WO1997044471A2 - Potato plants with reduced cytosolic starch phosphorylasis and modified germination - Google Patents

Potato plants with reduced cytosolic starch phosphorylasis and modified germination Download PDF

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Publication number
WO1997044471A2
WO1997044471A2 PCT/EP1997/002513 EP9702513W WO9744471A2 WO 1997044471 A2 WO1997044471 A2 WO 1997044471A2 EP 9702513 W EP9702513 W EP 9702513W WO 9744471 A2 WO9744471 A2 WO 9744471A2
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Prior art keywords
tubers
plants
cytosolic
wild
transgenic potato
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PCT/EP1997/002513
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German (de)
French (fr)
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WO1997044471A3 (en
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Jens Kossmann
Martin Steup
Elke Duwenig
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU28992/97A priority Critical patent/AU2899297A/en
Priority to EP97923093A priority patent/EP0906438A2/en
Publication of WO1997044471A2 publication Critical patent/WO1997044471A2/en
Publication of WO1997044471A3 publication Critical patent/WO1997044471A3/en

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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
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8267—Seed dormancy, germination or sprouting

Definitions

  • the present invention relates to transgenic potato plants which contain cells with a reduced activity of the cytosolic starch phosphorylase compared to wild-type plants.
  • the tubers of such potato plants show a different germination behavior compared to tubers of wild-type plants, which leads to the formation of an increased number of shoot ends and consequently to an increased number of stolens and tubers. Such plants also have an increased yield.
  • the yield from the agricultural cultivation of potatoes is primarily determined by the number of shoot ends that are formed per tuber. Normally, a germinating potato tuber forms only one, sometimes 2 to 3, shoot ends, the growth of further potentially existing side shoots being suppressed due to the apikaidominance of these shoot ends. Starting from the shoot ends, stolons are formed during the further growth of the potato plants, on which the tubers are later formed. Since the crop yield correlates with the number of stolons formed by the developed shoots, there is an effort to manipulate the germination of potato plants in such a way that the largest possible number of "eyes" germinate and develop into shoot ends.
  • One method is to break off the first sprout of a tuber, which suppresses the growth of further sprouts due to its apica dominance. This leads to the growth of further shoots.
  • Another method is to pre-germinate the potatoes to be laid out fine under regulated conditions in special containers (see e.g. Bouman, kannbau 47 (1996), 18-21; van de Waart, etcbau 44 (1993), 18-20).
  • Bouman, istbau 47 (1996), 18-21; van de Waart, cauliflower 44 (1993), 18-20 are very cost-intensive since, in addition to the special containers, they also require storage rooms in which both the light and the temperature conditions can be regulated.
  • a genetic engineering manipulation of potato plants was already proposed in DE-A1 42 13 444 in such a way that enzymes involved in starch metabolism are inhibited. Appropriate approaches for increased sprout formation of the potato tubers are not yet known. There is therefore a need for potato plants or processes in which the labor-intensive or cost-intensive steps described above are unnecessary and which lead to an increased number
  • the present invention is therefore based on the object of providing potato plants whose tubers form a large number of shoot ends when germinated.
  • the invention relates to transgenic potato plants that contain cells with a compared to wild-type plants, i.e. corresponding non-transformed plants, contain at least 60% reduced activity of the cytosolic starch phosphoryl.
  • the activity of the cytosolic starch phosphorylase is reduced by at least 80% and particularly preferably by at least 95% compared to wild-type potato plants.
  • cytosolic starch phosphorylase is understood to mean the isoform of starch phosphorylase (EC 2.4.1.1) localized in the cytoplasm of plant cells, which also is known as isoform H or I. This differs from the second, the plastid isoform, in that it shows, for example, a much higher affinity for highly branched glucans (Shimomura et al., J. Biochem. (Tokyo) 91 (1982), 703-717; Yang and Steup, Plant Physiol. 94, 960-969) and a low affinity for oligoglucans.
  • the enzyme catalyzes the reversible phosphorolysis of ⁇ -1, 4-glucans.
  • the activity of the starch phosphorylase can be determined, for example, as described in Parvin and Smith (Anal. Biochem. 27 (1969), 65-72), Conrads et al. (Biochim. Biophys. Acta 882 (1986), 452-463), Steup and Latzko (Planta 145 (1979), 69-75), Steup (In. Methods in Plant Biochemistry 3; Academic Press Limited (1990), 103- 128) or Sonnewald et al. (Plant Mol. Biol. 27: 567-576 (1995)).
  • the activity of the cytosolic starch phosphorylase is preferably reduced in all or in almost all cells of the plant. At least, however, in the bulbs and the shoot ends that develop from them.
  • tubers of potato plants which have such a reduced activity of the cytosolic starch phosphorylase show a drastically changed germination behavior compared to tubers of wild-type plants. Germination here means the growth of shoot ends from tubers.
  • tubers of wild-type plants usually form 1, at most 2 to 3, shoot ends during normal germination. To increase the number of shoots, either the elimination of the apikai shoot or a pre-germination under special conditions is necessary.
  • tubers of the potato plants according to the invention which contain cells with a reduced cytosolic starch phosphorylase, show a drastically increased number of shoot ends when germinated, in particular after storage at 20 ° C. in the dark. This leads to the formation of more stolons and consequently more tubers per plant. Overall, the tuber yield per plant increases. This concerns both the number of tubers per plant and the total fresh weight of tubers per plant.
  • the average number of shoots which are formed per tuber when germination takes place after storage for 5 months at 20 ° C. in the dark is at least doubled in comparison to wild-type plants ⁇ zen, ie corresponding non-transformed plants.
  • the number of shoot ends which are formed per budding eye when the germination takes place after storage for 5 months at 20 ° C. in the dark is at least doubled in comparison to tubers of wild-type Plants, ie corresponding non-transformed plants.
  • the plants according to the invention also have an increased yield with regard to the number of tubers and the weight of the tubers.
  • the number of tubers per plant is preferably at least 20%, preferably at least 50% and particularly preferably at least 100% higher than in corresponding ones non-transformed plants under the same growth conditions.
  • the fresh tuber weight of all tubers per plant is preferably at least 10%, preferably at least 15% and particularly preferably at least 20% higher than in corresponding non-transformed plants under the same growth conditions.
  • the activity of the cytosolic starch phosphorylase in the cells of the potato plants according to the invention can be reduced in principle by various methods known to the person skilled in the art.
  • the activity of the cytosolic starch phosphorylase is reduced by inhibiting the expression of endogenous genes which code for this enzyme.
  • Molecular biological techniques based on an antisense, ribozyme or a cosuppression effect are preferred.
  • a corresponding RNA is expressed in the antisense orientation. This preferably has a length of at least 30 nucleotides, preferably of at least 50 nucleotides and particularly preferably of at least 100 nucleotides.
  • the expressed antisense-RNA should have a high homology to the transcripts expressed endogenously in the plant, which encode cytosolic starch phosphorylase.
  • the homology is preferably at least 90%, preferably at least 95% and particularly preferably at least 99%.
  • an RNA is expressed which can specifically cleave transcripts of cytosolic starch phosphorylase.
  • the expression of ribozymes for reducing the activity of certain enzymes in cells is also known to the person skilled in the art and is described, for example, in EP-Bl 0 321 201.
  • the expression of ribozymes in plant cells was described, for example, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
  • the cosuppression effect is based on the expression of a sense RNA which expresses the expression suppressed by endogenous starch phosphorylase mRNA.
  • the implementation of these techniques is known to the person skilled in the art.
  • the method of cosuppression is described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al. (Curr. Top. Microbiol. Immuno. 197 (1995), 91-103), Flavell et al. (Curr. Top Microbiol. Immuno. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621) and other sources.
  • the reduction in the activity of a cytosolic starch phosphorylase in the cells is achieved by producing transgenic potato plants which contain a recombinant DNA molecule which is stably integrated into the genome and comprises the following elements:
  • the promoter of the patatin gene B33 from potato is suitable for expression in the tubers of the potato plants (Rocha-Sosa et al., EMBO J.8 (1989), 23-29).
  • the 35S promoter of the CaMV (Franck et al., Cell 21 (1980), 285-292) is suitable, for example, for constitutive expression.
  • DNA sequences which encode a potato cytosolic starch phosphorylase have already been described (see, for example, Mori et al., J. Biol. Chem. 266 (1991), 18446-18453). With the help of these DNA sequences it is possible for the person skilled in the art to isolate further sequences using standard methods which cyto- encode potato starch phosphorylase, if necessary.
  • the activity of the cytosolic starch phosphorylase can also be reduced by inactivating the endogenously present genes which code for this enzyme. Techniques for this are, for example, transposon mutagenesis or gene tagging. Alternatively, there is also the possibility of expressing antibodies in the cells which specifically recognize cytosolic starch phosphorylase.
  • the present invention relates to propagation material of the potato plants according to the invention, in particular seeds and particularly preferably potato tubers. These contain cells with a reduced activity of the cytosolic starch phosphorylase in comparison with tubers of wild-type plants and an altered germination behavior as described above.
  • the invention also relates to the use of nucleic acid molecules which encode a cytosolic starch phosphorylase or parts thereof for the production of transgenic potato plants with a reduced activity of the cytosolic starch phosphorylase, in particular by at least 60% in comparison to corresponding non-transfor ⁇ mated plants, preferably by at least 80% and particularly preferably by at least 95% and a changed germination behavior.
  • Figure 1 shows schematically the plasmid pBin-Anti-STPI K ⁇ r ⁇ .
  • cSTP 1.7 approx. 1.7 kb long DNA fragment which comprises part of the coding region for cytosolic starch phosphorylase from potato and is linked in antisense orientation to the 35S promoter.
  • FIG. 2 shows two polyacrylamide gels for detecting the activity of the cytosolic starch phosphorylase in leaf (A) and tuber tissue (B) transgenic potato plants which have been transformed with the plasmid pBin-Anti-STPI K ⁇ n .
  • Raw protein extracts from leaf and tuber tissue were separated in a non-denaturing polyacrylamide gel (discontinuous system; 12% and 4% (w / v) acrylamide).
  • the separating gel contained 2.4% (w / v) glycogen.
  • Approx. 20 ⁇ g protein was applied per lane.
  • the electrophoresis was carried out at 100 volts for 4 h. The direction of migration is from the top (cathode) to the bottom (anode).
  • the gels were incubated overnight in 20 mM glucose-1-phosphate / 100 mM citrate, pH 6.0 at room temperature. Protein bands with starch-synthesizing activity are visible as blue bands.
  • the cytosolic starch phosphorase (STPI) is strongly inhibited in its mobility by the immobilized polysaccharide.
  • the plastidic starch phosphorylase (STPII) is not so badly affected in its rate of migration.
  • FIG. 3 shows the germination behavior of tubers of wild-type plants (S. tuberosum L. cv Desiree; right) in comparison to tubers of transformed line cSTP 9 (left) after storage for 5 months at 20 ° C in the dark.
  • FIG. 4 shows the germination behavior of tubers from wild-type plants (center) in comparison to tubers of the transformed lines cSTP 6 (left) and cSTP 7 (right) after storage for 10 months at 20 ° C. in the dark .
  • FIG. 5 shows a statistical overview of the number of shoot ends formed on average per tuber or per 25 tuber in tubers of wild-type plants (cDesi) or the transformed lines (cSTP-6, -7, -9, -14, - 15, -16 and -18) after storage for 5 months at 20 ° C in the dark.
  • FIG. 6 shows a statistical overview of the average number of shoot ends formed per "eye" in tubers from wild-type plants (cDesi) and tubers in transformed lines (cSTP-6, -7, -9, -14, -15, -16 and -18).
  • the E.coli strain DH5 ⁇ (Bethesda Research Laboratories, Gaithersburgh, USA) was used for cloning.
  • the DNA was transferred by direct transformation using the Höfgen & Willmitzer method (Nucleic Acids Res. 16 (1988), 9877).
  • the plasmid DNA of transformed Agrobacteria was isolated by the method of Birnboim, & Doly (Nucleic Acids Res. 7 (1979), 1513-1523) and analyzed by gel electrophoresis after a suitable restriction cleavage.
  • the membrane was prehybridized in NSEB buffer for 2 h at 68 ° C. and then hybridized in NSEB buffer overnight at 6B ° C. in the presence of the radioactively labeled sample.
  • Potato plants are kept in the greenhouse under the following conditions:
  • the plants are kept in individual pots (200 cm 2 , 15 cm deep) and watered daily.
  • the tubers are harvested 4 months after the transfer of the tissue culture plants into the greenhouse.
  • Tubers with a fresh weight of 8-16 g are used for biochemical analyzes. The fresh weight is determined immediately after harvesting.
  • the harvested tubers are washed and stored in boxes at 20 ° C for 5 to 10 months in the dark. Examples
  • an antisense construct which encodes an anti-sense RNA to transcripts which encode cytosolic starch phosphorylase from potato
  • part of the coding region described in Mori et al. (loc. cit.) described cDNA amplified by means of PCR from a ⁇ ZAP cDNA library from tuber tissue.
  • a 1.7 kb Asp718 / Smal fragment was inserted with smooth ends into the Smal interface of the binary plant transformation vector pBIN19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). This contains the 35S promoter of the CaMV and the polyadenylation signal of the octopine synthase gene. Restriction digestion ensured that the coding region was arranged in an antisense orientation to the promoter.
  • the resulting construct was designated pBin-Anti-STPI Km (see FIG. 1).
  • the vector pBin-Anti-STP Km was introduced into the Agrobacterium tumefaciens strain C58C1: pGV2260 (Höfgen and Willmitzer, Nucl. Acid Res. 16
  • RNA from leaf or tuber material was isolated from various independent lines of the transgenic potato plants produced according to Example 2 and analyzed by means of Northern blot analysis for the expression of mRNA which encodes cytosolic starch phosphorylase.
  • mRNA which encodes cytosolic starch phosphorylase.
  • samples from wild-type plants Solanum tuberosum L. cv. Desiree
  • the detection of the activity of the cytosolic starch phosphorylase in tissues of the transformed potato plants was carried out by the method of Steup (In: Methods in Plant Biochemistry 3, Academic Press Limited (1990), 103-128).
  • crude protein extracts were first obtained from the tissues to be examined by freezing tissue in extraction buffer (100 mM HEPES-NaOH, pH 7.5; 1 mM EDTA, 10% (vol. / Vol.) Glycerol; 5 mM DTT , 200 mg Na 2 S0 3 ; 150 mg Na 2 S 2 0 5 ) homogenized. After centrifugation, the clear supernatant was separated in a polyacrylamide gel.
  • Electrophoresis was carried out as described in Steup and Latzko (Planta 145 (1979), 69-759). Here a non-de- natural discontinuous system used (12% or 4% (w / v) acrylamide).
  • the separating gel contained 2.4% (w / v) glycogen.
  • the gel was run after the gel run in a solution of 20 mM glucose-1-phosphate / 100 mM citrate; pH 6.0 incubated overnight at room temperature. The gel was then incubated in a Lugolian solution for 5 min. Decolorization was carried out by extensive washing with water over a longer period of time. Blue color indicates the presence of starch-synthesizing enzyme activities.
  • the activity of the cytosolic starch phosphorylase can subsequently be determined, for example, by densitometric analysis of the corresponding protein bands.
  • FIG. 2 shows a polyacrylamide gel in which seven independent transgenic potato lines produced according to Example 2 were tested for their activity on cytosolic starch phosphorylase.
  • tubers of the plants were stored at 20 ° C. and the germination behavior of the tubers was examined after various periods.
  • Figure 3 shows a comparison of tubers of Solanum tuberosum L. cv. Desiree (wild type) and tubers of the line cSTPI-9 transformed with the plasmid pBin-Anti-STPI Km , each of which had been stored at 20 ° C. for 5 months.
  • FIG. 4 shows a comparison of wild-type tubers and tubers from two other transformed lines (cSTPI-6 and cSTPI-7) which have been stored at 20 ° C. for 10 months. It is clear from the two figures that the tubers of the transformed lines, which have a reduced activity of the cytosolic starch phosphorylase, have a drastically changed germination behavior.
  • the bulbs of the transformed lines form on the one hand significantly more shoot ends per bulb and also more shoot ends per out of no eye. Furthermore, more eyes usually germinate in the tubers of the transformed plants with reduced activity of the cytosolic starch phosphorylase.
  • a statistical evaluation according to - is shown in FIGS. 5 and 6.
  • the potato plants growing from the tubers of the transformed plants furthermore show an increased yield (in fresh bulb weight / plant). This is shown in the following table using the example of the transformed lines cStP6, cSTP7, cSTP9, CSTP14, CSTP15, CSTP16 and cSTP18.
  • the table shows that plants with a reduced activity of the cytosolic starch phosphorylase on the one hand form more tubers per plant than Wild-type plants and, moreover, the fresh tuber weight per plant is significantly higher than in wild-type plants.
  • the transformed potato plants do not differ from wild-type plants with regard to the starch formed in the tubers.

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Abstract

The description relates to transgenic potato plants containing cells having a reduced cytosolic starch phosphorylasis activity in comparison with wild types of plant. By comparison with tubers of wild plant types, tubers of such potato plants exhibit a drastically altered germination behaviour, resulting in an increased number of sprout ends and hence of stolones and tubers. Plants from such tubers give higher yields.

Description

Kartoffelpflanzen mit einer verringerten Aktivität der cyto- solischen Stärkephosphorylase und einem veränderten Potato plants with a reduced activity of the cytosolic starch phosphorylase and a modified one
KeimungsverhaltenGermination behavior
Die vorliegende Erfindung betrifft transgene Kartoffelpflan¬ zen, die Zellen mit einer im Vergleich zu Wildtyp-Pflanzen verringerten Aktivität der cytosolischen Stärkephosphorylase enthalten. Die Knollen derartiger Kartoffelpflanzen zeigen im Vergleich zu Knollen von Wildtyp-Pflanzen ein verändertes Keimungsverhalten, das zur Bildung einer erhöhten Anzahl von Sproßenden und folglich zu einer erhöhten Anzahl von Stolo- nen und Knollen führt . Weiterhin weisen solche Pflanzen einen erhöhten Ertrag auf .The present invention relates to transgenic potato plants which contain cells with a reduced activity of the cytosolic starch phosphorylase compared to wild-type plants. The tubers of such potato plants show a different germination behavior compared to tubers of wild-type plants, which leads to the formation of an increased number of shoot ends and consequently to an increased number of stoles and tubers. Such plants also have an increased yield.
Der Ertrag beim landwirtschaftlichen Anbau von Kartoffeln wird in erster Linie bestimmt durch die Anzahl der Spro߬ enden, die pro angelegter Knolle gebildet werden. Normaler¬ weise bildet eine, auskeimende Kartoffelknolle lediglich ein, zeitweise 2 bis 3, Sproßenden, wobei aufgrund der Apikaido¬ minanz dieser Sproßenden das Wachstum weiterer potentiell vorhandener Seitensprosse unterdrückt wird. Ausgehend von den Sproßenden werden während des weiteren Wachstums der Kartoffelpflanzen Stolone gebildet, an denen später die Knollen gebildet werden. Da der Ernteertrag mit der Anzahl der durch die entwickelten Sproße gebildeten Stolone korre- liert, besteht ein Bestreben, die Keimung von Kartoffel- pflanzen derart zu manipulieren, daß eine möglichst große Anzahl von "Augen" auskeimen und sich zu Sproßenden ent¬ wickeln. Eine Methode besteht darin, den zuerst gebildeten Sproß einer Knolle, der aufgrund seiner Apikaidominanz das Wachstum weiterer Sproßenden unterdrückt, abzubrechen. Dies führt zum Wachstum weiterer Sproßenden. Eine weitere Methode besteht in dem Vorkeimen der zum Auslegen bestimmten Kartof- fein unter regulierten Bedingungen in speziellen Behältern (siehe z.B. Bouman, Kartoffelbau 47 (1996) , 18-21; van de Waart, Kartoffelbau 44 (1993) , 18-20) . Derartige Methoden sind jedoch sehr kostenintensiv, da sie neben den speziellen Behältern auch Lagerräume erfordern, in den sowohl die Licht- als auch die Temperaturbedingungen reguliert werden können. Im Zusammenhang mit der Unterdrückung der Knollen¬ keimung wurde bereits in der DE-Al 42 13 444 eine gentechni¬ sche Manipulation von Kartoffelpflanzen dahingehend vorge¬ schlagen, daß am Stärkemetabolismus beteiligte Enzyme inhi¬ biert werden. Entsprechende Ansätze für eine verstärkte Sproßbildung der Kartoffelknollen sind bisher jedoch nicht bekannt. Es besteht somit ein Bedarf an Kartoffelpflanzen bzw. Verfahren, bei denen sich die obenbeschriebenen ar- beits- bzw. kostenintensiven Schritte erübrigen und die zu einer erhöhten Sproßanzahl und somit erhöhtem Knollenertrag führen.The yield from the agricultural cultivation of potatoes is primarily determined by the number of shoot ends that are formed per tuber. Normally, a germinating potato tuber forms only one, sometimes 2 to 3, shoot ends, the growth of further potentially existing side shoots being suppressed due to the apikaidominance of these shoot ends. Starting from the shoot ends, stolons are formed during the further growth of the potato plants, on which the tubers are later formed. Since the crop yield correlates with the number of stolons formed by the developed shoots, there is an effort to manipulate the germination of potato plants in such a way that the largest possible number of "eyes" germinate and develop into shoot ends. One method is to break off the first sprout of a tuber, which suppresses the growth of further sprouts due to its apica dominance. This leads to the growth of further shoots. Another method is to pre-germinate the potatoes to be laid out fine under regulated conditions in special containers (see e.g. Bouman, Kartoffelbau 47 (1996), 18-21; van de Waart, Kartoffelbau 44 (1993), 18-20). However, such methods are very cost-intensive since, in addition to the special containers, they also require storage rooms in which both the light and the temperature conditions can be regulated. In connection with the suppression of tuber germination, a genetic engineering manipulation of potato plants was already proposed in DE-A1 42 13 444 in such a way that enzymes involved in starch metabolism are inhibited. Appropriate approaches for increased sprout formation of the potato tubers are not yet known. There is therefore a need for potato plants or processes in which the labor-intensive or cost-intensive steps described above are unnecessary and which lead to an increased number of shoots and thus an increased tuber yield.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, Kartoffelpflanzen zur Verfügung zu stellen, deren Knollen bei Keimung eine hohe Anzahl von Sproßenden bilden.The present invention is therefore based on the object of providing potato plants whose tubers form a large number of shoot ends when germinated.
Diese Aufgabe wird durch die in den Patentansprüchen be¬ zeichneten Ausführungsformen gelöst.This object is achieved by the embodiments described in the claims.
Somit betrifft die Erfindung transgene Kartoffelpflanzen, die Zellen mit einer im Vergleich zu Wildtyp-Pflanzen, d.h. entsprechenden nicht-transformierten Pflanzen, um mindestens 60 % verringerten Aktivität der cytosolischen Stärkephospho¬ rylase enthalten. In einer bevorzugten Ausführungsform ist die Aktivität der cytosolischen Stärkephosphorylase um min¬ destens 80 % und besonders bevorzugt um mindestens 95 % ver¬ ringert im Vergleich zu Wildtyp-Kartoffelpflanzen.Thus, the invention relates to transgenic potato plants that contain cells with a compared to wild-type plants, i.e. corresponding non-transformed plants, contain at least 60% reduced activity of the cytosolic starch phosphoryl. In a preferred embodiment, the activity of the cytosolic starch phosphorylase is reduced by at least 80% and particularly preferably by at least 95% compared to wild-type potato plants.
Unter dem Begriff "cytosolische Stärkephosphorylase" wird die im Cytoplasma von Pflanzenzellen lokalisierte Isoform der Stärkephosphorylase (EC 2.4.1.1) verstanden, die auch als Isoform H oder I bekannt ist. Von der zweiten, der pla- stidären Isoform unterscheidet sich diese dadurch, daß sie beispielsweise eine wesentlich höhere Affinität für hochver¬ zweigte Glucane zeigt (Shimomura et al . , J. Biochem. (Tokyo) 91 (1982) , 703-717; Yang und Steup, Plant Physiol . 94, 960- 969) und eine geringe Affinität zu Oligoglucanen. Das Enzym katalysiert die reversible Phosphorolyse von α-1, 4-Glucanen. Die Aktivität der Stärkephosphorylase läßt sich beispiels¬ weise bestimmen wie beschrieben in Parvin und Smith (Anal. Biochem. 27 (1969) , 65-72) , Conrads et al . (Biochim. Biophys. Acta 882 (1986) , 452-463) , Steup und Latzko (Planta 145 (1979) , 69-75) , Steup (In. Methods in Plant Biochemistry 3; Academic Press Limited (1990) , 103-128) oder Sonnewald et al. (Plant Mol. Biol . 27 (1995) , 567-576) .The term "cytosolic starch phosphorylase" is understood to mean the isoform of starch phosphorylase (EC 2.4.1.1) localized in the cytoplasm of plant cells, which also is known as isoform H or I. This differs from the second, the plastid isoform, in that it shows, for example, a much higher affinity for highly branched glucans (Shimomura et al., J. Biochem. (Tokyo) 91 (1982), 703-717; Yang and Steup, Plant Physiol. 94, 960-969) and a low affinity for oligoglucans. The enzyme catalyzes the reversible phosphorolysis of α-1, 4-glucans. The activity of the starch phosphorylase can be determined, for example, as described in Parvin and Smith (Anal. Biochem. 27 (1969), 65-72), Conrads et al. (Biochim. Biophys. Acta 882 (1986), 452-463), Steup and Latzko (Planta 145 (1979), 69-75), Steup (In. Methods in Plant Biochemistry 3; Academic Press Limited (1990), 103- 128) or Sonnewald et al. (Plant Mol. Biol. 27: 567-576 (1995)).
Erfindungsgemäß ist die Aktivität der cytosolischen Stärke¬ phosphorylase vorzugsweise in allen bzw. in fast allen Zel¬ len der Pflanze verringert. Zumindestens jedoch in den Knol¬ len und den sich aus diesen entwickelnden Sproßenden.According to the invention, the activity of the cytosolic starch phosphorylase is preferably reduced in all or in almost all cells of the plant. At least, however, in the bulbs and the shoot ends that develop from them.
Es wurde überraschend gefunden, daß Knollen von Kartoffel¬ pflanzen, die eine derart verringerte Aktivität der cytoso¬ lischen Stärkephosphorylase aufweisen, im Vergleich zu Knol¬ len von Wildtyp-Pflanzen ein drastisch verändertes Keimungs¬ verhalten zeigen. Unter Keimung wird hierbei das Auswachsen von Sproßenden aus Knollen verstanden.It has surprisingly been found that tubers of potato plants which have such a reduced activity of the cytosolic starch phosphorylase show a drastically changed germination behavior compared to tubers of wild-type plants. Germination here means the growth of shoot ends from tubers.
Das veränderte Keimungsverhalten zeigt sich darin, daß der¬ artige KnollenThe changed germination behavior is evident in the fact that such tubers
(a) zum einen im Durchschnitt pro Knolle mehr Sprosse aus¬ bilden; und/oder(a) on the one hand, form more shoots per tuber on average; and or
(b) im Durchschnitt pro Knolle mehr Augen (d.h. die an den Knollen vorhandenen Sproßknospen) auskeimen und zur Bil¬ dung von Sprossen führen; und/oder(b) on average more eyes per tuber (i.e. the buds present on the tuber) germinate and lead to the formation of sprouts; and or
(c) im Durchschnitt pro auskeimendem Knollenauge mehr Sproßenden gebildet werden, insbesondere, wenn die Kei¬ mung nach Lagerung für 5 Monate bei 20°C im Dunkeln er¬ folgt. Wie bereits oben erläutert, bilden Knollen von Wildtyp- Pflanzen beim normalen Auskeimen in der Regel 1, maximal 2 bis 3, Sproßenden. Zur Erhöhung der Anzahl der Sproßenden ist entweder die Eliminierung des Apikaisprosses oder eine Vorkeimung unter speziellen Bedingungen notwendig. Hingegen zeigen Knollen der erfindungsgemäßen Kartoffelpflanzen, die Zellen mit einer verringerten cytosolischen Stärkephos¬ phorylase enthalten, bei Keimung, insbesondere nach Lagerung bei 20°C im Dunkeln, eine drastisch erhöhte Anzahl von Sproßenden. Dies führt zur Ausbildung von mehr Stolonen und folglich zu mehr Knollen pro Pflanze. Insgesamt steigt damit der Knollenertrag pro Pflanze. Dies betrifft sowohl die An¬ zahl der Knollen pro Pflanze, als auch das Gesamtfrischge- wicht von Knollen pro Pflanze.(c) on average, more bud ends are formed per budding tuber eye, especially if the germination takes place after storage for 5 months at 20 ° C. in the dark. As already explained above, tubers of wild-type plants usually form 1, at most 2 to 3, shoot ends during normal germination. To increase the number of shoots, either the elimination of the apikai shoot or a pre-germination under special conditions is necessary. In contrast, tubers of the potato plants according to the invention, which contain cells with a reduced cytosolic starch phosphorylase, show a drastically increased number of shoot ends when germinated, in particular after storage at 20 ° C. in the dark. This leads to the formation of more stolons and consequently more tubers per plant. Overall, the tuber yield per plant increases. This concerns both the number of tubers per plant and the total fresh weight of tubers per plant.
In einer bevorzugten Ausführungsform ist bei den erfindungs- gemäßen Kartoffelpflanzen die durchschnittliche Anzahl der Sprosse, die pro Knolle gebildet werden, wenn die Keimung nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln er¬ folgt, mindestens verdoppelt im Vergleich zu Wildtyp-Pflan¬ zen, d.h. entsprechenden nicht-transformierten Pflanzen.In a preferred embodiment, in the potato plants according to the invention, the average number of shoots which are formed per tuber when germination takes place after storage for 5 months at 20 ° C. in the dark is at least doubled in comparison to wild-type plants ¬ zen, ie corresponding non-transformed plants.
In einer weiteren bevorzugten Ausführungsform ist bei den Knollen der erfindungsgemäßen Kartoffelpflanzen die Anzahl der Sproßenden, die pro auskeimendem Auge gebildet werden, wenn die Keimung nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln erfolgt, mindestens verdoppelt im Vergleich zu Knollen von Wildtyp-Pflanzen, d.h. entsprechenden nicht- transformierten Pflanzen.In a further preferred embodiment, in the tubers of the potato plants according to the invention, the number of shoot ends which are formed per budding eye when the germination takes place after storage for 5 months at 20 ° C. in the dark is at least doubled in comparison to tubers of wild-type Plants, ie corresponding non-transformed plants.
Neben dem veränderten Keimungsverhalten weisen die erfin¬ dungsgemäßen Pflanzen auch einen gesteigerten Ertrag hin¬ sichtlich der Knollenanzahl sowie des Knollengewichtes auf. Dabei ist die Anzahl der Knollen pro Pflanze vorzugsweise um mindestens 20 %, bevorzugt um mindestens 50 % und besonders bevorzugt um mindestens 100 % höher als in entsprechenden nicht-transformierten Pflanzen unter gleichen Wachstumsbe- dingungen.In addition to the changed germination behavior, the plants according to the invention also have an increased yield with regard to the number of tubers and the weight of the tubers. The number of tubers per plant is preferably at least 20%, preferably at least 50% and particularly preferably at least 100% higher than in corresponding ones non-transformed plants under the same growth conditions.
Ferner ist das Knollenfrischgewicht aller Knollen pro Pflanze vorzugsweise um mindestens 10 %, vorzugsweise um mindestens 15 % und besonders bevorzugt um mindestens 20 % höher als bei entsprechenden nicht-transformierten Pflanzen unter gleichen Wachstumsbedingungen.Furthermore, the fresh tuber weight of all tubers per plant is preferably at least 10%, preferably at least 15% and particularly preferably at least 20% higher than in corresponding non-transformed plants under the same growth conditions.
Die Verringerung der Aktivität der cytosolischen Stärkephos¬ phorylase in den Zellen der erfindungsgemäßen Kartoffel- pflanzen läßt sich prinzipiell durch verschiedene, dem Fach¬ mann bekannte Methoden erreichen.The activity of the cytosolic starch phosphorylase in the cells of the potato plants according to the invention can be reduced in principle by various methods known to the person skilled in the art.
In einer bevorzugten Ausführungsform erfolgt die Verringe¬ rung der Aktivität der cytosolischen Stärkephosphorylase durch die Inhibierung der Expression endogener Gene, die dieses Enzym codieren. Bevorzugt sind hierbei molekularbio¬ logische Techniken, die auf einem antisense-, Ribozym- oder einem Cosuppressionseffekt beruhen. Bei einem antisense- Effekt wird eine entsprechende RNA in antisense-Orientierung exprimiert . Diese hat vorzugsweise eine Länge von mindestens 30 Nucleotiden, bevorzugt von mindestens 50 Nucleotiden und besonders bevorzugt von mindestens 100 Nucleotiden. Die ex- primierte antisense-RNA sollte eine hohe Homologie zu den endogen in der Pflanze exprimierten Transkripten, die cyto- solische Stärkephosphorylase codieren, haben. Die Homologie beträgt vorzugsweise mindestens 90 %, bevorzugt mindestens 95 % und besonders bevorzugt mindestens 99 %. Bei einem Ri- bozym-Effekt wird eine RNA exprimiert, die spezifisch Transkripte cytosolischer Stärkephosphorylase spalten kann. Die Expression von Ribozymen zur Verringerung der Aktivität von bestimmten Enzymen in Zellen ist dem Fachmann ebenfalls bekannt und ist beispielsweise beschrieben in EP-Bl 0 321 201. Die Expression von Ribozymen in pflanzlichen Zel¬ len wurde z.B. beschrieben in Feyter et al . (Mol. Gen. Genet. 250 (1996) , 329-338) . Der Cosuppressions-Effekt be¬ ruht auf der Expression einer sense-RNA, die die Expression von endogener Stärkephosphorylase-mRNA unterdrückt. Die Ausführung dieser Techniken sind dem Fachmann bekannt. Das Verfahren der Cosuppression ist beispielsweise beschrieben in Jorgensen (Trends Biotechnol. 8 (1990), 340-344) , Niebel et al. (Curr. Top. Microbiol . Immuno1. 197 (1995) , 91-103) , Flavell et al . (Curr. Top Microbiol. Immuno1. 197 (1995) , 43-46) , Palaqui und Vaucheret (Plant. Mol. Biol . 29 (1995) , 149-159) , Vaucheret et al. (Mol. Gen. Genet . 248 (1995) , 311-317) , de Borne et al . (Mol. Gen. Genet. 243 (1994) , 613- 621) und anderen Quellen.In a preferred embodiment, the activity of the cytosolic starch phosphorylase is reduced by inhibiting the expression of endogenous genes which code for this enzyme. Molecular biological techniques based on an antisense, ribozyme or a cosuppression effect are preferred. In the case of an antisense effect, a corresponding RNA is expressed in the antisense orientation. This preferably has a length of at least 30 nucleotides, preferably of at least 50 nucleotides and particularly preferably of at least 100 nucleotides. The expressed antisense-RNA should have a high homology to the transcripts expressed endogenously in the plant, which encode cytosolic starch phosphorylase. The homology is preferably at least 90%, preferably at least 95% and particularly preferably at least 99%. In the case of a ribozyme effect, an RNA is expressed which can specifically cleave transcripts of cytosolic starch phosphorylase. The expression of ribozymes for reducing the activity of certain enzymes in cells is also known to the person skilled in the art and is described, for example, in EP-Bl 0 321 201. The expression of ribozymes in plant cells was described, for example, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338). The cosuppression effect is based on the expression of a sense RNA which expresses the expression suppressed by endogenous starch phosphorylase mRNA. The implementation of these techniques is known to the person skilled in the art. The method of cosuppression is described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al. (Curr. Top. Microbiol. Immuno. 197 (1995), 91-103), Flavell et al. (Curr. Top Microbiol. Immuno. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621) and other sources.
In einer besonders bevorzugten Ausführungsform der vorlie¬ genden Erfindung wird die Verringerung der Aktivität einer cytosolischen Stärkephosphorylase in den Zellen dadurch er¬ reicht, daß man transgene Kartoffelpflanzen erzeugt, die stabil ins Genom integriert ein rekombinantes DNA-Molekül enthalten, das folgende Elemente umfaßt:In a particularly preferred embodiment of the present invention, the reduction in the activity of a cytosolic starch phosphorylase in the cells is achieved by producing transgenic potato plants which contain a recombinant DNA molecule which is stably integrated into the genome and comprises the following elements:
(a) einen Promotor, der die Transkription zumindest in Zel¬ len der Knollen von Kartoffelpflanzen ermöglicht; und(a) a promoter which enables transcription at least in cells of the tubers of potato plants; and
(b) eine in antisense-Orientierung mit diesem Promotor ver¬ knüpfte DNA-Sequenz, deren Transkripte ganz oder teil¬ weise komplementär sind zu Transkripten eines endogen in Kartoffelpflanzenzellen vorliegenden Gens, das cytosoli- sche Stärkephosphorylase codiert .(b) a DNA sequence linked in antisense orientation to this promoter, the transcripts of which are wholly or partly complementary to transcripts of an endogenously present gene in potato plant cells which encodes cytosolic starch phosphorylase.
Promotoren, die die Expression in pflanzlichen Zellen ge¬ währleisten sind in großem Umfang beschrieben. Für eine Ex¬ pression in den Knollen der Kartoffelpflanzen bietet sich beispielsweise der Promotor des Patatingens B33 aus Kartof¬ fel an (Rocha-Sosa et al . , EMBO J.8 (1989) , 23-29) . Für eine konstitutive Expression eignet sich z.B. der 35S-Promotor des CaMV (Franck et al . , Cell 21 (1980) , 285-292) . DNA-Sequenzen, die eine cytosolische Stärkephosphorylase aus Kartoffel codieren sind bereits beschrieben (siehe z.B. Mori et al . , J. Biol. Chem. 266 (1991) , 18446-18453) . Mit Hilfe dieser DNA-Sequenzen ist es dem Fachmann möglich mittels gängiger Verfahren weitere Sequenzen zu isolieren, die cyto- solische Stärkephosphorylase aus Kartoffel codieren, falls dies erforderlich ist .Promoters which ensure expression in plant cells have been widely described. For example, the promoter of the patatin gene B33 from potato is suitable for expression in the tubers of the potato plants (Rocha-Sosa et al., EMBO J.8 (1989), 23-29). The 35S promoter of the CaMV (Franck et al., Cell 21 (1980), 285-292) is suitable, for example, for constitutive expression. DNA sequences which encode a potato cytosolic starch phosphorylase have already been described (see, for example, Mori et al., J. Biol. Chem. 266 (1991), 18446-18453). With the help of these DNA sequences it is possible for the person skilled in the art to isolate further sequences using standard methods which cyto- encode potato starch phosphorylase, if necessary.
Neben den obengenannten Möglichkeiten kann die Verringerung der Aktivität der cytosolischen Stärkephosphorylase auch durch Inaktivierung der endogen vorhandenen Gene, die dieses Enzym codieren, erreicht werden. Techniken hierfür sind bei¬ spielsweise Transposonmutagenese oder gene-tagging. Alternativ besteht auch die Möglichkeit, in den Zellen Anti¬ körper zu exprimieren, die spezifisch cytosolische Stärke¬ phosphorylase erkennen.In addition to the abovementioned possibilities, the activity of the cytosolic starch phosphorylase can also be reduced by inactivating the endogenously present genes which code for this enzyme. Techniques for this are, for example, transposon mutagenesis or gene tagging. Alternatively, there is also the possibility of expressing antibodies in the cells which specifically recognize cytosolic starch phosphorylase.
Verfahren zur Herstellung transgener Kartoffelpflanzen sind in der Literatur beschrieben, siehe z.B. (Rocha-Sosa (loc.cit.) . Bevorzugt wird die Transformation mittels Agro- bakterien angewandt .Methods for producing transgenic potato plants are described in the literature, see e.g. (Rocha-Sosa (loc.cit.). The transformation using agrobacteria is preferably used.
Ferner betrifft die vorliegende Erfindung Vermehrungsmate¬ rial der erfindungsgemäßen Kartoffelpflanzen, insbesondere Samen und besonders bevorzugt Kartoffelknollen. Diese ent¬ halten Zellen mit einer im Vergleich zu Knollen von Wildtyp- Pflanzen verringerten Aktivität der cytosolischen Stärke¬ phosphorylase und ein wie oben beschriebenes verändertes Keimungsverhalten auf.Furthermore, the present invention relates to propagation material of the potato plants according to the invention, in particular seeds and particularly preferably potato tubers. These contain cells with a reduced activity of the cytosolic starch phosphorylase in comparison with tubers of wild-type plants and an altered germination behavior as described above.
Schließlich betrifft die Erfindung auch die Verwendung von Nucleinsäuremolekülen, die eine cytosolische Stärkephospho¬ rylase codieren oder Teile davon zur Herstellung von trans- genen Kartoffelpflanzen mit einer verringerten Aktivität der cytosolischen Stärkephosphorylase, insbesondere um minde¬ stens 60 % im Vergleich zu entsprechenden nicht-transfor¬ mierten Pflanzen, vorzugsweise um mindestens 80 % und beson¬ ders bevorzugt um mindestens 95 % und einem verändertem Kei¬ mungsverhalten. Beschreibung der Figuren.Finally, the invention also relates to the use of nucleic acid molecules which encode a cytosolic starch phosphorylase or parts thereof for the production of transgenic potato plants with a reduced activity of the cytosolic starch phosphorylase, in particular by at least 60% in comparison to corresponding non-transfor¬ mated plants, preferably by at least 80% and particularly preferably by at least 95% and a changed germination behavior. Description of the figures.
Figur 1 zeigt schematisch das Plasmid pBin-Anti-STPIKτrι. cSTP 1,7: ca. 1,7 kb langes DNA-Fragment, das einen Teil der codierenden Region für cytosolische Stärkephosphorylase aus Kartoffel umfaßt und in antisense-Orientierung mit dem 35S-Promotor ver¬ knüpft ist.Figure 1 shows schematically the plasmid pBin-Anti-STPI Kτrι . cSTP 1.7: approx. 1.7 kb long DNA fragment which comprises part of the coding region for cytosolic starch phosphorylase from potato and is linked in antisense orientation to the 35S promoter.
Figur 2 zeigt zwei Polyacrylamidgele zum Nachweis der Ak¬ tivität der cytosolischen Stärkephosphorylase in Blatt- (A) und Knollengewebe (B) transgener Kartof¬ felpflanzen, die mit dem Plasmid pBin-Anti-STPIKτn transformiert sind. Proteinrohextrakte von Blatt- und Knollengewebe wurden in einem nicht-denaturie¬ renden Polyacrylamidgel (diskontinuierliches System; 12 % bzw. 4 % (Gew. /Vol.) Acrylamid) auf¬ getrennt. Das Trenngel enthielt 2,4 % (Gew. /Vol.) Glykogen. Es wurden ca. 20 μg Protein pro Spur aufgetragen. Die Elektrophorese wurde für 4 h bei 100 Volt durchgeführt. Die Wanderungsrichtung ist von oben (Kathode) nach unten (Anode) . Für die Ak¬ tivitätsfärbung wurden die Gele in 20 mM Glucose- l-Phosphat/100 mM Citrat, pH 6,0 bei Raumtempera¬ tur über Nacht inkubiert . Proteinbanden mit stär¬ kesynthetisierender Aktivität werden als blaue Banden sichtbar. Die cytosolische Stärkephospho¬ rylase (STPI) wird durch das immobilisierte Poly- saccharid stark in seiner Mobilität gehemmt . Die plastidäre Stärkephosphorylase (STPII) ist dagegen in ihrer Wanderungsgeschwindigkeit nicht so stark beeinträchtigt .FIG. 2 shows two polyacrylamide gels for detecting the activity of the cytosolic starch phosphorylase in leaf (A) and tuber tissue (B) transgenic potato plants which have been transformed with the plasmid pBin-Anti-STPI Kτn . Raw protein extracts from leaf and tuber tissue were separated in a non-denaturing polyacrylamide gel (discontinuous system; 12% and 4% (w / v) acrylamide). The separating gel contained 2.4% (w / v) glycogen. Approx. 20 μg protein was applied per lane. The electrophoresis was carried out at 100 volts for 4 h. The direction of migration is from the top (cathode) to the bottom (anode). For activity staining, the gels were incubated overnight in 20 mM glucose-1-phosphate / 100 mM citrate, pH 6.0 at room temperature. Protein bands with starch-synthesizing activity are visible as blue bands. The cytosolic starch phosphorase (STPI) is strongly inhibited in its mobility by the immobilized polysaccharide. The plastidic starch phosphorylase (STPII), on the other hand, is not so badly affected in its rate of migration.
Figur 3 zeigt im Vergleich das Keimungsverhalten von Knol¬ len von Wildtyp-Pflanzen (S. tuberosum L. cv Desiree; rechts) im Vergleich zu Knollen der transformierten Linie cSTP 9 (links) nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln.FIG. 3 shows the germination behavior of tubers of wild-type plants (S. tuberosum L. cv Desiree; right) in comparison to tubers of transformed line cSTP 9 (left) after storage for 5 months at 20 ° C in the dark.
Figur 4 zeigt im Vergleich das Keimungsverhalten von Knol¬ len von Wildtyp-Pflanzen (Mitte) im Vergleich zu Knollen der transformierten Linien cSTP 6 (links) und cSTP 7 (rechts) nach einer Lagerung von 10 Mo¬ naten bei 20°C im Dunkeln.FIG. 4 shows the germination behavior of tubers from wild-type plants (center) in comparison to tubers of the transformed lines cSTP 6 (left) and cSTP 7 (right) after storage for 10 months at 20 ° C. in the dark .
Figur 5 zeigt eine statistische Übersicht über die Anzahl der im Durchschnitt pro Knolle bzw. pro 25 Knollen gebildeten Sproßenden bei Knollen von Wildtyp- Pflanzen (cDesi) bzw. den transformierten Linien (cSTP-6, -7, -9, -14, -15, -16 und -18) nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln.FIG. 5 shows a statistical overview of the number of shoot ends formed on average per tuber or per 25 tuber in tubers of wild-type plants (cDesi) or the transformed lines (cSTP-6, -7, -9, -14, - 15, -16 and -18) after storage for 5 months at 20 ° C in the dark.
Figur 6 zeigt eine statistische Übersicht über die durch¬ schnittliche Anzahl der pro "Auge" gebildeten Sproßenden bei Knollen von Wildtyp-Pflanzen (cDesi) und Knollen von transformierten Linien (cSTP-6, -7, -9, -14, -15, -16 und -18) .FIG. 6 shows a statistical overview of the average number of shoot ends formed per "eye" in tubers from wild-type plants (cDesi) and tubers in transformed lines (cSTP-6, -7, -9, -14, -15, -16 and -18).
Die Beispiele erläutern die Erfindung.The examples illustrate the invention.
In den Beispielen werden unter anderem folgende Materialien und Techniken verwendet .The following materials and techniques are used in the examples.
1. Bakterienstämme1. Bacterial strains
Für Clonierungen wurde der E.coli-Stamm DH5α (Bethesda Research Laboratories, Gaithersburgh, USA) verwendet.The E.coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh, USA) was used for cloning.
Die Transformation der Plasmide in die Kartoffelpflanzen wurde mit Hilfe des Agrobacterium tumefaciens-Stammes C58C1 pGV2260 durchgeführt (Deblaere et al . , Nucl . Acids Res. 13 (1985) , 4777-4788) . 2. Transformation von Agrobacterium tumefaciensThe transformation of the plasmids into the potato plants was carried out using the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788). 2. Transformation of Agrobacterium tumefaciens
Der Transfer der DNA erfolgte durch direkte Transforma¬ tion nach der Methode von Höfgen & Willmitzer (Nucleic Acids Res . 16 (1988) , 9877) . Die Plasmid-DNA transfor¬ mierter Agrobakterien wurde nach der Methode von Birnboim, & Doly (Nucleic Acids Res. 7 (1979) , 1513- 1523) isoliert und nach geeigneter Restriktionsspaltung gelelektrophoretisch analysiert.The DNA was transferred by direct transformation using the Höfgen & Willmitzer method (Nucleic Acids Res. 16 (1988), 9877). The plasmid DNA of transformed Agrobacteria was isolated by the method of Birnboim, & Doly (Nucleic Acids Res. 7 (1979), 1513-1523) and analyzed by gel electrophoresis after a suitable restriction cleavage.
3. Transformation von Kartoffeln3. Transformation of potatoes
Zehn kleine mit dem Skalpell verwundete Blätter einer Kartoffel-Sterilkultur (Solanum tuberosum L.cv. Desiree) wurden in 10 ml MS-Medium (Murashige & Skoog, Physiol . Plant 15 (1962) , 473-497) mit 2 % Saccharose gelegt, welches 50 μl einer unter Selektion gewachsenen Agrobac¬ terium tumefaciens-Übernachtkultur enthielt. Nach 3-5 minütigem, leichtem Schütteln erfolgte eine weitere In¬ kubation für 2 Tage im Dunkeln. Daraufhin wurden die Blätter zur Kallusinduktion auf MS-Medium mit 1,6 % Glu¬ cose, 5 mg/1 Naphthylessigsäure, 0,2 mg/1 Benzylaminopu- rin, 250 mg/1 Claforan, 50 mg/1 Kanamycin und 0,80 % Bacto Agar gelegt. Nach einwöchiger Inkubation bei 25°C und 3000 Lux wurden die Blätter zur Sproßinduktion auf MS-Medium mit 1,6 % Glucose, 1,4 mg/1 Zeatinribose, 20 mg/1 Naphthylessigsäure, 20 mg/1 Giberellinsäure, 250 mg/1 Claforan, 50 mg/1 Kanamycin und 0,80 % Bacto Agar gelegt .Ten small leaves of a potato sterile culture (Solanum tuberosum L.cv. Desiree) wounded with the scalpel were placed in 10 ml MS medium (Murashige & Skoog, Physiol. Plant 15 (1962), 473-497) with 2% sucrose, which contained 50 μl of an overnight culture grown under selection Agrobacterium tumefaciens. After 3-5 minutes of gentle shaking, there was a further incubation for 2 days in the dark. Thereupon the leaves for callus induction on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 benzylaminopurine, 250 mg / 1 claforan, 50 mg / 1 kanamycin and 0.80 % Bacto agar laid. After incubation at 25 ° C. and 3000 lux for one week, the leaves were inducible to shoot on MS medium with 1.6% glucose, 1.4 mg / 1 zeatin ribose, 20 mg / 1 naphthylacetic acid, 20 mg / 1 giberellic acid, 250 mg / 1 Claforan, 50 mg / 1 kanamycin and 0.80% Bacto agar.
4. Radioaktive Markierung von DNA-Fragmenten4. Radioactive labeling of DNA fragments
Die radioaktive Markierung von DNA-Fragmenten wurde mit Hilfe eines DNA-Randon Primer Labelling Kits der Firma Boehringer (Deutschland) nach den Angaben des Herstel¬ lers durchgeführt. 5. Northern Blot-AnalyseThe radioactive labeling of DNA fragments was carried out with the aid of a DNA Randon Primer Labeling Kit from Boehringer (Germany) according to the manufacturer's instructions. 5. Northern blot analysis
RNA wurde nach Standardprotokollen aus Blattgewebe oder Knollengewebe von Pflanzen isoliert. 50 μg der RNA wur¬ den auf einem Agarosegel aufgetrennt (1,5 % Agarose, 1 x MEN-Puffer, 16,6 % Formaldehyd) . Das Gel wurde nach dem Gellauf kurz in Wasser gewaschen. Die RNA wurde mit 20 x SSC mittels Kapillarblot auf eine Nylonmembran vom Typ Hybond N (Amersham, UK) transferiert. Die Membran wurde anschließend bei 80°C unter Vakuum für zwei Stunden ge¬ backen.RNA was isolated from leaf or tuber tissue from plants according to standard protocols. 50 μg of the RNA were separated on an agarose gel (1.5% agarose, 1 x MEN buffer, 16.6% formaldehyde). The gel was washed briefly in water after the gel run. The RNA was transferred to a Hybond N nylon membrane (Amersham, UK) using 20 x SSC by capillary blot. The membrane was then baked at 80 ° C. under vacuum for two hours.
Die Membran wurde in NSEB-Puffer für 2 h bei 68°C prähy¬ bridisiert und anschließend in NSEB-Puffer über Nacht bei 6B°C in Gegenwart der radioaktiv markierten Probe hybridisiert .The membrane was prehybridized in NSEB buffer for 2 h at 68 ° C. and then hybridized in NSEB buffer overnight at 6B ° C. in the presence of the radioactively labeled sample.
6. Pflanzenhaltung6. Plant husbandry
Kartoffenpflanzen werden im Gewächshaus unter folgenden Bedingungen gehalten:Potato plants are kept in the greenhouse under the following conditions:
Lichtperiode 16 h bei 25000 Lux und 22°CLight period 16 h at 25000 lux and 22 ° C
Dunkelberiode 8 h bei 15°CDark period 8 h at 15 ° C
Luftfeuchte 60 %Humidity 60%
Die Pflanzen werden in einzelnen Töpfen (200 cm2, 15 cm tief) gehalten und täglich gewässert. Die Knollen werden 4 Monate nach dem Transfer der Gewebekulturpflanzen in das Gewächshaus geerntet. Für biochemische Analysen wer¬ den Knollen mit einem Frischgewicht von 8-16 g verwen¬ det . Das Frischgewicht wird unmittelbar nach dem Ernten bestimmt. Die gernteten Knollen werden gewaschen und in Kisten bei 20°C für 5 bis 10 Monate im Dunkeln gelagert. BeispieleThe plants are kept in individual pots (200 cm 2 , 15 cm deep) and watered daily. The tubers are harvested 4 months after the transfer of the tissue culture plants into the greenhouse. Tubers with a fresh weight of 8-16 g are used for biochemical analyzes. The fresh weight is determined immediately after harvesting. The harvested tubers are washed and stored in boxes at 20 ° C for 5 to 10 months in the dark. Examples
Beispiel 1example 1
Konstruktion des Plasmids pBin-Anti-STPIKm Construction of the plasmid pBin-Anti-STPI Km
Zur Herstellung eines antisense-Konstruktes, das eine anti¬ sense-RNA codiert zu Transkripten, die cytosolische Stärke¬ phosphorylase aus Kartoffel codieren, wurde ein Teil der co¬ dierenden Region der in Mori et al . (loc. cit . ) beschriebe¬ nen cDNA mittels PCR aus einer λZAP cDNA-Bibliothek aus Knollengewebe amplifiziert .To produce an antisense construct which encodes an anti-sense RNA to transcripts which encode cytosolic starch phosphorylase from potato, part of the coding region described in Mori et al. (loc. cit.) described cDNA amplified by means of PCR from a λZAP cDNA library from tuber tissue.
Ein 1,7 kb Asp718/Smal-Fragment wurde mit glatten Enden in die Smal-Schnittstelle des binären Pflanzentransformations¬ vektors pBIN19 (Bevan, Nucl . Acids Res. 12 (1984) , 8711- 8721) insertiert. Dieser enthält den 35S-Promotor des CaMV und das Polyadenylierungssignal des Octopinsynthase-Gens. Durch Restriktionsverdau wurde sichergestellt, daß die co¬ dierende Region in antisense-Orientierung zum Promotor ange¬ ordnet ist.A 1.7 kb Asp718 / Smal fragment was inserted with smooth ends into the Smal interface of the binary plant transformation vector pBIN19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). This contains the 35S promoter of the CaMV and the polyadenylation signal of the octopine synthase gene. Restriction digestion ensured that the coding region was arranged in an antisense orientation to the promoter.
Das resultierende Konstrukt wurde als pBin-Anti-STPI Km be¬ zeichnet (siehe Figur 1) .The resulting construct was designated pBin-Anti-STPI Km (see FIG. 1).
Beispiel 2Example 2
Herstellung transgener Kartoffelpflanzen mit einer verrin¬ gerten Aktivität der cytosolischen StärkephosphorylaseProduction of transgenic potato plants with a reduced activity of the cytosolic starch phosphorylase
Der Vektor pBin-Anti-STPKm wurde mittels direkter Transfor¬ mation in den Agrobacterium tumefaciens-Stamm C58C1: pGV2260 eingeführt (Höfgen und Willmitzer, Nucl. Acid Res. 16The vector pBin-Anti-STP Km was introduced into the Agrobacterium tumefaciens strain C58C1: pGV2260 (Höfgen and Willmitzer, Nucl. Acid Res. 16
(1988) , 9877) . Die Transformation und Regeneration transge¬ ner Kartoffelpflanzen erfolgte wie in Rocha-Sosa et al .(1988), 9877). The transformation and regeneration of transgenic potato plants was carried out as in Rocha-Sosa et al.
(EMBO J. 8 (1989) , 23-29) beschrieben. Es wurden mehrere un¬ abhängig voneinander erzeugte transgene Linien hinsichtlich ihrer Eigenschaften getestet, insbesondere die Linien mit den Bezeichnungen cSTPI-6, -7, -9, -14, -15, -16 und -18. Beispiel 3(EMBO J. 8 (1989), 23-29). A number of independently generated transgenic lines were tested for their properties, in particular the lines with the designations cSTPI-6, -7, -9, -14, -15, -16 and -18. Example 3
Analyse transgener Kartoffelpflanzen mit einer verringerten Aktivität der cytosolischen StärkephosphorylaseAnalysis of transgenic potato plants with a reduced activity of the cytosolic starch phosphorylase
(a) Northern-Blot-Analyse(a) Northern blot analysis
Von verschiedenen unabhängigen Linien der gemäß Beispiel 2 hergestellten transgenen Kartoffelpflanzen wurde Ge- samt-RNA aus Blatt- bzw. Knollenmaterial isoliert und mittels Northern-Blot-Analyse hinsichtlich der Expres¬ sion von mRNA analysiert, die cytosolische Stärkephos¬ phorylase codiert. Im Vergleich zu Proben von Wildtyp- Pflanzen (Solanum tuberosum L. cv. Desiree) , die ein starkes Signal bei der Hybridisierung, mit einer für cy¬ tosolische Stärkephosphorylase spezifischen Probe zeig¬ ten, konnten in fast allen der untersuchten transfor¬ mierten Pflanzen keine bis fast keine Transkripte für cytosolische Stärkephosphorylase nachgewiesen werden.Total RNA from leaf or tuber material was isolated from various independent lines of the transgenic potato plants produced according to Example 2 and analyzed by means of Northern blot analysis for the expression of mRNA which encodes cytosolic starch phosphorylase. In comparison to samples from wild-type plants (Solanum tuberosum L. cv. Desiree), which showed a strong signal during hybridization with a sample specific for cyto-solic starch phosphorylase, none could be found in almost all of the transformed plants examined until almost no transcripts for cytosolic starch phosphorylase are detected.
(b) Nachweis der Aktivität der cytosolischen Stärkephospho¬ rylase(b) Detection of the activity of the cytosolic starch phosphorylase
Der Nachweis der Aktivität der cytosolischen Stärkephos¬ phorylase in Geweben der transformierten Kartoffelpflan¬ zen wurde nach der Methode von Steup (In: Methods in Plant Biochemistry 3, Academic Press Limited (1990) , 103-128) durchgeführt. Hierzu wurden zunächst Protein¬ rohextrakte aus den zu untersuchenden Geweben gewonnen, indem gefrorenes Gewebe in Extraktionspuffer (100 mM HEPES-NaOH, pH 7,5; 1 mM EDTA, 10 % (Vol. /Vol.) Glyce- rin; 5 mM DTT, 200 mg Na2S03; 150 mg Na2S205) homogeni¬ siert. Nach Zentrifugation wurde der klare Überstand in einem Polyacrylamidgel aufgetrennt. Die Elektrophorese wurde durchgeführt wie in Steup und Latzko (Planta 145 (1979) , 69-759) beschrieben. Hierbei wurde ein nicht-de- naturierendes diskontinuierliches System verwendet (12 % bzw. 4 % (Gew. /Vol.) Acrylamid) . Das Trenngel enthielt 2,4 % (Gew. /Vol.) Glycogen. Zum Nachweis der Aktivität der cytosolischen Stärkephosphorylase wurde das Gel nach dem Gellauf in einer Lösung von 20 mM Glucose-1-Phos- phat/100 mM Citrat; pH 6,0 über Nacht bei Raumtemperatur inkubiert. Anschließend wurde das Gel in lugolscher Lö¬ sung für 5 min inkubiert. Entfärbung erfolgte durch ex¬ tensives Waschen mit Wasser über einen längeren Zeit¬ raum. Blaufärbung zeigt die Anwesenheit stärkesyntheti¬ sierender Enzymaktivitäten an. Die Aktivität der cytoso¬ lischen Stärkephosphorylase kann beispielsweise an¬ schließend durch densitometrische Analyse der entspre¬ chenden Proteinbanden bestimmt werden.The detection of the activity of the cytosolic starch phosphorylase in tissues of the transformed potato plants was carried out by the method of Steup (In: Methods in Plant Biochemistry 3, Academic Press Limited (1990), 103-128). For this purpose, crude protein extracts were first obtained from the tissues to be examined by freezing tissue in extraction buffer (100 mM HEPES-NaOH, pH 7.5; 1 mM EDTA, 10% (vol. / Vol.) Glycerol; 5 mM DTT , 200 mg Na 2 S0 3 ; 150 mg Na 2 S 2 0 5 ) homogenized. After centrifugation, the clear supernatant was separated in a polyacrylamide gel. Electrophoresis was carried out as described in Steup and Latzko (Planta 145 (1979), 69-759). Here a non-de- natural discontinuous system used (12% or 4% (w / v) acrylamide). The separating gel contained 2.4% (w / v) glycogen. To demonstrate the activity of the cytosolic starch phosphorylase, the gel was run after the gel run in a solution of 20 mM glucose-1-phosphate / 100 mM citrate; pH 6.0 incubated overnight at room temperature. The gel was then incubated in a Lugolian solution for 5 min. Decolorization was carried out by extensive washing with water over a longer period of time. Blue color indicates the presence of starch-synthesizing enzyme activities. The activity of the cytosolic starch phosphorylase can subsequently be determined, for example, by densitometric analysis of the corresponding protein bands.
Figur 2 zeigt ein Polyacrylamidgel in dem sieben unab¬ hängige gemäß Beispiel 2 hergestellte transgene Kartof¬ fellinien hinsichtlich ihrer Aktivität an cytosolischer Stärkephosphorylase getestet wurden.FIG. 2 shows a polyacrylamide gel in which seven independent transgenic potato lines produced according to Example 2 were tested for their activity on cytosolic starch phosphorylase.
(c) Untersuchung des Keimungsverhaltens von Knollen der er¬ zeugten transgenen Kartoffelpflanzen(c) Investigation of the germination behavior of tubers of the transgenic potato plants produced
Zur Untersuchung des Einflusses der Verringerung der Ak¬ tivität der cytosolischen Stärkephosphorylase auf das Keimungsverhalten wurden Knollen der Pflanzen bei 20°C gelagert und das Keimungsverhalten der Knollen nach ver¬ schiedenen Zeiträumen untersucht.In order to investigate the influence of the reduction in the activity of the cytosolic starch phosphorylase on the germination behavior, tubers of the plants were stored at 20 ° C. and the germination behavior of the tubers was examined after various periods.
Figur 3 zeigt im Vergleich Knollen von Solanum tuberosum L. cv. Desiree (Wildtyp) , sowie Knollen der mit dem Plasmid pBin-Anti-STPIKm transformierten Linie cSTPI-9, die jeweils 5 Monate bei 20°C gelagert worden waren. Figur 4 zeigt im Vergleich Wildtyp-Knollen und Knollen von zwei anderen transformierten Linien (cSTPI-6 und cSTPI-7) , die für 10 Monate bei 20°C gelagert worden sind. Aus den beiden Figuren wird deutlich, daß die Knollen der transformierten Linien, die eine verringerte Aktivi¬ tät der cytosolischen Stärkephosphorylase aufweisen, ein drastisch verändertes Keimungsverhalten aufweisen. Die Knollen der transformierten Linien bilden zum einen wesentlich mehr Sproßenden pro Knolle und auch mehr Sproßenden pro auskeinemdem Auge aus. Ferner keimen bei den Knollen der transformierten Pflanzen mit verringer¬ ter Aktivität der cytosolischen Stärkephosphorylase in der Regel mehr Augen aus. Eine statistische Auswertung lt-- ist in den Figuren 5 und 6 dargestellt.Figure 3 shows a comparison of tubers of Solanum tuberosum L. cv. Desiree (wild type) and tubers of the line cSTPI-9 transformed with the plasmid pBin-Anti-STPI Km , each of which had been stored at 20 ° C. for 5 months. FIG. 4 shows a comparison of wild-type tubers and tubers from two other transformed lines (cSTPI-6 and cSTPI-7) which have been stored at 20 ° C. for 10 months. It is clear from the two figures that the tubers of the transformed lines, which have a reduced activity of the cytosolic starch phosphorylase, have a drastically changed germination behavior. The bulbs of the transformed lines form on the one hand significantly more shoot ends per bulb and also more shoot ends per out of no eye. Furthermore, more eyes usually germinate in the tubers of the transformed plants with reduced activity of the cytosolic starch phosphorylase. A statistical evaluation according to - is shown in FIGS. 5 and 6.
Die aus den Knollen der transformierten Pflanzen wach¬ senden Kartoffelpflanzen zeigen ferner einen erhöhten Ertrag (in Knollenfrischgewicht/Pflanze) . Dies ist in der folgenden Tabelle dargestellt am Beispiel der trans¬ formierten Linien cStP6, cSTP7, cSTP9, CSTP14 , CSTP15, CSTP16 und cSTP18.The potato plants growing from the tubers of the transformed plants furthermore show an increased yield (in fresh bulb weight / plant). This is shown in the following table using the example of the transformed lines cStP6, cSTP7, cSTP9, CSTP14, CSTP15, CSTP16 and cSTP18.
Tabelle ITable I
Pflanze Anzahl der Knollen Knollenfrischcrewicht (q)Plant number of tubers fresh tubers weight (q)
Wildtyp 5,6 ± 1,5 69,0 + 9,0Wild type 5.6 ± 1.5 69.0 + 9.0
CSTP6 13,5 + 2,7 95, 0 + 12, 0CSTP6 13.5 + 2.7 95.0 + 12.0
CSTP7 10,8 + 1,9 90,5 + 8,6CSTP7 10.8 + 1.9 90.5 + 8.6
CSTP9 10, 0 1,4 92,5 + 9,1CSTP9 10.0 1.4 92.5 + 9.1
CSTP14 9,6 + 3,5 93,8 + 12,4CSTP14 9.6 + 3.5 93.8 + 12.4
CSTP15 12,8 + 5, 0 94, 5 ± 8,8CSTP15 12.8 + 5.0 94.5 ± 8.8
CSTP16 8,8 + 0,8 92,5 + 5,6CSTP16 8.8 + 0.8 92.5 + 5.6
CSTP18 11,3 + 1,3 82,5 ± 8,3CSTP18 11.3 + 1.3 82.5 ± 8.3
Die Daten wurden von unabhängigen Pflanzen erhalten, die von individuel ¬ len Saatgutknollen propagiert worden waren . Knollen mit 10 -40 g Frisch¬ gewicht von nicht seneszenten Pflanzen wurden geerntet . Die Werte sind Mittelwerte von unabhängigen Pflanzen (n = 4- 8 ) mit Angabe der Standard¬ abweichung .The data were obtained from independent plants that had been propagated from individual seed tubers. Tubers with a fresh weight of 10-40 g from non-senescent plants were harvested. The values are mean values of independent plants (n = 4-8) with indication of the standard deviation.
Aus der Tabelle geht hervor , daß Pf lanzen mit einer ver¬ ringerten Aktivität der cytosolischen Stärkephospho¬ rylase zum einen mehr Knollen pro Pf lanze ausbilden als Wildtyp-Pflanzen und darüber hinaus das Knollenfrischge¬ wicht pro Pflanze wesentlich höher ist als bei Wildtyp- Pflanzen.The table shows that plants with a reduced activity of the cytosolic starch phosphorylase on the one hand form more tubers per plant than Wild-type plants and, moreover, the fresh tuber weight per plant is significantly higher than in wild-type plants.
Die transformierten Kartoffelpflanzen unterscheiden sich hinsichtlich der in den Knollen gebildeten Stärke nicht von Wildtyp-Pflanzen. The transformed potato plants do not differ from wild-type plants with regard to the starch formed in the tubers.

Claims

P a t e n t a n s p r ü c h e Patent claims
1. Transgene Kartoffelpflanze, die Zellen mit einer im Ver¬ gleich zu Wildtyp-Pflanzen um mindestens 60 % verringer¬ ten Aktivität der cytosolischen Stärkephosphorylase ent¬ hält.1. Transgenic potato plant which contains cells with an activity of the cytosolic starch phosphorylase which is at least 60% lower than that of wild-type plants.
2. Transgene Kartoffelpflanze nach Anspruch 1, deren Knol¬ len im Vergleich zu Knollen von Wildtyp-Pflanzen ein verändertes Keimungsverhalten aufweisen.2. Transgenic potato plant according to claim 1, the tubers of which have a different germination behavior than tubers of wild-type plants.
3. Transgene Kartoffelpflanze nach Anspruch 1 oder 2, deren Knollen sich bei Keimung von Knollen von Wildtyp-Pflan- zen dadurch unterscheiden, daß3. Transgenic potato plant according to claim 1 or 2, the tubers of which differ in the germination of tubers from wild-type plants in that
(a) im Durchschnitt pro Knolle mehr Sprosse gebildet werden; und/oder(a) more shoots are formed on average per tuber; and or
(b) im Durchschnitt pro Knolle mehr Augen auskeimen und zur Bildung von Sprossen führen; und/oder(b) germinate more eyes per tuber and lead to sprout formation; and or
(c) im Durchschnitt pro auskeimenden Knollenauge mehr Sproßenden gebildet werden.(c) On average, more shoot ends are formed per budding tuber eye.
4. Transgene Kartoffelpflanze nach einem der Ansprüche 1 bis 3, wobei die durchschnittliche Anzahl der Sprosse, die pro Knolle gebildet werden, wenn die Keimung nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln er¬ folgt, mindestens verdoppelt ist im Vergleich zu Knollen von Wildtyp-Pflanzen.4. Transgenic potato plant according to one of claims 1 to 3, wherein the average number of sprouts, which are formed per tuber when the germination takes place after storage for 5 months at 20 ° C in the dark, is at least doubled compared to Bulbs of wild-type plants.
5. Transgene Kartoffelpflanze nach einem der Ansprüche 1 bis 4, wobei die Anzahl der Sproßenden, die pro auskei¬ mendem Auge gebildet werden, wenn die Keimung nach einer Lagerung von 5 Monaten bei 20°C im Dunkeln erfolgt, min¬ destens verdoppelt ist im Vergleich zu Knollen von Wild¬ typ-Pflanzen. 5. Transgenic potato plant according to one of claims 1 to 4, wherein the number of shoot ends, which are formed per eye auskei¬ when the germination takes place after storage for 5 months at 20 ° C in the dark, at least doubled in Comparison to tubers of wild type plants.
6. Transgene Kartoffelpflanze nach einem der Ansprüche 1 bis 5, die im Vergleich zu Wildtyp-Pflanzen eine erhöhte Anzahl an Knollen sowie ein gesteigertes Knollenfrisch¬ gewicht aufweist.6. Transgenic potato plant according to one of claims 1 to 5, which has an increased number of tubers and an increased tuber fresh weight compared to wild-type plants.
7. Transgene Kartoffelpflanze nach einem der Ansprüche 1 bis 6, wobei die Verringerung der Aktivität der cytoso¬ lischen Stärkephosphorylase in den Zellen erreicht wird durch die Inhibierung der Expression endogener Gene, die dieses Enzym codieren.7. Transgenic potato plant according to one of claims 1 to 6, wherein the reduction in the activity of the cytosolic starch phosphorylase in the cells is achieved by inhibiting the expression of endogenous genes which encode this enzyme.
8. Transgene Kartoffelpflanze nach Anspruch 7, wobei die Inhibierung mittels eines antisense-, Ribozym- oder Co- suppressions-Effektes erreicht wird.8. The transgenic potato plant according to claim 7, wherein the inhibition is achieved by means of an antisense, ribozyme or cosuppression effect.
9. Transgene Kartoffelpflanze nach Anspruch 8, die stabil ins Genom integriert ein rekombinantes DNA-Molekül ent¬ hält, das folgende Elemente umfaßt:9. Transgenic potato plant according to claim 8, which contains a recombinant DNA molecule stably integrated into the genome and which comprises the following elements:
(a) einen Promotor, der die Transkription zumindest in Zellen der Knollen von Kartoffelpflanzen ermöglicht; und(a) a promoter which enables transcription at least in cells of the tubers of potato plants; and
(b) eine in antisense-Orientierung mit diesem Promotor verknüpfte DNA-Sequenz, deren Transkripte ganz oder teilweise komplementär sind zu Transkripten eines endogen in Kartoffelpflanzenzellen vorliegenden Gens, das cytosolische Stärkephosphorylase codiert.(b) a DNA sequence linked in antisense orientation to this promoter, the transcripts of which are wholly or partly complementary to transcripts of an endogenously present gene in potato plant cells which encodes cytosolic starch phosphorylase.
10. Vermehrungsmaterial einer transgenen Kartoffelpflanze nach einem der Ansprüche 1 bis 9, das die Reproduktion von Pflanzen nach einem der Ansprüche 1 bis 9 erlaubt.10. propagation material of a transgenic potato plant according to one of claims 1 to 9, which allows the reproduction of plants according to one of claims 1 to 9.
11. Vermehrungsmaterial nach Anspruch 10, das eine Knolle mit den in einem der Ansprüche 1 bis 6 beschriebenen Eigenschaften ist . 11. Propagation material according to claim 10, which is a tuber with the properties described in one of claims 1 to 6.
12. Verwendung von Nucleinsäuremolekülen, die eine cytosoli¬ sche Stärkephosphorylase codieren oder Teile davon zur Herstellung von transgenen Kartoffelpflanzen mit einer verringerten Aktivität der cytosolischen Stärkephospho¬ rylase und einem veränderten Keimungsverhalten. 12. Use of nucleic acid molecules which encode a cytosolic starch phosphorylase or parts thereof for the production of transgenic potato plants with a reduced activity of the cytosolic starch phosphorylase and a changed germination behavior.
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CN114085838B (en) * 2021-12-02 2023-08-15 甘肃农业大学 Potato stu-miRn220 and application thereof

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DE19619917A1 (en) 1997-11-20

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