NO885091L - SPLEED GENES AND MANUFACTURING THEREOF. - Google Patents

Abstract

The invention relates to a new method, which,. based on homologous recombination of natural or artificially modified genes, gene fragments or other useful DNa sequences with homologous ONa sections within the plant genome, enables targeted splicing of genetic material at precisely defined sites in the plant's genome, as well as a targeted, predictable modification of genes within the plant genome. In addition, the method according to the invention enables a targeted identification of the function of individual genes within the total genome of the plant. A further subject of the present invention comprises transgenic plants and their descendants as well as the plant products thereof obtained by this process.

Description

The present invention relates to a new method which, based on the homologous recombination of natural or artificially modified genes, gene fragments or otherwise useful DNA sequences with homologous DNA sections within the p1ante genome, allows a targeted splicing of genetic material at precisely defined locations in the plant's genome and thus a targeted, predictable modification of genes within the p 1 ante-genome (in situ modification). Moreover, the method according to the invention enables a targeted identification of the function of individual genes within the plants' total genome. The invention also relates to transgenic plants obtained according to this method, as well as their successors and the plant products thereof.

A prerequisite for a precise genetic manipulation of plants is a precise and predictable change of the plant genome by targeted splicing of new genes in the genome of plant cells, which are then regenerated into cell, tissue or tissue structures or into whole plants and thus lead to new plants that exhibit a new trait.

Intensive research on recombinant DNA technology no 1 and in the area of plants has in recent years led to significant progress. This primarily concerns the production of transgenic plants, for which purpose in the meantime a number of gene transfer methods are available, which are already used routinely in many laboratories.

Without a doubt, the Agrobacterium transformation system belongs to the best established and most frequently used gene transfer methods.

Agrobacterium cells have on their Ti plasmid a large DNA fragment, the so-called T-DNA region, which is integrated into the plant genome during the transformation of plant cells.

This natural gene transfer system can, after the execution of various modifications, be used as a gene vector system for the artificial transformation of plants.

In addition to this, there are also other effective methods for splicing genetic material in plants, which are mostly based on recent developments. This includes e.g. the direct gene transfer in protop1 esters, the fusion of DNA-containing, membrane-coated vesicles, so-called liposomes, with the protoplast membrane, the intranuclear microinjection of protop1 esters, and possibly the macroinjection of genetic material in the meristematic areas of the flowers.

These previously more closely characterized gene transfer procedures do indeed lead to a splicing of genetic material in the plant cell and to an integration in the plant genome; but they have the decisive disadvantage that the placement of the spliced genetic material in the nuclear genome of the plant occurs non-specifically and thus randomly, so that the genomic localization of foreign DNA cannot be predetermined (Potrykus I et al., 1985; Wallroth M et al. 1986; Ambros PF et al., 1986).

On the other hand, however, it is known that the integration site within the plant genome has a decisive influence on the efficiency and strength of the expression of the integrated foreign genes. Thus, e.g. the expression rate of a particular integrated foreign gene depending on the site of interaction within the genome differs considerably in two different independently produced transgenic plants.

The same dependence on the integration site could also be observed in the context of the regu1 gene, especially in the reaction of integrated foreign genes to induction and/or suppressor signals. In this case too, clear differences are found between independently produced transgenic plants.

This dependence on integration site can be overcome e.g. by carrying out an in-situ modification of genes within the plant genome.

Within the framework of such in-situ modification of specific target genes, the gene remains in its natural, fixed position within the plant genome so that optimal expression and regulation of the respective target gene is ensured.

Until now, however, no method has been known which enables an in-situ modification of genes within the plant genome.

Against the background of the general efforts in the field of p1antigen engineering 1 and in one to increase the effect of regulation and expression of spliced foreign genes in the plant cell, it must be considered an urgent task to develop methods that allow the targeted integration of foreign genes at defined, predeterminable sites within the plant genome and which thus enables e.g. an i n- s i t u modification of plant genes.

With yeast, some other fungi, as well as with the slime fungus D i ctyo-ste1 i um d i sco i deum (Hinnen H et al., 1978; Miller BL et al., 1987; De Lozanne A and Spudich SA., 1987) it could naturally very effective homologous recombination of transforming DNA is used in investigations of the natural genomic environment of transformed and modified genes, as well as of a direct modification of genes within the genome.

However, this method cannot be applied to conditions of taxonomically higher organisms due to the higher comp 1 exisity.

In mammals, e.g. next to the aforementioned homologous recombination, one also finds a very efficient, non-homologous or illegitimate recombination of foreign DNA in the genome, which makes it difficult to target splicing of foreign DNA at a specific desired location within the genome.

Also in plants, due to the high transformation rates that can be achieved with DNA molecules, which show no homology whatsoever to sections of the plant genome (Shillito R et al., 1985; Negrutiu I et al., 1987) it is not possible with security assume that the proportion of illegitimate, non-homologous recombination is likewise very high.

Until now, it has been completely unclear whether homologous recombination exists in plants at all and, if so, how efficiently it works.

Consequently, it has so far also not been possible to integrate genetic material targeted at precisely defined locations within the plant genome.

This task was now surprisingly solved within the framework of the present invention by means of certain precautions.

The present invention relates to a method for the production of transgenic plants which is characterized by integrating a gene, a gene fragment or other useful DNA sequences targeted at a desired, predetermined location in the plant genome by means of homologous recombination.

Hereby, the term plant genome within the scope of this invention shall not be limited by definition to the nuclear genome, but also includes the genome sections present in the mitochondria and plastids which, with the help of the method according to the invention, also become available for a targeted modification.

Thus, within the scope of this invention, it is now possible for the first time to modify targeted and effective n-situ determined target genes within the plant genome (targeted mutagenesis).

The in-situ modification of genes has decisive advantages compared to the hitherto practiced methods of modifying plant genes, where a desired gene is first isolated, which is then modified in situ and then reintegrated into the genome.

The re-integration of a first isolated and virtually modified gene into the plant genome occurs purely by chance, so that, depending on the integration site, difficulties may arise in the regulation and/or expression of the integrated gene. In the case of the in-situ modification made possible for the first time within the framework of this invention, the gene, on the other hand, remains in its natural, fixed position within the plant genome, as optimal regulation and expression for the respective gene must be determined.

In addition to the targeted and thus very effective i ns i t u modification of plant genes (targeted mutagenesis), the method according to the invention opens up a number of further application possibilities such as e.g. splicing of additional gene copies in regions with a known high expression rate as well as elimination and thus switching off of unwanted genes, etc.

In detail, it is thus by means of the method according to the invention, e.g. possible to exchange dominant or semi-dominant alleles which give the plant an undesirable characteristic with regard to cultivation and/or commercial points of view, for recessive alleles with useful and desirable characteristics.

A further concrete application possibility of the method according to the invention comprises a targeted splicing of a desired gene in the plant genome by means of homologous recombination with genes or gene fragments or with otherwise useful DNA sequences which are first artificially integrated into the plant genome by means of known methods.

The first integrated gene or gene fragment or the first integrated DNA sequence can be used, e.g. as probe mo1eky 1 , with the help of which it is possible to track down regions within the plant genome that exhibit advantageous properties, e.g. good expression or good regulability of the spliced gene.

With the help of the method according to the invention, a desired gene or gene fragment or an otherwise useful DNA sequence can then be directed very easily via homologous recombination into this preferred region within the plant genome for gene expression or regulatability.

Particularly preferred for use as probe molecules are genes or gene fragments or otherwise useful DNA sequences that are characterized by easy excisionability, such as e.g. genes that lead to phenotypic selection traits in the transformed cell or plant, in particular to resistance to antibiotics or to certain herbicides.

In the same way, it is possible to link genes or gene fragments or other useful DNA sequences with genes or gene fragments that were first artificially integrated into the plant genome using known methods.

Thus, e.g. a gene that codes for a desired characteristic such as e.g. an insect resistance, is linked with another, already in advance artificially in the genome of an ornamental plant integrated gene that codes e.g. a herbicide resistance.

However, the method according to the invention is not limited to the replacement or the linking of genes or other DNA sequences with genes or DNA sequences previously artificially integrated into the plant genome.

It is of course possible, with the help of the method according to the invention, to replace desired genes or gene fragments as well as other useful DNA sequences with natural genes already present in the plant genome or to connect these to each other.

In this way, you can e.g. linking a desired and useful gene from a breeding point of view with a marker gene which can be easily followed in gene generations due to its phenotypic distinctiveness.

In this way, it is very easy for a grower to pursue a desired gene through the breeding generations, as he orients himself by the phenotypic marker gene.

A further object of the present invention thus consists in recombinant DNA molecules which, due to their specific construction, are capable of purposefully integrating genes or DNA sequences which possibly encode new and desired properties, by means of homologous recombination at defined locations within the plant genome .

Furthermore, the present invention comprises transgenic plants with new and/or improved properties, which have been transformed with said recombinant DNA molecule.

The present invention also relates to transgenic plants that are regenerated from transformed plant cells containing the aforementioned recombinant DNA molecule, as well as their seeds, and also the descendants of transgenic plants as well as the mutants and variants thereof.

The present invention likewise includes chimeric genetic constructions, also k1 on ing carriers and host organisms, as well as methods for the targeted transfer of desired properties to plants.

In the following description, a number of terms will be used which are common in recombinant DNA technology as well as in plant genetics.

In order to ensure a clear and uniform understanding of the description and requirements as well as the scope of the said expression, the following definitions shall be given: Heterologous gene(s) or DNA: The DNA sequence that codes for a specific product or products or fulfills a biological function and which originates from a different species than the one to be spliced into said gene; said DNA sequence is also referred to as foreign gene or foreign DNA.

Homologous gene(s) or DNA: A DNA sequence that codes for a specific product or products or fulfills a biological function and that originates from the same species as the said gene to be spliced into.

Synthetic gene(s) or DNA: A DNA sequence that encodes a specific product or products or fulfills a biological function and that has been produced synthetically.

Anti-promoter: A control sequence of DNA expression, which ensures the transcription of any homologous or heterologous DNA gene sequence in a plant, provided that said gene sequence is linked in an operable manner to such a promoter.

Termination sequence: DNA sequence at the end of a transcription unit that signals the end of the transcription process.

Overproducing Plant Promoter (OPP): P 1 ante promoter capable, in a transgenic plant cell, of causing the expression of any operably linked functional gene sequence(s) to the extent (measured in terms of RNA or poly.pept in the amount) that it is clearly higher than can be observed in a natural way in host cells that have not been transformed with said OPP.

3'/5' non-translated region: DNA section located upstream/upstream of the coding region, which is indeed transcribed into mRNA, but is not translated into a polypeptide. The ne region contains regulatory sequences such as e.g. r ibosome binding sites (5<1>) or po 1yadeny1 er ings signal et (3 1 ) •

Plant material: in culture or as such viable plant parts such as protopes, cells, callus, tissue, embryos, vegetative organs, buds, seeds, etc., as well as whole plants.

P1 antece 1 ler: Structural and physiological unit of the plant, consisting of a protoplast and a cell wall.

Protopl ast: "naked" plant cell without a cell wall, which is isolated from plant cells or plant tissue, with the potential to regenerate into a cell clone or a whole plant.

Ce 11 ek 1 on : Population of genetically similar cells arising from a cell by successive mitoses.

Plant tissue: A group of plant cells that are organized in the form of a structural and functional unit.

P1anteorgan: A structural and functional unit, composed of several tissues, such as e.g. root, stem, leaf or embryo.

DNA expression vector: K1 on ing carrier, e.g. a plasmid or bacteriophage containing all signal sequences necessary for expression of an inserted DNA in a suitable host cell.

DNA transfer vector: Transfer carrier, e.g. a ti plasmid or a virus that enables the splicing of genetic material in a suitable host cell.

Homologous recombination: reciprocal exchange of pieces between homologous, double-stranded DNA molecules.

Mutants, variants of transgenic plants: progeny of a transgenic plant that has arisen spontaneously or artificially, using known procedural precautions, such as e.g. UV treatment, treatment with mutagenic agents, etc., which still have the distinctive features and properties according to the invention of the transgenic starting plant which has acquired these due to transformation with exogenous DNA.

Within the framework of the present invention, it has now for the first time succeeded in developing a method which enables plants to integrate targeted genetic material at defined predetermined positions within the plant genome and to express there.

The method according to the invention is based on a targeted integration of genetic material into the genome of the plant by means of the homologous recombination of natural or artificially modified and/or synthetic genes or gene fragments or otherwise useful DNA sequences with homologous DNA sections within the plant genome, said homologous DNA sections are a natural component of the plant genome or, if necessary, are first artificially spliced into the plant genome using known procedural precautions.

An essential component of the present inventions are thus recombinant DNA molecules which, due to their specific construction, enable the splicing of genes in the genome of a target plant by means of homologous recombination.

The present invention relates in particular to recombinant DNA molecules which enable a targeted integration of genes, gene fragments or otherwise useful DNA sequences at precisely defined locations within the plant genome, and which are characterized by the fact that they contain DNA to be integrated, and that said DNA to a certain extent exhibits homologies corresponding to DNA regions within the plant genome or is flanked by such homologous DNA sequences that upon transformation of the plant cell containing the homologous DNA regions, by homologous recombination a targeted integration of said DNA takes place which must be integrated at precisely defined locations within the plant genome.

This is preferably about recombinant DNA molecules that consist of a DNA sequence to be integrated, which is optionally linked in an operable manner with expression signals that are functional in the plant cell, as well as with flanking DNA sections that show homologies to a specific region within the plant genome. In the first line, the present invention relates to recombinant DNA molecules that are characterized by a) said recombinant DNA molecules have a structural gene that codes for a useful and desirable property, b) said structural gene is optionally linked in an operable manner with expression signals that are functional in

p1antece1 laughs,

c) said functional unit, consisting of structural gene and possibly expression signals, is somehow flanked by

DNA sequences which to such an extent show homologies to corresponding DNA regions within the plant genome, that upon transformation of the plant cell containing said homologous region, a targeted integration of the gene sequence flanked by homologous DNA sequences takes place by homologous recombination, on defined locations within the p1 a ntegenome.

In addition to structural genes, any other genes or gene fragments as well as DNA sequences can also be used.

Furthermore, the broad concept of the present invention includes genetic constructions, consisting of genes or gene fragments or otherwise useful DNA sequences, which are possibly under the control of functional expression signals in plant cells and which have a sufficiently large homology to corresponding DNA regions within the plant genome, so that a direct replacement of these genes or gene fragments or otherwise useful DNA sequences against said homologous genomic DNA by means of homologous recombination can take place.

In addition to double-stranded DNA, single-stranded DNA and partially single-stranded DNA can also be used in the method according to the invention.

There is also the possibility of using DNA/protein complexes where the target DNA to be integrated is associated with a specific functional protein.

Here, e.g. some DNA/protein comp 1 exes are mentioned, such as e.g. a DNA/chromate binding protein comp 1 ex , a DNA/ p1 ante v i rus protein comp 1 ex or a DNA/protein compiex including heterologous proteins such as e.g. recombination proteins of bacteria or yeast (eg RecA, B, C, D). For use in the method according to the invention, all the genes that are expressed in the plant cell and that give the plant a useful and/or desired characteristic, such as e.g. increased resistance or tolerance to pathogens

(e.g. phytopathogenic insects, fungi, bacteria, viruses, etc.)

in relation to herbicides, insecticides or other biocides, in relation to climatic influences and local peculiarities (e.g. heat, cold, wind, drought, humidity, especially extreme soil conditions, osmotic stress, etc.) or an increased formation of reserve and 1aging substances in leaf, seed, tuber, root, stem, etc. Likewise, the present invention includes genes that code for pharmaceutically acceptable active substances, such as e.g. alkaloids, steroids, hormones, immunomodulators, and other physiologically active substances.

In addition to naturally occurring genes that encode a useful and desired characteristic, within the scope of this invention genes that were purposefully modified in advance using chemical or genetic engineering methods can also be used.

Moreover, the present invention also covers such genes which are produced completely by chemical syntheses.

As an example, mention should be made here of the 1 ante 1 agr ing proteins. Any modifications to the protein genes primarily aim at their amino acid composition, which, as is known, is in most cases only of minor nutritional, physiological and nutritional value. Thus, e.g. the zein storage protein of maize, whose protein-coding DNA sequence is known (Wienand K et. al.

Mo 1. Ge n- Genet., 1982: 440 - 444, 1981) a high proline content, while the lysine and tryptophan content is very small.

By replacing plant-specific zein genes with a first modified, now a lysine and/or tryptophan-rich protein or an otherwise improved zein protein coding gene using the method according to the invention, it has now become possible to improve the targeted nutritional-physiological value of specific target plants.

In addition to this, the high background of plant-specific agricultural proteins with little nutritional physiological and nutritional value can be reduced by targeted integration of disruptive genes that inactivate the aforementioned plant-specific genes.

An example of such a disruptive 1ses gene is the opaque-2 gene of maize which inhibits the expression of zein proteins.

In addition, other useful DNA sequences, especially non-coding DNA sequences with regulatory functions, are suitable for use in the method according to the invention.

For use in the method according to the invention, homologous as well as heterologous gene(s) or DNA, as well as synthetic genes or DNA according to the definition within the scope of the present invention, are thus taken into consideration.

The DNA sequences to be integrated can here be constructed exclusively from genomic, cDNA or synthetic DNA. Another possibility consists in the construction of hybrid DNA sequences, consisting of both cDNA and genomic DNA and/or synthetic DNA.

In this case, the cDNA can originate from the same gene or DNA section as the genomic DNA, or both the cDNA and the genomic DNA can originate from different genes or DNA sections. In each case, however, both the genomic DNA and/or the c DNA, separately, can be produced from the same or different genes or DNA sections.

When a DNA sequence contains portions of more than one gene or DNA segment, these can originate either from one and the same organism, from several organisms belonging to different strains or variations of these species or different species of the same family, or they can originate from organisms belonging to more than one of these families or another taxonomic unit.

In order to ensure the expression of a structural gene in the plant cell, the coding gene sequences can optionally be linked together first in an operable manner with expression sequences capable of functioning in plant cells.

The hybrid gene constructs in the present invention thus usually contain, next to the structural gene(s), expression signals that include both promoter and terminator sequences as well as additional regulatory sequences from 3' and 5' untranslated regions.

Each promoter and each terminator capable of effecting an induction of the expression of a coding DNA sequence (structural gene) can be used as a component of the chimeric gene sequence. Particularly suitable are expression signals originating from genes of plants or plant viruses. Examples of suitable promoters and terminators are e.g. nopa1 in synthase genes (nos), octopin synthesis genes (oes) and eau i ir i oi/er mosaic virus genes (CaMV). Preferred within the scope of the present invention are the 35S and 19S expression signals from the CaMV genome, which by means of molecular and biological methods, as described e.g. in Maniatis et al., 1982, can be isolated from said genome and linked together with the coding DNA sequence.

According to the invention, e.g. The Scal fragment from the CaMV strain "S" comprising nucleotides 6808-7632 in the gene card (Frank G et al., 1980).

The 19S promoter and 5' untranslated region is located on a genome fragment between the Pstl site (position 5386) and the H in nd 111 site (position 5850) in the CaMV gene map (Hohn et. al., 1982). The corresponding terminator and 3' untranslated region is located on an EcoRV/Bg111 fragment between positions 7342 and 7643 in the CaMV genome.

Moreover, within the scope of the present invention, the expression signal from the CaMV strain CM 1841, whose complete nucleotide sequence is described in Gardner RC et a 1. 1981, is preferred.

A further active representative of a p1 antepromoter that can be used is an overproducing p1 antepromoter. This form of promoter should, provided it is operatively linked to the gene sequence encoding a desired gene product, be able to mediate the expression of said gene sequence.

Overproducing p 1 antepromoters that can be used within the scope of the present invention include the promoter of small subunits (small subunit; ss) from r i bu 1 ose-1,5-bisphosphate-carboxy1 ase from soybeans [Berry-Lowe et al. , J. Molecular and App. Gen. , J_: 483^498 ( 1982 )] as well as the promoter from the k1orophy1-a/b-binding protein network. These two promoters are known to be induced by light in eukaryotic plant cells [cf. e.g. Genet i c Engi neer i ng of Plants, an Agriculturai Perspective, A. Cashmore, Plenum, New York 1983, pp. 29-38; Coruzzi G. et al., The

Journal of Biological Chemistry, 258: 1399 (1983) and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2: 285 (1983)].

The thus formed functional units, consisting of a gene, a hybrid gene construct or otherwise useful DNA sequences which are possibly under the regulatory control of expression signals active in plant cells, are then flanked with DNA segments which to such an extent exhibit homologies corresponding to DNA the regions within the plant genome that upon transformation of the plant cell containing the aforementioned homologous region, by homologous recombination, a targeted integration of the gene sequence that is flanked by homologous DNA sequences takes place at defined locations within the plant genome.

By choosing suitable flanking DNA sequences that have sufficiently high homology to corresponding sections within the plant genome and thus enable an exchange of genetic material via homologous recombination, it is now possible for the first time to integrate genes targeted at predetermined locations in the plant genome and possibly to express these.

The extent of homology required for a replacement via homologous recombination between flanking DNA and the corresponding genomic DNA region depends on various parameters, such as the specific chromate instructor, etc., and must therefore, depending on the DNA sequences used, be adapted to the corresponding needs.

For use as flanking DNA, within the scope of this invention, first-line DNA sequences that have the homology of such plant-specific genes and/or gene sequences, whose nucleotide sequences are fully or partially known, with such genes or gene sequences being preferred,

which are located in the areas, which naturally already have a high expression efficiency.

As an example, the p1 ante 1ager protein genes should be mentioned here -

without thereby limiting inventing 1 see again the situation - which are known to achieve very high expression rates,

depending on the plant's stage of development.

Additional genes, whose nucleotide sequence is at least partially already known and which can thus be used in the method according to the invention, include e.g. the nitrate-reductase gene, the psbA gene (Gressel J., Oxford Surv. Plant Mol. Cel 1 Biol. , 2: 321 - 328, 1985; Zurawski

G. et al., Proe. Nati. Acad. Sei, USA, 79: 7699 - 7703, 1982), the gene in the small subunit (ss) of

r i bu 1 ose - 1,5-bisphosphate-kar bok sy 1 a sen, as well as a 1 pha-amy1ase and sucrose synthetase gene. In addition to plant-specific genes, within the scope of the present invention, all those genes which, due to strong conservatism in the course of evolution, comprise large areas which show homology to the corresponding p1 antespecic genes can also be used.

Here belongs, for example, the histone genes, the actin genes as well as g 1 ut that in on-S-transfer a se genes in mammals, moreover various microbial genes, such as e.g. the trypophan and glutamine synthase genes.

These exemplary enumerations of possible and for application

genes suitable in the method according to the invention only serve to better illustrate the present invention and shall in no way limit the object of the invention.

Also suitable for use in the method according to the invention are such DNA sequences which have homology to genes or gene fragments or to otherwise useful DNA sequences which were first artificially integrated into the genome of a target plant using known process conditions and whose nucleotide sequences are wholly or partly known. Particularly preferred are such DNA sequences which are located in areas which naturally ensure a high expression efficiency.

In a specific embodiment of the present invention, the integrative DNA and the artificially integrated DNA already present in the plant genome are mutually complementary DNA sequences which, after homologous recombination, form a functional unit within the plant genome.

The aforementioned functional unit may be a structural gene that codes for a desired and useful protein product or a control sequence that fulfills a specific regulatory function within the plant genome.

Particularly preferred within the scope of the present invention are mutually complementary DNA sequences which, after recombination, form a functional structural gene which codes for a specific phenotypic marker or an otherwise easily detectable protein product.

Thus, within the framework of the present invention, it has become possible, by producing corresponding hybrid gene constructs in the previously described manner, to make predictable modifications of plant genes within the plant genome.

An essential component of the present invention thus forms a method for the production of transgenic plants, which is characterized by transforming plant material with one of the recombinant DNA molecules described above, the genes contained in said recombinant DNA molecule, the hybrid gene constructs or otherwise useful DNA -sequences are purposefully integrated by homologous recombination at a desired predetermined location in the p 1 antegenome.

In a first process step, the recombinant DNA molecules described above are first constructed, such as e.g. consists of one or more structural genes that encode a useful and desired gene product and that are linked together in an operable manner with functional expression signals in the plant cell, as well as flanking sequence sections that have homology to plant-specific genome sections, and are spliced into suitable cloning vectors that are able to again form a functional gene by means of homologous recombination.

In addition to structural genes, any other desirable genes or gene fragments or otherwise useful DNA sequences can also be used.

Plasmid or virus (backed in phage) vectors with replication and control sequences, which originate from species that are compatible with the host cell, are generally used as cloning vectors.

The K1 expansion vector usually carries an origin of replication, and also specific genes that lead to phenotypic selection characteristics in the transformed host cell, especially to resistance to antibiotics or to certain herbicides. The transformed vectors can be selected in a host cell with the help of these phenotypic markers after a transformation.

Selectable phenotypic markers that can be used within the scope of the present invention include e.g. resistances to ampicillin, tetracycline, hygromycin, G418, kanamycin and neomycin, without thus limiting the state of invention.

As a host cell, prokaryotes, including bacterial host cells, such as e.g. A. tumefaci ens, E. co 1 i , S. typhimurium 0 g Serrat i a marcescens, as well as cyanobacteria in consideration. Moreover, within the scope of the present invention, eukaryotic host cells such as yeast, mycelium-forming fungi and plant cells can also be used. 1 nnsp 1 the isolation of the chimeric gene construct according to the invention into a suitable cloning vector takes place using standard methods as described by e.g. Maniatis et al. , 1982.

In this way, the vector and the DNA sequence to be spliced in with suitable restriction enzymes are usually cut in the first instance. Suitable restriction enzymes are e.g. such as give fragments with smooth ends, such as e.g. Smal, HpaI and EcoRV, or enzymes that form cohesive ends, such as e.g. EcoRI, SacI and BamHI.

Fragments with blunt ends as well as those with cohesive ends, which are mutually complementary, can again be linked to a uniform continuous DNA molecule with the help of suitable DNA ligases.

Blunt ends can also be produced by treating DNA fragments that have overhanging cohesive ends with the Klenow fragment of the E.coli DNA polymerase by filling in the gaps with the corresponding complementary nuc 1 eotides.

On the other hand, cohesive ends can also be produced artificially, e.g. by adding complementary homopolymeric tails to the ends of a desired DNA sequence and the cut vector template using a terminal doxynucleotidyl transerase, or by adding synthetic o 1 i gon uk 1eotide sequences (Linker), which carry a restriction site, and subsequent cutting with the corresponding enzyme.

The cloning vector and the host cells transformed with this vector are used according to the invention to increase the vector's copy number. With an increased copy number, it is possible to isolate the vector carrying the hybrid gene construct according to the invention, and to use it e.g. by splicing the chimeric gene sequence into the plant cell.

An essential component of the present invention thus relates to the construction of plasmids which display said chimeric gene constructs, consisting of one or more structural genes or otherwise desirable genes or gene fragments or otherwise useful DNA sequences, which are possibly linked in an operable manner to functional expression signals in plant cells, as well as flanking DNA sequences that have homology to plant-specific genome sections.

In a further process step, the plasmids can be used to splice the chimeric genetic construction described above, which possibly contains a structural gene that codes for a desired gene product, into the plant cell and to integrate it into the plant genome.

The present invention thus also relates to the production of plant recipients which receive the aforementioned structural gene or otherwise desired genes or gene fragments or otherwise useful DNA sequences spliced into their genome.

In this case, the creation of the chimeric genetic construction preferably takes place in p 1 anteprototypes using known gene transfer methods.

There are, however, a number of very effective methods for splicing DNA into plant cells, based on the use of gene transfer vectors or direct gene transfer methods.

One possibility consists, for example, of in that you bring plant cells into contact with viruses or with agrobacterium. This can be done by infection of sensitive plant cells or by co-cultivation of protoplasts derived from plant cells. The Cauliflower Mosaic virus (CaMV) can, within the scope of the present invention, also be used as a vector for splicing the chimeric genetic construction according to the invention into the plant (Hohn et al., in "Molecular Biology of Plant Tumors", academic Press, New York, 1982, pp. 549-560; Howell, US Patent No. 4,407,956). The total viral DNA genome of CaMV is thereby integrated into a bacterial fore 1drep 1 asmid during the design of a recombinant DNA molecule that can be propagated in bacteria. After cloning, the recombinant plasmid is cleaved with the help of restriction enzymes either case by case or at completely specific, non-essential sites within the viral part of the recombinant plasmid, e.g. within the gene that codes for the transmissibility of the virus by aphids, in order to be able to insert the hybrid gene construct.

A small oligonucleotide, a so-called linker, which has a single, specific restriction site, can likewise be integrated. The thus modified recombinant plasmid is cloned again and further modified by splicing the hybrid gene construct at a restriction site that occurs only once.

The modified viral portion in the recombinant plasmid is cut out from the bacterial precursor and used for inoculation of the plant cell or the entire plant.

Another method for splicing the chimeric gene construct into the cell uses the infection of the plant cell with Agrobacterium tumefaciens, which has been transformed with said gene construct. The transgenic plant cells are then cultured under suitable conditions known to the person skilled in the art such that they form shoots and roots and

finally resulting in the total plant.

The chimeric gene construct can be transferred e.g. using the ti plasmid of Agrobacterium tumefaciens in suitable plant cells [DeCleene et al., Bot. Rev., 47: 147-194 (1981); Fine. Fox. , 4_2: 389-466 (1976)]. The Ti plasmid is transferred during the infection by Agrobacterium tu mefaciens t in 1 the plant and is stably integrated into the plant genome. [Horsch et al. al., Science, 233: 496-498 (1984); Fraley et al., et al., Proe. Nati. Acad. Pollock. USA, 80: 4803 (1983)].

Ti plasmids have two regions that are essential for the production of transformed cells. One of these, namely the transfer DNA region, is transferred onto the plant and leads to the induction of tumours. The second, (vir) region conferring virulence is only essential for the formation of the tumor, but not for its maintenance. The dimensions of the transfer DNA region can be increased by splicing the chimeric gene construct, without affecting the ability to transfer. By removing tumor-causing genes, whereby the transgenic plant cells become non-tumorous, and by splicing a selectable marker, the modified ti plasmid can be used as a vector for transferring the gene construct according to the invention into a suitable plant cell.

The vir region causes the transfer of the T-DNA region of Agrobacterium to the genome of the plant cell, regardless of whether the T-DNA region and the vir region are present on the same vector or on different vectors within the same Agrobacterium cell. A vir region on a chromosome likewise induces transfer of T-DNA from a vector to a plant cell.

A system for transferring a T-DNA region from an Agrobacterium to plant cells is preferred, which is characterized in that the vir region and the T-DNA region are located on different vectors. Such a system is known under the term "binary vector system" and the vector containing T-DNA is referred to as a "binary vector".

Any vector containing T-DMA, which can be transferred into plant cells and which allows a selection of the transformed cells, is suitable for use within the scope of this invention.

Using newly developed transformation techniques, however, such p1 antespecies can also be transformed in vitro, which are not natural host plants for Agrobacter ium. Thus, e.g. monocotyledonous plants, especially cereals and grasses, no natural host cells for Agrobacteri um.

In the meantime, there are several references to the fact that monocotyledons can also be transformed with Agrobacteria, so that with the use of new experimental feeding sites that are now becoming available, grain and grass species are also available for transformation [Grimsley NH, et al ., Nature, 325: 1 77- 1 79 ( 1 987 )].

Preferred within the scope of the present invention is the direct splicing of the gene constructs according to the invention into p1 anteprotop1 asters, for which a number of methods also already exist.

Thus, the genetic material contained by a vector can be injected directly into the plant cell, e.g. using micropipettes for the mechanical transfer of the recombinant DNA. (Neuhaus et al., 1987).

Further possibilities for the direct transfer of genetic material to plant cells consist in the treatment of protoplasts with process precautions that modify the plasma membrane, such as e.g. po 1 yety1 eng 1yko1 treatment [Paszkowski et al., EMBO J., 3: 2717-22 (1984))], heat shock treatment1 in ngen or e1ectroporation as well as a combination of these procedural precautions [Shillito et al., Bio Technology, 3: 1099-1103 (1985); Fromm et al., Proe. Nati. Acad. Pollock. USA, 82: 5824 (1985)].

In the electroporation technique, the p1 anteprotop 1 astes together with the plasmids containing the hybrid gene construct are exposed to electrical impulses with high field strength. This leads to a reversible increase in the permeability of bio-membranes and thus enables the entry of the plasmids. E1ectroporated p1 anteprotop1 asters renew their cell walls, divide and form callus tissue. The selection of the transformed plant cell can take place with the help of the above-described phenotypic marker.

A further method for the direct introduction of genetic material into plant cells, which within the scope of the present invention is particularly preferred, is described by Negrutiu I et al., 1987.

This concerns a method which is based on purely chemical process conditions and enables a very efficient and rapid implementation of the transformation.

In detail, this method consists in

- isolate p1 antiprotop1 esters from any plant tissue and optionally cultivate in a nutrient medium that is usually used for cultivating p1 ateprotop1 esters; - pre-incubates said protoplasts before the actual transformation for 20 minutes to 6 hours in an incubation medium, containing alkaline earth and/or a 1ka 1 icate ions, preferably Ca^+-, K+ and/or Na<+-> ions, as well as a suitable C source, at a temperature of 4° to 10°C; - then separate the protop1 astes from for incubation forged in a and resuspend in the actual transformation medium, containing as an essential component 0.1 to 60 mM, preferably 10 to 30 mM Mg^<+-> ions, in the presence or absence of Ca^<+- >ions; - adds to the transformation solution immediately thereafter a DNA sample, containing one or more genes, under the control of expression signals active in plants, as well as a carrier DNA; - adds a few seconds to up to 20 minutes later, preferably 0.1 to 10 minutes later, an agent which modifies the plasma membrane; - incubates the protoplast and the DNA sample in said transformation sopp 1 ladle for a period of time which ensures the uptake of DNA in the protop 1 ladles; and - if desired, regenerate entire plants from the transformed protop1 aster.

Particularly preferred within the scope of the present invention is the direct gene transfer using Co-transformation (Schocher R.J. et al., Bio/Technology, 4: 1093-1096, 1086).

Co-transformation involves a method that relies on the simultaneous uptake and integration of different DNA molecules (non-see 1 sections bare and see 1 sections bare gene) in the plant genome and thus makes it possible to find the cells that have been transformed with non-se1ectable genes.

The above-given exemplary listing of possible transformation methods does not provide completeness and should in no way limit the object of the invention.

The 11 genes that have the hybrid gene construct inserted into the genome are determined using conventional selection, screening and detection methods and are used for the regeneration of transgenic plants.

The regeneration of protoplasts maintained in culture into whole plants is described in Evans, et al., "Protoplast Isolation and Culture", in Handbook of Plant Cell Culture, J_: 124-176 (MacMilan Publishing Co. New York 1983 ); M.R. Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts", Protoplasts, 1 983, Lecture Proceedings, pp. 19-29, (Birkhåuser, Basel 1983); P. J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops", in Protoplasts 1983 - Lecture Proceedings, pp. 31-41), (Birkhåuser, Basel 1983); and H. Binding, "Regeneration of Plants", in P1 ant Protoplasts, pp. 21-37, (CRC Press, Boca Raton 1985) and Potryhus I and Sh i 11 i to RD, Methods in Enzymology, Vol 118, Plant Molecular Biology, eds A. and H. Weissbach, Academic Press, Orlando, 1986.

The regeneration procedures differ from plant species to plant species. In general, meanwhile, the protop1 cells are stimulated to divide and ce11 wall formation in one of the known culture media. In the end, there are unexpected trips that are induced with specific active substances, such as e.g. auxins and cytokinins, to root and shoot form 1 se.

The small plants produced in this way can then be transferred to soil and further cured in the same way as normal seed-a v 1.

An effective regeneration depends primarily on the medium, on the genotype and on the previous history of the culture. If these three variables are adequately controlled, regeneration becomes completely reproducible and repeatable.

The regenerated transgenic plants which contain a structural gene expressible in the plant cell of the hybrid gene construct described above as an integral component of the plant genome are preferably propagated vegetatively in the form of sterile sprouts.

The stable integration of a functionally expressible gene into the plant genome of the regenerated transgenic plant is determined by means of the mitotic stability of the integrated gene as well as by its appearance as a Mendelian trait during meiosis and during use 1 se . a v "Southern blot" analysis (Southern EM, 1975).

A further object of the present invention thus relates to the production of transgenic plants with improved and/or desired properties by targeted splicing of DNA at predetermined locations within the plant genome, especially those that ensure a high expression efficiency and advantageous regulability.

The invention also includes transgenic plants that have been transformed using the above-described method according to the invention, as well as their asexual and/or sexual descendants that still exhibit the new and desired properties that are required by the mother plant's transformation.

Under the terms asexual and/or sexual descendants of transgenic plants, in addition to the definition within the scope of this invention, all the mutants and variants that can be extracted using known methods, such as e.g. by cell fusions or mutant selections, and which have the characteristic properties of the transformed starting plants, as well as all crossing and fusion products with the transformed plant material. The invention also relates to the propagation material of transgenic plants.

Propagation material of transgenic plants is understood within the scope of this invention to be any plant material that can be propagated sexually or asexually or in n-v in vo or in-vitro. Particularly preferred within the scope of the present invention are primarily protop1 asters, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, as well as any other formative material that can be obtained from transgenic plants.

Plant parts, such as flowers, stems, fruit, leaves, roots, which originate from transgenic plants or their successors which have previously been transformed using the method according to the invention and which are thus at least partially built up from transgenic cells, are likewise the subject of the present invention.

The method according to the invention is suitable for transformation of all plants, especially of the systematic groups Angiospermae and Gymnospermae.

Among the Gymnospermae, the plants of the class Con i ferae are of special interest.

Among Ang iospermae, next to deciduous trees and shrubs, there are plants of the family So 1anaceae, Cruc iferae, Compos itae, L i 1 i aceae, V itaceae, Chenopod i aceae , Rutaceae , A11 i aceae , Amary11idaceae, Asparagaceae, Orch idaceae, P a 1mae, B rome - 1 i aceae, Rub i aceae, Theaceae, Mu s aceae or Gram i neae and the family Leguminosae, above all the family Papilionaceae of particular interest. Preference is given to representatives of the families Solanaceae, Cruciferae and Gram i neae.

Target cultures according to the present invention are e.g. also the following: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycoperison, Nicotinana, Solanum, Petunia , Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Ant irrhinium, Hemeroca11 is , Nemesia, Pelargonium, Panicum, Pennisetum, Ranuculus, Senecio, Sa 1pig 1 ossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum and Sorghum, as well as from the series: Ipomoea, Passiflora, Cyclamen, Mal us, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Archis, Phaseolus and Pisum.

Due to recent developments in the field of in vitro cultivation of plants, preferably within the planter generation, it has occasionally become possible from p1 anteprotop1 asters to regenerate entire plants within the family Gramineae. Examples of successful regeneration sec s experiments at Gramineer are i.a. described in Abdullah, R et al., Bio/ Teehno 1 ogy , 4: 1 087- 1 090, 1 986, Fujimura, T., et al., Plant Tissue Culture Lett, 2_:74-75, 1985, Toriyama, K., et al., Theor Appl Genet, 73: 16-19, 1986, Yamada, Y., et al., Plant Cell Rep, 5:85-88, 1986 (Reis) as well as in Rhodes et al. (1988) for rna ice protoplasts.

Within the framework of the present invention, the following plants can thus also be used: Lolium, Zea, Triticum, Sorghum, Saccharum, Bromus, Oryzae, Avena, Hordeium, Secale and Setar i a.

The mature plants grown from transformed plant cells are crossed with themselves for seed production. Some of the seeds contain the genes that encode a useful and desired trait in a relationship that obeys exactly the established laws of heredity. These seeds can be used in the production of transgenic plants.

Hymozygous lines can be obtained by repeated self-pollination and production of inna v 1 -1 injers. These innav 1 -1 injers can again be used for the development of hybrids. In this method, an innav line containing said foreign gene is crossed for production with another innav 1 -1 inje.

After the general description of the present invention, reference must now be made to give a better understanding

specific working examples which are included to illustrate the invention, but which are not limiting

character, unless this is specifically referred to.

Non-limiting execution examples:

General recombinant DNA techniques

Because many of the recombinant DNA techniques used in the present invention are routine for the person skilled in the art, only a brief description of these generally used techniques shall be given here. When nothing is specified

otherwise, all these methods are described by Man i at i s et al. (1982).

A. Cutting with a restriction endonuclease

In the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA., there is typically approx. 50 to 500 yjg/ml DNA. For each pg of DNA, 2 to 5 units of restriction endonucleases are added and the reaction site is incubated for one to three hours at the temperature recommended by the manufacturer. The reaction is terminated by heating to 65°C for 10 minutes or by extraction with phenol, followed by precipitation of DNA with ethanol. This technique is also described on pages 104 to 106 of Maniatis et al.

B. Treatment of DNA with polymerase to produce blunt ends. 50 to 500 µg/ml DNA fragments are added to reaction st in 1 guide site in a buffer recommended by the manufacturer, New England Biolabs. Reaction st in 1 before ing site contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction takes place within 30 minutes at 15<0>C and ends after 10 minutes of heating to 65°C. For fragments obtained by cutting with restriction endonucleases that produce 5' protruding ends, such as EcoRI and BamHI, the large fragment, or the Klenow fragment in the DNA polymerase is used. For fragments obtained by endonucleases that produce 3 1-progressing ends, such as Pstl and SacI, the T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of Man i at i s et al. C. Agarose electrophoresis and purification of DNA fragments from gels.

Aga rose-ge1e1 ectrophoresis is carried out in a horizontal apparatus which is described on pages 150 to 163 of Maniatis et al. The buffer used is the tris-borate buffer described there. The DNA fragments are stained with 0.5 µg/ml of an idium bromide which is present either in the gel or tank buffer during the electrophoresis, or which is added after the electrophoresis. DNA is made visible by means of illumination with long-wavelength u11ra v io 1 ett 1 ys. When the fragments are not to be separated from the gel, an agarose which gels at low temperature and which is supplied by Sigma Chemical, St. Louis, Missouri, should be used. After electrophoresis, the desired fragment is cut out, placed in a small plastic tube, heated approx. 15 minutes at 65°C, extracted three times with phenol and precipitated twice with ethanol. This procedure can easily be changed in relation to the one described on 170 by Maniatis et al.

Alternatively, DNA can be isolated from the agarose using Geneclean Kits (Bio 101 Inc., La Jolla, CA, USA). D. Addition of synthetic 1 inker fragments to DNA ends.

If a new endonuclease is desired at an insertion site added at the end of a DNA molecule, the molecule is likewise first treated with DNA polymerase to produce smooth ends, as described in the section above. About. 0.1 to 1.0 ug of this fragment is added to approx. 10 ng of phosphorylated linker DNA, supplied by New England Biolabs, in a volume of 20 to 30 µl with 2 µl of T4 DNA ligase from New England Biolabs, and 1 mM ATP in the buffer recommended by the manufacturer. After overnight incubation at 15°C, end

the reaction by heating to 65°C for 10 minutes. Reaks ionsti 1 f eye in the ngstedet is diluted to approx. 100 µl in a buffer appropriate for the restriction endonuclease that cuts the synthetic primer sequence. About 50 to 200 units of this endonuclease are added. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose-gel effect rotoresis and purified as described above. The resulting fragment will now have ends with ends produced by cutting with the restriction endonuclease. These ends are usually cohesive so that the resulting fragment can be easily linked to other fragments with the same cohesive ends.

E. Removal of 5'-terminal phosphates from DNA fragments.

During p1 asmi dk 1 on ingstr innes, treatment of the vector plasmid with phosphatase reduces vector recirculation (discussed on page 13 of Maniatis et al.). After cutting the DNA with the appropriate restriction endonuclease, one unit of calf intestinal alkaline phosphatase, supplied by Boehringer-Mannheim, Mannheim, is added. The DNA is incubated for one hour at 37°C and then extracted twice with phenol and precipitated with ethanol.

F. Joining of the DNA fragments.

When fragments with complementary cohesive ends are to be linked together, incubate approx. 100 ng of each fragment in a reaction volume of 20 to 40 µl with approx. 0.2 units of T4 DNA ligase from New England Biolabs in a buffer recommended by the manufacturer. The incubation is carried out for 1 to 20 hours at 150 C. When the DNA fragments are to be joined together with smooth ends, these are incubated as indicated above, except that the amount of T4 DNa ligase is increased from 2 h to 1 4 units.

G. The transformation of DNA in E. co 1 i .

E. co 1 i Stamm HB101 is used for most experiments. DNA is introduced into E. coli by the calcium chloride method as described by Maniatis et al., pp. 250 - 251.

H. Screening of E. co 1 i on plasmids.

After the transformation, the resulting colonies of E. coli are tested for the presence of the desired plasmid by means of a rapid p1 asmidisolation method. Two common methods are described on pp. 366 - 369 of Maniatis et al. I. Isolation of plasmid DNA on a large scale.

Methods for isolating plasmids from E_. coli on a large scale is described on pp. 88 - 94 by Maniatis et.

il-

J. Cloning in M13 phage vectors.

The following description should be understood as meaning that the double-stranded replicative form of subject M13 progeny is used for routine procedures, such as cutting with

restriction endonuclease, cross-linking etc.

Enzymes are supplied, when not indicated otherwise, from

Boehringer Biolabs (BRL). They are used according to the manufacturer's instructions, unless otherwise stated.

Example 1: Construction of plasma using pABDI

The freely available plasmids pkm 21 and pkm 244 [Beck, E., et al., Gene _19, 327-336 (1982)] are cut with the restriction endonuclease Pstl. The plasmid fragments used for recombination are purified by electrophoresis in 0.8% agarose gel. The plasmid obtained from the junction of fragments pkm 21244 contains a combination of 5'- and 3'-Bal 31 deletion of the NPT-11 gene, as described by Beck et al. in Gene J_9, 327-336 (1982). In order to connect the promoter igna1 et i Cau1 i f 1ower-mos a ikk-v i rus et with the Hind111 fragment in the plasmid pkm 21 244, the linking plasmid pJPAX is constructed. Kob1 ingsp 1 asmi det pJPAX is obtained from plasmids pUC8 and pUC9 [Messing, J. and J. Vieira, Gene j_9, 269-276 (1982)]. 10 base pairs of the connecting sequence of the plasmid pUC9 are separated by cutting the Hindi 11 and Sa 11 positions and the subsequent filling of the adhesive ends with the polymerase-1-K1enow fragment [Jacobsen, H., et al., Eur. J. Biochem. 45, 623 (1974)] and the connection of the po lynuk 1 eoti d chain, the Hind III position being established again. A synthetic linker of 8 base pairs (Xhol) is inserted at the SmaI position of this separated linker sequence. The recombination of the appropriate Xor1 and Hind111 fragments in the plasmid pUC8 and the modified plasmid pUC9 gives the plasmid pJPAX with a partially asymmetric binding sequence with the following restriction sites: EcoRI, SmaI, BamHT, SalT, PstI, HindHI, BamHI, Xhol and EcoRT. The CaMv gene VI promoter region linked to the NPT II structure gene originates from the genome of CaMV strain CM 4-184, a variant of CaMV strain CM 1841, whose complete nucleotide sequence is described in Gardner RC et al. The CaMV promoter region is cloned in the cosmid pHC 79, which comprises 6 kb, a progeny of the E. coli plasmid pBR322, [Hohn B and Co11ing J, 1980], the CaMV genome and the cosmid pHC79 being cut with Bstll and the resulting fragments joined together . The connection of the 5'-expression signal 1 et in Cau1 i11ower-mosaic-v in the rus-gene VI and the HindI I 1 fragment in the NPT-II gene is effected in the plasmid pJPAX by inserting the promoter region in Cau1 in f 1ower-mosaic-v in the rus gene VI between the Pstl and Hind 111 positions. The plasmid thus obtained is cut up at the only Hind 111 position and the Hind111 fragment in the plasmid pkm 2 1 244 is built into the cut site in both directions of orientation, obtaining the plasmids pJPAX Cakm<+>and pJPAX Cakm". To create a EcoRV sequence in the vicinity of the 3 1 termination signal in the NPT-11 hy brid gene, a BamHI fragment of the plasmid pJPAX Cakm<+> is built into the BamHI position of the plasmid pBR 327 [Soberon, X. et al ., Gene 9, 287-305 (1980)]. Plasmid pBR 327 Cakm is obtained. The EcoRV fragment in plasmid pBR

327 Cakm containing the new DNA constructs is used to replace the EcoRV region in the Cau1 i f 1ower mosaic ikk-v i rus gene VI which is cloned at the Sa 11 position in the plasmid pUC8, setting the protein coding DNA sequence in the NPT-II gene under the control of the 5' and 3<1> expression signals in Cau1 in the f 1ower mosaic gene VI. The plasmids thus obtained are designated pABDI and pABDII (cf. fig. 1).

Example 2: Construction of the plasmid pHP23A The construction of the plasmid pHP23 takes place after inserting an EcoRV fragment into the plasmid pABDI, which contains the APH-(3')11 structural gene as well as part of the 19S RNA promoter sequence of CaMV, into the Smal- since in the plasmid pDH51 (cf. Fig. 2).

The plasmid pDH51 is described in Pietrzak et al., 1986 (26). This plasmid contains the 35S promoter region and the terminator region of CaMV which are separated by an insert poly 1 inker region containing numerous k 1 on ing sites.

As starting material a 1e for the 35S promoter/terminator region serves a Sea I fragment of CaMV "S" comprising nucleotide 6808-7632 in the gene map (Frank G et al., Cel1, 2J_: ( 285-294, 1980 ), and which is spliced into the SmaI site of pUC 18 (Norrander J. et al., Gene, 26: 101-106, 1983), forming plasmid pDB21. This is then digested with the restriction enzyme Hph1. The cohesive ends are removed by treatment with T4 DNA polymerase.

A fragment containing the 35S promoter region [p U C18 pos. 21 (Hph I)-412 (Narrow) plus CaMV pos. 6808-7437 (Hphl), ligated with KpnI linkers, cut with NcoI (pos. 6909 CaMv), the cohesive ends filled using the DNA polymerase's Klenow fragment, ligated with EcoRI - 1 inkers and finally digested with EcoRI and KpnI.

The resulting EcoRI/KpnI fragment containing the CaMv promoter sequence is then spliced into the plasmid pUC18 between the KpnI and EcoRI restriction sites. In this way, the plasmid pDG2 is obtained.

Another fragment carrying the termination signal [CaMV pos. 7439 (Hph1)-7632 plus pUC18 pos. 413-1550 (Hphl)], cut with EcoRI, the cohesive ends filled with Klenow, provided with Hindi 11 linkers and digested with Hindi 11 and Sphl.

The resulting sphl-hindlll fragment containing the CaMV terminator sequence is then spliced into the plasmid pDG2, namely into the Hindi 11 and Sphl restriction sites.

pHP23 is modified by cleavage of part of the polylinker

the region. After cutting the plasmid with BamHI and SphI and deletion of the BamHI-SphI fragment, the cohesive ends are removed with the help of T4 polymerase and the broken pieces are linked together to form pHP23/\.

The construction of p H P 2 3 Zl is completed by recloning an EcoRI fragment carrying the hybrid gene in the plasmid p U C19 (Roberts RJ, 1984), which makes the production of deletion mutants easier.

Example 3: Construction of the plasmid pABDH

The BamHI fragment of the plasmid pLG88, which is described in Blochlinger and Diggelmann, Mol. Cel 1. Biol., 4 ( 12): 2929 - 293 1, 1984 and containing the region of the hygromycin phosphotransferase (hph) encoding the protein, spliced into the BamHI site in p D0B612 (Balazs E et al., Gene, 40 : 343-348, 1985) to form pABDH (cf. Fig. 2).

Example 4: Gene targeting

2 Plasmids with different structures of the A P H(3 ' ) 11 hybrid marker gene are used in the production of deletion mutants which are mutually complementary.

N j stem

1. Plasmid pABDI (cf. fig. la) which is described by Paszkowski J et al., Embo J,3<_>, 27 17 -2722, 1984) is cut with the restriction enzymes Ball and Smal. By deleting the Ba 11-sma I fragment in pABDI, the 1-ession mutants A-Ball are obtained (cf. fig. 1b). 2. Alignment of this plasmid at the NdeI restriction site.

3. Integration of the linearized plasmid into it

nuclear genome in tobacco by co-transformation with

pABDH. In this way, the Nj strain is obtained in the Target tobacco.

Rp, R^ strain

1. Cutting pABDI with Narl. By deleting the Nar1 fragment in the plasmid pABDI, the 1-ession mutants A Nar1a are obtained (cf. fig. 1c). 2. Linear visualization of the plasmid at the Ndel restriction site. 3. Co-transformation of A Narl in the tobacco genome. One obtains the Target strains R2 and R^.

Jj , -stem

1. Cutting pABDI with Pstl. By deleting the Pstl fragment in the plasmid pABDI, the 1-ession mutants A Pst I are obtained (cf. fig. Id). 2. Line near sighting of the plasmid at the Ndel restorative splice site. 3. Introduction of A Pstl into the tobacco genome gives the Target strains Jjog J^.

Deletion mutants

A Sphl ( p 19SphI):

Cutting plasmid pHP23 A with sphl and deletion of the Sp hI fragment leads to the deletion mutants Sphl (cf. fig.

1 f ).

A Nar Ib ( p! 9NarIb) :

Cutting of plasmid pHP23 A with Narl and deletion of the Narl fragment leads to the deletion mutants A Narlb (cf. fig. 1g).

The methods used for the p1 asmid construction are described in Maniatis et al., 1982.

Example 5: Production of tobacco protopI öster Tobacco protop1 öster is produced according to conventional methods from a ta bac-sus pension cur11ur (Potrykus I and Shillito RD, Methods in Enzymology, Vol. 118, Plant Molecular Biology, eds. A and H. Weissbach, Academic Press , Orlande, 1986). Fully unfolded leaves of 6-week-old sprouts are removed under sterile conditions and thoroughly moistened with an enzyme solution of the following composition:

Enzyme solution

H20 70 mg

Sucrose 13 mg

Macerozymes 1 mg

R 10

Cellulose 2 g

'Onozuka' Rjq

Drisellase 0.13 g

MES 0.5 ml

pH 6.0

The leaves are then cut into 1-2 cm square pieces and allowed to float in the above-mentioned enzyme solution. Incubation takes place overnight at a temperature of 26°C in the dark. The mixture is then gently shaken and incubated for a further 30 minutes until complete digestion.

This suspension is then filtered for separation of undigested tissue residues through a steel sieve with a mesh size of 100 µm, rinsed with 0.6 M sucrose 1 tablespoon (MES, pH 5-6) and then centrifuged for 10 minutes at 100 g. Due to this treatment, protoplasts accumulate on the surface of the medium which are then pulled by unbder protop1 åster, e.g. using a sterilized injection syringe.

The protoplasts are then resuspended in a K3A medium containing 0.4 M sucrose and which is washed in the manner indicated above 3 x by flotation and subsequent removal of the medium to remove the attached enzyme. The purified protoplasts are then suspended in a solution of 0.4 M mannitol and 1 g/liter MES so that there is a protoplast density of 1.2 to 1.6 x 10<4>ml.

Example 6: Co-transformation of protoplasts of Nicotiana tabacum cv. Petit Havanna SRI using Mg^+/ PEG treatment (Negrutiu, I, et al. 1987)

The co-transformation of the 1 estion p1 asmid DNA and pABDH DNA is carried out according to the instructions of Schocher et al., 1986. To carry out the transformation experiments, the protoplasts are first washed, counted and then resuspended in a W5 medium [154 mM NaCl, 125 mM CaCl 2

2H 2 O, 5 mM KCl, 5 mM glucose, pH 5.6; Menczel, L. et al.

(1981)], which ensures a high survival rate of the isolated protoplasts, in a cell density of 1-2.5 x 10 cells per ml. The protoplasts are then used after an incubation time of 30 minutes at 6°C to 8°C for the transformation experiments.

Shortly before the actual transformation of the isolated protoplasts, the W^ medium is replaced with the actual transformation medium. This concerns a Mannitol/magnesium solution [MaMg-Lsg: 0.4-0.5 mM Mannitol, 0.1% MES (Mo rfo1 in netansulphon sy re), pH 5.6] with a Mg^ + -concentration of 12 to 16 mM. For this, the protoplasts are first separated from the W^ solution by centrifugation at 100 g for 5 minutes and resuspended in the MaMg medium (0.3 ml). To this suspension is added 65 µl of an aqueous DNa solution containing 5 - 10 µg of deletion plasmid DNA, 2 µg of the plasmid pABDH and 40 µg of vehymus ca rr i er DNA. The latter involves a neutral carrier DNA without transforming inserts that is added in excess to this mixture, in order to protect against muc1ease digestion of the DNA to be transformed. Plasmid and carrier DNA are previously sterilized by ethanol replication as described by Paszkowski und Saul, 1986. After a period of approx. 0.1 to 10 minutes after the DNA-1i 1 set, the application of an aqueous 40% polyethylene glycol solution (w/v) takes place, until a final concentration of 24 to 28% (w/v) is achieved. The use of PEG of the CMS type has proven to be particularly advantageous. This involves a Ca^<+->containing [0.1 M Ca(N03 ) 2-4H20 ] 0.4 M rnani11solution which contains

PEG with a molecular weight of approx. 1000 to approx. 6000 in a final concentration of 40% (w/v). The pH value of this solution is at pH 7 to 9 fNegrutiu, I. et al.

(1986a ) ].

In the case of Nicotiana tabacum cv. Petit Havanna SRI preferably uses PEG CMS 4000. The pH value of the transformation medium is then adjusted to a value of pH 7. This mixture is incubated with occasional shaking for 30 minutes at 26°C.

When using high PEG concentrations of >20%, a stepwise dilution of 3 to 5 times the volume of a 0.2 M CaCl2 solution is advantageous. Then off-center grouting

the protoplasts treated in the indicated manner (5 minutes at 100 g) and resuspended in fresh K^ medium (0.3 ml protoplast solution in 10 ml fresh K^ medium). The further incubation takes place in 10 ml portions in Petri dishes of 10 cm diameter at 24°C in the dark, the population density being 4 to 8x10 4 protoplasts per

ml. After 3 days, the culture medium is diluted with 0.3 parts by volume of fresh K^ medium per dish and incubated for a further 4 days at 24°C and 3000 lux artificial lighting. After a total of 7 days, the clones developed from the protoplasts are introduced into nutrient medium solidified in 1% agarose and containing 12 mg/l hygromycin and cultivated according to the "bead type" culture method [Shillito, R.D. et al, Plant Cell Reports, 2, 244-247 (1983)] at 24°C in the dark. Ne ingsmed i et is replaced every 5 days with a fresh similar ne ing solution.

After 6 weeks, the hygromycin-resistant clones, which have reached a diameter of 1-2 mm, are transferred to an agar solidified ant ibiot ikaho1dig medium without hygromycin.

Cow 11i ver ingsbet inge1 tendon as well as the regeneration protocol is described in Saul et al., 1987.

Example 7: Examination of hygromycin's tested clones

on splicing of APH(3') 11 deletion mutants A total of 7 hygromycin-resistant clones were tested by means of Southern blot Hybridization on splicing of APH(3')II de 1 ession mutants in the plant genome.

Five of the tested clones that have the expected gene construction (3 different APH(3') II-gene-de 1 esions) are used in the further experiments.

The copy number of the target deletion varies in a range of two to ten. (Strain Jj- 3 copies, strain J~- 7 copies. Strain Nj- 5 copies, strain R2- 2 copies, strain R^

- 10 copies).

From these cell lines, the transgenic plants are regenerated according to the regeneration protocol described by Saul et al., 1987 and further propagated in the form of sterile sprout cultures.

After several subcutures, leaves are obtained which give a good yield of pro 1 in fertilizing protoplasts. The integration of the gene structure in the nuclear genome is verified by means of the mitotic stability and inheritance as a singular Mendelian trait by means of Southern blot hybridization.

Example 1 8: "Gen Targeting" experiments

For "gene targeting" experiments, protoplasts of the 5 preselected lines are used. Plasmid DNA, which contains complementary sections of the chimeric APH(3 1 ) 11 gene, is spliced using the direct gene transfer methods described in example 6 into the protoplasts of the corresponding plant strains, the following combinations being selected.

pl9NarIb with stem Nj

p 19 S p h I with stem R^, R3, Jjog J^.

Example 9: Selection of kanamycin-resistant cell clones After 3 to 4 weeks of continued cultivation in kanamycin-rich medium, the resistant calli with a diameter of 2 to 3 mm are planted on LS culture medium solidified with agar [Linsmaier EM and Skoog F, Phys i 01. Plant. 18: 1 00 -1 27, 1 965 ], containing 0.05 mg/l 2,4-dic1orphenoxy - acetic acid, 2 mg/l 1-naphthy 1 acetic acid , 0.1 mg/l 6-benzyl-aminopurine, 0.1 mg/l kinetin and 75 mg/l kanamycin. By sprout induction on LS medium containing 150 mg/l kanamycin and 0.2 mg/l 6-benzy1aminopurine and subsequent root formation on T medium [Nitsch, YP and Nitsch C, Science, 163: 85-87 1969 ], kanamycin- res istente Nicotiana tabacum plants of the variety Petit Havanna SRI.

Example 10: Detection of the NPT-II gene in the p 1 ancestral heritage 0.5 g callus of the transformed cell cultures or leaf tissue from the plant regenerated therefrom is homogenized at 0°C in 15 1 sucrose solution, containing 50 mM 1/1 ethylenediamine N,N,N',N'-tetraacetic acid, EDTA), 0.25 mol/l sodium chloride and 50 mM a,a,a-tris-(hydroxymethyl)-methylamine-hydrochloride (TRIS -HCl) at pH 8.0. By centrifuging the homogeneous mixture for 5 minutes at 1,000 g, the cell nuclei are coarsely separated. The cell nuclei are resuspended in 15% sucrose solution, containing 50 mMol/1 EDTA and 50 mMol/1 TRIS-HCl, mixed at pH 8.0, with sodium dodecyl sulfate to a final concentration of 0.2% and heated for 10 minutes at 70 °C. After cooling to 20-25°C, potassium acetate is added to the mixture up to a concentration of 0.5 Mol/l. This mixture is incubated for 1 hour at 0°C. The precipitate is centrifuged by centrifugation (15 minutes at 4°C in a

microcentrifuge ). From the remaining liquid, DNS is precipitated by mixing with 2.5 times the amount of ethanol at 20-25°C.

The separated DNS is dissolved in a solution of 10 mMol TRIS-HC1, containing 10 µg/ml ribonuclease A, incubated for 10 minutes at 37°C, mixed with proteinase K until a concentration of 250 µg/ml and incubated for a further hour at 37°C. Proteinase K is removed by phenol and chloroform/isoamyl alcohol extractions. From the aqueous phase, DNS is precipitated by the addition of 0.6 parts by volume of 0.6 molar isopropanoic sodium in urn acetate solution and dissolved in a solution containing 10 mMol/1 TRIS-HC1 and 5 mMol/1 EDTA at pH 7, 5. This preparation results in DNS sequences that preferably contain more than 50,000 base pairs. Restriction of this DNA with EcoRV endonuclease, hybridization of the fragments with radioactively labeled HindIII fragments of the NPT-II gene and comparison with plasmid pABDI shows in a "Southern Blot" analysis the presence of the NPT-II gene in the nuclear DNA of the transformed' Nicotiana tabacum cells.

Example 11: Southern blot analysis:

The analysis of plant DNA is carried out according to one of the methods described by Paszkowski und Saul, 1983.

About 5 µg of genomic DNA is optionally cut with EcoRV or Hind 111 restriction enzymes and the fragments obtained are resolved by electrophoresis in a 1% standard agarose gel.

(Maniatis T et al., 1982).

The Southern blot analysis is carried out according to the instructions of Paszkowski und Saul, 1986.

The nick transition is thereby replaced by the "random primer" method described by Feinberg und Vogelstein, 1983.

This leads to a reproducible activity of more than

IO<8>Cpm/jug DNA.

Results

From 3 independently conducted experiments, in which a total of 1.4 x 10 Q protoplasts participated, 8 kanamycin-resistant cell clones were obtained.

In the control experiments carried out at the same time where Wildtype SRI protoplasts were used and where no complementary de1estion mutants were offered, no resistant clones could be observed.

Kanamycin-resistant cell lines were obtained in the first line from the recipient strains J 2 0 9 R 3 •

The frequency of the formation of kanamycin-resistant clones can be compared with the transformation frequency of parallel conducted experiments where uninterrupted hybrid gene constructs were used.

The used 3' and 5' APH(3' ) 11 gene-de 1 esions originate from two hybrid genes that differ in their promoter and terminator region.

Thus, a homologous recombination of these deletion mutants leads to a marker gene that carries new combined flanking DNA sequences which, in comparison to the pre-existing gene constructs, lead to different restriction fragments.

The Southern blot analysis confirms that a homologous recombination has taken place at the central homo 1 and ie regions in the two APH(31 ) 11 de 1 ession mutants and thus at the same time excludes the possibility of a transformation by contaminating parental plasmids.

DNA analysis

DNA from 4 J2complementing clones was examined.

An EcoRV fragment (1247 bp) which stands in connection with the new marker genes was detected in all examined kana-myc in-resistant clones.

At the same time, the Southern blot analysis makes it clear that not all integrated APH(3 1 ) 11 gende1 esions could be corrected.

The appearance of DNA fragments corresponding to the newly created complete gene copy comes simultaneously with a removal of the deleted gene copies. This gives a reference to the fact that homologous recombination within the genome results in a specific correction of the mutated gene copy by the spliced DNA.

A genomic mapping shows that a HindIII fragment of 805 bp which carries the region in the APH(3')II gene which codes for the proteins, is exactly reconstructed.

This means that the hybrid gene correction is most likely to proceed exactly.

Demonstration of the transmission of the transformed gene to the sexual offspring as well as its inheritance as a normal plant gene.

Extensive genetic cross-breeding and detailed molecular and biochemical studies (e.g. "Southern blot" analysis of DNS in the plant genome; Investigations of the enzyme activity in aminoglycoside id-phos transfer, i.e. the enzyme for kanamycin - spes i f fish fos fory1 er ing ) with the genetically altered plants (first generation and the successors) have given the following results: 1. The bacterial gene is stably spliced into the p 1 antegenome; 2. It is usually transmitted unchanged and regularly to the offspring of the cross; 3. The course of inheritance corresponds to that in a natural, simple, dom i nant pl antegen ; 4. The molecular analysis using DNS hybridization and enzyme testing confirms the results of the genetic cross analysis; 5. The genetically modified plants have retained their normal, natural phenotype during the treatment; thus, no undesirable changes were determined.

These results show that the method according to the invention for direct gene transfer in protoplasts has found the best way for a targeted genetic change of plant material. The genetic change is stable and unwanted changes to the plant's genotype do not occur.

From the kanamycin-resistant tobacco plant obtained according to a), homozygous lines are extracted according to known methods, namely by themselves and by selection. Thus, Ni cot in ana-tabacum plants become available, where each haploid genome contains only a single copy of the neomycin transferase gene NPT-II.

D epon in ng:

The plasmid p 19 S ph I according to the present invention was, with a view to patenting, in accordance with the requirements of the Budapest Convention, deposited at the "Deutsche Sammlung von Mikroorganismen" (DSM), Federal Republic of Germany, which is recognized as an international depository. A declaration on the viability of the deposited samples was issued by

said international depository.

Microorganism Deposit- Deposit- Date for date number<*>viability certificate ;p19 Sph I 27.1 0. 1987 DSM 4289 4.1 1.1987 ;( Escheri ch i a;coli trans -;multiplied with;p 1 9Sph I;plasmid DNA;<* >Deposit number, issued by the above-mentioned international depository.

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Claims (30)

1. Recombinant DNA molecule which enables a targeted integration of genes, gene fragments or other DNA sequences at precisely defined locations within the plant genome, characterized in that said recombinant DNA molecule contains DNA to be integrated, and said DNA exhibits homologies corresponding to DNA regions within the plant genome or flanked by such homologous DNA sequences to the extent that upon transformation of the plant cells containing the homologous DNA regions, by homologous recombination a targeted integration of said DNA to be integrated takes place at precisely defined locations within the plant genome. 2. Recombinant DNA molecule as stated in claim 1, characterized in that the DNA to be integrated, which is possibly under the control of expression signals that are functional in the plant cell, is linked together with flanking DNA sections that show homologies with a specific region within the plant genome. 3. Recombinant DNA molecule as stated in claim 1, characterized in that the DNA to be integrated, which is possibly under the control of expression signals that are functional in the plant cell, exhibits a sufficiently large homology to corresponding DNA regions within the plant genome, so that by means of of the homologous recombination, a direct replacement of said DNA to be integrated takes place, against said homologous genomic DNA. 4. Recombinant DNA molecule according to one of claims 1 to 3, characterized in that said DNA to be integrated a) consists either of genomic DNA, of a cDNA or of synthetic DNA; b) is composed of genomic DNA as well as cDNA and/or synthetic DNA; c) is composed of a gene(s) or other DNA fragments of several organisms belonging to different families; d) is composed of a gene(s) or other DNA fragments of organisms belonging to different strains, or varieties of a species or different species of a family; or e) is composed of shares of more than one gene of the same organism. 5. Recombinant DNA molecule as stated in one of claims 1 to 3, characterized in that the said DNA to be integrated is a structural gene which gives the transformed plant a useful and desired property. 6. Recombinant DNA molecule as stated in claim 5, characterized in that said structural gene a) gives the plant an increased resistance or tolerance to pathogens, herbicides, insecticides or other biocides; b) improves the formation and quality of reserve and storage substances in leaves, seeds, tubers, roots and stems; c) is a p1 ante1agring protein gene; or d) codes pharmaceutically acceptable active substances. 7. Recombinant DNA molecule as set forth in claim 1, characterized in that the DNA to be integrated revolves around non-coding DNA sequences with a regulatory function. '8. Recombinant DNA molecule as stated in claim 2, characterized in that said expression signals originate from genes of plants or plant viruses. 9. Recombinant DNA molecule as stated in claim 8, characterized in that a) said expression signals originate from a plant gene which encodes the smallest subunit of r i bu 1 ose -1 ,5-bisphos fatkarbok - sylase or the chlorophyll a/b binding protein; b) derived from genes from Cauliflower mosaic virus (CaMV). 10. Recombinant DNA molecule as stated in claim 1, characterized in that the said flank end e' DNA sequences are those which have homology to plant specific genes or genome sections. 11. Recombinant DNA molecule as set forth in claim 1, characterized in that said flanking DNA sequences are those which show homology to genes or gene fragments or other useful DNA sequences, which have previously been artificially integrated into the genome of a target plant using known procedures precautions, and whose nucleotide sequences are fully or partially known. 12. "Recombinant DNA molecule as set forth in claim 11, characterized in that the said flanking DNA and the DNA that is artificially integrated into the plant genome are mutually complementary DNA sequences which, after homologous recombination, form a functional unit within the plant genome . 13. Recombinant DNA molecule as stated in one of claims 10 to 12, characterized by named genes or gene fragments or other useful DNA sequences are located in areas within the plant genome, which naturally exhibit a high expression rate. 14. Recombinant DNA molecule as stated in claim 1, characterized in that a) said recombinant DNA molecule contains a structural gene according to one of claims 5 or 6, b) said structural gene is optionally linked in an operable manner with the expression signal functional in plant cells 1 is according to one of claims 8 or 9, c) said functional unit, consisting of a structural gene and possibly expression signals, is in a way flanked by DNA sequences which to such an extent exhibit homologies to corresponding DNA regions within the plant genome, that upon transformation of the plant cell containing said homologous region, by homologous recombination takes place a targeted integration of the gene sequence that is flanked by homologous DNA sequences, at defined locations within the p 1 a ntegenome. 15. Method for the production of transgenic plants, characterized in that one transforms a gene, a gene fragment or other useful DNA sequences using a recombinant DNA molecule according to one of claims 1 to 14, by means of known methods into a plant cell and integrates it is targeted into the plant genome at a predetermined location via homologous recombination. 16. Method as stated in claim 15, characterized in that the transformation of the plant material containing the homologous DNA sections is carried out using a) The Agrobacterium transformation system; b) the CaMV transformation system; c) direct gene transfer; or d) a Co-transformation. 17. Method as stated in claim 15, characterized in that natural or modified genes or gene fragments or other useful DNA sequences are integrated into the plant genome by recombination with homologous DNA sections within the plant genome. 18. Method as stated in claim 15, characterized in that the integration takes place in a region of the plant genome, which ensures a high expression efficiency and/or an advantageous controllability of the integrated gene. 19. Method as stated in claim 17, characterized in that said homologous genomic DNA sections are a natural component of the plant genome or have been artificially integrated into the plant genome using known method precautions. 20. A DNA transfer vector, characterized in that it contains a recombinant DNA molecule according to one of claims 1 to 14. 21. A DNA expression vector, characterized in that it contains a recombinant DNA molecule according to one of claims 1 to 14. 22. A host cell, characterized in that it a) contains a DNA transfer vector according to claim 21; or b) contains a DNA expression vector according to claim 21. 23. A host cell as stated in claim 22, characterized in that it concerns a) a microorganism or b ) a pl antece11e. 24. Plant material consisting of a transgenic plant and its seed, characterized in that said plant material has been transformed with a recombinant DNA molecule according to one of claims 1 to 14. 25. A plant cell, characterized in that it contains a recombinant DNA molecule according to one of claims 1 to 14. 26. Plant material consisting of a transgenic plant and its seed, characterized in that said plant material contains plant cells according to claim 25. 27. Plant material consisting of a transgenic plant and its seed, characterized in that said plant material has been regenerated from plant cells according to claim 25. 28. Descendants of a transgenic plant according to one of claims 24, 26 and 27, wherein said descendants also include mutants and variants. 29. Forming material of transgenic plants according to one of claims 24 and 26 to 28, characterized in that it concerns protoplasts, cells, calli, tissues, organ seeds, pollen, egg cells, zygotes, embryos or other forming material that can be obtained from a transgenic plant. 30. Plant parts, such as e.g. flowers, stems, fruits, leaves, roots, which originate from transgenic plants or their descendants, which have previously been transformed by means of the method according to the invention and which are built up at least partially from transgenic cells.