DE19914792A1 - Production of transgenic plants by transforming metabolically defective plants with DNA that can complement the defect, eliminating need for e.g. antibiotic resistance markers - Google Patents

Abstract

Preparation of transgenic plant cells, plant tissues or plants (A) comprises: (i) transforming (A) that has a metabolic defect with a DNA molecule (I) that includes a sequence able to complement the defect; and (ii) selection of transformants on suitable media. Independent claims are also included for the following: (1) (A) transformed this way; (2) harvested and replicative materials from the products of (a); (3) a method for producing a selection marker for plants; (4) a recombinant DNA (Ia) comprising regulatory sequences of a promoter functional in plants linked to (I), optionally also to regulatory sequences that function in plants as transcription terminator and/or polyadenylation signal; (5) a vector containing (Ia); (6) a host cell containing (Ia) or the vector of (e); and (7) a kit comprising (Ia) or the vector of (e), optionally also a suitable plant selection medium.

Description

The present invention relates to the use of DNA sequences which in the Able to complement a metabolic defect in a plant Selection markers in plant toes for selection of transformed plants. Furthermore, this invention relates to recombinant DNA molecules that such Contain DNA sequences, these with regulatory sequences one in Plant active promoter and optionally transcription termination and / or polyadenylation signals are operatively linked. Furthermore Vectors, host cells and kits described such recombinant Contain DNA molecules, as well as with the recombinant DNA molecules transformed plant cells and plants. The present invention relates to likewise processes for the production of transgenic plants which, owing to the Introduction of the recombinant DNA molecules described above to selective ones Media can be selected. The present invention further relates to transgenic plants, plant cells and tissues that the invention Contain DNA molecule or obtained by the method described above be, as well as crop products and propagation material of the described transgenic plants.

In the following, documents from the prior art are cited, the Disclosure content is hereby incorporated by reference into this application.

Using genetic engineering, it is possible to target foreign genes into the to transmit plant genome. This process is called transformation and the resulting plants referred to as transgenic. The primary goals are to one of crop protection and the other an increase in quality of Harvest products. Examples of crop protection measures are: (i) herbicide tolerant Plants (Stalker (1988) Science 242, 419), (ii) insect-resistant plants (Vaek  (1987) Plant Cell 5, 159-169), (iii) Virus Resistant Plants (Powell (1986) Science 232, 738-743) and (iv) ozone-resistant plants (Van Camp (1994) BioTech. 12, 165-168). Examples of quality improvements are: (i) Increasing the durability of Fruit (Oeller (1991) Science 254, 437-439), (ii) increase in starch production in potato tubers (Stark (1992) Science 242, 419), (iii) change in starch (Visser (1991) Mol. Gen. Genet. 225, 289-296) and lipid composition (Voelker (1992) Science 257, 72-74) and (iv) production of non-plant polymers (Poirer (1992) Science 256, 520-523). Basic requirement for production transgenic plants is the availability of suitable transformation systems and the presence of selection markers that make the identification successful enable transformed plant cells.

The development of new selection markers wins through the growing Use of plants in biotechnology is becoming increasingly important. So far were primarily antibiotic or herbicide resistance, for example Kanamycin, hygromycin or BASTA, used to select transgenic plants, which, depending on the type of plant, is not always effective and often the regeneration of plants influence negatively. In addition, the use of genes that Providing resistance to antibiotics, undesirable in the food sector. Furthermore is the manipulation of several to control complex metabolic processes enzymatic steps necessary. Genes are another selection marker overexpressed, the resistance to methotrexate or 2-difluoromethylornithine generate or the use of non-metabolic substrates, for example Allow indole / thryptophane, histinol / histidine or mannose.

The present invention is therefore based on the object, means and methods to be made available for the production of transgenic plant cells and Plants can be used.

This task is accomplished by the provision of the claims characterized embodiments solved.

The present invention thus relates to a method for producing transgenic plant cells, plant tissue or plants comprising:

• a) transformation of the plant cells, plant tissue or plants that a Have a metabolic defect with a DNA molecule that has a DNA sequence contains which is able to complement the metabolic defect or repeal;
• b) selection of the transformed plant cells, plant tissue or plants on a suitable selection medium.

In connection with the present invention, a Metabolic defect caused by a naturally or artificially created mutation in the genome understood in the plant or plant cell caused by the Plant cell or plant is no longer able to do an essential one for them Metabolites in sufficient amount to synthesize themselves, so that they on the Supply of this metabolite or suitable precursors is dependent on the outside, to compensate for the phenotype caused by the lack of metabolites or preferably to cancel. The term "complement" is used in the present invention understood the ability to perform the aforementioned through the Compensate for or cancel the phenotype caused by the metabolic defect.

Examples of "metabolites" in the sense of the present invention include Amino acids or their precursors, for example the amino acids methionine and threonine or homocysteine. "Phenotype" is the expression of a characteristic understood that by means of obvious observation, biochemical analyzes or preferably selective conditions can be read out. In the sense of the In the present invention, the phenotype caused is expressed in one slowed growth of the plants up to the death of the plant.

The present invention is based on the surprising finding that genetic complementation of metabolic defects in plants, for example within the amino acid biosynthesis of the aspartate family, by means of expression homologous or heterologous genes as a selection system for the production can serve transgenic plants. The metabolic mutants can by Different methods have been achieved and by means of metabolite supplementation in vitro  be propagated. By applying this procedure to others Selection markers can be dispensed with by changing the metabolic defect to Wild type status is attributed with simultaneous expression of the new desired property. Using homologous genes for complementation the defect is advantageously the proportion of foreign DNA in the generated reduced transgenic lines. The inventive method is in the Examples based on the generation and complementation of a Methionine auxotrophic potato plant explained. The gene for the cystathionin beta-lyase (CbL; EC 4.4.1.8), which play an essential role in the biosynthesis of Amino acid methionine plays by complementation Methionine auxotrophic E. coli mutant GUC41 isolated. Starting from a λZapII cDNA library of potato leaves were plasmids by in vivo excision (pBluescript SK-), which contain different cDNAs, isolated in their entirety and established in the mutant. At first it was only brought in by the Resistance from the plasmid selected and in a second run to the Auxotrophy of the mutant on a suitable selection medium. The analysis of the Colonies that emerged from this survey gave a uniform picture with cDNA fragments of one size. The protein derived from the cDNA sequence shows high homology to the derived proteins from E. coli (Belfazia, Proc. Natl. Acad. Sci. USA 83 (1986), 867-871) and Arabidopsis thaliana (Ravanel, Plant Mol. Biol. 29 (1995), 875-882). By inserting the cDNA for Cystathionin beta-lyase in inverse orientation with respect to the promoter could Potato plants are regenerated that have no complementary medium Show change to wild type. However, if the transgenic lines are in Bringing pots into the greenhouse is a broad spectrum of phenotypes visible. As a rule, one finds depending on the degree of the antisense effect a retardation of growth until the loss of the plant; see also Application examples 1 and 3, the latter for the enzyme threonine synthase. The at phenotype observed in the antisense plants of use example 1 by supplementation (pour or spray with water containing methionine) to Wild type can be reverted. In a similar experiment, expression of the E. coli CbL gene, the genetic defect of a tobacco mutant that affects the has the same phenotype as the antisense plants, successfully complemented be (application example 2).  

For other toxic compounds that act as selection markers in the Plant transformation are used, have multiple effects on the Metabolism of the plant described. So z. B. the addition of some Antibiotics are often used to regenerate transgenic plants, the physiological and morphological changes compared to the phenotype of the wild type have and / or are sterile. However, when using the The inventive method in connection with the selection of suitable Complementation media phenotypically normal and fertile transgenic plants regenerates.

DNA sequences used to generate metabolic defects in plants can, or their complementation, can be from natural sources are isolated, preferably plants, for example by complementing corresponding auxotrophic bacterial or yeast mutants, preferably E. coli and Saccharomyces cerevisae. Examples of such DNA sequences are described in Kim, Plant Mol. Biol. 32 (1996), 1117-1124; Ravanel et al., Biochem. J 331 (1998) 639-648; Curien et al., FEBS Letters 390 (1996), 85-90; Leggewie et al., Plant Cell 9 (1997), 381-392.

Using common molecular biological techniques, it is possible (see, for example, Sambrook, 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) to introduce various types of mutations into the aforementioned DNA sequences , which leads to the synthesis of RNA and possibly proteins with possibly changed biological properties. On the one hand, it is possible to generate deletion mutants in which the synthesis of correspondingly shortened proteins can be achieved by progressive deletions from the 5 'or from the 3' end of the coding DNA sequence. Furthermore, it is possible to specifically produce enzymes which are localized in certain compartments of the plant cell by adding corresponding signal sequences. Such sequences are known (see, for example, Braun, EMBO J. 11 (1992), 3219-3227; Wolter, Proc. Natl. Acad. Sci. USA 85 (1988), 846 850; Sonnewald, Plant J 1 (1991), 95 -106). Preferred signal sequences are the transit peptide from Rubisco: Krebbers et al., Plant Mol. Biol. 11 (1988), 745-759 and signal sequences for targeting in mitochondria: Boutry et al., Nature 328 (1987), 340-342. On the other hand, the introduction of point mutations is also conceivable at positions in which a change in the amino acid sequence has an influence, for example on the enzyme activity or the regulation of the enzyme. In this way, e.g. B. Mutants are produced which have a changed K _m value or are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification. Furthermore, mutants can be produced which have an altered substrate or product specificity. Furthermore, mutants can be produced which have a changed activity-temperature profile.

For genetic engineering manipulation in prokaryotic cells, the DNA molecules or parts of these molecules are introduced into plasmids, which are a Mutagenesis or a sequence change by recombination of Allow DNA sequences. Using standard procedures (see Sambrook, 1989, Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, USA) can be base exchanged or natural or synthetic sequences can be added. For connecting the DNA fragments among themselves can be adapters or linkers to the fragments can be scheduled. Manipulations can also be the appropriate Provide restriction interfaces or the superfluous DNA or Remove restriction interfaces, are used. Where insertions, deletions or substitutions can be considered, in vitro mutagenesis, "primer repair", Restriction or ligation can be used. As an analysis method in generally sequence analysis, restriction analysis and others biochemical-molecular biological methods carried out. The degeneration of the genetic codes offers u. a. the possibility that Nucleotide sequence of the DNA sequence to the codon preference of the respective host, preferably plants.

The desired DNA sequence can be made synthetically or naturally be won or a mixture of synthetic and natural Contain DNA components. Generally synthetic  Generated DNA sequences with codons that are preferred by plants. This Plant-preferred codons can be made from triplets with the highest Protein frequency can be determined in the most interesting Plant species are expressed. When preparing an expression cassette Different DNA fragments can be manipulated to create a DNA sequence to get that reads in the correct direction and those with a correct one Reading frame is equipped. For connecting the DNA fragments together adapters or linkers can be attached to the fragments.

Process for reducing the activity or the amount of a specific enzyme, that can be used to create metabolic defects in plants, are common state of the art. For this purpose, a generally chimeric gene is inserted into the Plant introduced. It consists of a promoter active in the plant, one in the antisense direction of the coding sequence bound to the promoter Enzyme whose activity is to be reduced (3 'end of the coding sequence on 3 'end of the promoter) and a termination signal functional in the plant for transcription. The production of metabolic mutants can of course also through other common techniques such as cosuppression effects and expression of Ribozymes occur, see for example Hans Heldt, plant biochemistry, Spectrum, Akad.-Verl., 1996; Benjamin Lewin, Molecular Biology of Genes, Akad. Publ., 1998; William Dashek, Methods in plant biochemistry and molecular biology, CRC Press, 1997. Stand for the introduction of such genes in plant cells different procedures are available. From the transformed cells intact transgenic plants are regenerated, among which those with the desired phenotype (reduction in the activity of the target enzyme) become. The aforementioned metabolic defects are preferably involved Plants around auxotrophic mutants, for example due to the external supply of (a) amino acid (s) or their precursors.

In a preferred embodiment of the method according to the invention, the Metabolic defect within the amino acid biosynthesis before, preferably in the Amino acid biosynthesis of the aspartate family.

In a particularly preferred embodiment of the invention  The metabolic defect lies in the biosynthesis of the amino acid cysteine, Methionine or threonine.

In general, the metabolic defect in the plant cells and plants for the method according to the invention by mutation, antisense, co-suppression, or a dominant mutant effect. Such techniques are known to the expert; see, for example, Negrutiu et al., Mol. Gen. Genet. 199 (1985), 330-337, or the references cited above.

The DNA sequences described above also include fragments, derivatives and allelic variants of the DNA sequences described above, which are used to generate of the metabolic defect or its complementation can be used. “Fragments” mean parts of the DNA sequence that are long are enough to encode one of the proteins described. The expression In this context, "derivative" means that the sequences differ from the DNA sequences described above at one or more positions but distinguish a high degree of homology to these sequences exhibit. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations from those in the prior art DNA sequences described can, for example, by deletion, Substitution, insertion and / or recombination have arisen.

For the DNA sequences that are homologous to the sequences described above and represent derivatives of these sequences, they are usually Variations on these sequences that represent modifications that are the same perform biological function. It can be both natural occurring variations are, for example, sequences from others Organisms, or around mutations, these mutations occurring naturally may have occurred or were introduced through targeted mutagenesis. Further the variations can be synthetically produced sequences. The allelic variants can be both naturally occurring Act variants, as well as synthetically produced or by recombinant Variants generated by DNA techniques. Of course, the expert is also aware that the gene used to complement the genetic defect is not  necessarily homologous to or identical to the corresponding defective gene in the Must be a plant. It only has to encode a gene product and express that in is able to complement the gene defect, for example a protein that at least as one of its enzymatic activities that of the protein instructs that due to the genetic defect no longer or not sufficiently Amount or is formed in inactive form. With CbL, for example, a gene is out E. coli (MaIY, Zdych et al., J. Bacteriol. 177 (1995), 5035-5039), which for a Enzyme encoded from another metabolic pathway and yet significant CbL Has activity and thus potentially to complement the genetic Defects can be used. As further in the present application described, methods are also provided for suitable selection markers isolate who are able to overcome the genetic defects described above complement.

That of the different variants in the recombinant DNA molecules contained DNA sequences encoded proteins, preferably have certain common characteristics such as enzyme activity, molecular weight, immunological reactivity or conformation or physical properties, such as the running behavior in gel electrophoresis, chromatographic behavior, Sedimentation coefficient, solubility, spectroscopic properties, stability; pH optimum, temperature optimum.

For expression of the recombinant DNA molecules according to the invention DNA sequence contained in plant cells is this with regulatory Linked sequences that ensure transcription in plant cells. These include promoters in particular. Generally everyone comes for expression promoter active in plant cells.

The promoter can preferably be chosen so that the expression constitutive or only in a certain tissue, to a certain one Time of plant development or due to external influences determined time. With respect to the plant, the promoter can be homologous or be heterologous. Useful promoters are e.g. B. the promoter of the 35S RNA of Cauliflower Mosaic Virus and the ubiquitin promoter from maize for a constitutive Expression. A promoter that is particularly preferred during the  Plant transformation, plant regeneration or certain stages of this Processes is active, e.g. B. Cell division specific promoters; see for example the meristematic promoter meri-5 (Medford et al., Plant Cell 3 (1991), 359-370). An overview of further promoters in question is e.g. B. in Ward (1993) Plant Mol. Biol. 22, 361-366.

Furthermore, there is preferably a transcription termination sequence which serves the correct termination of the transcription and the addition of a Poly-A-tail can serve the transcript, which has a function in the Stabilization of the transcripts is attributed. Such elements, e.g. B. the Terminators of the octopine synthase gene from agrobacteria are in the literature described (cf. Gielen, EMBO J. 8 (1989), 23-29) and are interchangeable.

In a preferred embodiment, the aforementioned DNA sequences originate from bacteria or plants.

As described in the examples, metabolism allowed auxotrophic plants can be successfully complemented by the expression of the CbL gene from E. coli. Phenotypic and physiological studies of these transgenic lines showed no difference to the control plants also examined. This The method therefore does not lead to any change in the plants, as it does with Application of this technique in biotechnology is desirable. An influence on other metabolites can therefore be excluded.

Therefore coded in a preferred embodiment of the invention Method the DNA sequence an enzyme, preferably cystathionine beta lyase or threonine synthase.

DNA molecules according to the invention are introduced into plant cells preferably using vectors such as plasmids that are stable Ensure the integration of the inserted DNA into the plant genome. For the For example, the present invention can use binary vectors BIN 19 and Derivatives are used. The vector BIN 19 was developed by Bevan (Nucl. Acids Res. 12 (1984), 8711-8721) and is commercially available (Clontech Laboratories, Inc., USA). However, it is everyone else  Plant transformation vector suitable in which an expression cassette is integrated and the integration of the expression cassette into the plant genome guaranteed.

To prepare for the introduction of foreign genes into higher plants there is a large number of cloning vectors are available that provide a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC derivatives, M13mp derivatives, pBluescript derivatives, pACYC184, etc. The desired DNA sequence can be a suitable restriction interface into the vector. The The plasmid obtained is used for the transformation of E. coli cells. Transformed E. coli cells are cultivated in a suitable medium, then harvested and lysed. The plasmid is made using standard methods recovered. As an analysis method to characterize the obtained Plasmid DNA is generally used for restriction analysis and sequence analysis used. After each manipulation, the plasmid DNA can be cleaved and resulting DNA fragments can be linked to other DNA sequences.

In a preferred embodiment, the vector according to the invention contains at least one further recombinant DNA molecule. By providing the DNA molecules and vectors according to the invention can contain any information, contained in another recombinant DNA molecule, on plants or Plant cells are transferred and then selected using suitable media for the desired information. Naturally the person skilled in the art knows that the recombinant DNA molecule which is the additional Contains information, not necessarily in the vector that carries the selection marker, must be present, but can also be co-transformed with them (Lyznik (1989) Plant Mol. Biol. 13, 151-161; Peng (1995) Plant Mol. Biol. 27, 91-104). This This is an option, for example, if there is no physical coupling of the Marker gene and the information to be transmitted is desired. In this case after selection of the primary transgenic plant, the marker gene and the desired information in subsequent crossings independently of each other segregate.  

In a preferred embodiment, the further recombinant DNA Molecule a DNA sequence that is a peptide, protein, antisense, sense RNA, viral RNA, or ribozyme encoded. Some examples of heterologous (over) expression and for antisense inhibition with the aim of metabolic flows in transgenic plants can be manipulated in Herbers & Sonnewald (1996) TIBTECH 14, 198-205 summarized. An example of ribozymes has been published by Feyter (1996) Mol. Gen. Genet. 250, 329-338. By expression of a "hammerhead" ribozyme the authors managed to achieve TMV resistance in transgenic tobacco plants produce. Diverse applications of transgenic plants with Generated with the aid of the recombinant DNA molecules and vectors according to the invention can also be found in TIPTEC Plant Product & Crop Biotechnology, 13 (1995), 312-397.

The vectors according to the invention can have further functional units which stabilize the vector in the host organism, like a bacterial one Origin of replication or the 2 micron DNA for stabilization in Saccharomyces cerevisiae. Furthermore, "left border" and "right border" sequences Agrobacterial T-DNA can be included, which ensures stable integration into the Genetic material of plants is made possible. Another strategy, marker-free transgenic Generating plants is the use of sequence-specific Recombinases. For this purpose, e.g. B. Two strategies can be used: (i) Re-transforming a starting line expressing recombinase and Outcrossing of the recombinase after removal of the selection marker, which was associated with the desired gene. (ii) Co-transform with subsequent outcrossing. The prerequisites for this recombinase strategy are (i) Flanking the selection marker with recognition sequences for the Recombinase and (ii) a recombinase that is active in plants and none Plants own sequences used for recombination. Such methods can the expert can take from the prior art, for. B. RecA (Reiss (1996) Proc. Natl. Acad. Sci. USA 93, 3094-3098), Cre / Iox (Bayley (1992) Plant Mol. Biol. 18, 353-361), FLPIFRT (Lloyd (1994) Mol. Gen. Genet. 242, 653-657), Gin (Maeser (1991) Mol. Gen. Genet. 230, 170-176) and RIRS (Onouchi (1991) Nucl. Acids Res. 19, 6373-6378).  

There are a large number of for the introduction of DNA into a plant host cell Techniques available. These techniques include transformation plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacferium rhizogenes as a transformation agent, the fusion of Protoplasts, injection, electroporation of DNA, introduction of DNA using the biolistic method and other possibilities.

When injecting and electroporation of DNA into plant cells are in themselves no special requirements were placed on the plasmids used. It can simple plasmids such as B. pUC derivatives can be used. But should out Whole plants are regenerated in such a transformed cells Presence of a selectable marker gene necessary.

Depending on the method of introducing desired genes into plant cells, others can DNA sequences may be required. Are z. B. for the transformation of Plant cell that uses the Ti or Ri plasmid, at least the right one Limitation, but often the right and left limitation of the Ti and Ri plasmid T-DNA as a flank region with the genes to be introduced.

If Agrobacteria are used for the transformation, the one to be introduced DNA can be cloned into special plasmids, either in one intermediate vector or into a binary vector. The intermediate vectors Due to sequences that are homologous to sequences in the T-DNA, by homologous recombination in the Ti or Ri plasmid of the agrobacteria to get integrated. This also contains those for the transfer of T-DNA necessary vir region. Intermediate vectors cannot be found in agrobacteria replicate. The intermediate vector can be found by means of a helper plasmid Agrobacterium tumefaciens are transmitted (conjugation). Binary vectors can replicate in both E. coli and Agrobacteria. They contain one Selection marker gene and a left or polylinker, different from the right and left T-DNA border region to be framed. You can go straight to the Agrobacteria are transformed (Holsters et al., Mol. Gen. Genet. (1978), 181-187). The plasmids used to transform the agrobacteria also contain a selection marker gene, for example the NPT II gene, the  allows the selection of transformed bacteria. That serving as the host cell Agrobacterium is said to contain a plasmid that carries a vir region. The vir region is necessary for the transfer of T-DNA into the plant cell. Additional T-DNA can be present. The agrobacterium transformed in this way becomes Transformation of plant cells used.

The use of T-DNA for the transformation of plant cells is intensive examined and sufficient in EP 120516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46; Ann et al., EMBO J. 4 (1985), 277-287) been. Binary vectors are already e.g. T. commercially available.

Plant explants can be used to transfer the DNA into the plant cell Agrobacterium fumefaciens or A. rhizogenes can be cocultivated. From the infected plant material (e.g. leaf pieces, stem segments, roots, but also Protoplasts or suspension-cultivated plant cells) can then be used in one suitable medium, which transformed antibiotics or biocides for selection Cells can contain, whole plants can be regenerated again. The so obtained Plants 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 in there usually stable and also remains in the offspring of the original obtained transformed cell. It usually contains a selection marker, the resistance of the transformed plant cells to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or Phosphinotricin and the like. a. mediated. The individually chosen marker should therefore be the Selection of transformed cells against cells that lack the inserted DNA allow.

The metabolic defects used for the method according to the invention Plant cells, plant tissues or plants are preferred on Complementation medium cultivated, especially if the Metabolic defect is lethal to the plant. The transformed cells grow within the plant in the usual way (see also McCormick et al., Plant Cell  Reports 5 (1986), 81-84). For example, the metabolically defective plants attracted with the addition of methionine. After transformation with one of the DNA molecules described above, which are able to Complementing or eliminating metabolic defects can resulting plant cells, plant tissue and plants cultivated on media be the missing metabolic product in the metabolically defective plants or no longer contain a corresponding intermediate. A corresponding one Selection medium can be transformed right at the beginning of cultivation transgenic plant cells, plant tissue and plants are used or at any stage of transgenic plant regeneration. The choice of When the selective medium is used, for example, may depend on the choice depend on the promoters who are jointly responsible for metabolic defects and / or which control the DNA sequence which is capable of the corresponding one Complement or cancel metabolic defect.

Two or more generations should be attracted to ensure that the phenotypic trait is stably maintained and inherited. As Host plants are all plant species, especially cultivated plant species, suitable.

Among crops are z. B. sugar beet, potato, tobacco, tomato, rapeseed, To understand wheat and corn, but also other crop species, especially those species that are used as agricultural auxiliary plants are usable.

By providing the method according to the invention, it is possible to create new ones To use selection agents for plant transformation, preferably for Selection of transformed plants derived from high-performance varieties for which the selection markers described in the prior art often only in limited dimensions are useful. The present invention thus also relates transgenic plant cells which have a recombinant DNA molecule according to the invention or contain a vector according to the invention or by the inventive Procedures have been obtained as well as transgenic plant cells that are of such transformed plant cells. Such cells can be from  distinguish naturally occurring plant cells in that they contain at least one recombinant DNA molecule according to the invention, the does not occur naturally in these cells or because such Molecule is integrated at a location in the genome of the cell in which it is does not occur naturally, d. H. in a different genomic environment.

In a preferred embodiment, the plant cell according to the invention contains at least one other foreign gene. As already mentioned in the introduction to this Patent application mentioned is more complex for the possibility of control Metabolic processes require the manipulation of several enzymatic steps and therefore the possibility to transform transgenic plants several times essential. With the help of the recombinant DNA molecules and The person skilled in the art can now multiply vectors for new markers for selection use transformed plant cells.

The transgenic plant cells can be used according to techniques known to those skilled in the art to be regenerated into whole plants. The regeneration of the Plants obtainable from transgenic plant cells according to the invention are also Subject of the present invention. The invention also relates to.

Plants and plant tissues that are transgenic as described above Plant cells included. In principle, the transgenic plants can be Trade plants of any plant species, d. H. both monocotyledon also dicotyledonous plants. They are preferably useful plants such as wheat, Barley, rice, rapeseed, pea, corn, sugar beet, sugar cane or potato.

The invention also relates to propagation material and crop products of the plants according to the invention, for example fruits, seeds, bulbs, Rhizomes, seedlings, cuttings etc.

In a further aspect, the present invention relates to a method for producing selection markers for plants comprising

• a) Transformation of an auxotrophic due to a metabolic defect Microorganism with a DNA library;  
• b) cultivating the microorganism under suitable conditions Allow complementation of the metabolic defect,
• c) screening the microorganism on a suitable selection medium;
• d) Isolation of the (s) DNA molecules (s) that (the) the metabolic defect complement (can); and if necessary
• e) Linking the (s) DNA molecules (s) from (d) with the regulatory ones Sequences of a promoter active in plants

The basis for this process is the production of suitable DNA libraries, the so-called expression banks. As a donor, both serve eukaryotic as well as prokaryotic organisms. Starting from RNA, which is overwritten in cDNA (copy-DNA) or by genomic DNA DNA fragments cloned into expression vectors. Then stand in these vectors the fragments under the control of a host-specific promoter which u. a. the Expression of the protein / enzyme sought allows. Bacteria (e.g. E. coli) and yeast (S. cerevisiae) mutants that have a defect in one Have biosynthetic step, for example in amino acid biosynthesis. Become then transformed microorganisms with the prepared DNA libraries Plated medium that contains only basic components (e.g. for bacteria M9- Medium, for yeasts SD medium) only those cells that have the gene will survive express that complements the defect of the mutant used. Material and Methods that can be used in the sense of the method according to the invention are described, for example in Kim, Leustek, Plant. Mol. Biol. 32 (1996), 1117 - 1124; Curien et al., FEBS Letters 390 (1996), 85-90; Leggewie et al., Plant Cell 9 (1997), 381-392.

The metabolic defect preferably relates to a metabolic defect as in one of the above embodiments was defined.

In a preferred embodiment of the method according to the invention, the Microorganism E. coli.

As already stated above, the present invention provides new selection markers for plants. The present invention therefore also relates to selection markers in the form of recombinant DNA molecules

• a) regulatory sequences of a promoter active in plants;
• b) functionally linked to one in one of the above embodiment defined or obtained by a method according to the invention Molecule; and if necessary
• c) functionally linked regulatory sequences that act as transcription Termination and / or polyadenylation signals can serve in plants.

Suitable promoters and termination or polyadenylation signals are in the State of the art described; see also above.

The invention further relates to vectors which contain recombinant DNA Contain molecules. These are preferably plasmids, cosmids, Viruses, bacteriophages and other vectors common in genetic engineering. For Preparing for the introduction of foreign genes into higher plants is a major challenge Number of cloning vectors available that provide a replication signal for E. coli and contain a marker gene for the selection of transformed bacterial cells. Examples for such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be inserted at a suitable restriction site in the Vector are introduced. The plasmid obtained is used for the transformation of E. coli cells used. Transformed E. coli cells are in a suitable Medium grown, then harvested and lysed. The plasmid will recovered. As an analysis method to characterize the obtained Plasmid DNA are generally used for restriction analysis, gel electrophoresis and other biochemical-molecular biological methods used. After every Manipulation can cleave the plasmid DNA and recover DNA fragments with it other DNA sequences. Each plasmid DNA sequence can be in the same or different plasmids can be cloned.

In a further embodiment, the invention relates to host cells that recombinant DNA molecules or vectors according to the invention transient or stable included. A host cell is understood to mean an organism that is in the Is able to record in vitro recombined DNA and, if necessary, that in the DNA sequence contained recombinant DNA molecules according to the invention  express.

These are preferably prokaryotic or eukaryotic cells. In particular The invention relates to plant cells containing the vector systems according to the invention or derivatives or parts thereof. These preferably have due to the Inclusion of the vector systems, derivatives or parts of the invention Vector systems on one of the metabolic defects described above. This Plant cells or plants regenerated from them can then in turn in one of the inventive method described above can be used. The cells according to the invention are preferably characterized in that the introduced recombinant DNA molecule according to the invention either heterologously with respect to the transformed cell, i. H. naturally not in these cells occurs or is located at a different location in the genome than that corresponding naturally occurring sequence.

In a further embodiment, the invention relates to kits comprising a recombinant DNA molecule according to the invention or one according to the invention Contain vector and optionally a suitable selection medium for the plants described above. The DNA molecules described above and vectors can be packed in containers in the kit according to the invention, for example in vessels, possibly in buffers and / or solutions. The Kits according to the invention can be used in a variety of ways. Exemplary Areas of application such as breeding have been indicated above. The The kits themselves are preferably produced using standard processes.

As stated above, the present invention provides recombinant DNA Molecules and vectors are available that act as selection markers in plant cells for the selection of transformed plant cells and transgene derived therefrom Plants, plant cells and / or tissues are available. So that affects present invention also the use of an inventive recombinant DNA molecule, a vector according to the invention or the Selection markers produced according to the invention for the production of transgenic Plants, plant cells and / or tissues and their use as selectable Markers in plant cell and tissue culture and / or plant breeding.  

These and other embodiments are disclosed to those skilled in the art obvious and encompassed by the description and examples of the present invention. Further literature can be found in one of the above the methods, means and uses mentioned within the meaning of the present Invention can be applied, taken from the prior art be, e.g. B. from public libraries under z. B. the use of electronic aids. For this purpose, there are, among others, public ones Databases such as the "Medline", which are available on the Internet, e.g. B. at the address http://www.ncbi.nim.nih.gov/PubMed/medline.html. Further Databases and addresses are familiar to the person skilled in the art and can be extracted from the Internet can be removed, e.g. B. at http://www.lycos.com. A Overview of sources and information on patents and patent applications in biotechnology is given in Berks, TIBTECH 12 (1994), 352-364.

Methods used in the examples (unless otherwise stated):

1. Cloning procedure

The vector pBluescript SK- (Stratagene) was used for cloning in E. coli. For the plant transformation, the gene constructions were converted into binary Vectors BIN 19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8720). If not mentioned otherwise, those in Sambrook, 1989 or Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley and Sons, USA Techniques used.

2. Bacterial strains

The E. coli strain XL-1 Blue (Stratagene) was used for the plasmid vectors mentioned under 1. and the auxotrophic E. coli strain GUC41 (Gutermann, J. Bact. 114 (1973), 1225-1230) for complementation. used. A cDNA bank of potato leaves was used to complement the E. coli mutant. The cDNA library was cloned in a phage vector (λZAP II, Stratagene, USA). The phage vector contains the plasmid Bluescript SK ^- (Stratagene, USA) integrated, into which the cDNA is cloned. With the help of a helper phage, the plasmid was cut out and used for the transformation. For complementation, the bacteria were plated on a minimal medium (M9 medium) (Sambrook et al., 1989) and incubated for three days at 37 ° C.

The transformation of the binary plasmids into the potato plants was done with the help of the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere, Nucl. Acids Res. (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transformation using the method of Höfgen & Willmitzer (Nucl. Acids Res. 7 (1989), 1513-1523) isolated and after suitable restriction cleavage analyzed by gel electrophoresis.

4. Transformation of potato

Ten small leaves of a potato sterile culture wound with the scalpel (Solanum tuberosum L. cv.Desiree) were dissolved in 10 ml MS medium (Murashige & Skoog, Physiol. Plans. 15 (1962), 473-497) with 2% sucrose, which 50 µl of an Agrobacterium tumefaciens overnight culture grown under selection contained. After gentle shaking for 3-5 minutes, another incubation for 2 days in the dark. The leaves were then opened for callus induction MS medium with 1.6% glucose, 5 mg / l naphthylacetic acid, 0.2 mg / l Benzylaminopurine, 250 mg / l claforan or 125 mg / l b-bactyl, 50 mg / l kanamycin or 1 mg 1 mg / l, hygromycin B, and 0.8% Bacto agar. After a week Incubation at 25 ° C and 3000 lux was used to express the leaves on the shoot MS medium with 1.6% giucose, 1.4 mg / l zeatin ribose, 20 mg / l naphthylacetic acid, 20 mg / l giberellic acid, 250 mg / l claforan or 125 mg / l b-bactyl, 50 mg / l Kanamycin or 3 mg / l hygromycin B and 0.8% Bacto agar.

5. Radioactive labeling of DNA fragments

The radioactive labeling of DNA fragments was carried out using a  DNA random primer labeling kits from Boehringer (Germany) according to the Manufacturer's information carried out.

6. Plant husbandry

Potato plants were kept in the greenhouse under the following conditions:
Light period 16 h at 25,000 lux and 22 ° C
Dark period 8 h at 15 ° C
Humidity 60%
Complementation medium of the bacteria: M9 medium (Sambrook et al., 1989), mixed with 1 mM IPTG, 40 mg / l threonine, 40 mg / l leucine.
Supplementation medium for antisense plants: MS medium (Murashige, Skoog, Plant Physiol. 15, 573-497, 1962), mixed with 2% sucrose, 125 mg / l β-bactyl, 45 mg / l methionine, 200 mg / l Casein hydrolyzate

7. Analysis of genomic DNA from transgenic potato plants

Genomic plant DNA was isolated according to Rogers & Bendich (Plant Mol. Biol. 5 (1985), 69-76). With the help of "Southern Blot" analyzes 10-30 µg DNA after appropriate restriction cleavage of the introductory DNA sequences examined.

The examples illustrate the invention.

Application example 1 Production of metabolism of auxotrophic plants

The gene for the cystathionin beta lyase has been complemented by the Methionine auxotrophen-E. coli mutant GUC41 isolated. Starting from a λZapII cDNA library of potato leaves were analyzed using in vivo excision plasmids (pBluescript SK-), which contain different cDNAs, isolated in their entirety and established in the E. coli mutant. Initially, only the dated inserted plasmid selected outgoing resistance and in a second Passage on the auxotrophy of the mutant on an appropriate Selection medium (M9 without methionine). The analysis of this  Colonies emerged from screening gave a uniform picture with cDNA fragments of one length. Parts of the sequence of the genomic DNA were cut by standard methods using the dideoxynucleotide method (Sanger, Proc. Natl. Acad. Sci. USA (1977), 5463-5467) partially determined. A comparison of the determined Sequences with the determined Arabidopsis sequence clearly demonstrated that it was the DNA insertion is a fragment of nuclear DNA. That from the Protein derived from cDNA sequence exhibits high homology to the derived Proteins from E. coli and Arabidopsis thaliana.

By inserting the cDNA for cystathionin beta-lyase in inverse Potato plants were able to orient themselves with regard to the promoter (CaMV 35S) be regenerated on the complementation medium no change to the wild type exhibit. However, the transgenic lines are in pots in the greenhouse brought, a broad spectrum of phenotypes is visible. Usually takes place depending on the degree of the antisense effect, a retardation of the Growth to lethal effects.

About the importance of cystathionin beta-lyase in the metabolic pathway have so far only been speculated. Enzyme data are available that are cytosolic Do not rule out isoform. However, more recent findings can support this hypothesis not support. So far, Arabidopsis thaliana and Solanum tuberosum only plastid localized variants can be isolated. Furthermore could successfully a tobacco mutant that has a defect for the cystathionin beta-lyase by plastid expression of the E. coli metc gene (encoding cystathionin beta-lyase) can be complemented. This mutant tobacco has so far been the one the only mutant described within the methionine pathway. The The observed phenotype in the antisense plants therefore supports the Results of the mutant tobacco. This explains the essentiality of cystathionin Derive beta-lyase for the metabolic pathway and consequently for the plant.  

Example of use 2 Complementation of an auxotrophic tobacco mutant 1. Plant growing conditions

N. plumbaginifolia plants were grown in tissue culture on 2MS medium (Murashige and Skoog, Plant Physiol. 15 (1962), 573-497) with 0.3 mM methionine (Negrutiu et al., Int. J. Dev. Biol. 36 (1992), 73-84) and casein hydrolyzate (200 mg / l,) supplemented. The plants were illuminated for 16 hours a day.

2. Binary vector construction

The E. coli metC gene was generated by PCR from genomic DNA from E. coli K12 (Laber et al., FEBS Letter 379 (1996), 94-96) with primers which isolated a BamHI Interface included. The amplified DNA fragment with a length of 1187 bp was digested with BamHI and into the BamHI site of the pBinAR vector (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230). The binary vector contains the CaMV 35S promoter and the OCS terminator, as well as the nptII gene for a kanamycin selection of the transformed plants. For the plastid Localization of the E. coli CbL was the transit peptide of the small subunit of the Spinach Rubisco merged with the open reading frame of the CbL. That from it resulting plasmid was named pEc-TP-metC.

3. Plant transformation

Leaf pieces of the N. plumbaginifolia mutant were extracted using the Agrobacterium tumefaciens strain GV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1985), 4777-4788) according to a protocol by Rosahl et al. (EMBO J. 6 (1987), 1155-1159) transformed. For selection, the kanamycin concentration was 50 µg / ml. Methionine was adjusted to 0.3 mM until the plants were examined for metC expression were.

4. DNA and RNA analysis

Standard techniques were used for molecular biological work (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ^and Cold Spring Harbor Laboratory Press, NY, 1989). The complete metC gene was used as a hybridization probe for Southern and Northern blot analyzes. The 1.2 kb fragment cut out of pMet33 was separated in a 0.8% agarose gel, then electroeluted and used for the radioactive labeling with ³² P-dCTP according to the manufacturer's instructions (Rediprime, Amersham).

N. plumbaginifolia genomic DNA was obtained by the method of Dellaporta et al. (Plant Mol. Biol. Reporter 1 (1983), 19-21). The Southern blot was with 10 µg DNA per restriction digest performed. Positively charged nylon membranes were purchased from Boehringer. Total RNA was changed after one Protocol according to Rerie et al. (Mol. Gen. Genet. 225 (1991), 148-157) starting from two g fresh leaf material isolated. For the Northern blots, 10 µg RNA each plotted per track. An RNA ladder (Gibco BRL) was used as a marker. Uniform loading of the RNA gels was achieved after transfer to the membrane controlled by staining with methylene blue (Brown and Mackey, Current Protocols in Molecular Biology, Vol. 1, John Wiley and Sons, USA (1995), 4.9.1-4.9.16).

5. Partial purification of the cystathionin beta lyase

All steps were carried out at 4 ° C. Fifteen g of frozen leaf material was ground in liquid nitrogen and then homogenized in buffer E (100 mM KHPO ₄ , pH 8.0, 1 mM EDTA, 5 mM thiourea, 20% glycerol, 10 mM β-mercaptoethanol). The extract was filtered through Miracloth (Calbiochem) and then centrifuged for 15 min at 10,000 rpm. The supernatant was brought to 65% saturation within one hour by the successive addition of ammonium sulfate. After centrifugation for 20 min at 10,000 rpm, the pellet was resuspended in 1 ml buffer D (50 mM KHPO ₄ , pH 7.5, 1 mM EDTA, 20% glycerol, 1 mM DTT). The protein fraction was desalted using a Sephadex G-25 column, which had previously been equilibrated with buffer D. Cystathionin beta-lyase activity co-elutes with the dark green fraction. The fractions were collected and stored at -20 ° C until used. The protein content was measured by the Bradford method (Anal. Biochem. 72 (1976), 248-254) using the BIO-Rad protein assay with bovine serum as the standard.

6. Cystathionin beta lyase activity

To determine the CbL activity, partially purified enzyme extracts were used Cystathionin incubated. The pyruvate released in the reaction was converted into a  Implemented hydrazone in alkaline solution and determined the absorption (Sharma and Mazumber, J. Biol. Chem. 245 (1970), 3008-3014).

The 200 µl reaction mix consists of 60 µl desalted protein extract, 200 mM Tris Buffer, pH 8.6, 5mM cystathionin. After an hour of incubation at 37 ° C the reaction was stopped by adding 120 μl of TCA. The denatured proteins were pelleted by centrifugation at 13,000 rpm for 10 min. The The keto acid content in the supernatant was determined after adding 80 µl Dinitrophenylhydrazone (0.05% in HCl) and incubation for 10 minutes Room temperature determined. After adding 800 µl of 0.4 N NaOH and another 30 After incubation, the absorbance was then measured at 505 nm. The Pyruvate concentration was determined using a pyruvate calibration line for the Concentration range between 0 and 800 µM determined. CbL activity is expressed in pmol Pyruvate given per min and mg protein.

7. Cystathionin beta-lyase activity detection in native PAGE

Protein samples were collected in a vertical small gel according to Laemmli (Nature 227 (1970), 680-685) separated in native 7.5% acrylamide PAGE gels at 4 ° C. After electrophoresis, the gel was washed twice in 10 mM Tris / HCl, pH 8.6 washed and then at 37 ° C with a reaction mixture consisting of 10mM Tris / HCl, pH 8.6, 5mM cystathionin, 0.8% (w / v) tetraphenylborate (TPB). The ammonium released during the reaction forms an insoluble with TPB Precipitate that is well detectable on a dark background (Turner, Ph.D. thesis, University of Lancaster, UK (1994)). Controls without cystathionin were performed involved.

8. Free amino acid extraction

Between 100 and 500 mg of plant material were homogenized in the mortar and mixed with a mixture of methanol: chloroform: water (12: 5: 1) (Bieleski and Turner, Anal. Biochem. 17 (1966), 278-293). The homogenate was centrifuged and the supernatant transferred to a new tube. The pellet was three Extracted once in a row and the supernatants combined. Chlorophyll was through Add two volumes of chloroform and a portion of water removed. The aqueous phase containing amino acids was completely evaporated to dryness. The rest was taken up in water and passed through a 1.3 µm filter.  

HCl was added ad 2 N and then for the hydrolysis of the amides at 110 ° C. incubated for 2 h. After a second drying the rest was in one Sample buffer (Na citrate buffer, pH 2.2) was added and passed through a 0.45 µm Filter filtered. The amino acids were derivatized using ninhydrin post-column measured on a Beckman System Gold system.

9. Results Transformation of the auxotrophic mutant and its development

With the aim of restoring the autotrophy of the met ^- (10.1 / 9) N. plumbaginifolia mutant, a chimeric gene consisting of the coding region of the metC gene and the transit peptide of the small subunit of Rubisco was constructed and cloned into a binary vector. Agrobacterium-mediated gene transfer in N. plumbaginifolia mutant 10.1 / 9 with the construct led to callus development and shoot formation on 2MS medium without addition of methionine in the presence of kanamycin. In contrast, only shoots of the untransformed mutant could be regenerated when methionine was added to the culture medium. Transformed shoots also took root under these conditions. Five primary transformants, designated EcCbL 1-5, showed a normal phenotype and were selected for further analysis.

To check the phenotypic reversion under greenhouse conditions the five regenerated lines were planted in soil. EcCbL resemble 1-5 plants in the phenotype the wild type of N. plumbaginifolia. They also showed the same fertility and seed formation like the control plants.

Based on preliminary analysis of Northern blots and enzyme activity were two lines (EcCbL 1 and 4) with approximately the same expression and Activity selected for further detailed investigation.

Molecular analysis

Primary transformants were based on integrating the construct into the DNA and on Expression examined. Total RNA was obtained from control plants and the transgenic lines EcCbL 1 and 4 isolated and analyzed by Northern blot. Strength Expression signals with a size of 1.7 kb were only for the transgenic Plants obtained while no signal was detected in the control plants.  

Genomic DNA was digested with different restriction enzymes Integration of the E. coli metC gene into the plant genome Primary transformants examined. The result of the Northern blot Analysis to be confirmed. A cross reaction between the E. coli DNA sample and the vegetable gene could not be observed. The size of the received Signals coincides with the calculated sizes of the corresponding ones Restriction sites in the T-DNA match, with the BamHI and HindIII bands around 0.3 kb deviate due to the termination signal. Through the EcoRI digestion one copy was found for EcCbL 1 and two copies for EcCbL 4.

CbL enzyme activity

Partially purified protein from control plants, the mutant and the transformed Lines EcCbl 1 and 4 were used for the analysis of the CbL activity. In the Extracts from control plants could show little activity in the auxotroph Mutant no activity can be determined. In contrast, for the two transgenic lines up to 500 times higher activities than in the control plants be determined. This increase could also be seen in native PAGE gels Activity staining can be shown. Equal amounts of protein were loaded and only in the transgenic lines could a white, thick precipitation band be detected. This could be due to the low for the wild type Occurrence are not shown. Further purification would be necessary in order to to show this.

Free amino acid content

To see the effect of overexpression of CbL on amino acid biosynthesis show the free amino acids of leaves taking into account the Amino acids of the aspartate family of transgenic plants grown in vitro analyzed. The amino acid content of the transgenic lines has been compared to the control plants including the methionine content were not changed. It the amount of methionine could be reduced to the wild type level, but it there was no increase. During the entire plant development, none Changes in free amino acid levels were observed in the different lines become. Also for S-adenosylmethionine and methionine sulfoxide contents resulting from the No change was found in methionine metabolism  become. The methionine content of the auxotrophic mutant stems from the uptake of the Amino acid from the supplemented culture medium.

Example of use 3

Another example of using this technique is the enzyme Threonine synthase. In an antisense approach, it was also possible to use transgenic Plants are produced that have a CbL-like phenotype. The The first analysis allows the statement that here, as in the case of the CbL, another Gene is available as a selection marker.

Example of use 4

Currently there are mutable supplements that are already available different processes for the production of plant material with defined Metabolic defects available to apply the technique described; see, for example, Negrutiu, Mol. Gen. Genet. 199: 330-337 (1985). On the one hand They are mutagenesis methods in which protoplasts are used alkylating agents are modified so that they subsequently on special Selection media must be complemented. On the other hand allows one Process by "gene knock out" using RNA / DNA hybrids to target genes change. The subsequent selection is the same as for a mutagenesis. Further is the use of the antisense technique without antibiotic markers due to the high Transformation efficiency possible. The selection of lethal mutants is in the Greenhouse feasible and can by metabolite complementation in vivo be propagated.

Claims (23)

1. A method for producing transgenic plant cells, plant tissue or plants comprising

• a) transformation of the plant cells, plant tissue or plants which have a metabolic defect with a DNA molecule which contains a DNA sequence which is capable of complementing or eliminating the metabolic defect;
• b) Selection of the transformed plant cells, plant tissue or plants on a suitable selection medium.

2. The method of claim 1, wherein the metabolic defect within the Amino acid biosynthesis is present. 3. The method of claim 2, wherein the amino acid biosynthesis Aspartate family affects. 4. The method according to claim 2 or 3, wherein the amino acid cysteine, methionine or is threonine. 5. The method according to any one of claims 1 to 4, wherein the metabolic defect through a mutation, antisense, cosuppression, or a dominant Mutant effect is generated. 6. The method according to any one of claims 1 to 5, wherein the DNA sequence to the regulatory sequence of a promoter active in plants functional is linked. 7. The method of claim 6, wherein the promoter is a constitutive promoter. 8. The method according to any one of claims 1 to 7, wherein the DNA sequence from Bacteria or plants.   9. The method according to any one of claims 1 to 8, wherein the DNA sequence Enzyme encoded. 10. The method of claim 9, wherein the enzyme is cystathionin beta-lyase or Is threonine synthase. 11. The method according to any one of claims 1 to 10, wherein the DNA molecule in a vector is included. 12. The method of claim 11, wherein the vector is another recombinant DNA molecule that contains a DNA sequence that contains a peptide, protein, Antisense, sense RNA, viral RNA or ribozyme encoded. 13. Transgenic plant cell, plant tissue or plant manufactured according to the method according to any one of claims 1 to 12 containing a according DNA molecule defined according to one of claims 1 to 10. 14. crop products or propagation material of a plant according to claim 13 containing plant cells according to claim 13. 15. A method for producing selection markers for plants comprising

• a) transformation of a microorganism auxotrophic due to a metabolic defect with a DNA library;
• b) cultivating the microorganism under suitable conditions which allow the metabolic defect to be complemented;
• c) screening the microorganism on a suitable selection medium;
• d) isolation of the DNA molecule (s) which can (may) complement the metabolic defect; and if necessary
• e) Linking the (s) DNA molecules (s) from (d) with the regulatory sequences of a promoter active in plants.

16. The method of claim 15, wherein the metabolic defect as in one of the Claims 2 to 5 is defined.   17. The method according to claim 15 or 16, wherein the microorganism is E. coli. 18. Including recombinant DNA molecule

• a) regulatory sequences of a promoter active in plants;
• b) functionally linked to a DNA molecule defined in one of claims 1 to 10 or obtained according to one of claims 15 to 17; and if necessary
• c) functionally linked regulatory sequences which can serve as transcription termination and / or polyadenylation signals in plants.

19. A vector comprising a recombinant DNA molecule according to claim 18. 20. The vector of claim 19, which at least one further recombinant DNA Molecule with a DNA sequence that contains a peptide, protein, antisense, Sense RNA, viral RNA or ribozyme encoded. 21. A host cell containing a recombinant DNA molecule according to claim 18 or a vector according to claim 19 or 20. 22. Kit comprising a recombinant DNA molecule according to claim 18 or one Vector according to claim 19 or 20 and optionally a suitable one Selection medium for plants. 23. Use of a DNA molecule according to claim 18 or as in one of claims 1 to 10 is defined, a vector according to claim 19 or 20 or as defined in claim 11 or 12 or the selection marker obtainable by the process according to any one of claims 15 to 17 Selection marker for the production of transgenic plants, plant cells and / or tissue or in plant cell and tissue culture and / or Plant breeding.