DE4344929A1 - In vitro transformation complex for plants - Google Patents

Abstract

In vitro transformation complex (A) comprises \- 1 each of: (a) DNA binding or encapsulating protein (I); (b) DNA sequence (II) that facilitates integration into a plant genome; and (c) any selected DNA sequence (III) coupled to, or integrated within, (II). Also claimed are: (1) transformed plant cells, contg. a DNA sequence from (A); and (2) plants (or their descendants) contg. such cells.

Description

The invention relates to an in vitro transformation complex.

It has long been known that Agrobacterium tumefaciens after infestation higher plants in the trunk area produced tumors that are considered "Crown galls" are called. It will be a whole special part of the tumor-inducing plasmid (Ti plasmid) from Bacteria transferred into the plant cell and there as T-DNA Integrated core genome. This process is used by some chromosomal Genes and in the nopaline type of a total of six virulence operons controlled, which are also on the Ti plasmid.

This natural transformation system, in which a piece of DNA from the Bacteria transferred to a higher plant has been around for several Years successfully used to use genetic engineering to produce transgenic plants. There are a number of them today known further methods with which DNA sequences in plant Cells, protoplasts or plants can be introduced, e.g. B.  direct gene transfer via PEG treatment or through Electroporation, particle bombardment or the use of cationic liposomes. These methods are as follows, for example Publications revealed: Zambryski et al. (1983), EMBO J 12: 2143-2150; Deblaere et al. (1985), Nucleic Acid Research, Vol. 13, No. 13, 4777; Hain et al. (1985) Molec Gen Genet 199: 161-168; Paszkowski et al. (1984) EMBO J 3: 2717-2722; Negrutiu et al. (1987) Plant Mol. Biol. 8: 363-373; Fromm et al. (1987), Methods Enzymol. 153: 351-366; Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417; Christou (1993), Curr. Opin. Biotechnol. 4: 135-141.

Each of the transformation systems mentioned above has certain advantages and disadvantages. The sometimes small ones are disadvantageous Transformation rates. In the Agrobacterium system it is advantageous that usually only a few or only a copy of the foreign DNA in it Plant genome is integrated and that the transferred DNA is only one shows slight modification. On the other hand, the host range is from Agrobacterium restricted so that, for. B. Grain not transformed can be.

The object of the invention is a means of transformation specify with the most possible all types of plants with good efficiency can be transformed.

The task is solved by an in vitro transformation complex, the at least one DNA binding or enveloping protein, at least one integration into a plant genome mediating DNA sequence and has at least any DNA sequence which on or in the integration-mediating DNA sequence is coupled or integrated.  

Through the in vitro production of such a transformation complex and its subsequent transfer into a plant cell, a Protoplasts, a part of a plant or in the plant itself becomes a Transformation system provided that all higher plants with high efficiency can be transformed.

For the in vitro transformation complex, for example Components from Agrobacterium tumefaciens or from others Agrobacterium species are used that are also on the natural Transformation system are involved. The at least one DNA binding or enveloping protein can be the VirD2 and / or the VirE2 protein include. The right-hand sequence can be used as an integration-mediating DNA sequence and / or the left border sequence of the T-DNA from Agrobacterium tumefaciens or from other Agrobacterium species.

In the induction of virulence (vir) gene expression by plant Phenols are the T-ONA Borders specific by a protein complex, which carries VirD1 and VirD2 protein subunits, cut (sting et al. (1986) Nature 322: 706-712; Zambryski (1988), Annu. Rev. Genet. 22: 1-30). VirD2 catalyzes with the participation of VirD1 to the Borders the T-DNA a nicking reaction and then binds itself covalently to the 5-end of the nodded T-DNA. In addition, the T-DNA is still encased by a single strand binding protein (VirE2). So far on T-complex no other proteins have been identified. On the way from The T complex must be bacterial and bacterial to the plant cell nucleus vegetable membranes happen, which is not without specific Interactions with other proteins can take place. One takes that the T complex is protected from DNAsen by the protein envelope, VirE2 maintains an elongated shape and thus the core pores can happen because both proteins (VirD2 and VirE2) are over  Nuclear localization signals feature (Zambryski (1992), Annu. Rev. Plant Physiol. Mol. Biol. 43: 465-490).

By cloning z. B. the VirD1, VirD2 and VirE2 genes in one Expression vector and the overproduction of the gene products in E. coli with subsequent purification of the corresponding proteins can be used for Production of the in vitro transformation complex can be used. When DNA substrate can be single-stranded T-DNA, e.g. B. the right one T-DNA border region of the Ti plasmid C58 can be used. It was demonstrated that the VirD2 protein undergoes the same cleavage reaction between the third and fourth bases of the 25 bp T-DNA border sequence in vitro explains how this was previously determined in vivo (Dürrenberger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 9154-9158). Further was demonstrated that the VirD2 protein after cleavage of the 25 bp T-DNA border sequence remains associated with the 5'-end of the T strand and that added VirE2 protein envelops the T complex.

Such an in vitro transformation complex can be used for transformation used by higher plants. A reporter gene can be sent to the integration-mediating DNA sequence are coupled. This For example, the reporter gene can contain the GUS gene (β-glucuronidase) Intron can be used (Töpfer et al. (1987), Nucl. Acids Res. 15: 5890; Vancanneyt et al. (1990) Mol. Gen. Genet. 220: 245-250).

In this case, the in vitro transformation complex exists after the Invention from that coupled to the right border sequence of the T-DNA GUS-INT gene and the two proteins VirD2 and VirE2. This in vitro Transformation complex can be used for the in vitro production Transformation of higher plants can be used.  

By making the in vitro transformation complex in vitro and is transferred directly into the plant, the diverse chemical interactions between the agrobacteria and the wounded Plant cells or the complicated mobilization of the T complex excluded. The in vitro transformation complex protects the DNA degradation and introduces the DNA into the cell nucleus, the DNA in transcription-active areas is integrated. With the in vitro Transformation complexes according to the invention can all be Take advantage of the Agrobacterium gene ferry and the disadvantages, e.g. B. Avoid restricted host area. The invention thus leads to following improvements: All plants can be transformed; in usually just a copy of the foreign DNA into the plant genome integrated; the transformation rate in some cases significantly exceeds all of them previously common methods of so-called direct gene transfer; the DNA transferred shows the minor modification customary for Agrobacterium and the DNA is preferably integrated into transcribable areas.

For the extraction, presentation and purification of the for in vitro Transformation complex proteins and DNA sequences to be used the known biochemical methods in the field of protein and Nucleic acid chemistry can be used. The production of the in vitro Transformation complex happens under such in vitro conditions, that the at least one protein or protein fragment the at least one DNA sequence envelops and / or binds to it. The corresponding Conditions are known or can be determined in a simple manner become. For example, purified VirD2 and VirE2 protein can also single-stranded DNA in 20-100 mM Tris-Cl (pH 7-9), 2-50 mM Mgcl₂, 0.5-2 mM dithiothreitol, 20-200 µM EDTA, 0-200 µg / ml Bovine serum albumin at 0-37 ° C for about 30 minutes to 24 hours be incubated. For example, 200 µg protein and 1 ng DNA  be used. This incubation approach can then be used directly for Transformation. Alternatively, it is possible to use the in vitro Transformation complex by ion exchange chromatography or by To clean density gradient centrifugation. The one formed in vitro Transformation complex can be used for transformation experiments plant cells, protoplasts or plant tissues become. To control a successful transfer into the plant cell a single-stranded substrate DNA is used, for example in addition to the lower strand of the right border of the T-DNA encoding Sequences of a reporter gene, e.g. B. the GUS reporter gene contains. The in vitro transformation complex according to the invention can by various Methods are transferred into the plant cells.

Leaf mesophyll protoplasts, e.g. B. from N. tabacum cv. cultiva petite Havana SR1, or protoplasts from a suspension culture of Barley variety Golden Promise can be added after in vitro Transformation complex with polyethylene glycol (PEG) according to the method by Negrutiu et al. (Plant Mol. Biol. 8: 363-373, 1987) or according to Maas and Werr (Plant Cell Report 8: 148-151, 1989). Further can the electroporation process (Joersbo and Brunstedt (1991), Physiol. Plans. 81: 256-264) are used, here the in vitro Transformation complex together with leaf mesophyll protoplasts electrical field is exposed.

The transformation can also be carried out using the cationic liposome method be carried out (Spörlein and Koop (1991), Theor. Appl. Genet. 83, 1-5). The in vitro transformation complex will do this with liposomes "packed" and added to protoplasts.

The in vitro transformation complex can also, for. B. on gold particles  bound and with a pneumatic particle cannon on plant tissue, like suspension cultures of tobacco and barley, parts of plants like leaves, Leaf bases, roots, dry ripe seeds, swelling seeds, unripe Embryos and in particular scutellum and epiplast of cereals, be shot. Particle bombardment is described, for example by Christou in Curr. Opin. Biotechnol. 4: 135-143 (1993).

All parts of the plant mentioned above can in principle also by Microinjection of the in vitro transformation complex transformed are according to Reich et al. (Bio / Technol. 4: 1001-1004, 1986) or Neuhaus et al. (Theor. Appl. Genet. 75: 279-290, 1987).

The transformation of plant tissue or plant cells or Protoplasts with an in vitro transformation complex can also by additional components are supported that support the integration process optimize. So the plant cells with restriction enzymes to be treated before or during the transformation Open plant DNA. By means of chemicals, UV and / or X-rays can induce repair mechanisms that are too can lead to increased installation rates. To increase the Transformation rates can also be used: electric shock, Ultrasound, microwaves, heat shock, cold shock, dry stress, Wilting stimuli or the induction of stress proteins. The cell cultures, whose cells are to be transformed can also be synchronized the DNA transfer at a specific point in time Cell cycle occurs. Furthermore, the transformation with an in vitro Transformation complex and simultaneous coculture with z. B. Agrobacterium tumefaciens, Agrobacterium rhizogenes or others Agrobacterium species are carried out. When transforming with the in vitro transformation complex it is also possible to remove the tissue  incubate simultaneously with enzymes such as kinases, transferases, Helicases, nucleases, topoisomerases, ligases or ONA / RNA polymerases. The use of substances that promote integration, such as ATP, is also possible.

Instead of the VirE2 and / or VirD2 protein described above, the DNA of the in vitro transformation complex also with other DNA-binding and / or enveloping proteins. They are all proteins capable of performing at least one or more of the following functions exhibit: They bind to DNA, protect the DNA from degradation, transport the DNA into the cell nucleus and mediate the integration of DNA in the plant genome. Such suitable proteins are listed for example in claim 3. These proteins can be used individually or mixed, with essential protein parts are sufficient if they can perform one of the above functions.

The integration-mediating DNA sequence of the in vitro Transformation complex can be any DNA sequence, provided that it is a Integration into the plant genome mediated. Such sequences are listed for example in claims 5 and 6.

Any on or in the integration-mediating DNA sequence DNA sequence can be coupled or integrated. Any DNA sequence can e.g. B. the coding sequence of a reporter gene and / or one Resistance gene. Any DNA sequence can also be a have regulatory sequence.  

Embodiment Cloning of the GUS-INT gene and the right border fragment the T-DNA from Agrobacterium tumefaciens

The bacterial gene for β-glucuronidase (GUS) is widely used as a reporter gene in plant systems. By inserting an intron from a potato gene, expression in bacteria is prevented, so that the measured enzyme activity is solely due to expression in the transformed plant. The GUS gene with intron is called GUS-INT. The GUS-INT is in plasmid p35S GUS-INT in the plant expression cassette of the vector pRT102 (Töpfer et al. (1987), Nucl. Acids Res. 15: 5890). The 35S promoter was removed by cutting with Bam HI and HindIII, but the polyadenylation site of the cassette was left. The binary Agrobacterium vector was also cut with the two restriction enzymes, the polyadenylation site of the nos gene being deleted. By ligation of the GUS-INT fragment with the vector, the promoterless GUS-INT with its 5′-end was placed directly next to the right border of the vector and the plasmid pPCV6NF GUS-INT was created. The GUS-1NT gene, together with the right border, was cut out of this plasmid using Sph I and inserted into the corresponding interface of the vector pUC118. The newly formed plasmid pGIRB 118 is shown in FIGS . 1 and 2.

In the figures, LB and RB denote the left and right borders of the T-DNA from Agrobactium tumefaciens, Ap the gene for β-lactamase, P den 35S cauliflower mosaic virus (CaMV) promoter, pA die Polyadenylation site, GUS-INT with the gene for the β-glucuronidase Intron.  

From the plasmid pGIRB118, helper phage from E. coli single-stranded circular DNA can be obtained as a substrate for the production of the in vitro transformation complex serves.

An E. coli strain with the plasmid pGIRB118 was named GIRB118 at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Mascheroder Weg 1b, 38124 Braunschweig, Federal Republic Germany, in accordance with the provisions of the Budapest Contract deposited and has the deposit number. . . receive.

Cloning and overexpression of the VirD1, VirD2 and VirE2 genes in E. coli

The genes VirD1, VirD2 and VirE2 were isolated from the plasmid pGV0361 (Depicker et al. (1980), plasmid 3: 193-211), a pBR322 vector which is a 15.83 kb fragment with the VirD and VirE region and carries parts of the VirC region of the nopalin-type Ti plasmid pTIC58. For better handling the Bam HI fragments were No. 13 (3.78 kb) with the Vir genes VirD1 and VirD2 and No. 15 (3.39 kb) with the VirE2 gene respectively into the vector pBSK ^-.. Cloned. The numbers indicate the Bam HI fragments of the pTIC58 sorted by size. The newly formed plasmids were given the names Ba 13 BS and Ba 15 BS. For overexpression in Escherichia coli, the Vir genes were cloned into the expression vector pET3b (Rosenberg et al (1987), Gene 56: 125-135). The start codon ATG of the VirE2 gene was removed from fragment No. 15 by cutting with the restriction endonuclease Bam HI. A translational fusion of the VirE2 gene with the codons of the first 12 amino acids of gene 10 of the T7 bacteriophage was produced by ligating the fragment in the correct orientation with the Bam Hi interface of the vector pET3b. Such an amino terminal fusion does not lead to loss of the DNA binding activity of the VirE2 protein (Citovsky et al. (1988), Science 240: 501-504). The plasmid was named pET Ba15. For the cloning of the VirD1 and VirD2 genes, the polymerase chain reaction (PCR; Mullis and Faloona (1987) Methods Enzymol. 155: 335-350) was used using Taq polymerase (Saiki et al. (1988) Science 239: 487- 491) used to ensure optimal expression in the expression vector pET3b without translational fusion. A fusion at the N-terminus of the VirD1 protein leads to the loss of the activity of the protein in the heterologous E. coli system. The primer oligonucleotides for the PCR were designed so that only the open reading names of the VirD1 and VirD2 genes or both genes were amplified together as a transcription unit, but without additional sequences at the 5 'and 3' ends. In addition, the interfaces for Nde I and Bam HI (read from the translation start) were added to the primers that define the 5'-end of the PCR products, and the interface for for the primers that define the 3'-end Bam HI. The PCR was carried out in three independent approaches for each construct. The size of the PCR products was 0.44 kb for VirD1, 1.34 kb for VirD2 and 1.82 kb for VirD1 / VirD2. After cutting with Bam HI, the PCR products were first ligated to the Bam HI site of the vector pTTQ18 (Starck (1987) Gene 51: 255-267). The PCR products with Nde I and Bam HI were cut out of this vector and inserted into the expression vector pET3b, which had also been cut. As a result, the initiation codon of the translation was at an optimal distance from the Shine-Dalgarno sequence of the expression vector. The constructs formed were called pET VirD1, pET VirD2 and pET VirD1 / D2.

The cloning of the genes VirD1, VirD2 and VirE2 into the expression vector pET3b is shown in FIG. 3. The upper section of FIG. 3 shows a section of the Bam HI restriction map of pTiC58. The numbers indicate the Bam HI fragments of the Ti plasmid, sorted according to size. The arrows marked "D" and "E above the restriction map mark the region of the VirD and VirE operon. PCR denotes the polymerase chain reaction, Ap the β-lactamase gene, while P, S and T the promoter Characterize Shine / Dalgarno sequence and the transcription terminator of the T7 RNA polymerase expression vector.

• E. coli strains with the plasmids pET VirD2 and pET Ba 15 were identified as ET VirD2 and ET Ba 15 at the German Collection of Microorganisms and Zellkulturen GmbH (DSM), Mascheroder Weg 1b, 38124 Braunschweig, Federal Republic of Germany, in accordance with the provisions of Budapest contract and have the deposit numbers receive.

The bacteria (E. coli BL21 (DE3); Studier et al. (1990) Methods Enzymol. 185: 60-89) with the corresponding expression plasmids up to an optical density (2 = 600 nm) of 0.5 and then added by adding IPTG a final concentration of 0.5 mM induced and for another three hours incubated before they were harvested.

Extraction and purification of the proteins

The E. coli inclusion bodies were isolated and cleaned using the method of Schmidt et al. (Proc. Natl. Acad. Sci. USA 83: 9581-9585, 1986). The cell sediment from 250 ml culture volume was in 20 ml  50 mM Tris-Cl (pH 8.0) was added. Then the bacterial cells by ultrasound treatment with the micro tip of a Branson Sonifier in 30 second intervals with cooling breaks for a total of 5-10 minutes open minded. The mixture was then run at 10,000 rpm for 30 minutes and 4 ° C centrifuged (JA 20 from Beckman). The sediment was in 10-20 ml of 20% sucrose, 3 mM EDTA (pH 7.6) added and again Centrifuged for 30 minutes at 11000 rpm and 4 ° C. The Inclusion body sediment was stored at -20 ° C until use.

The three Vir proteins VirD1, VirD2 and VirE2 were subjected to the renaturation procedure according to Buchner and Rudolph (Bio / Technol. 9: 157-162, 1991). The inclusion bodies of a 250-500 ml bacterial culture were dissolved in 500 μl solubilization buffer, transferred into an Eppendorf reaction vessel and incubated for 30 minutes at 37 ° C. with occasional mixing (vortex). After centrifugation for five minutes in an Eppendorf centrifuge at 13000 rpm, the supernatant was diluted in 50 ml renaturation buffer (1: 100) (Falcon 2070). For VirD2 the solubilization buffer contained 100 mM dithiothreitol (final concentration for renaturation thus 1 mM). Incubation in the renaturation buffer was carried out at 22 ° C. overnight. For VirD1 and VirE2, the renaturation buffer contained 5 mM / 0.5 mM glutathione reduced / oxidized. Incubation was at 8 ° C for two days. All three proteins were then dialyzed overnight against dialysis buffer.
Solubilization buffer: 20mM Tris-Cl (pH 8.0), 1mM EDTA, 6M guanidinium hydrochloride.
Renaturation buffer: 20 mM Tris-Cl (pH 7.6), 1 mM EDTA, 0.4 M arginine, adjusted to pH 7.6 with HCl.
Dialysis buffer:
for VirD1: 25 mM Tris-Cl (pH 7.6), 50 µM EDTA.
for VirD2: 25 mM Tris-Cl (pH 7.6), 50 µM EDTA, 1 mM dithiothreitol.
for VirE2: 25 mM Tris-Cl (pH 8.0), 1 mM EDTA.

The dialysis tubes were made according to the method of Pohl (Methods Enzymol. 182: 68-83, 1990) before use. For VirD1 Dialysis tubing with pores with a molecular weight cutoff of 6-8 kDa (Spectra / Porl) related, for VirD2 and VirE2 hoses with Pores of 12-14 kDa exclusion limit (Servapor).

After dialysis, the samples were kept at 8000 rpm and 4 ° C. for 30 minutes JS13 (Beckman) centrifuged. The sediment was in the Solubilization solution resumed. The protein content in the Sediment and in the supernatant were against the solubilization solution or determined the dialysis buffer and to the original total volume certainly. The solubility after the Renaturation procedure, expressed in% of the soluble protein from the total amount of protein used.

The protein remaining in solution after dialysis was removed using a P1 peristaltic pump on a HiTrap heparin-Sepharose column (Pharmacia Biotech) with a bed volume of 1 ml, that with 10 ml Dialysis buffer had been equilibrated. The column was then washed with 5 ml Dialysis buffer washed. The proteins were eluted in a NaCl Step gradients in the dialysis buffer. First there was a salt concentration placed on the column where some of the contaminating proteins but the overproduced protein has not yet been eluted from the column. Then the overproduced protein became at higher salt concentration  (380 mM for VirD2; 250 mM NaCl for VirD1) eluted in 3 ml dialysis buffer. Then any that were still attached to the column material Proteins eluted with 2 M NaCl. The flow rate was 2 ml / minutes.

Production of the in vitro transformation complex

For the production of the in vitro transformation complex purified VirD2 protein and VirE2 protein used. A single-stranded DNA fragment consisting of the GUS INT gene and the right border sequence of T-DNA from Agrobacterium tumefaciens, was with known methods from the plasmid pGIRB118. 100 µg each Proteins were mixed with 1 ng DNA for one hour at 37 ° C in 20 mM Tris-HCl (pH 9.0), 5 mM MgCl₂, 1 mM dithiothreitol, 100 µM EDTA, 100 µg / ml Bovine serum albumin incubated.

The in vitro transformation complex produced in this way became Transformation of plant protoplasts used. In some Cases, the in vitro transformation complex was developed Ion exchange chromatography or density gradient centrifugation cleaned.

It could be shown that VirD2 is a sequence and strand specific (lower strand of the T-DNA border sequence) Cleavage of a single strand DNA substrate catalyzed at the 25 bp border sequences of the T-DNA. This was confirmed with in vitro transformation complexes in which only VirD2 or VirD2 and VirD1 were used. For this, the DNA substrates labeled at their 5 'ends using T7 polynucleotide kinase. To the connection between the 5'-end of the T-strand and VirD2 too  show was a T-DNA border fragment at its 3'-end of the lower Stranges marked by DNA polymerase Klenow fragment. For this Known molecular biological methods have been used (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Lab. Press, Plainview, NY, USA). there has been a averaged 50% cleavage of the T-strand DNA, whereby a saturation balance was observed. The VirD2 protein remains covalent, at least very firmly at the 5 'end of the T-DNA bound. It has also been shown that the VirE2 protein on the Transformation complex is liable.

Transformation of tobacco and barley protoplasts with the in vitro transformation complex

Tobacco mesophyll protoplasts, Petite SR1, were made by known methods isolated (Pröls et al. (1988) Plant Cell Report 17: 221-224). Approx. 2.5-3 g of leaf material was dissolved in 20 ml of enzyme solution (1.5% cellulase R10; 0.5% Mazerozym R10 (SERVA), pH 5.7, in the culture medium) for 16 hours in Incubated in the dark. After shaking at 100 rpm for 20 minutes Tissue residues during filtration through steel sieves with 300 and 100 µm mesh size separated. Cell debris became by centrifugation and floating protoplasts separated. The protoplasts were washed three times in the culture medium and centrifuged. The Protoplast yield was determined using a Neubauer counting chamber.

The extraction of barley protoplasts of the Golden Promise variety or their culture was carried out according to known methods (Lührs and Lörz (1987), Theor. Appl. Genet. 75: 16-25; Lührs and Lörz (1988), Planta 175: 71-81).  

The protoplasts were transformed using the methods of Negrutiu et al. (Plant Mol. Biol. 8: 363-373, 1987), Fromm et al. (Methods Enzymol. 153: 351-366, 1987) and Maas and Werr (Plant Cell Report 8: 148-150, 1989).

By expressing the GUS reporter gene, the efficiency of the Gene transfers are tracked. By means of the substrate X-Gluc Enzyme activity of β-glucuronidase based on the blue color of the transformed cells detected. Here was a reporter gene without Promoter used so that the detection of enzyme activity is a sign for the integration of the transferred DNA in the vicinity of a promoter is in the plant genome. This gene also allows quantitative colorimetric determination according to Jefferson et al. (EMB0 J 6: 3901-3907, 1987). The comparative experiments were carried out with "naked" DNA, i.e. H. with the corresponding DNA fragment from pGIRB118 or with this Plasmid itself.

In the case of transformed tobacco protoplasts, the cultures which the in vitro transformation complex had been transformed, after 7-day culture duration increased at least 5 to 10 times Transformation rate compared to the cultures with "naked" DNA had been transformed. Likewise, a blue color from with the in vitro transformation complex transformed barley protoplasts to be shown.

The use of the in vitro transformation complex for the Transformation of higher plants thus leads to higher ones Transformation rates for tobacco and barley protoplasts or for tobacco and barley plants.  

The grain transformation is common today, e.g. B. with rice (Toriyama et al. (1988) Bio / Technol. 6: 1072-1074; Zhang and Wu (l988), Theor. Appl. Genet. 76: 135-140; Peng et al. (1992), Theor. Appl. Genet. 83: 855-863), wheat (Vasil et al. (1991) Bio / Technol. 10: 667-674; Weeks et al. (1983) Plant Physiol. 102: 1077-1084), Maize (Dhalluin et al. (1992) Plant Cell 4: 1495-1505; Gordon-Kamm et al. (1990), Plant Cell 2: 603-618) or oats (Somers et al. (1992), Bio / Technol. 10: 1589-1594).

Claims (20)

1. In vitro transformation complex, characterized in that it
at least one DNA-binding or enveloping protein,
at least one DNA sequence mediating the integration into a plant genome and
has at least any DNA sequence that is coupled to or integrated into the integration-mediating DNA sequence. 2. In vitro transformation complex according to claim 1, characterized characterized in that the at least one DNA-binding or enveloping Protein the VirD2 and / or the VirE2 protein from Agrobacterium tumefaciens or analog proteins from other Agrobacterium species includes. 3. In vitro transformation complex according to claim 1 or 2, characterized  characterized in that the at least one DNA-binding or enveloping Protein is selected from the group consisting of the coat proteins Plant RNA viruses, transcription factors, heat shock proteins, Proteins from animal and human viruses, proteins from bacteriophages, proteins bacterial conjugative systems, proteins from gemini viruses, Polyviruses or the Paramyxoviridae family, core proteins of the Xenopus Oocytes, the SSB protein from Escherichia coli, histones, RecA, biotin, Avidin, influenza PBI protein or a mixture of these proteins. 4. In vitro transformation complex according to one of claims 1 to 3, characterized in that essential parts of the in claims 2 and 3 listed proteins individually or mixed the at least one Represent protein. 5. In vitro transformation complex according to one of claims 1 to 4, characterized in that the at least one integrating DNA sequence the right and / or the left border sequence of the T-DNA Agrobacterium tumefaciens or from other Agrobacterium species represents. 6. In vitro transformation complex according to one of claims 1 to 4, characterized in that the at least one integrating DNA sequence is selected from the group comprising the border sequences the T-DNA from Agrobacterium rhizogenes, DNA sequences from plants, direct repetitions of flanking regions, sequences that procarytic, replicative or conjugative processes are involved, Initiation sequences of viral replication of geminiviruses or a Mixture thereof includes. 7. In vitro transformation complex, characterized in that it  at least any single-stranded DNA sequence, the right one and / or the left, single-stranded border sequence of the T-DNA Agrobacterium tumefaciens, with any DNA sequence at least a border sequence is coupled, and the VirD2 protein and the VirE2 protein from Agrobacterium tumefaciens having. 8. In vitro transformation complex according to claim 7, characterized characterized in that it additionally contains at least one protein according to claim 3 or a substantial part of it. 9. In vitro transformation complex according to claim 7, characterized characterized in that the VirD2 protein and / or the VirE2 protein by at least one protein according to claim 3 or by an essential part of it is replaced. 10. In vitro transformation complex according to one of claims 7 to 9, characterized in that it is only an essential part of the VirD2 and / or the VirE2 protein. 11. In vitro transformation complex according to one of claims 7 to 10, characterized in that he additionally has at least one integration-mediating sequence according to claim 6 or an essential Has part of it. 12. In vitro transformation complex according to one of claims 7-11, characterized in that the right or left border sequence of the T-DNA from Agrobacterium tumefaciens according to at least one sequence  Claim 6 or is replaced by a substantial part thereof. 13. Process for the production of genetically transformed Plant cells, characterized in that an in vitro Transformation complex according to one of claims 1 to 12 into one Plant cell, a plant protoplast, a plant part or in a plant is introduced. 14. A process for the production of genetically transformed plants, the process being characterized by the following steps:

• a) the DNA sequence of the in vitro transformation complex is inserted into the genome of a plant cell or a protoplast.
• b) the transformed plant cells obtained express at least one gene product, the information of which lies on any DNA sequence, and
• c) the transformed cells are regenerated to genetically transformed plants.

15. The method according to claim 14, characterized in that the transformed cells and / or the regenerated plants for Determination of the transformation to be tested. 16. The method according to claim 14 or 15, characterized in that the transformed plants are propagated. 17. Transformed plant cell, characterized in that it a DNA sequence from the in vitro transformation complex according to one of the  Claims 1 to 12. 18. Transformed plant, characterized in that it transformed cells according to claim 17. 19. Plant progeny, obtainable by multiplying the transformed plants according to claim 18. 20. Plant progeny, characterized in that at least a parent plant of the progeny a plant according to claim 18 is.