein the total glucan phosphorylase activity measured as ⁇ mol NADPH produced mg "1 protein "1 h "1 in tubers of plants stored at 4°C for 189 days is as much as 70% lower than the total glucan phosphorylase activity in tubers of untransformed plants stored under the same conditions.
  • Another relatively direct measure of improved cold-storage characteristics is a reduction in sweetening of potatoes observed after a period of cold-storage.
  • Transformed potato varieties have been developed wherein the sum of the concentrations of glucose and fructose in tubers stored at 4°C for 91 days is 39% lower than the sum of the concentrations of glucose and fructose in tubers of an untransformed plant stored under the same conditions.
  • Yet another measure of improved cold-storage characteristics is a reduction in darkening of a potato chip during processing (cooking).
  • the accumulation of sugars in potatoes during cold-storage contributes to unacceptable darkening of the fried product.
  • Reduced darkening upon frying can be quantified as a measure of the reflectance, or chip score, of the fried potato chip. Techniques for measuring chip scores are discussed herein.
  • Transformed potato varieties of the present invention have been developed wherein the chip score for tubers of plants stored at 4°C for 124 days was as much as 89% higher than the chip scores for tubers of untransformed plants stored under the same conditions.
  • the present invention allows for storage of potatoes at cooler temperatures than would be possible with wildtype potatoes of the same cultivar.
  • storage of potatoes at cooler temperatures than those traditionally used could result in increased storage life, increased dormancy through reduced respiration and sprouting, and reduced incidence of disease. It will be apparent to those skilled in the art that such additional benefits also constitute improved cold-storage characteristics and may be measured and quantified by known techniques.
  • Figure 1 is a schematic diagram of the tuber L-type ⁇ glucan phosphorylase antisense sequence inserted into the pBI121 transformation vector
  • Figure 2 is a schematic diagram of the tuber H-type ⁇ glucan phosphorylase antisense sequence inserted into the pBI121 transformation vector
  • Figure 3 shows the basic structure of the three isolated isoforms of glucan phosphorylase.
  • the transit peptide (TS) and insertion sequence (IS) are characteristic of the L-type phosphorylases and are not found in the H-type phosphorylase.
  • Figure 4 is a schematic diagram of carbohydrate interconversions in potatoes (Sowokinos 1990);
  • Figure 5 is a comparison of the amino acid sequences of the three isoforms of phosphorylase found in potato for the region targeted by the antisense GLTP construct used in the Examples herein. Highlighted amino acids are identical.
  • the leaf L-type glucan phosphorylase amino acid sequence is on top (amino acids 21 - 238 of SEQ ID NO: 6), the tuber L-type a glucan phosphorylase amino acid sequence is in the middle (amino acids 49 - 266 of SEQ ID NO: 2), and tuber H-type glucan phosphorylase amino acid sequence is on the bottom (amino acids 46 - 264 of SEQ ID NO: 4);
  • Figure 6A and 6B are a comparison of the nucleotide sequences of the three isoforms of phosphorylase found in potato for the region targeted by the antisense GLTP construct used in the Examples herein. Highlighted nucleotides are identical.
  • leaf L-type glucan phosphorylase nucleotide sequence is on top (nucleotides 389 - 1045 of SEQ ID NO: 5), the tuber L-type ⁇ glucan phosphorylase nucleotide sequence is in the middle (nucleotides 338 - 993 of SEQ ID NO: 1), and tuber H-type glucan phosphorylase nucleotide sequence is on the bottom (nucleotides 147 - 805 of SEQ ID NO: 3);
  • Figure 7 is a northern blot of polyadenylated RNA isolated from potato tubers of wild type and lines 3,4,5, and 9 transformed with the tuber L-type ⁇ glucan phosphorylase.
  • Figure 8 is a northern blot of total RNA isolated from potato tubers of wild type and lines 1 and 2 transformed with the H-type ⁇ -glucan phosphorylase.
  • Figure 10 shows the activity gel and western blot of L-type and H-type isozymes of ⁇ 1,4 glucan phosphorylase extracted from wild type tubers and tubers transformed with the antisense construct for the L-type isoform; and
  • Figure 11 shows the activity gel and western blot of L-type and H-type isozymes of ⁇ 1 ,4 glucan phosphorylase extracted from wild type tubers and transformed with the antisense construct for the H-type isoform.
  • Potato plants having a reduced level of ⁇ glucan L-type tuber phosphorylase (GLTP) or ⁇ glucan H-type tuber phosphorylase (GHTP) activity in tubers produced by the plants O 98/35051
  • reduction in ⁇ glucan phosphorylase activity is accomplished by transforming a potato plant with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene.
  • the DNA sequence is inserted in the expression cassette in the antisense orientation, a reduction in ⁇ glucan phosphorylase activity can be achieved with the DNA sequence inserted in the expression cassette in either a sense or antisense orientation.
  • Homology-dependent silencing appears to be a general phenomenon that may be used to control the activity of many endogenous genes.
  • genes exhibiting reduced expression after the introduction of homologous sequences include dihydroflavanol reductase (Van der Krol 1990), polygalacturonidase (Smith et al 1990), phytoene synthase (Fray and Grierson 1993), pectinesterase (Seymour et al. 1993), phenylalanine ammonia-lyase (De Carvalho et al. 1992), ⁇ -l,3-glucanase (Hart et al. 1992), chitinase (Dorlhac et al.
  • RNA which is of complementary sequence to the mRNA produced by the target gene. It is theorized that the complementary RNA sequences form a duplex thereby inhibiting translation to protein.
  • the theory underlying both sense and antisense inhibition has been discussed in the literature, including in Antisense Research and Applications (CRC Press, 1993) pp. 125-148.
  • the complementary sequence may be equivalent in length to the whole sequence of the target gene, but a fragment is usually sufficient and is more convenient to work with. For instance, Cannon et al. (1990) reveals that an antisense sequence as short as 41 base pairs is sufficient to achieve gene inhibition.
  • RNA molecules can be altered (promoters, polyadenylation signals, post-transcriptional processing sites) or used to alter the expression levels (enhancers and silencers) of a specific mRNA.
  • Another strategy to reduce expression of a gene and its encoded protein is the use of ribozymes designed to specifically cleave the target mRNA rendering it incapable of producing a fully functional protein (Hasseloff and Gerlach, 1988). Identification of naturally occurring alleles or the development of genetically engineered alleles of an enzyme that have been identified to be important in determining a particular trait can alter activity levels and be exploited by classical breeding programs (Oritz and Huaman, 1994). Site-directed mutagenesis is often used to modify the activity of an identified gene product.
  • the structural coding sequence for a phosphorylase enzyme can be mutagenized in E. coli or another suitable host and screened for reduced starch phosphorolysis. Alternatively, naturally occurring alleles of the phosphorylase with reduced affinity and/or specific activity may be identified. Additionally, the activity of a particular enzyme can be altered using various inhibitors. These procedures are routinely used and can be found in text books such as Sambrook et al. (1989).
  • the foregoing variants may include GLTP and GHTP nucleotide sequence variants that differ from those exemplified but still encode the same polypeptide due to codon degeneracy, as well as variants which encode proteins capable of recognition by antibodies raised against the GLTP and GHTP amino acid sequences set forth in SEQ ID NO's. 2 and 4.
  • homology dependent silencing of GLTP and/or GHTP in potato plants may be accomplished with sense or antisense sequences other than those exemplified.
  • the region of the GLTP or GHTP cDNA sequence from which the antisense sequence is derived is not essential.
  • the length of the antisense sequence used may vary considerably.
  • the sense or antisense sequence need not be identical to that of the target GLTP or GHTP gene to be suppressed.
  • the inventors have observed that transformation of potato plants with antisense DNA sequences derived from the GHTP gene not only substantially suppresses GHTP gene activity, but causes some degree of suppression of GLTP gene activity.
  • the GHTP and GLTP genes antisense sequences have 56.8% sequence identity.
  • the sequence identity between the GLTP antisense sequence and the corresponding leaf type ⁇ glucan phosphorylase squence described by Sonnewald et al. (1990) is 71.3%.
  • Useful sense or antisense sequences may differ from the exemplified antisense sequences or from other sequences derived from the endogenous GHTP or GLTP gene sequences by way of conservative amino acid substitutions or differences in the percentage of matched nucleotides or amino acids over portions of the sequences which are aligned for comparison purposes.
  • United States Patent 5,585,545 (Bennett et al., December 17, 1996) provides a helpful discussion regarding techniques for comparing sequence identity for polynucleotides and polypeptides, conservative amino acid substitutions, and hybridization conditions indicative of degrees of sequence identity. Relevant parts of that discussion are summarized herein.
  • Percentage of sequence identity for polynucleotides and polypeptides may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and, (c) multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFTT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection.
  • Polypeptides which are substantially similar share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • nucleotide sequences are substantially identical if two molecules specifically hybridize to each other under stringent conditions.
  • Stringent conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • the T m of a hybrid which is a function of both the length and the base composition of the probe, can be calculated as described in Sambrook et al. (1989).
  • stringent conditions for a Southern blot protocol involve washing at 65 °C with 0.2XSSC. For preferred oligonucleotide probes, washing conditions are typically about at 42°C in 6XSSC.
  • the steps involved in preparing antisense ⁇ glucan phosphorylase cDNAs and introducing them into a plant cell include: (1) isolating mRNA from potato plants and preparing cDNA from the mRNA; (2) screening the cDNA for the desired sequences; (3) linking a promoter to the desired cDNAs in the opposite orientation for expression of the phosphorylase genes; (4) transforming suitable host plant cells; and (5) selecting and regenerating cells which transcribe the inverted sequences.
  • DNA derived from potato GLTP and GHTP genes is used to create expression cassettes having a plant promoter sequence operably linked to an antisense DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene.
  • Agrobacterium tumefaciens is used as a vehicle for transmission of the DNA to stem explants of potato plant shoots.
  • a plant regenerated from the transformed explants transcribes the antisense DNA which inhibits activity of the enzyme.
  • the recombinant DNA technology described herein involves standard laboratory techniques that are well known in the art and are described in standard references such as Sambrook et al. (1989). Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications.
  • GHTP and GLTP cDNA cDNA are prepared from isolated potato tuber mRNA by reverse transcription.
  • a primer is annealed to the mRNA, providing a free 3' end that can be used for extension by the enzyme reverse transcriptase.
  • the enzyme engages in the usual 5'-3' elongation, as directed by complementary base pairing with the mRNA template to form a hybrid molecule, consisting of a template RNA strand base-paired with the complementary cDNA strand.
  • a DNA polymerase is used to synthesize the complementary DNA strand to convert the single-stranded cDNA into a duplex DNA.
  • the double stranded cDNA is inserted into a vector for propagation in E. coli.
  • identification of clones harbouring the desired cDNA's would be performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used.
  • the matter is simplified in that the DNA sequences of the GLTP and GHTP genes are known, as are the sequences of suitable primers (Brisson et al., 1990; Fukui et al., 1991).
  • the primers used hybridize within the GLTP and GHTP genes.
  • the amplified cDNA's prepared represent portions of the GLTP and GHTP genes without further analysis.
  • E. coli transformed with pUC19 plasmids carrying the phosphorylase DNA insert were detected by color selection.
  • Strains transformed with pBluescript plasmids carrying inserts grow as white colonies. Plasmids isolated from transformed E. coli were sequenced to confirm the sequence of the phosphorylase inserts. '
  • the cDNAs prepared can be inserted in the antisense or sense orientation into expression cassette in expression vectors for transformation of potato plants to inhibit the expression of the GLTP and/or GHTP genes in potato tubers.
  • the desired recombinant vector will comprise an expression cassette designed for initiating transcription of the antisense cDNAs in plants. Additional sequences are included to allow the vector to be cloned in a bacterial or phage host.
  • the vector will preferably contain a prokaryote origin of replication having a broad host range.
  • a selectable marker should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers include resistance to antibiotics such ampicillin.
  • DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
  • T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
  • the recombinant expression cassette will contain in addition to the desired sequence, a plant promoter region, a transcription initiation site (if the sequence to be transcribed lacks one), and a transcription termination sequence.
  • Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector. Sequences controlling eukaryotic gene expression are well known in the art. Transcription of DNA into mRNA is regulated by a region of DNA referred to as the promoter.
  • the promoter region contains sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of RNA.
  • Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream (by convention -30 to -20 bp relative to the transcription start site) of the transcription start site. In most instances the TATA box is required for accurate transcription initiation.
  • the TATA box is the only upstream promoter element that has a relatively fixed location with respect to the start point.
  • the CAAT box consensus sequence is centered at -75, but can function at distances that vary considerably from the start point and in either orientation.
  • Another common promoter element is the GC box at -90 which contains the consensus sequence GGGCGG. It may occur in multiple copies and in either orientation. Other sequences conferring tissue specificity, response to environmental signals, or maximum efficiency of transcription may also be found in the promoter region. Such sequences are often found within 400 bp of transcription initiation size, but may extend as far as 2000 bp or more.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. However, some variation in this distance can be accommodated without loss of promoter function.
  • the particular promoter used in the expression cassette is not critical to the invention. Any of a number of promoters which direct transcription in plant cells is suitable.
  • the promoter can be either constitutive or inducible.
  • a number of promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumour-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and the figwort mosaic virus 35S-promoters, the light-inducible promoter from the small subunit of ribulose-l,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the chlorophyll a/b binding protein gene promoter, etc.
  • NOS nopaline synthase
  • OCS octopine synthase
  • promoters which are contemplated to be useful in this invention include those that show enhanced or specific expression in potato tubers, that are promoters normally associated with the expression of starch biosynthetic or modification enzyme genes, or that show different patterns of expression, for example, or are expressed at different times during tuber development.
  • promoters examples include those for the genes for the granule-bound and other starch synthases, the branching enzymes (Blennow et al., 1991 ; WO 9214827; WO 9211375), disproportionating enzyme (Takaha et al., 1993) debranching enzymes, amylases, starch phosphorylases (Nakano et al., 1989; Mori et al., 1991), pectin esterases (Ebbelaar et al., 1993), the 40 kD glycoprotein; ubiquitin, aspartic proteinase inhibitor (Stukerlj et al, 1990), the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shahar et al, 1992; GenBank Accession Numbers M95196 and M95197), putative trypsin inhibitor and other tuber cDNAs (Stiekema et al., 1988), and for amylases and spora
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the nopaline synthase NOS 3' terminator sequence (Bevan et al. 1983) was used.
  • Polyadenylation sequences are also commonly added to the vector construct if the mRNA encoded by the structural gene is to be efficiently translated (Alber and Kawasaki, 1982). Polyadenylation is believed to have an effect on stabilizing mRNAs.
  • Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., 1984) or the nopaline synthase signal (Depicker et al., 1982).
  • the vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Typically, the marker gene encodes antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamycin. In the exemplified case, the marker gene confers resistance to kanamycin. After transforming the plant cells, those cells containing the vector will be identified by their ability to grow in a medium containing the particular antibiotic.
  • the DNA may also be introduced into plant cells by electroporation, wherein plant protoplasts are electroporated in the presence of plasmids carrying the expression cassette.
  • the exemplified case uses vectored transformation using Agrobacterium tumefaciens.
  • Agrobacterium tumefaciens is a Gram- negative soil bacteria which causes a neoplastic disease known as crown gall in dicotyledonous plants.
  • Induction of tumours is caused by tumour-inducing plasmids known as Ti plasmids.
  • Ti plasmids direct the synthesis of opines in the infected plant.
  • the opines are used as a source of carbon and/or nitrogen by the Agrobacteria.
  • the bacterium does not enter the plant cell, but transfers only part of the Ti plasmid, a portion called T-DNA, which is stably integrated into the plant genome, where it expresses the functions needed to synthesize opines and to transform the plant cell.
  • Vir (virulence) genes on the Ti plasmid, outside of the T-DNA region are necessary for the transfer of the T- DNA.
  • the vir region is not transferred.
  • the vir region although required for T-DNA transfer, need not be physically linked to the T-DNA and may be provided on a separate plasmid.
  • tumour-inducing portions of the T-DNA can be interrupted or deleted without loss of the transfer and integration functions, such that normal and healthy transformed plant cells may be produced which have lost all properties of tumour cells, but still harbour and express certain parts of T-DNA, particularly the T-DNA border regions. Therefore, modified Ti plasmids, in which the disease causing genes have been deleted, may be used as vectors for the transfer of the sense and antisense gene constructs of the present invention into potato plants (see generally Winnacker, 1987). Transformation of plants cells with Agrobacterium and regeneration of whole plants typically involves either co-cultivation of Agrobacterium with cultured isolated protoplasts or transformation of intact cells or tissues with Agrobacterium.
  • cauliflower mosaic virus may be used as a vector for introducing sense or antisense DNA into plants of the Solanaceae family.
  • CaMV cauliflower mosaic virus
  • United States Patent No. 4,407,956 Howell, October 4, 1983
  • a selectable marker such as antibiotic resistance
  • transformed plant cells were selected by growing the cells on growth medium containing kanamycin. Other selectable markers will be apparent to those skilled in the art.
  • the presence of opines can be used to identify transformants if the plants are transformed with Agrobacterium.
  • Expression of the foreign DNA can be confirmed by detection of RNA encoded by the inserted DNA using well known methods such as Northern blot hybridization.
  • the inserted DNA sequence can itself be identified by Southern blot hybridization or the polymerase chain reaction, as well (See, generally, Sambrook et al. (1989)).
  • whole plants are regenerated.
  • stem and leaf explants of potato shoot cultures were inoculated with a culture of Agrobacterium tumefaciens carrying the desired antisense DNA and a kanamycin marker gene.
  • Transformants were selected on a kanamycin-containing growth medium. After transfer to a suitable medium for shoot induction, shoots were transferred to a medium suitable for rooting. Plants were then transferred to soil and hardened off. The plants regenerated in culture were transplanted and grown to maturity under greenhouse conditions.
  • Starch synthesis by the tuber L-type and H-type isoforms was determined by iodine staining of the gel after incubation with glucose- 1 -phosphate and a starch primer (Steup, 1990).
  • Western analysis was performed by blotting the protein from an identical unincubated native gel to nitrocellulose and probing with polyclonal antibodies specific for tuber type L and type H glucan phosphorylase isoforms.
  • Levels of reducing sugars (glucose and fructose) in tuber tissues were quantified by HPLC (Tables 2, 3 and 4).
  • the extent of Maillard reaction, which is proportional to the concentration of reducing sugars in tubers was examined by determining chip scores after frying (Table 5 and Figure 6).
  • the term: - "about three months”, “about four months” and “about six months” refer, respectively, to periods of time of three months plus or minus two weeks, four months plus or minus two weeks, and six months plus or minus two weeks; - "antisense orientation” refers to the orientation of nucleic acid sequence from a structural gene that is inserted in an expression cassette in an inverted manner with respect to its naturally occurring orientation.
  • chip score of a tuber means the reflectance measurement recorded by an Agtron model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc.
  • an improvement in a cold-storage characteristic refers to a difference in the described characteristic relative to that in a control, wildtype or unmodified potato plant; - "modified" or variants thereof, when used to describe potato plants or tubers, is used to distinguish a potato plant or tuber that has been altered from its naturally occurring state through: the introduction of a nucleotide sequence from the same or a different species, whether in a sense or antisense orientation, whether by recombinant DNA technology or by traditional cross-breeding methods including the introduction of modified structural or regulatory sequences; modification of a native nucleotide sequence by site -directed mutagenesis or otherwise; or the treatment of the potato plant with chemical or protein inhibitors.
  • nucleic acid sequence or “nucleic acid segment” refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and non- functional DNA or RNA; - “operably linked” refers to functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates transcription of RNA corresponding to the second sequence; - "plant " includes whole plants, plant organs (e.g.
  • promoter refers to a region of DNA upstream from the structural gene and involved in recognition and binding RNA polymerase and other proteins that initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells;
  • reduced activity when used in reference to the level of GLTP or GHTP enzyme activity in a potato tuber includes reduction of GLTP or GHTP enzyme activity resulting from reduced expression of the GLTP or GHTP gene product, reduced substrate affinity of the GLTP or GHTP enzyme, and reduced catalytic activity of the GLTP or GHTP enzyme;
  • - “reduced” or variants thereof may be used herein with reference to, without limitation, activity levels of GLTP or GHTP enzyme in potato tubers, accumulation of sugars in potato tubers and darkening of potato chips upon frying.
  • reduced levels or reduced activity refers to a demonstrable statistically significant difference in the described characteristic relative to that in a control, wildtype or unmodified potato plant;
  • stress or variants thereof, when used in relation to stresses experienced by potato plants and tubers, includes the effects of environment, fertility, moisture, temperature, handling, disease, atmosphere and aging that impact upon plant or tuber quality and which may be experienced by potato plants through all stages of their life cycle and by tubers at all stages of the growth and development cycle and during subsequent harvesting, transport, storage and processing;
  • stress resistance or variants thereof, shall mean reduced effects of temperature, aging, disease, atmosphere, physical handling, moisture, chemical residues, environment, pests and other stresses;
  • suitable host refers to a microorganism or cell that is compatible with a recombinant plasmid, DNA sequence or recombinant expression cassette and will permit the plasmid to replicate, to be incorporated into its genome, or to be expressed; and
  • EXAMPLE 1 This example describes the reduction of GHTP and/or GLTP activity in tubers of potato plants by transforming potato plants with expression cassettes containing DNA sequences derived from the GLTP and GHTP gene sequences linked to the promoter in the antisense orientation.
  • a Isolation of Potato Tuber mRNA Potato total RNA was purified at 4°C using autoclaved reagents from lg of tuber tissue ground to a fine powder under liquid nitrogen with a mortar and pestle.
  • the powder was transferred to a 30ml corex tube and 3 volumes were added of 100 M Tris-Cl, pH 8.0, 100 mM NaCl, and 10 mM EDTA (lOx TNE) containing 0.2% (w/v) SDS and 0.5% (v/v) 2- mercaptoethanol.
  • An equal volume of phenol-chloroform (1:1) was added and the sample gently vortexed before centrifugation at 4 °C in a SS34 rotor at 8,000 rpm for 5 min.
  • the organic phase was reextracted with 0.5 volume of lOx TNE containing 0.2% (w/v)SDS and 0.5% (v/v) 2-mercaptoethanol and the combined aqueous phases were extracted with chloroform.
  • Nucleic acids were precipitated from the aqueous phase with sodium acetate and absolute ethanol, pelleted by centrifugation, and resuspended in 3 ml of lx TNE. An equal volume of 5 M LiCl was added and the sample stored at -20 °C for at 4 h before centrifuging at 8,000 rpm in a SS34 rotor at 4°C for 10 min. The RNA pellet was washed with 70% ethanol, dried, and resuspended in DEPC-treated water. Poly (A + ) RNA was isolated using oligo (dT) cellulose (Boehringer Mannheim) column chromatography.
  • RNA was isolated from total RNA resuspended in RNAse free water. Columns were prepared using an autoclaved Bio-Rad Poly-Prep 10 ml column to which was added 50 mg of oligo (dT) cellulose suspended in 1 ml of loading buffer B which contains 20 mM Tris-Cl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.1% (w/v) SDS. The column was washed with 3 volumes of 0.1 M NaOH with 5 mM EDTA and then DEPC- treated water until the pH of effluent was less than 8, as determined with pH paper.
  • loading buffer B which contains 20 mM Tris-Cl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.1% (w/v) SDS.
  • RNA samples were heated to 65 °C for 5 min at which time 400 ⁇ of loading buffer A, prewarmed to 65 °C, was added. The sample was mixed and allowed to cool at room temperature for 2 min before application to the column. Eluate was collected, heated to 65 °C for 5 min, cooled to room temperature for 2 min, and reapplied to the column. This was followed by a 5 volume washing with loading buffer A followed by a 4 volume wash with loading buffer B.
  • RNA was eluted with 3 volumes of 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, and 0.05% (w/v) SDS. Fractions were collected and those containing RNA were identified in an ethidium bromide plate assay, a petri dish with 1 % agarose made with TAE containing EtBr. RNA was precipitated, resuspended in 10 ⁇ l, and a 1 ⁇ l aliquot quantitated with a spectrophotometer.
  • GLTP and GHTP DNA Sequences The nucleotide sequences utilized in the development of the antisense construct were randomly selected from the 5' sequence of GLTP (SEQ D NO: 1) and GHTP (SEQ ID NO: 3). DNA sequences used to develop the antisense constructs were obtained using reverse transcription-polymerase chain reaction. GLTP (SPL1 and SPL2)- and GHTP (SPH1 and SPH2)-specific primers were designed according to the published sequences (Brisson et al. 1990, Fukui et al.
  • SPL1 Primer 5 ⁇ TTCGAAAAGCTCGAGATTTGCATAGA3 ' (SEQ ID NO: 7) (additional CG creates Xho I site);
  • SPL2 Primer 5'GTGTGCTCTCGAGCATTGAAAGC3' (SEQ ID NO: 8) (changed C to G to create Xho I site);
  • SPH1 Primer 5'GTTTATTTTCCATCGATGGAAGGTGGTG3' (SEQ ID NO: 9) (added CGAT to create Cla I site);
  • Reverse transcription was performed in a volume of 15 ⁇ l containing 1 x PCR buffer (10 mM Tris-Cl pH 8.2, 50 mM KC1, 0.001% gelatin, 1.5 mM MgCL), 670 ⁇ M of each dNTP, 6 ⁇ g of total potato tuber cv. Russet Burbank RNA, 1 mM each primer (SPH1 and SPL2, or SPH1 and SPH2) and 200 U of Maloney urine leukemia virus reverse transcriptase (BRL).
  • the reaction was set at 37°C for 30 minutes, then heat-killed at 94°C for 5 minutes and snap cooled on ice.
  • the polylinker and T3 and T7 RNA polymerase promoter sequences are present in the N-terminal portion of the lacZ gene fragment.
  • pUC19 plasmids without inserts in the polylinker grow as blue colonies in appropriate bacterial strains such as DH5 ⁇ . Color selection was made by spreading 100 ⁇ l of 2% X-gal (prepared in dimethyl formamide) on LB plates containing 50 ⁇ g/ml ampicillin 30 minutes prior to plating the transformants. Colonies containing plasmids without inserts will be blue after incubation for 12 to 18 hours at 37C and colonies with plasmids containing inserts will remain white. An isolated plasmid was sequenced to confirm the sequence of the phosphorylase inserts.
  • Sequences were determined using the ABI Prism Dye Terminator Cycle Sequencing Core Kit (Applied Biosystems, Foster City, CA), M13 universal and reverse primers, and an ABI automated DNA sequencer.
  • the engineered plasmid was purified by the rapid alkaline extraction procedure from a 5 ml overnight culture (Birnboim and Doly. 1979). Orientation of the SPL and SPH fragments in pUC19 was determined by restriction enzyme digestion.
  • the recombinant pUC19 vectors and the binary vector pBI121 (Clonetech) were restricted, run on a agarose gel and the fragments purified by gel separation as described by Thuring et al (1975). Ligation fused the antisense sequence to the binary vector pBI121.
  • the ligation contained pBI121 vector that had been digested with Bam I and S ⁇ cl, along with the SPL or SPH phosphorylase DNA product, that had been cut from the pUC 19 subclone with JS ⁇ mHI and S ⁇ cl.
  • Ligated DNA was transformed into SCE E. coli DH5 ⁇ cells, and the transformed cells were plated on LB plates containing ampicillin.
  • the nucleotide sequences of the antisense DNA SPL and SPH are nucleotides 338 to 993 of SEQ ID NO: 1 and nucleotides 147 to 799 of SEQ D NO: 3, respectively. Selection of pBI121 with phosphorlylase inserts was done with CAMV and NOS specific primers.
  • Samples 1 and 2 representing the tuber L-type and tuber H-type phosphorylase DNA fragments were picked from a plate after overnight growth. These samples were inoculated into 5 ml of LB media and grown overnight at 37 °C. Plasmids were isolated by the rapid alkaline extraction procedure, and the DNA was electroporated into Agrobacterium tumefaciens. Constructs were engineered into the pBI121 vector that contains the CaMV 35S promoter (Kay et al. 1987) and the NOS 3' terminator (Bevan et al. 1983) sequence. The pBI121 plasmid is made up of the following well characterized segments of DNA.
  • the chimeric gene consists of the 0.35 kb cauliflower mosaic virus 35S promoter (P-35S) (Odell et al., 1985), the 0.83 kb neomycin phosphotransferase type II gene (NPTII), and the 026 kb 3' non- translated region of the nopaline synthase gene (NOS 3') (Fraley et al., 1983).
  • P-35S 0.35 kb cauliflower mosaic virus 35S promoter
  • NPTII 0.83 kb neomycin phosphotransferase type II gene
  • NOS 3' non- translated region of the nopaline synthase gene
  • the next segment is a 0.75 kb origin of replication from the RK2 plasmid (ori-V) (Stalker et al., 1981). It is joined to a 3.1 kb Sail to Pvul segment of pBR322 which provides the origin of replication for maintenance in E.
  • the stem explants were co-cultured for 2 days at 20° C on S 1 medium (De Block 1988). Following co-culture, the explants were transferred to S4 medium (MS medium without sucrose, supplemented with 0.5 g/1 MES pH 5.7, 200 mg/1 glutamine, 0.5 g/1 PVP, 20 g/1 mannitol, 20 g/1 glucose, 40 mg/1 adenine, 1 mg/1 trans zeatin, 0.1 mg/1 NAA, 1 g/1 carbenicillin, 50 mg/1 kanamycin, solidified with 6 g/1 phytagar) for 1 week and then 2 weeks to induce callus formation.
  • S4 medium MS medium without sucrose, supplemented with 0.5 g/1 MES pH 5.7, 200 mg/1 glutamine, 0.5 g/1 PVP, 20 g/1 mannitol, 20 g/1 glucose, 40 mg/1 adenine, 1 mg/1 trans zeatin, 0.1 mg/1 NAA, 1 g/1 carbenicillin, 50 mg/1 kanamycin,
  • the explants were transferred to S6 medium (S4 without NAA and with half the concentration (500 mg/1) of carbenicillin). After another two weeks, the explants were transferred to S8 medium (S6 with only 250 mg/1 carbenicillin and 0.01 mg/1 gibberellic acid, GA3) to promote shoot formation. Shoots began to develop approximately 2 weeks after transfer to S8 shoot induction medium. These shoots were excised and transferred to vials of S 1 medium for rooting. After about 6 weeks of multiplication on the rooting medium, the plants were transferred to soil and are gradually hardened off. Desiree plants regenerated in culture were transplanted in 1 gallon pots and were grown to maturity under greenhouse conditions. Tubers were harvested and allowed to suberize at room temperature for two days. All tubers greater than 2 cm in length were collected and stored at 4°C under high humidity.
  • Tubers were stored at 4°C and were not allowed to recondition at room temperature prior to sugar analysis.
  • An intact longitudinal slice (1 cm thick, width variable and equal to the outside dimensions of the tuber) was cut from the central portion of each tuber, thus representing all of the tuber's tissues.
  • the central slices from four tubers per clone (3 replicates) were collectively diced into 1-cm cubes and 15 g was randomly selected from the pooled tissue for analysis.
  • Glucan phosphorylase see below
  • sugars were extracted with 15 mL of Tris buffer (50 mM, pH 7.0) containing 2 mM sodium bisulfite, 2 mM EDTA.
  • proteins were electrophoresed on glycogen-containing polyacrylamide gels as described above. The proteins were electroblotted to nitrocellulose and blots were probed with polyclonal antibodies raised against GHTP and GLTP. Immunoblots were developed with alkaline phosphatase conjugated anti-rabbit secondary antibodies (Sigma).
  • H Chip Color Determination Five transgenic potato lines expressing the GLTP antisense sequence, two transgenic lines expressing the GHTP antisense sequence, non-transgenic Desiree control lines, and two control lines transformed with the pBI121 vector T-DNA, were grown under field conditions in Alberta, Canada. Tubers were harvested and stored at 10°C and 4°C. Chip color was determined for all potato lines by taking center cuts from representative samples from each line and frying at 205 °F in soybean oil for approximately 3 minutes until bubbling stops.
  • Chip color which correlated with sugar content, was determined prior to cold storage and after 86 and 124 days of cold storage.
  • the chip color of tubers from all transgenic plants expressing the antisense GLTP transcript was significantly improved (lighter) relative to that of control tubers (darker) stored under identical conditions (Table 5 and Figure 7).
  • Chip scores of tubers from "Desiree " potato plants expressing the GLTP transcript were improved by at least 4.3 points and 8.9 points as determined with an Agtron model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431) following storage at 10°C and 4°C, respectively, for 86 days.
  • Chip scores of GLTP transformants measured after 124 days of storage at 4°C were improved by 44% to 89% relative to wildtype (Table 5).
  • the Desiree cultivar is not a commercially desirable potato for chipping due to its high natural sugar content and propensity to sweeten rapidly in cold storage. Nevertheless, significant improvements in fried chip color were noted with the transformed "Desiree" potatoes. It is expected that superior color lightening would be achieved if the methods of the invention were applied to commercial processing potato varieties. Analysis of tubers stored at 10°C and 4°C shows that those expressing the antisense GHTP transcript sometimes provided chips that fried lighter than control tubers, indicating a lower buildup of reducing sugars (Table 5).
  • Results showing heterologous and homologous reduction in phosphorylase activity indicate that the improvement may be a result of reducing one or both tuber phosphorylases.
  • these results suggest that the L-type phosphorylase plays a more important role in the catabolism of starch into reducing sugars.
  • the results show that the difference in reducing sugar levels (Table 2) and chip scores (Table 5) between tubers wildtype plants and those expressing tuber phosphorylase antisense RNA, are sustained during long-term storage. As shown in Table 5, the chip scores are approximately the same at 86 days and 124 days. No further increases in reducing sugar concentrations were evident after 49 and 91 days storage at 4°C (Table 2).
  • Reduced sugar accumulation relates to the observed chip score improvements, and also reflects improved specific gravity of tubers, another important commercial measure of tuber quality. Even at harvest, substantial improvements in chip score and reduced sugar accumulation were noted for transformed lines relative to wildtype. Thus, the benefits of the invention are not limited to improvements that arise only after extended periods of cold storage, but that are present at the time of harvest. In this sense, the invention is not limited only to improvements in cold-storage characteristics but encompasses improvements in tuber quality characteristics such as chip score or sugar accumulation which are present at the time of harvest, resulting in earlier maturity.
  • GLTP- type transformants (ATL3 - ATL9) exhibited up to a 66%, 70% and 69% reduction in ⁇ glucan phosphorylase activity relative to wildtype, at harvest, and after storage for 91 and 189 days, respectively. Most also exhibited improvements in excess of 10% and 30% relative to wildtype at harvest and after storage for 91 and 189 days. After storage for 91 and 189 days, the GHTP-type transformants (ATHl and ATH2) exhibited, respectively, up to 28% and 39% relative improvement over wildtype and generally showed at least 10% improvement. The GLTP-type transformants exhibited up to 80% and 39% reduction of sugar accumulation relative to wildtype at harvest and at 91 days, respectively.
  • all GLTP-type transformants exhibited at least 10% and at least 30% relative improvement.
  • all GLTP-type transformants exhibited at least 10% and most exhibited at least 30% relative improvement.
  • the GLTP-type transformants exhibited up to 46%, 89% and 89% chip score improvement relative to wildtype at harvest, and after storage for 86 days and 124 days, respectively. Almost all exhibited at least 10% and 30% relative improvement at harvest, and after storage for 86 and 124 days.
  • At least one of the GHTP-type transformants exhibited at least 5% and at least 10% improvement relative to wildtype at harvest, and after storage for 86 and 124 days. After 124 days storage, at least one of the GHTP-type transformants exhibited up to 25% relative improvement in chip score.
  • results clearly demonstrate that substantial improvements in tuber cold-storage characteristics may be readily obtained through the methods of the invention.
  • Results will vary due to, among other things, the location within the plant genome where the recombinant antisense or sense DNA is inserted, and the number of insertion events that occur. It is important to note that despite the variability in the results amongst the various transformed lines, there was little variation in the results amongst the samples within a single transformed potato line (see footnotes to Tables 1 to 5). Results are presented in Table 6 for all potato plant lines which were successfully transformed with the GHTP or GLTP antisense DNA.
  • Chip color rating was assigned using an Agtron meter similar to that used by industry to measure color of fried potatoes. In this index, the higher the number the lighter the chip product but color does not represent a linear relationship to the index.
  • ATL3 C 25 37.4 26.7 30.8 ATL4 35 43.7 29.1 32.3 ATL5 36 29.6 24.7 24.6 ATL9 38 38.7 24.3 26.6
  • d ATH tubers transformed with the tuber H-type ⁇ * glucan phosphorylase.
  • e GMP negative control, tubers transformed with pBI121 T-DNA. Table 6
  • MOLECULE TYPE DNA (genomic)
  • AAA AAC CTT GGC CAC AAT CTA GAA AAT GTG GCT TCT CAG GAA CCA GAT 583 Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser Gin Glu Pro Asp 115 120 125 130
  • MOLECULE TYPE DNA (genomic)
  • GAT GCT TTA AAC AAA CTG GGT CAG CAG CTT GAG GAG GTC GTT GAG CAG 386 Asp Ala Leu Asn Lys Leu Gly Gin Gin Leu Glu- Glu Val Val Glu Gin 110 115 120 125
  • MOLECULE TYPE DNA (genomic)
  • GCT GAA AAT TGG CTC GAG ATG GGA AAT CCA TGG GAA ATT GTG AGG AAT 833 Ala Glu Asn Trp Leu Glu Met Gly Asn Pro Trp Glu He Val Arg Asn 155 160 165 GAT ATT TCG TAT CCC GTA AAA TTC TAT GGG AAG GTC ATT GAA GGA GCT 881 Asp He Ser Tyr Pro Val Lys Phe Tyr Gly Lys Val He Glu Gly Ala 170 175 180
  • GGT ATG GAG GCT AGT GGA ACC AGC AAC ATG AAA TTT TCA ATG AAT GGC 2561 Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ser Met Asn Gly 730 735 740
  • Trp Glu He Thr Gin Arg Thr Val Ala Tyr Thr Asn His Thr Val Leu 340 345 350
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Cette invention se rapporte à des cultures de pommes de terre, qui présentent une réduction des niveaux d'activité des enzymes tubercule-phosphorylase de type L α-glucan (GLTP) ou tubercule-phosphorylase de type H α-glucan (GHTP) dans le tubercule de la pomme de terre. La conversion des amidons en sucres dans les tubercules des pommes de terre, en particulier lorsqu'ils sont conservés à des températures inférieures à 7 °C, est réduite dans les tubercules présentant une réduction d'activité des enzymes GLTP ou GHTP. La réduction de l'adoucissement au froid des pommes de terre permet une conservation des pommes de terre à des températures plus froides, ce qui permet d'obtenir une dormance prolongée, une incidence réduite des maladies et une durée de conservation accrue. Cette invention décrit également des procédés pour produire des cultures de pommes de terre qui génèrent des tubercules présentant une réduction d'activité des enzymes GLTP ou GHTP. La réduction de l'activité des GLTP ou GHTP dans le tubercule de la pomme de terre peut être obtenu par des techniques telles que la suppression de l'expression génique, au moyen d'ARN anti-sens homologue, l'utilisation de la co-suppression, de séquences silencieuses régulatoires, d'inhibiteurs chimiques et protéiques et l'utilisation de la mutagenèse dirigée sur site ou de l'isolation d'allèles alternatifs, pour obtenir des variants de GLTP ou de GHTP avec une affinité réduite à l'amidon ou une activité réduite de l'amidon.
PCT/CA1998/000055 1997-02-10 1998-02-05 Pommes de terre transgeniques avec reduction des niveaux d'activite de la tubercule-phosphorylase de type l ou h alpha-glucan et avec reduction de l'adoucissement au froid WO1998035051A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
BR9807214-5A BR9807214A (pt) 1997-02-10 1998-02-05 Método para melhoramento das caracterìsticas de armazenamento a frio do tubérculo de batata
AU58493/98A AU724942B2 (en) 1997-02-10 1998-02-05 Transgenic potatoes having reduced levels of alpha glucan L- or H-type tuber phosphorylase activity with reduced cold-sweetening
NZ336766A NZ336766A (en) 1997-02-10 1998-02-05 Transgenic potatoes having reduced levels of alpha glucan l- or h- type tuber phosphorylase activity with reduced cold-sweetening
CA002275885A CA2275885C (fr) 1997-02-10 1998-02-05 Pommes de terre transgeniques avec reduction des niveaux d'activite de la tubercule-phosphorylase de type l ou h alpha-glucan et avec reduction de l'adoucissement au froid
EP98901894A EP1009839A1 (fr) 1997-02-10 1998-02-05 Pommes de terre transgeniques avec reduction des niveaux d'activite de la tubercule-phosphorylase de type l ou h alpha-glucan et avec reduction de l'adoucissement au froid
JP53344998A JP2001511007A (ja) 1997-02-10 1998-02-05 低温誘発性甘味増加の減少を伴うαグルカンＬ−タイプまたはＨ−タイプ塊茎ホスホリラーゼのレベルの低下したトランスジェニックポテト
PL98334962A PL334962A1 (en) 1997-02-10 1998-02-05 Method of obtaining transgenous potatoes exhibiting reduced activity level of l- or h-type alpha glucane phosphorylase in their bulbs
HU0000542A HUP0000542A3 (en) 1997-02-10 1998-02-05 Transgenic potatoes having reduced levels of alpha glucan l-or h-type tuber phosphorylase activity with reduced cold-sweetening

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US3694697P 1997-02-10 1997-02-10
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US08/868,786 US5998701A (en) 1997-06-04 1997-06-04 Potatoes having improved quality characteristics and methods for their production
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US6495740B1 (en) 1996-06-27 2002-12-17 The Austrailian National University Manipulation of cellulose and/or β-1,4-Glucan
FR2868080A1 (fr) * 2004-03-29 2005-09-30 Genoplante Valor Soc Par Actio Procede d'amelioration des plantes
US8143477B2 (en) 2002-02-20 2012-03-27 J. R. Simplot Company All-native recombinant plant
US8273949B2 (en) 2002-02-20 2012-09-25 J.R. Simplot Company Precise breeding
CN102770017A (zh) * 2009-10-26 2012-11-07 艾格文册尔有限公司 杂交种用马铃薯的育种
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LU502613B1 (en) * 2022-08-01 2024-02-01 Plant Bioscience Ltd Methods of altering the starch granule profile in plants
WO2024027967A1 (fr) * 2022-08-01 2024-02-08 Plant Bioscience Limited Procédés de modification du profil de granules d'amidon dans des plantes

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NZ336766A (en) 2000-08-25
AU724942B2 (en) 2000-10-05
CA2275885A1 (fr) 1998-08-13
AU5849398A (en) 1998-08-26
HUP0000542A2 (hu) 2000-06-28
CN1246894A (zh) 2000-03-08
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CN1300338A (zh) 2001-06-20
AR011121A1 (es) 2000-08-02

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