WO1996017069A2 - Transgenic plants with improved biomass production - Google Patents

Abstract

Transgenic plant cells are disclosed in which the introduction and expression of particular DNA molecules results in the formation of easily mobilised phosphate pools outside the vacuole. Transgenic plants with cells of this type show an enhanced biomass production and/or altered flowering behaviour. Processes are also disclosed by which the plant yields can be improved and/or plant flowering behaviour modified.

Description

 Transgenic plants with increased biomass production

The present invention relates to transgenic plant cells in which the introduction and expression of certain DNA molecules leads to the formation of easily mobilizable phosphate pools outside the vacuole. Transgenic plants which contain such plant cells show an increased biomass production and / or a changed flowering behavior in comparison to non-transformed plants. The invention further relates to methods for increasing yields or changing the flowering behavior in plants, in which plants are changed in such a way that easily mobilizable phosphate pools outside the vacuole are formed in the cells.

In order to ensure the supply of food to the constantly growing world population, efforts must be made to increase the yield of crops. In recent decades, it has been possible to continuously increase crop yields due to the industrialization of agriculture. The mechanization of agriculture, modernized plant breeding, plant protection, but also improved plant nutrition through the use of various fertilizers contributed in particular to this.

An increase in earnings through an improvement of the above-mentioned measures can currently hardly be achieved. For example, the supply of plants with nutrients limiting the growth of plants in the biosphere, for example nitrogen and phosphorus, is generally ensured nowadays. This means that even further fertilization no longer leads to an increase in yield. The development of new plant varieties with higher yields through the processes of plant breeding is very time consuming and often the negative properties of the resulting plant elements, for example a lower resistance to disease, are accepted. This in turn leads to the increased use of pesticides.

One possibility for the production of plant varieties with an increased yield is the targeted genetic modification of plants.

For example, the expression of a prokaryotic asparagine synthetase in plant cells is described, which among other things leads to an increase in biomass production in the plants (EP-Bl 0 511 979). Other processes attempt to change the expression of genes whose products have a function in storage pathways in plants, or attempt to change the distribution of the photoassimilates formed in the course of photosynthesis in such a way that “sink” organs are preferred , in particular storage organs, are supplied with photoassimilates. On the one hand, attempts are made to improve the absorption capacity of the "sink" organs for the respective form of transport of the photoassimilates, usually sucrose, or to influence the transport directly. EP 0 442 592 e.g. describes the expression of an invertase in potato plants which leads to a change in the yield in plants manipulated in this way. The use of the sucrose transporter (Riesmeier et al., EMBO J. 13 (1994), 1-7) to influence the sucrose transport is also considered (see also WO 94/00574).

The present invention is therefore based on the object of making available further genetic engineering methods which can generally be used in plants to increase the biomass production or the yield.

This object was achieved by the provision of the embodiments described in the patent claims. It has surprisingly been found that by the introduction and expression of DNA molecules that encode proteins, their enzymatic activity leads to the generation of phosphate pools outside the vacuole in transgenic plant cells, an increase in biomass production and thus an increase in yield can be achieved in plants which contain such cells. Plants of this type can also have a different flowering behavior.

The invention thus relates to transgenic plant cells in which at least one easily mobilizable phosphate pool is formed outside the vacuole due to the introduction and expression of a DNA molecule which codes for a protein which is involved in the synthesis of a phos - is involved in a molecule containing phosphate.

Increasing the biomass production in plants is understood in the context of this invention to mean an increase in the biomass of the entire plant (measured as dry weight) and / or individual parts in comparison with wild-type plants, preferably an increase of at least 5% and in particular an increase of more than 10%.

The increase in biomass production thus also includes the increase in the yield of agriculturally usable parts of plants, for example storage organs, such as potato tubers, beets, seeds, fruits, or of leaves, stems, etc., in plants in which phosphate pools outside the vacuole were produced compared to wild-type plants, preferably by at least 5%, and in particular by more than 10%.

Such increases exceed increases which are usually achieved by conventional breeding methods.

In the context of this application, a change in the flowering behavior or premature flower formation means that transformed plants compared to non-transformed plants at least a few days, preferably one to several weeks, in particular 1-2 weeks early bloom here. In the context of this invention, the term “phosphate pool” is understood to mean a class of phosphate-containing molecules which contain phosphate covalently bound and from which phosphate is easily mobilized, ie released or onto other molecules, by generally reversible enzymatic reactions can be transferred. This increases the availability of phosphate in the cells for various phosphate-dependent reactions. The term “easily mobilisable” is understood to mean that the phosphate is available more quickly in the cell's cytosol than is usually possible by transporting phosphate from the vacuole into the cytosol. The release of phosphate from the vacuum into the cytosol in the event of a phosphate deficiency in the cytosol generally takes place in the region of several hours (Woodrow et al., Planta 161 (1984), 525-530).

The phosphate pools are preferably pools of phosphate-containing molecules which do not impair the cytosolic homeostasis necessary for metabolic processes with regard to the phosphate concentration, but from which phosphate can be easily released or transferred to other molecules, i.e. which ensure an increased availability of phosphate.

Particularly suitable for this purpose are phosphate-containing compounds which do not normally occur in higher plants. These include polyphosphate (Wood, Ann. Rev. Biochem. 57 (1988), 235-260), a compound which is synthesized by most organisms, except for higher plants, as a storage substance for phosphate, and e.g. also acetyl phosphate, which is used in bacteria for ATP regeneration (see e.g. Matsuyama et al., J. Bacteriol. 171 (1989), 577-580). According to the invention, the phosphate pools can also consist of trehalose-6-phosphate, phosphoenolpyruvate or fructose-1-phosphate.

Such phosphate pools outside the vacuum are preferably produced by the fact that in vegetable Cells DNA molecules are introduced and expressed, which encode proteins whose enzymatic activity leads to the generation of the respective phosphate pool in transgenic plant cells.

In a preferred embodiment, the DNA molecules introduced into the plant cells code for proteins with the enzymatic property of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenolpyruvate mutase or a ketohexokinase.

Polyphosphate kinases (ATP: polyphosphate phosphotransferase; E.C. 2.7.4.1) catalyze the reaction:

Polyphosphate _n + ATP s Polyphosphate _{n + 1} + ADP

The product of this reversible reaction is a linear polymer of orthophosphate residues. The length of the polymers can range from 3 to over 1000 phosphate residues. The enzyme has already been described in various organisms, including E. coli (Ahn and Kornberg, J. Biol. Chem. 265 (1990), 11734-11739; Akiyama et al., J. Biol. Chem. 267 (1992), 22556-22561), Klebsiella aerogenes (Kato et al ., Gene 137 (1993), 237-242), S. cerevisiae (Felter and Stahl, Biochimie 55 (1973), 245-251), Propionibacterium shermanii (Robinson and Wood, J. Biol. Chem. 261 (1986), 4481-4485; Robinson et al., J. Biol. Chem. 262 (1987), 5216-5222), Cor neJacteriiun xerosis (Muhammed, Biochim. Biophys. Acta 54 (1961), 121-132) and A fchoJbacterium atrocyaneus ( Levinson et al., J. Gen. Microbiol. 88 (1975), 65-74).

Acetate kinases (ATP: acetate phosphotransferase; E.C. 2.7.2.1.) Catalyze the reaction:

Acetate + ATP s acetyl phosphate + ADP The enzyme has been identified in a number of microorganisms and DNA sequences encoding acetate kinases have also been isolated, e.g. the ack gene from E. coli K-12 (Matsuyama et al., J. Bacteriol. 171 ( 1989), 577-580) and the acλ gene from Methanosarcina thermophila (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829).

Phosphotransacetylases (acetyl-CoA: orthophosphate acetyl transferase; E.C. 2.3.1.8.) Catalyze the following reaction:

Acetyl Coenzyme A + Orthophosphate = Acetyl Phosphate + Coenzyme A

DNA sequences which code for this enzyme are also described, e.g. the pta gene from Methanosarcina thermophila (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829)

Trehalose-6-phosphate synthase catalyzes the synthesis of trehalose-6-phosphate. Such an enzyme from yeast is described, for example, in US Pat. No. 5,422,254.

Phosphoenolpyruvate mutase catalyzes the synthesis of phosphoenolpyruvate. Such an enzyme from Tetrahymena pyriformis is described, for example, in Seidel et al. (Biochemistry 31 (1995), 2598-2608).

Ketohexokinase catalyzes the synthesis of fructose-1-phosphate. Such rat and human enzymes are described, for example, in Donaldson et al. (Biochem. J. 291 (1993), 179-186) or in Bonthron et al. (Hum. Mol. Genet. 3 (1994), 1627-1631).

In the case of the DNA molecules which code for proteins whose enzymatic activity leads to the formation of phosphate pools outside the vacuole, in particular proteins with the enzymatic see property of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenolpyruvate mutase or a ketohexokinase, it can be genomic or cDNA molecules from any organism, preferably around DNA molecules from prokaryotic, in particular bacterial organisms. The DNA molecules can be isolated from cells with the aid of common molecular biological methods or produced synthetically. Furthermore, the molecules can be modified according to methods known to those skilled in the art in such a way that they contain plant-specific codons in order to improve expression in plant cells.

DNA molecules are preferably used for introduction and expression in plant cells which encode proteins which have the enzymatic property of a polyphosphate kinase and which have a low K - value for ADP and a high 1 ^ value for ATP.

The activity of these enzymes is usually influenced by the ratio of ATP to ADP. This means that a phosphate pool is only formed if there is an excess of ATP compared to ADP. This leads to a depletion of the cytosolic phosphate concentration under conditions which prefer the formation of ATP and to a constant reflux of phosphate from the vacuole. These newly formed pools have the advantage over the vacuolar phosphate pool that they are easier to mobilize. The release of phosphate from the vacuole into the cytosol in the event of a phosphate deficiency in the cytosol generally takes place in the region of several hours (Woodrow et al., Planta 161 (1984), 525-530). In particular, this means that, due to the enzymatic properties of the above-described enzymes, these plant-foreign compounds can be broken down relatively quickly and phosphate can thus be mobilized if the ratio of ATP to ADP drops. In a further preferred embodiment, the DNA molecules which encode a polyphosphate kinase and which are introduced into the plant cells are DNA molecules from E. coli, Klebsiella aerogenes, Neisseria meningi tidis, or Synechocystia sp. , It is particularly preferably the pp gene from E. coli (Akiyama et al., J. Biol. Chem. 267 (1992), 22556-22561), the ppk gene from Klebsiella aerogenes (Kato et al., Gene 137 (1993), 237-242), by the sequence from Neisseria meningi tidis (Tinsley and Gottschlich; 1995) stored in the GenBank database under the access number U16262 or by the sequence stored in the GenBank database under the access number D64005 Synechocystia sp. Strain PCC6803 (Kaneko et al .; 1995).

In a further preferred embodiment, DNA molecules are introduced which code for an acetate kinase and originate from Methanosarcina thermophila, E. coli, Haemophilus influenza, Bacillus subtilis or Mycoplasma geni taliu. It is preferably the ac gene from E. coli K-12 (Matsuyama et al., J. Bacteriol. 171 (1989), 577-580), the ack gene from Methanosarcina thermophila, which is described in Fleischmann et al. (Science 269 (1995), 496-512) published sequence from Haemophilus influenza to the in Grundy et al. \ J. Bacteriol. 175 (1993), 7348-7355) published sequence from Bacillus subtilis or the sequence described in Fraser et al. (Science 270 (1995), 397-403) published sequence from Mycoplasma geni talium. DNA molecules that encode a phosphotransacetylase preferably originate from Methanosarcina thermophila, Escherichia coli or Mycoplasma geni talium. It is particularly preferably the pta gene from Methanosarcina thermophila

(Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829) to convert a DNA molecule from E. coli that is described in Kakuda et al. (J. Biochem. (1994), 916-922) or those described in Matsuyama et al.

(Biochim. Biophys. Acta (1994), 559-562) has published sequence, or a DNA molecule from Mycoplasma geni talium, which the in Fraser et al. (Science 270 (1995), 397-403).

DNA molecules that encode a trehalose-6-phosphate synthase _¬ preferably come from yeast. The DNA molecule described in US Pat. No. 5,422,254 is preferably used. DNA molecules that encode a phosphoenolpyruvate mutase are preferably derived from Tetrahymena pyriformis, such as that in Seidel et al. (see above) described molecule. DNA molecules that encode a ketohexokinase are preferably from humans or rats, such as those in Bonthron et al. (see above) or Donaldson et al. (see above) molecules described.

In a further preferred embodiment, DNA molecules which encode a protein which leads to the synthesis of polyphosphate, DNA molecules which encode a protein with the enzymatic activity of an acetate kinase and a DNA sequence coding for a protein with the enzymatic activity of a phosphotransacetylase. The enzymatic activities of acetate kinase and phosphotransacetylase lead to the conversion of acetyl-coenzyme A and ADP to acetate and ATP. The resulting ATP can then be used by the polyphosphate kinase for the synthesis of polyphosphate.

The mobilization of the phosphate pools formed by the expressed proteins in the cells outside the vacuole, in particular polyphosphate, can also be carried out by other enzymes in addition to the enzymes which catalyze the reversible reactions described above. Enzymes using polyphosphate as a substrate are known. DNA sequences which encode such enzymes must then also be introduced into the plant cells and expressed. For example, polyphosphate glucokinase, which has already been described in a number of microorganisms, is known to for example in Mycobacterium phlei (Szymona, Bull. Acad. Pol. Sei. Ser. Sei. Biol. 5 (1956), 379-381; Szymona and Ostrowski, Biochim. Biophys. Acta 85 (1964), 283-295), Propionibacterium shermanii (Pepin and Wood, J. Biol. Chem. 261 (1986), 4476-4480), Corynebacterium xerosis (Dirheimer and Ebel, Bull. Soc. Chim. Biol. 50 (1968), 1933-1947) and Mycobacterium tuberculosis ( Szymona et al., Acta Biochim. Pol. 24: 133-42 (1977). Other enzymes which use polyphosphate as a substrate and are therefore suitable for mobilizing polyphosphate are, for example, also polyphosphate: AMP phosphotransferase (Bonting et al., J. Bacteriol. 173 (1991), 6484-6488; Dirheimer and Ebel, CR Acad. Sci. Paris 260 (1965), 3787-3790), 1,3-diphosphoglycerate: polyphosphate phosphotransferase (Kulaev et al., Biokhimiya 33 (1968), 419-434; Wood and Goss, Proc. Natl. Acad. Sci. USA 82 (1985), 312-315), the polyphosphate-dependent NAD kinase (Murata et al., Agric. Biol. Chem. 44 (1980), 61-68), exopolyphosphatase (see e.g. Akiyama et al., J. Biol. Chem. 268 (1993), 633-639; Wurst and Kornberg, J. Biol. Chem. 269 (1994), 10996-11001) and endopolyphosphatase (EC 3.6 .1.10 .; see e.g. Kowalczyk and Phillips, Analyt. Biochem. 212 (1993), 194-205).

There is basically the possibility that the protein expressed in the transgenic plant cell, which due to its enzymatic activity causes the formation of a phosphate pool outside the vacuole, in particular polyphosphate or acetylphosphate, is localized in any compartment of the plant cell can. In order to achieve localization in a specific compartment, the coding region must be linked to DNA sequences which ensure localization in the respective compartment. The protein to be expressed will preferably be located in the cytosol, the plastids or the mitochondria. No special signal sequence is required for localization in the cytosol. When using prokaryotic DNA molecules, care must be taken that the coding regions have no signal sequences which cause secretion of the protein to be expressed. Signal sequences which ensure localization, for example in the mitoehondria or plastids, are known (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sei. USA 85: 846-850 (1988).

Plant cells into which the DNA molecules described above are introduced can generally originate from any plant species, in particular from any monocotyledonous or dicotyledonous plants. Plant cells of agricultural useful plants are preferably used, in particular of cereals (such as barley, rye, oats, millet, rice, corn, wheat etc.), types of fruit, potatoes, rape, sugar beet, soybeans, vegetables (such as peas, Bean, tomato etc.) or from ornamental plants. Photosynthetically active cells are particularly preferred.

The invention also relates to transgenic plants which contain transgenic plant cells according to the invention. These can be obtained, for example, by regeneration from the transgenic plant cells described, using methods known to the person skilled in the art.

It has been found that such plants, compared to non-transformed (wild-type) plants, have an increased biomass production and / or a changed flowering behavior, in particular premature flowering and flowering.

The invention also relates to propagation material of a plant according to the invention. This means for example seeds, fruits, tubers, cuttings, rhizomes etc.

Furthermore, the present invention relates to methods for increasing the biomass product or the yield and / or changing the flowering behavior in plants, characterized in that easily mobilizable phosphate pools are generated in plant cells outside the vacuole, which guarantee an increased availability of phosphate in the cells.

Such a procedure usually comprises the following steps:

(a) Preparation of an expression cassette which comprises the following DNA sequences:

(i) a promoter which is functional in plant cells and which ensures the transcription of a subsequent DNA sequence;

(ii) at least one DNA sequence which encodes a protein, the enzymatic activity of which leads to the formation of an easily mobilizable phosphate pool outside the vacuole, and which is in sense orientation at the 3 'end of the promoter is coupled; and

(iii) optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the resulting transcript, which is coupled to the 3 'end of the coding region;

(b) Transformation of plant cells with that in step

(a) produced expression cassette and stable integration of the expression cassette into the plant genome; and

(c) Regeneration of whole, intact plants from the transformed plant cells.

In principle, any promoter which is functional in plants is suitable for the promoter mentioned under (i) in the processes according to the invention. The expression of the said DNA sequences can generally take place in any tissue of a transformed plant and at any time. For example, the 35S promoter of the Cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) is suitable, which ensures constitutive expression in all tissues of a plant. continuous However, promoters can also be used which lead to an expression of subsequent sequences in a certain tissue of the plant, preferably in photosynthetically active tissue (see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245- 2251) or only at a point in time determined by external influences (see for example WO / 9307279).

Any sequence of the DNA molecules described above in connection with transgenic cells can be considered for the DNA sequence mentioned under step (ii).

The termination sequence serves to correctly terminate the transcription and to add a poly-A tail to the transcript, which is believed to have a function in stabilizing the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be interchanged as desired.

The expression cassette produced in the course of the method according to the invention is also a subject of the invention. It is preferably located on a plasmid, in particular on the plasmids p35S-PPK (FIG. 1) or p35S-ACK (FIG. 3), and is preferably used using a plasmid which is suitable for the transformation of plant cells is introduced into plant cells. The binary vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66 (1990), 221-230) was used in the examples of the present invention. This vector is a derivative of the binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721), which is commercially available (Clontech Laboratories, Inc., USA). However, any other plant transformation vector into which an expression cassette can be inserted and which ensures the integration of the expression cassette into the plant genome is also suitable. In principle, cells of all plant species, in particular monocotyledonous or dicotyledonous plants, are suitable for the transformation according to process step (b) with the expression cassette constructed according to process step (a). Cells of agricultural crops are preferably used in the process, in particular cereals (such as barley, rye, oats, millet, rice, corn, wheat etc.), fruit varieties, potatoes, rapeseed, sugar beet, soybeans, Vegetable types (such as peas, beans, tomatoes etc.) or cells from ornamental plants.

The invention also relates to the transgenic plant cells and transgenic plants resulting from the processes according to the invention. The transgenic plants are characterized in that in cells of these plants, due to the introduction and stable integration of an expression cassette constructed according to the method according to the invention into the genome, there is expression of a protein which, owing to its enzymatic activity, forms polyphosphate, Acetyl phosphate, trehalose-6-phosphate, phosphoenol pyruvate or fructose-1-phosphate outside the vacuole in the transgenic cells.

In particular, the invention relates to transgenic plants containing at least one DNA sequence which contains a protein with the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenolpyruvate mutase, or a ketohexokinase encoded, this DNA sequence being linked to regulatory DNA regions for transcription and translation in plant cells, being stably integrated into the genome and, owing to the expression of the said DNA sequence, forming polyphosphate, acetylphosphate, trehalose-6 Phosphate, phosphoenolpyruvate or fructose-1-phosphate comes outside the vacuole in plant cells. Another object of the invention is the use of DNA molecules which encode proteins which, owing to their enzymatic activity, form phosphate pools which can be easily mobilized, in particular polyphosphate, acetylphosphate, trehalose-6-phosphate, fructose-1 Phosphate or phosphoenol pyruvate, outside the vacuole in plant cells, lead to the transformation of plant cells and to expression in plant cells, in particular to improve the phenotype of these cells. In particular, the invention relates to the use of DNA molecules, the proteins with the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase. encode a phosphoenolpyruvate mutase or a ketohexokinase, for expression in plant cells and for the production of polyphosphate, acetylphosphate, trehalose-6-phosphate, phosphoenolpyruvate or fructose-1-phosphate outside the vacuole in plant cells. These DNA molecules include in particular the DNA molecules described above, which encode proteins with the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenolpyruvate mutase or a ketohexokinase.

To prepare the introduction of foreign genes into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be introduced into the vector at a suitable restriction site. The plasmid obtained is used for the transformation of E. coli cells. Transformed E. coli cells are grown in a suitable medium, then harvested and lysed. The plasmid is recovered. Restriction analyzes are generally used as the analysis method for characterizing the plasmid DNA obtained, Gel electrophoresis and other biochemical-molecular biological methods are used. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids. A variety of techniques are available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and More options.

When DNA is injected and electroporated into plant cells, there are no special requirements for the plasmids used. Simple plasmids such as e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. Depending on the method of introducing desired genes into the plant cell, further DNA sequences may be required. E.g. If the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, must be linked to the genes to be introduced .

If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate vector or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate both in E. coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells. The use of T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4 (1985), 1-46 and An et al. , EMBO J. 4 (1985), 277-287.

For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells . The plants thus obtained can then be examined for the presence of the introduced DNA.

Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and remains in the offspring of the originally transformed cell. It normally contains a selection marker which gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and others. The individually selected marker should therefore allow the selection of transformed cells over cells which lack the introduced DNA.

The transformed cells grow within the plant in the usual way (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and crossed with plants which have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties. Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.

Media and solutions used

20 x SSC 175.3 g NaCl

88.2 g sodium citrate ad 1000 ml with ddH ₂ 0 pH 7.0 with 10 N NaOH

10 x MEN 200 mM MOPS

50mM sodium acetate 10mM EDTA pH 7.0

NSEB buffer 0.25 M sodium phosphate buffer pH 7.2

7% SDS 1 mM EDTA 1% BSA (w / v) Description of the pictures

1 shows the plasmid p35S-PPK

Construction of the plasmid:

A = Fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294)

B = fragment B: DNA fragment from Escherichia coli coding for polyphosphate kinase;

Nucleotides 187-2253 of the polyphosphate kinase (ppk) gene (Akiyama et al., J. Biol. Chem. 1992, 267 (22556-22561)); Orientation to the promoter: sense

The arrow indicates the direction of transcription of the ppk gene

C = fragment C: nt 11748-11939 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846)

Figure 2 shows the plasmid pACK

The thin line corresponds to the plasmid pUC19, the strong line represents a DNA insert which comprises the coding region of the ac gene from Methanosarcina thermophila (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829). The arrow indicates the direction of transcription of the ac gene.

Figure 3 shows the plasmid p35S-ACK

Construction of the plasmid:

A = Fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294) B = fragment B: DNA fragment from Methanosarcina thermophila coding for acetate kinase; Nucleotides 1314-2748 of the acetate kinase (ack) gene (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829);

Orientation to the promoter: sense

The arrow indicates the direction of transcription of the ack gene

C = fragment C: nt 11748-11939 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846)

4 shows transformed potato plants in comparison with non-transformed potato plants.

Three plants of the potato line JP1 35, which were transformed with the plasmid p35S-PPK (rear), are shown in comparison with two non-transformed potato plants (front). The plants are about 84 days old and were kept in the greenhouse.

The examples illustrate the invention. The following methods are used in the examples:

1. Cloning procedure

The vector pUC19 was used for cloning in E. coli. For the plant transformation, the gene constructions were cloned into the binary vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66 (1990), 221-230).

2. Bacterial strains

E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh, USA) was used for the pUC19 vector and for the pBinAR constructs. The transformation of the plasmids into the potato plants was carried out using the Agrobacterium tumefaciens-Sta es C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transformation according to the method of Höfgen & Willmitzer (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.

4. Transformation of potatoes

Ten small leaves of a potato sterile culture (Solanu tuberosum L.cv. Desiree) wounded with the scalpel were placed in 10 ml of MS medium (Murashige & Skoog, Physiol. Plant. 15 (1962), 473) with 2% sucrose, which Contained 50 μl of an Agrobacterium tumefaciens overnight culture grown under selection. After 3-5 minutes of gentle shaking, another incubation was carried out for 2 days in the dark. The leaves for callus induction were then 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 or 1 mg / 1 hygromycin B, and 0.80% Bacto agar. After a week's incubation at 25 ° C. and 3000 lux, the leaves were sprouted for induction 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 or 3 mg / 1 hygromycin B, and 0.80% Bacto agar. 5. Radioactive labeling of DNA fragments

The radiocative labeling of DNA fragments was carried out using a DNA random primer labeling kit from Boehringer (Germany) according to the manufacturer's instructions.

6. Northern blot analysis

RNA was isolated from leaf tissue of 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 type nylon membrane (Amersham, UK) using 20 × SSC using capillary blot. The membrane was then baked at 80 ° C under vacuum for two hours.

The membrane was prehybridized in NSEB buffer for 2 h at 68 ° C. and then hybridized in NSEB buffer overnight at 68 ° C. in the presence of the radioactively labeled sample.

7. Plant husbandry

Potato plants were kept in the greenhouse under the following conditions:

Light period 16 h at 2500 lux and 22 ° C

Dark period 8 h at 15 ° C

Humidity 60%

example 1

Construction of the plasmid p35S-PPK and introduction of the plasmid into the genome of potato plants

For the construction of the plasmid p35S-PPK, a DNA fragment which codes for the polyphosphate kinase from E. coli was first amplified using the PCR technique ("polymerase chain reaction"). Genomic DNA from E. coli cells of the DH5α strain were isolated by standard methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using the two oligonucleotides

Oligo 1 5 '-AAGGATCCAGGAACCCGGGCCATGGGTCAGGAAAAG-3' (Seq ID No. 1) and

Oligo 2 5 '-GGGGATCCCGGGCCATGGGTTATTCAGGTTG-3' (Seq ID No. 2)

an approximately 2.1 kb long DNA fragment was amplified by the PCR reaction and contains nucleotides 187 to 2253 of the type described in Akiyama et al. (J. Biol. Chem. 267 (1992), 22558) comprises the DNA sequence (ppk gene) which encodes polyphosphate kinase from E. coli. Through the oligonucleotide 1, which is partially complementary to the 5 'end of the pp gene, restriction sites for the restriction endonucleases BamH I and an Sma I are introduced at the 5' end of the amplified DNA fragment. Oligonucleotide 2, which is partially complementary to the 3 'end of the ppk gene, introduces interfaces for BamH I, Sma I and Nco I at the 3' end of the fragment. The DNA fragment resulting from the PCR reaction was cut with BamH I and ligated into the binary vector pBinAR cut with BamH I (Hδfgen and Willmitzer, Plant Sei. 66 (1990), 221-230). This is a derivative of the binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). pBinAR was constructed as follows:

A 529 bp fragment comprising nucleotides 6909-7437 of the 35S promoter of the Cauliflowermosaic virus (Franck et al., Cell 21 (1980), 285-294) was selected as the EcoRI / Kpn I fragment the plasmid pDH51 (Pietrzak et al., Nucl. Acids Res. 14, 5857-5868) isolated and between the EcoR I and the Kpn J sites of the polylinker from pBinl9 were ligated. The plasmid pBin19-A was formed.

A 192 bp fragment was isolated from the plasmid pAGV40 (Herrera-Estrella et al., Nature 303, 209-213) with the aid of the restriction endonucleases Pvu II and Hind III which contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3, 835-846) comprises (nucleotides 11749-11939). After addition of Sph I linkers to the Pvu J site, the fragment was ligated between the Sph I and Hind HI sites pBin19-A. This created pBinAR.

With the aid of restriction and sequence analyzes, recombinant vectors were identified in which the amplified DNA fragment was inserted into the vector in such a way that the coding region for the polyphosphate kinase from E. coli in sense orientation with the 35S promoter is linked. The resulting plasmid, p35S-PPK, is shown in FIG. 1.

The insertion of the amplified DNA fragment creates an expression cassette which is composed of fragments A, B and C as follows:

Fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV). The fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck et al., 1980, Cell 21 (285-294)).

Fragment B contains the coding region for polyphosphate kinase from E. coli. The fragment comprises nucleotides 187-2253 of the polyphosphate kinase gene (Akiyama et al., J. Biol. Chem. 267 (1992), 22556-22561). This coding region was ligated to the 35S promoter in sense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846). The size of the plasmid p35S-PPK is approximately 13 kb. Since the coding region of the gene for polyphosphate kinase from E. coli used did not comprise a signal sequence, the expressed protein should be present in the cytoplasm of transformed cells.

The plasmid was transferred into cells of potato plants using Agrobacterium-mediated transformation as described above. Whole plants were regenerated from the transformed cells. In this way, 40 lines of transformed plants were produced, of which 7 lines, in particular lines JP1 16, JP1 26, JP1 29, JP1 31, JP1 33, JP1 34 and JP1 35, were analyzed in more detail.

As a result of the transformation, transgenic potato plants showed the expression of the gene for the polyphosphate kinase from E. coli. The expression was verified with the help of Northern blot analyzes. For this purpose, RNA was isolated from tissue from transgenic plants, separated by gel electrophoresis, transferred to a nylon membrane and hybridized with the radioactively labeled coding region of the ppk gene from E. coli. Of the 7 transgenic potato lines mentioned above, one plant was examined with regard to the expression of the ppk gene. All showed a clear expression of the ppk gene from E. coli. In contrast, no transcripts of this gene could be detected in wild-type plants.

In comparison to wild-type plants, the plants showed a substantially higher yield per plant (measured as g bulbs / plant), as shown in the table below. Table I ng / plant

JP1 16 8 113.88

JP1 26 8 115.13

JP1 29 8 111.63

JP1 31 8 132.50

JP1 33 8 122.38

JP1 34 8 122.50

JP1 35 8 117.25

Wild type 8 102.38

The tuber yield is shown in g / plant of plants which have been transformed with the plasmid p35S-PPK, in comparison to non-transformed plants (wild type). Plants that were cultivated in the greenhouse, lines JP1 16, JP1 26, JP1 29, JP1 31, JP1 33, JP1 34 and JP1 35 and wild-type plants of the potato variety Desiree were examined. The potatoes were harvested 11 weeks after plant exposure. n = number of plants used for analysis g / plant = average tuber weight / plant

In comparison to wild-type plants, the transformed plants no longer formed, but instead significantly larger bulbs.

The starch content of the tubers of transformed plants corresponded to the starch content of tubers of wild-type plants (measurement by determining the density of the tubers). Furthermore, the transformed plants showed premature flowering compared to wild-type plants, as shown in the following tables: Table II n% of plants

JP1 16 14 33.3

JP1 26 14 46.6

JP1 29 14 28.6

JP1 31 14 35.7

JP1 33 14 57.1

JP1 34 14 35.7

JP1 35 14 64.3

Wild type 14 0.0

Table III n% of plants

JP1 16 14 66.7

JP1 26 14 46.6

JP1 29 14 35.7

JP1 31 14 71.4

JP1 33 14 61.5

JP1 34 14 42.9

JP1 35 14 64.3

Wild type 14 8.3

Tables II and III illustrate the premature flower formation of plants which were transformed with the plasmid p35S-PPK in comparison with non-transformed plants (wild type).

Transformed potato plants of the lines JP1 16, JP1 26, JP1 29, JP1 31, JP1 33, JP1 34 and JP1 35 as well as wild-type plants of the potato variety Desiree were cultivated under greenhouse conditions. Table II shows the percentage of the plants examined in each line or of the wild-type plants which, after 80 days after the exposure of the Plants bloomed. Table III shows the percentage of plants examined in each line or of the wild-type plants which bloomed after 84 days after the plants had been exposed. n = number of plants analyzed

% Plants = percentage of plants that bloom

In the case of transformed plants, flowering started on average 1-2 weeks earlier than in the case of wild-type plants which were kept under the same conditions.

Example 2

Construction of the plasmid p35S-ACK and introduction of the plasmid into the genome of potato plants

For the construction of the plasmid p35S-ACK, a DNA fragment was first isolated which encodes the acetate kinase from Methanosarcina thermophila (ack gene). The gene is inserted in the Sma J site of a pUC19 plasmid. The construction of this pUC plasmid is described in detail in Latimer and Ferry (J. Bacteriol. 175 (1993), 6822-6829; see page 6823, right column). The plasmid used, which is called pACK in the context of this invention, is shown in FIG. 2.

An approximately 1.45 kb DNA fragment was isolated from this plasmid by restriction digestion with the restriction endonucleases Asp718 and BamH I and ligated into the binary vector pBinAR cut with Asp718 and BamH I. The resulting plasmid was designated p35S-ACK and is shown in FIG. 3.

The insertion of the isolated Asp718 / BamH I DNA fragment creates an expression cassette which is composed of fragments A, B and C as follows:

Fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV). The fragment includes the Nucleotides 6909 to 7437 of CaMV (Franck et al., 1980, Cell 21, 285-294).

Fragment B contains the coding region for acetate kinase from Methanosarcina thermophila. The fragment comprises nucleotides 1314-2748 of the sequence shown in Latimer and Ferry (J. Bacteriol. 175 (1993), 6822-6829) (acetylkinase iack) gene). This coding region was ligated to the 35S promoter in sense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The size of the plasmid p35S-ACK is approximately 12.5 kb. As described above, the plasmid was transferred into cells of potato plants using agrobacterium-mediated transformation. Whole plants were regenerated from the transformed cells.

As a result of the transformation, transgenic potato plants showed the expression of the acetate kinase from Methanosarcina thermophila in the cytosol of the cells.

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Institute for Genetic Research Berlin

GmbH

(B) STREET: Ihnestrasse 63

(C) LOCATION: Berlin

(E) COUNTRY: DE

(F) POSTAL NUMBER: 14195

(G) TELEPHONE: (0 30) 8 30 00 70 (H) TELEFAX: (0 30) 83 00 07 36

(ii) NAME OF THE INVENTION: Transgenic plants with increased biomass production

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE VERSION:

<A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(vi) DATA OF THE URN REGISTRATION:

(A) REGISTRATION NUMBER: DE P 44 44 460.5

(B) REGISTRATION DAY: 29-N0V-1994

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAGGATCCAG GAACCCGGGC CATGGGTCAG GAAAAG 36 (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GGGGATCCCG GGCCATGGGT TATTCAGGTT G 31

Claims

Patent claims 1. Transgenic plant cell, characterized in that there is an easily mobilizable phosphate pool in the cell outside the vacuole due to the introduction and expression of at least one DNA molecule that encodes a protein that is involved in the synthesis of a phosphate-containing molecule outside of the vacuole is involved. 2. Transgenic plant cell according to claim 1, wherein the phosphate pool consists of polyphosphate, acetyl phosphate, trehalose-6-phosphate, phosphoenolpyruvate or fructose-1-phosphate. 3. Transgenic plant cell according to claim 1 or 2, wherein the DNA molecule encodes a protein with the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenolpyruvate mutase or a ketohexokinase. 4. Transgenic plant cell according to claim 3, wherein the DNA molecule encoding a polyphosphate kinase from E. coli, Klebsiella aerogenes, Neisseria meningitidis or Synechocystia sp. comes from. 5. Transgenic plant cell according to claim 3, wherein the DNA molecule encoding an acetate kinase is derived from E. coli, Methanosarcina thermophila, Haemophilus influenza, Bacillus subtilis or Mycoplasma genitalium. 6. The transgenic plant cell according to claim 3, wherein the DNA molecule encoding a phosphotransacetylase is derived from Methanosarcina thermophila, E. coli or Mycoplasma genitalium. 7. The transgenic plant cell of claim 3, wherein the DNA molecule encoding a trehalose-6-phosphate synthase is derived from yeast. 8. The transgenic plant cell according to claim 3, wherein the DNA molecule encoding a phosphoenolpyruvate mutase is derived from Tetrahymena pyriformis. 9. The transgenic plant cell of claim 3, wherein the DNA molecule encoding a ketohexokinase is derived from human or rat. 10. Transgenic plant cell according to one of claims 1 to 9, which is a photosynthetically active cell. 11. Transgenic plant, characterized in that it contains transgenic plant cells according to one of claims 1 to 10. 12. Transgenic plant according to claim 11, which has an increased biomass production compared to non-transformed plants. 13. Transgenic plant according to claim 11 or 12, which has a different flowering behavior compared to non-transformed plants. 14. Transgenic plant according to one of claims 11 to 13, which is a useful plant. 15. Transgenic plant according to claim 14, which is a potato plant. 16. Propagation material of a plant according to one of claims 11 to 15 containing cells according to one of claims 1 to 10. 17. Use of DNA molecules which encode proteins whose enzymatic activity leads to the synthesis of polyphosphate, acetyl phosphate, trehalose 6-phosphate, phosphoenol pyruvate or fructose 1-phosphate, for transformation and expression in plant cells.