DE3382838T2 - Transgenic dicotyledonous plant cells and plants - Google Patents

Abstract

(1) Acceptor tumour-inducing (Ti) plasmid comprises (a) the 2 border sequences of the T-region of the wild-type Ti plasmid; (b) non-oncogenic DNA segment derived from a cloning vector located between the 2 border sequences contg. a DNA sequence homologous with at least part of a DNA sequence in an intermediate cloning vector permitting a single cross-over event; and (c) segment of the wild-type Ti plasmid contg. DNA sequences essential for the transfer of Agrobacterium of the T-region of the T-region of wild-type Ti plasmids into plant cell genomes.

Description

This invention relates to dicotyledons Plant cells or plants that have foreign DNA sequences in their Genome included. The foreign DNA sequences envisaged are characterized by sequences encoding products, e.g. B. amino acids or Polypeptides that are useful are for the growth of the plant or for improving their quality as food or for manufacturing of valuable metabolic products (e.g. alkaloids or steroid precursors).

The following terms are used in the description:
bom site: DNA area in which mob functions interact specifically and initiate autonomous DNA transfer.
Edge sequence: DNA sequence which contains the ends of the T-DNA.
Wide host range replicon: a DNA molecule that can be transferred to and maintained in many different host cells.
Callus tissue: a mass of disorganized and undifferentiated cells.
Cloning: A method of obtaining a population of organisms or DNA sequences derived from such an organism or sequence by asexual reproduction.
or better:
Process for isolating a certain organism or part thereof and multiplying this sub-fraction as a homogeneous population.
Cloning vehicle: a plasmid, phage DNA or other DNA sequences capable of replication in a host cell and characterized by one or a small number of endonuclease recognition sites at which such DNA sequences can be cleaved in a fixable manner, without an associated loss of an essential biological function of the DNA, e.g. B. replication, production of coat proteins or loss of promoter or binding sites, and containing a marker suitable for use in the identification of transformed cells, e.g. B. tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Coding sequence: DNA sequence which determines the amino acid sequence of a polypeptide.
Cointegrated structure: the structure that arises from a simple crossover event between two circular DNA molecules.
Complementation in trans: Process whereby a DNA molecule (replicon) that is not physically linked to another replicon can provide a diffusible substance that is missing from and required by the other non-linked replicon.
Conjugation: The process by which DNA is transferred from bacteria of one type to another type during contact from cell to cell.
Crossover: process of exchanging genetic material between homologous DNA sequences.
Deletion substitution: Removal of a DNA sequence and its replacement by another DNA sequence.
Differentiation: The process by which the descendants of a cell achieve and maintain the specialization of structure and function.
DNA sequence or segment: a linear series of nucleotides that are connected to each other by phosphorus diester bonds between the 3 'and 5' carbon atoms of the adjacent pentoses.
Double crossover: Process in which a cointegrated structure is dissolved to form two circular DNA molecules. This process is used to exchange genetic information. One of the DNA rings has two regions homologous to the target DNA through which recombination can occur; these two areas enclose a non-homologous DNA sequence that is to be exchanged with the target DNA. If this second crossover takes place in the same DNA area as the first, the original DNA rings will be formed. If the second crossover takes place in the second homologous area, the result will be a genetic exchange between the two rings.
Expression: the process of making a polypeptide that a structural gene has undergone. It is a combination of transcription and translation.
Expression control sequence: a nucleotide sequence that controls and regulates the expression of structural genes when it is functionally linked to such genes.
F-type plasmid: plasmid that carries the F (fertility) factor, which enables the transfer of a copy of the plasmid to a host that does not carry the F factor.
Gene: a DNA sequence composed of two parts: (1.) the sequence encoding the gene product and (2.) the sequences in the promoter region which control whether the gene is expressed or not.
Genome: the entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptide (s) and also the operator, promoter and sequences which bind to and interact with the ribosomes, comprising sequences such as, for. B. the Shine-Dalgarno sequences.
Genotype: the total amount of genetic information contained in the organism.
Homologous recombination: recombination between two DNA regions that contain homologous sequences.
I-type plasmid: a group of autotransferable Plasmids of an incompatibility group different from F.
Incompatibility: when two DNA molecules are unable to coexist in the same cell in the absence of selective pressure.
Insertion: insertion of a DNA sequence into the DNA sequence of another molecule.
Leader sequence: the region of an mRNA molecule that extends from the 5 'end to the beginning of the first structural gene; it also includes sites that are important for initiating translation of the coding sequence of the structural gene.
Meiosis: two successive divisions that reduce the initial number of 4 n chromosomes to 1 n in each of the four cells formed. This process plays an important role in sexual reproduction.
mob (mobilization functions): a set of products that only promote DNA transmission in combination with tra functions. mob can promote the transfer of plasmids that contain a bom site.
Mobilization: The process by which a DNA molecule that is not capable of being transferred to another cell is assisted in the transfer by another DNA molecule.
Mobilization helper plasmid: a plasmid that is capable of providing diffusible products that another plasmid lacks for transfer to another host cell.
Non-conjugative recombinant plasmid: a DNA molecule that cannot be transferred from its host cell to another host cell by itself during cell / cell contact. For the transfer, additional functions will be required which are provided by another DNA, e.g. B. by (a) helper plasmid (s).
Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar residue (pentose), a phosphate and a heterocyclic nitrogen base. The base is attached to the sugar residue via a glycosidic bond (1'-carbon atom of the pentose), and this combination of base and sugar is a nucleotide. The base identifies the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").
Phenotype: the characteristics of an individual to be observed that arise from the interaction between the genotype and the environment in which the development takes place.
Plasmid: a non-chromosomal double-stranded DNA sequence comprising an intact "replicon" so that the plasmid is replicated in a host cell. When the plasmid is introduced into a unicellular organism, the characteristics of that organism are changed or transformed as a result of the plasmid DNA. For example, a plasmid carrying the gene for tetracycline resistance (Tc ^R ) transforms a cell that was previously sensitive to tetracycline into a cell that is resistant to tetracycline. A cell transformed by a plasmid is called a "transformant".
Polypeptide: a linear series of amino acids linked by peptide bonds between the α-amino and carboxyl groups of the neighboring amino acids.
Promoter region: DNA sequences upstream of the start of the coding sequence which regulate the transcription of the gene:
Promoter sequence: sequence to which RNA polymerase binds and which promotes the reliable transcription of downstream sequences.
Recombinant DNA molecule or hybrid DNA: a hybrid DNA sequence comprising at least two nucleotide sequences, the first sequence not normally occurring together with the second in nature.
Recombination: the creation of a new compilation of DNA molecules or parts of DNA molecules.
Homology region: a DNA region that comprises the same nucleotide sequence as that which is present in a region of another DNA.
Replicon: a self-replicating genetic element that has a site for the initiation of DNA replication and genes that designate the functions necessary for the control of replication.
Restriction fragment: a DNA molecule that results from double-stranded cleavage by an enzyme that recognizes a specific target DNA sequence.
RNA polymerase: enzyme that causes the transcription of DNA into RNA.
Selectable marker gene: a DNA sequence that, when expressed, gives this cell a growth advantage over cells that do not contain this DNA sequence when all cells are in a growth medium that distinguishes the two cell types can. Commonly used genes for a selectable marker are those that encode antibiotic resistance.
Simple crossover: The process of recombining two circular DNA molecules to form a cointegrated larger ring.
Structural gene: a gene that encodes a polypeptide.
T-DNA: part of the Ti plasmid that is found stably integrated in the plant cell genome.
T region: part of the Ti plasmid that contains the DNA sequences that are transferred to the plant cell genome.
Ti plasmid: large plasmids found in strains of Agrobacterium tumefaciens and which contain the genetic information for the induction of (root neck gall) tumor in susceptible plants.
TL-DNA and TR-DNA: Octopin bile duct tumor cells can contain two T-DNA sequences, a left T-DNA, ie TL-DNA, and a right one TR-DNA. TL-DNA contains sequences that are also found together in T-DNA from nopaline tumor cells, but TR-DNA does not.
tra (transfer functions): both plasmid-encoded diffusible products and sites of action used during DNA transfer between cells, ie products required to bridge two cells and the site at which initiates the DNA transfer.
Transcription: Process of producing mRNA from a structural gene
or: A process involving base pairing whereby the genetic information contained in the DNA is used to place a complementary base sequence in an RNA chain.
Transformation: genetic modification induced by the incorporation of exogenous DNA into a cell's DNA complement.
Translation: Process of making a polypeptide from mRNA
or: Process whereby the genetic information present in an mRNA molecule determines the arrangement of specific amino acids during the synthesis of a polypeptide.
Undifferentiated phenotype: a homologous appearance of cells in a tissue without any specialized parts.
Vector: a DNA molecule designed for transfer between different host cells.

The development of DNA recombination techniques the genetic engineering of microorganisms is a challenge made. These techniques could extended to multicellular eukaryotes, provided complete organisms could be regenerated from individual somatic cells. The Cells from some higher plants own extraordinary Skills regeneration, and therefore they make a good material for genetic engineering Producing higher Organisms.

A major problem of genetic engineering Plant production is the availability of a system for introduction of exogenous (foreign) DNA into the plant genome. Such a system is caused by the Gram-negative soil bacterium Agrobacterium tumefaciens-borne tumor-inducing (Ti) plasmids are provided. It has been shown that this Organism in many different dicotyledonous plants neoplastic transformation, the so-called "root neck bile", caused by wounded tissue. The rampant neoplasms synthesize novel, Ti-specific metabolites, the so-called opines. The molecular basis of this transformation consists of the transfer and the stable incorporation of a well-defined T-DNA (transferred DNA) fragment of the Ti plasmids in the plant cell genome. In other words included Root neck gall tumors in their chromosomal DNA a DNA segment, called the T-DNA, which is used to induce the DNA sequences in the Tumor plasmid used is homologous. Corresponds in all cases this T-DNA and is colinear with a continuous range of Ti plasmid DNA, which is therefore called the T region.

The Ti plasmids are of the type of the opine synthesized in the root graft cells. Agrobacterium strains the synthesis of nopaline [N-α- (1,3-dicarboxypropyl) -L-arginine] induce nopaline strains called and tribes, which the synthesis of octopine [N-α- (N-1-carboxyethyl) -L-arginine] induce are called octopin strains. These are the most common used Agrobacterium strains.

The use of T-DNA as a Vector for Genetic engineering of plants was carried out in a pilot project shown in which the bacterial 14 kb transposon Tn7 in vivo in nearby the right edge of the T-DNA from the Ti plasmid of the strain Agrobacterium T37 was installed. The tumors lacked nopaline synthesis, which are stimulated by the Agrobacteria carrying this Ti plasmid were. Moreover the Southern blot hybridizations made it clear that the whole Tn7 in the chromosomal DNA of these tumors as part of an otherwise normal T-DNA sequence was present (Hernalsteens et al., Nature, 287 (1980), 654-656; Holsters et al., Mol. Gen. Genet. 185: 283-289 (1982). Hence the introduction of this 14 kb DNA fragment in the 23 kb T-DNA the ability of the latter to transfer into plant cell genomes to be changed.

The edges of the T-DNA from the Ti plasmid of the Nopalin strain Agrobacterium T37 are located very precisely Service. It represents only a part, approximately 23 kb, of the entire Nopalin-Ti plasmid. Moreover are the edges The T-DNA is known: the nucleotide sequences that make up the edges of the T-DNA define, are located and with the same area of the Nopalin-Ti plasmids have been compared (Zambryski et al., Science 209: 1385-1391 (1980); Zambryski et al., J. Mol. Appl. Genet. 1: 361-370 (1982). The margins the T region are most likely involved in the integration of T-DNA into the plant cell genome.

Knowing the T-DNA sequences that define the edges of the transferred DNA is a basic requirement for using the Ti plasmid as a vector for the transfer of DNA to plant cells. In this way, foreign DNA can be inserted within these edges, which ensures their transfer to the plant cell genome. It is also important, if you want to use this system, that the transformed plant cells are normal rather than tumorous in their growth properties. In order to produce normal cells after T-DNA transfer, knowledge is that encoded by the T-DNA itself Features required. Therefore, the T region of the Ti plasmid has been subjected to intensive genetic analysis to determine which areas are responsible for the tumor phenotype.

The T-DNA encodes functions that responsible for the root neck phenotype are. The genes were localized to specific areas of the T-DNA (Leemans et al., EMBO J. 1 (1982), 147-152; Willmitzer et al., EMBO J. 1 (1982), 139-146). Generally there are at least 4 genes that make up the undifferentiated phenotype control of tumor callus tissue. Mutants of these genes can too lead transformed tissues, which is either sprout-like or Root-like are. The latter results are particularly important if you plan to Transfer DNA to plants and prefer to express in normal plant tissue than in tumor tissue.

Recently a mutated Ti plasmid was found, the transformed rung induced that to. Regeneration in completely normal plants. These plants were fertile and transmitted T-DNA specific Sequences even through meiosis, i.e. H. the offspring plants still contained T-DNA-specific sequences (Otten et al., Mol. Gene. Genet. 183: 209-213 (1981). However, the transformed plant tissue contained in its chromosomal DNA a T-DNA, the size of which is very was greatly reduced, which was due to the formation of a large deletion was based, whereby the one controlling the tumor phenotype Area of T-DNA was removed. It is not known if the deletion while of the original Transformation event or as a subsequent event, that for sprout formation led, took place.

The Ti plasmids are large (200 kb) and many genes located at different locations on the Ti plasmid are involved in the transformation of plant cells. thats why it is not possible a small, unique cloning vector derived from the Ti plasmid Endonuclease recognition sites at appropriate locations in the T region to construct that has all the functions necessary for the transmission and the stable incorporation of the T-DNA into the plant cell genome is necessary. A well-known way, a chosen one DNA fragment in specific restriction enzyme sites of the T region insert a Ti plasmid in constructing cloning plasmids necessary for replication are capable of both Agrobacterium and Escherichia coli and the one selected Contain restriction fragment of T-DNA. Such cloning plasmids were referred to as "intermediate vectors". Such intermediate Vectors were used to analyze the functions encoded by the T region used (Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164).

The present invention relates to one embodiment, as in the claims is characterized. It describes a procedure for the introduction of expressible genes in plant cell genomes of dicotyledonous plants. Modified acceptor Ti plasmids which introduce any (any) desired Enabling genes (genes) in these plasmids are described. The to be introduced desired) Gene (s) is (are) contained in a novel intermediate cloning vector, that carries an area that is homologous to a corresponding region in the acceptor Ti plasmid is.

The introduction of the desired one Gens (genes) in the acceptor Ti plasmids are identified by a simple Crossover event achieved within the two homologues DNA segments of the Agrobacterium-housed acceptor Ti plasmid and the intermediate Cloning vector takes place. The intermediate cloning vector is made from Escherichia coli mobilized from where it is on Agrobacterium Use of helper plasmids is spread. Such helper plasmids and their functions for mobilization are known (Finnegan et al., Mol. Gen. Genet. 185: 344-351 (1982); Figurski et al., Proc. Natl. Acad. Sci. USA 76 (1979), Ditta et al., Proc. Natl. Acad. Sci. USA 77: 7347 (1980).

The result of the simple crossover event in Agrobacterium is a hybrid Ti plasmid vector.

The emerging ones in Agrobacterium harbored hybrid plasmid vectors (hereinafter referred to as vector composition) can be used directly to infect dicotyledonous plant cells, which are then subsequently the expression of the product (s) of the desired one Genes are searched. This strategy is on everyone plant-transferable plasmids of Actrobacterium applicable.

1 illustrates an embodiment of an acceptor Ti plasmid of the invention which is accomplished by deleting the internal portion of the T region except for the edge sequences ( 1 ) and ( 2 ) comes about. The marginal sequences are essential for the integration of the T region into a plant cell genome. The area ( 3 ) between the edge sequences ( 1 ) and ( 2 ) represents the DNA segment that will be transferred to the plant. The acceptor Ti plasmid contains a DNA segment ( 3 ) with a DNA sequence that is homologous to at least part of a DNA sequence in an intermediate cloning vector, which enables the integration of the intermediate cloning vector with the aid of a simple crossover event. The Ti plasmid region ( 4 ) encodes functions that are essential for the transfer of the T region into plant cell genomes by Agrobacterium. This area was called the vir area.

2 illustrates an intermediate reference cloning vector that is inserted into the acceptor Ti plasmid by a simple crossover event 1 should be introduced. It contains a DNA segment ( 3 ' ) from a cloning vehicle with a DNA sequence that connects to at least part of the DNA segment ( 3 ) of the acceptor Ti plasmid is homologous, which enables the desired simple crossover event. In addition, the intermediate reference cloning vector contains a desired gene or group of desired genes ( 5 ) that contain its natural promoter sequence. Plant genes can generally be used in this setup because they are more likely to be expressed. In principle, however, any desired natural genes can be inserted. The intermediate reference cloning vector can also contain a gene for a selectable marker ( 6 ) contain. This gene should contain a promoter sequence which allows expression of the gene in plant cells. Plant cells containing this marker gene should have a selective growth advantage over cells without this gene; in this way, plant cells which have been transformed by the DNA containing this marker gene can be distinguished from untransformed plant cells.

3 illustrates another embodiment of an intermediate cloning vector that corresponds to the intermediate reference cloning vector of 2 is similar and by a simple crossover event into the acceptor Ti plasmid of 1 should be introduced. It contains the DNA segment ( 3 ' ) of the cloning vehicle, an exogenous promoter sequence ( 8th ), which regulates the expression of the desired gene (s) ( 7 ) and, if desired, a gene for a marker ( 6 ).

4 illustrates schematically the construction of hybrid Ti plasmid vectors from the acceptor Ti plasmid of 1 and the intermediate cloning vectors of 2 and 3 through a simple crossover event.

5 represents the steps involved in the genetic transfer of an E. coli intermediate cloning vector to Agrobacterium containing an acceptor Ti plasmid. The first step involves conjugation of the E. coli strain ( 1 ) containing the intermediate cloning vector to another E. coli strain ( 2 ), which contains two helper plasmids for later conjugation to Agrobacterium. One helper plasmid contains DNA sequences that are important for plasmid transfer (tra) and the other helper plasmid contains sequences that are important for mobilization (mob). If these helper plasmids are conjugated into E. coli strain ( 1 ) are introduced, this enables the transfer of the intermediate cloning vector contained therein to other bacterial strains. The tra and mob helper plasmids also contain brands for antibiotic resistance Ab ^r2 and Ab ^r3 , which by the. markers found in the intermediate cloning vector (Ab ^r1 ) are different. In this way, the presence of all plasmids on selective media can be monitored. A mobilization strain ( 3 ) is preserved. This molecularization strain ( 3 ) is transferred to an A. tumefaciens strain ( 4 ) conjugating the acceptor Ti plasmid from 1 contains, followed by the selection for antibiotic resistance marker (s) of the intermediate cloning vector. Since the intermediate cloning vector cannot replicate in Agrobacterium, it can only be maintained if it has formed a cointegrated structure with the accepting Ti plasmid; this cointegrated structure in Agrobacterium ( 5 ) represents the final hybrid Ti plasmid used for the transfer of DNA into plant cell genomes.

6 explains the construction of a model of the acceptor Ti plasmid (A) that corresponds to that in 1 shown is analog. There is a double crossover event between a Ti plasmid and another plasmid that contains a DNA sequence that is intended to replace part of the original Ti plasmid. In particular, the smaller plasmid contains the edge sequences of the T region ( 1 ; 2 ) in a cloning vehicle ( 3 ). A double crossover deletes the internal part T of the T region and replaces it with the cloning vehicle. The acceptor Ti plasmid (A) obtained can be the between the edge sequences ( 1 ; 2 ) transferred contained DNA into plant cell genomes. The resulting transformed plant DNA will not produce a tumorous bile duct tissue because the genes controlling neoplastic growth are deleted in the Ti plasmid (A). The Ti plasmid (A) is a very generalized acceptor Ti plasmid for any intermediate cloning vector with homology to cloning vehicles ( 3 ). The cloning vehicle ( 3 ) can be a common plasmid such. B. pBR322 (or its derivatives).

7 schematically illustrates the steps leading to the construction of an intermediate cloning vector in E. coli host cells. A desired gene ( 5 ), which is bracketed by the restriction site R1, and a gene for a selectable marker ( 6 ), which is bracketed by the restriction site R2, are placed in a cloning vehicle ( 3 ' ), which contains restriction sites for the enzymes R1 and R2 each simply. All three molecules are cleaved with restriction enzymes R1 and / or R2 and linked together using DNA ligase to form the intermediate cloning vector. The cloning vehicle 3 ' must also contain other DNA sequences encoding antibiotic resistance (Ab ^r1 ) so that they can be used as a selectable marker for bacterial genetics. The desired gene ( 5 ) is under the control of an exogenous promoter, as described in 3 is shown.

8th explained. the construction of another embodiment of an acceptor Ti plasmid (B). Here a double crossover event takes place between a Ti plasmid and a cloning v ehikel ( 3 ) that the DNA sequences ( 9 ) and ( 10 ) that includes the just outside the border sequences ( 1 ) respectively. ( 2 ) lying Ti sequences are homologous. The double crossover event leads to the deletion of the entire T region T including the edge sequences ( 1 ) and ( 2 ) and their replacement by the cloning vehicle ( 3 ). Ti plasmid (B) is an acceptor for intermediate cloning vectors that clones the desired gene between the edge sequences ( 1 ) and ( 2 ) contain; (please refer 9 ).

9 illustrates an intermediate cloning vector by a simple crossover event in the acceptor Ti plasmid (B) from 8th to be inserted. It contains the edge sequences ( 1 ) and ( 2 ) that have the desired gene (s) ( 5 ) flank. It also contains a cloning vehicle sequence ( 3 ' ) that is at least partially homologous to the cloning vehicle sequence ( 3 ) in the acceptor Ti plasmid (B), which enables homologous recombination between the two plasmids.

10 explains schematically the construction of hybrid Ti plasmid vectors from the acceptor Ti plasmid (B) of 8th and the corresponding intermediate cloning vector of 9 , A simple crossover event carries the intermediate cloning vector from 9 into the acceptor Ti plasmid (B) from 8th on.

11 explains the insertion of the 5.2 kb HindIII fragment AcgB in pBR322 (see also Zambryski et al., Science 209 (1980), 1385-1391). This AcgB fragment contains the right and left margins of the T-DNA of the nopaline Ti plasmid. This clone pAcgB is used in the construction of acceptor plasmid pGV3850, as in 6 "A-like" acceptor plasmid shown. It will be apparent to those skilled in the art that a clone analogous to clone pAcgB can be obtained using the cloned restriction fragments containing the left and right margins of a wild-type Ti plasmid.

12 illustrates the T region of nopaline Ti plasmid pGV3839. The HindIII restriction endonuclease sites are indicated by (H). The mutated HindIII fragment 19 is with ( 19 ' ) designated. The acetylphosphotransferase gene that provides kanamycin or neomycin resistance is designated apt and is in the black area. The edges of the T region are indicated by arrows. The nopaline synthase gene is designated nos. The numbers refer to the respective sizes of the restriction fragments according to Depicker et al., Plasmid 3 (1980), 193-211. The construction of the Ti plasmid pGV3838 can be found in Example 1 and the two cited documents.

13 explains the construction of acceptor Ti plasmid pGV3850. The plasmid pBR322-pAcgB ( 11 ) is shown in a linearized form. The pBR322 sequences are characterized by the hatched area and the ampicillin resistance gene of pBR322 by Ap ^R. Here is part of the T region of the in 12 pGV3839 shown; the HindIII fragments involved in the homologous recombination with pAcgB ( 10 ) and ( 23 ) and the apt gene are labeled. Duplicate crossover events lead to the construction of pGV3850 and another replicon, which contains the lost apt gene.

14 explains schematically the construction of the intermediate cloning vector pGV700, which is shown in detail in Example 2. The following abbreviations are used to indicate the restriction endonuclease sites: B, BamHI; Bg, BglII; E, EcoRI; H, Hind III; S, SalI; Sm, SmaI. The following abbreviations are used to denote antibiotic resistance; Ap, ampicillin; Cm, chloramphenicol; Sm, streptomycin; Tc, tetracycline. The numbers at the bottom of the figure relating to TL-DNA indicate the RNA transcripts of this area (Willmitzer et al., EMBO J. 1 (1982), 139-146).

15 explains the structure of the intermediate cloning vector pGV750. Its construction is described in Example 2. The restriction endonuclease sites are labeled with their relative positions, which are given in numbers as kilobase pairs (kb). PstI sites are not identified, but there are three in the Km ^R / Nm ^R area and one in the Cb ^R gene. The right and left margins are also shown. The BglII / BamHI and HpaI / SmaI sites used in the construction of pGV750 are designated, but are not available in pGV750. The tinted area corresponds to the TL-DNA, the dark area to the Km ^R / Nm ^R area, the white area to the neighboring Ti plasmid sequences and the line to the cloning vehicle pBR325. Other abbreviations used are: Ocs, octopine synthase; Cm ^R , chloramphenicol resistance; Cb ^R , resistance to carbenicillin (analogous to ampicillin) and Km ^R / Nm ^R , kanamycin resistance / neomycin resistance.

16 explains the construction of the intermediate vector pGV745 described in detail in Example 3.

pGV745 is used in the construction of the acceptor plasmid pGV2260, an in 8th "B-like" acceptor plasmid shown. The restriction endonuclease sites are labeled as follows: B, BamHI; H, Hind III; R, EcoRI. The gene for ampicillin resistance is designated Ap ^R. The hatched area denotes DNA that is homologous to the left side of the T-DNA area of the octopine-Ti plasmid, and the white area denotes DNA that is homologous to the right side of the T-DNA area of the octopine-Ti plasmid plasmid; the physical position determination and the description of the starting plasmids pGV0219 and pGV0120 can be found in De Vos et al., Plasmid 6 (1981), 249-253.

17 explains the construction of the acceptor plasmid pGV2260. The deletion substitution in pGV2217 is marked as a black box containing the acetylphosphotransferase gene (labeled apt) which provides resistance to neomycin and kanamycin. The intermediate vector pGV745 ( 16 ) is shown in a linearized form; it is at the HindIII position of the in 16 pGV745 shown has been opened. The pBR322 sequence is shown as a hatched section and the gene for ampicillin resistance by Ap ^R. Double crossover events lead to the construction of pGV2260 and the loss of the apt gene. Restriction endonuclease sites are identified as follows: B, BamHI; H, Hind III; R, EcoRI.

18 illustrates the construction of a plasmid pLGV2381 for expression of the genes downstream of the promoter of the nopaline synthase gene (nos). 5 ' and 3 ' refer to the beginning and end of transcription, and ATG and TAA to the codons used to start and stop translation. The thick, sharp line denotes the nos promoter area and the white area the nos coding area. Ap ^R means ampicillin resistance and Km ^R kanamycin resistance.

19 illustrates the insertion of plasmid pAGV10, which contains the complete sequence coding for the octopine synthase (ocs), into plasmid pLGV2381 (see also 18 ) behind the nos promoter, which leads to plasmid pNO1 / 2. The thick black lines refer to the promoter areas and the white area to the ocs coding area. Other names are as in 18 ,

20 illustrates the nucleotide sequences around the promoter region of the nopaline synthase (nos) gene and the nucleotide sequences around the same region following fusion with the coding region of the octopine synthase gene. The fusion point is marked with an asterisk (*). Several restriction endonuclease sites are designated, BamHI, HindIII, SacII. 5 ' and 3 ' relate to the start and stop of transcription. ATG refers to the first codon used in translation and TAA to the stop codon used in translation. The large arrows indicate the coding areas of the nopalin gene in white and the octopine gene in stripes.

Referring to 1 we presented a simplified diagram of an acceptor Ti plasmid. This acceptor Ti plasmid contains the two edge sequences ( 1 ; 2 ) or areas of the tumor-inducing wild-type (Ti) plasmid. The marginal sequences are essential for the integration of the T region of the Ti plasmid into a plenum cell genome. In other words, they are for the integration of any DNA sequence ( 3 ) or the T region that lies between these sequences is essential in a plant cell genome.

The DNA sequence ( 3 ) of the acceptor Ti plasmid contains a DNA segment which is part of at least part of the DNA sequence ( 3 ' ) one in the 2 and 3 intermediate cloning vector shown is homologous. This homology is necessary for cointegration through a simple crossover event (homologous recombination) of the intermediate cloning vector with the acceptor Ti plasmid. The frequency with which cointegrated structures are obtained is largely determined by the length of the homology area. In order to cause a homologous recombination event with a good frequency, ranges from 1 to 4 kb are normally used (Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164).

The acceptor Ti plasmid also contains sequences ( 4 ), the. are essential for the transfer of the T region of the Ti plasmid by Agrobacterium into plant cell genomes.

The construction of such acceptor Ti plasmids and their cointegration with those in the 2 and 3 intermediate cloning vectors presented below is in connection with the discussion of 4 described.

In the 2 and 3 Simplified diagrams of intermediate cloning vectors for cloning any desired prokaryotic or eukaryotic gene (s) to be expressed, that is, to be transcribed and translated in plant cells under the control of a promoter, have been presented. , These intermediate cloning vectors contain a DNA segment ( 3 ' ) from a cloning vehicle containing a DNA sequence which is homologous to at least a part of the DNA segment (3) of the acceptor Ti plasmid, whereby a simple crossover event is made possible. In addition, the intermediate cloning vectors contain at least one desired gene ( 5 ; 7 ), which contains either its natural (reference examples) or an exogenous promoter sequence. The promoter sequence enables expression of the inserted gene sequence (s). The use of an exogenous promoter sequence (tailor-made promoter) can be useful to regulate the expression of the inserted gene (s) in a regulated manner.

Desired genes for the intermediate cloning vectors are for example: DNA fragments or sequences with the genetic Information that supports the synthesis of products such as B. amino acids or Sugar controls to the nutritional or growth potential to modify a plant.

The 2 and 3 each show intermediate cloning vectors which may contain genes for selectable markers (6). Selectable marker genes are, for example, those which code for resistance to antibiotics or toxic analogs (e.g. amino acid analogs), or genes which have a defect in of the receiving host cell. can.

4 explains the structures involved in the construction of the hybrid Ti plasmid vectors and 5 describes the actual conjugation levels involved in the isolation of Agrobacterium carrying the hybrid Ti plasmid vectors. Because the intermediate cloning vector is constructed in E. coli, these steps are necessary to transfer the intermediate cloning vector to the acceptor Ti plasmid in Agrobacterium.

• a) Ersatz der Sequenz des pBR-Clonierungsvehikels durch ein anderes Clonierungsvehikel mit breitem Wirtsbereich wie z. B. das Mini-Sa-Plasmid (Leemans et al., Gene 19 (1982), 361–364), das auch zur Replikation in Agrobacterium fähig ist. Die Manipulationen werden in E. coli ausgeführt. Ein intermediärer Clonierungsvektor wird erhalten.
• b) Konjugation des E. coli-Stammes, der den die gewünschte DNA enthaltenden intermediären Clonierungsvektor trägt, mit einem anderen E. coli-Stamm, der ein Helferplasmid trägt, das sich nicht in Agrobacterium replizieren kann, das aber seine eigene Übertragung und die von anderen DNAs auf Agrobacterium vermitteln kann.
• c) Konjugation des in Stufe (b) erhaltenen E. coli mit Agrobacterium, das ein Ti-Plasmid enthält. Das Helferplasmid geht verloren.
• d) Da der intermediäre Clonierungsvektor sich in Agrobacterium als ein getrenntes Replicon replizieren und vorliegen kann, stellen die in Stufe (c) erhaltenen Konjuganten ein Gemisch aus Zellen die entweder die kointegrierte Struktur zwischen dem intermediären Clonierungsvektor und dem Ti-Plasmid enthalten, oder anderen Zellen dar, die den intermediären Clonierungsvektor und das Ti-Plasmid enthalten, bei denen keine Kointegration stattgefunden hat. Damit nur die kointegrierten Strukturen spezifisch isoliert werden können, muß eine zweite Konjugation an einen anderen Agrobacterium-Stamm ohne ein Ti-Plasmid durchgeführt werden. Diese Übertragung wird durch von dem Ti-Plasmid selbst codierten Funktionen vermittelt. Die Übertragung des intermediären Clonierungsvektors in den zweiten Agrobacterium-Stamm wird nur in Form einer kointegrierten Struktur mit dem Ti-Plasmid erreicht.
• e) Um das endgültige modifizierte Ti-Plasmid mit dem gewünschten Ersatz zu erhalten, wird ein zweites Crossover-Ereignis ausgeführt (Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149–164).

The known transfer method used to produce modified Ti plasmids in which part of the T region has been replaced by an altered sequence comprises many stages. Typically, most recombination manipulations in DNA are carried out in specially designed cloning vehicles such as B. pBR322 (Bolivar, Gene 2 (1977), 75-93). However, these cloning vehicles cannot transfer themselves to Agrobacterium. In the known method, this problem is solved by:

• a) Replacement of the sequence of the pBR cloning vehicle by another cloning vehicle with a broad host range such as e.g. B. the Mini-Sa plasmid (Leemans et al., Gene 19 (1982), 361-364), which is also capable of replication in Agrobacterium. The manipulations are carried out in E. coli. An intermediate cloning vector is obtained.
• b) conjugation of the E. coli strain which carries the intermediate cloning vector containing the desired DNA with another E. coli strain which carries a helper plasmid which cannot replicate in Agrobacterium, but which is capable of replicating itself and of can mediate other DNAs on Agrobacterium.
• c) Conjugation of the E. coli obtained in step (b) with Agrobacterium, which contains a Ti plasmid. The helper plasmid is lost.
• d) Since the intermediate cloning vector can replicate and be present in Agrobacterium as a separate replicon, the conjugants obtained in step (c) constitute a mixture of cells which either contain the cointegrated structure between the intermediate cloning vector and the Ti plasmid, or other cells which contain the intermediate cloning vector and the Ti plasmid, in which no cointegration has taken place. So that only the cointegrated structures can be specifically isolated, a second conjugation to another Agrobacterium strain must be carried out without a Ti plasmid. This transfer is mediated by functions encoded by the Ti plasmid itself. The transfer of the intermediate cloning vector into the second Agrobacterium strain is only achieved in the form of a cointegrated structure with the Ti plasmid.
• e) In order to obtain the final modified Ti plasmid with the desired replacement, a second crossover event is carried out (Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164).

The only other known method is essentially the same as that described above with except that as Step (d) another plasmid associated with the intermediate cloning vector is not compatible, is introduced in Agrobacterium; in this case be selected for cointegration (simple crossover event), because all intermediaries Cloning vectors that remain as independent replicons are lost (Matzke et al., J. Mol. Appl. Genet. 1 (1981), 39-49).

We describe a novel and highly simplified procedure for the introduction of intermediate cloning vectors in Agrobacterium acceptor Ti plasmids. Explained in a few words, based this method on the discovery that helper plasmids from E. coli the transfer of many of the common ones used cloning plasmids (such as pBR322) directly on Agrobacterium enable. Since none of these plasmids can replicate in Agrobacterium, only those with the acceptor Ti plasmid will be preserved can form a cointegrated structure. We also use them cointegrated structure in Agrobacterium as a vector composition directly for the infection of plant cells. In this way, the above Described stages (d) and (e) are eliminated, which reduces the possibilities for using the acceptor Ti plasmids as vectors for DNA transfer on plant cell genomes have been greatly improved by using both the time required to construct modified hybrid Ti plasmids is reduced and the flexibility of the possible constructions is increased.

In this way, as in 5 shown, the introduction of the intermediate cloning vector in the acceptor Ti plasmid achieved in two stages. First, conjugation of E. coli strain ( 1 ) carrying the intermediate cloning vector to another E. coli strain ( 2 ) which carries two plasmids which will support the mobilization of the intermediate cloning vector on Agrobacterium. Typical and preferred examples of these helper plasmids are R64drd11, which contains mob functions, and pGJ28, which contains tra functions (Finnegan et al., Mol. Gen. Genet. 185 (1982), 344-351). A bom site (Warren et al., Nature 274 (1978), 259-261) on the cloning vehicle of the intermediate cloning vector is recognized by the functions encoded by the other two plasmids, thereby enabling transmission. Preferably, all plasmids should contain antibiotic resistance markers to demonstrate their presence. Second, the E. coli strain obtained, ie the mobilization strain ( 3 ), which carries all three plasmids, is conjugated to Agrobacterium, which is an acceptor Ti plasmid with a region which is to egg is homologous to the region of the intermediate cloning vector. A simple crossover event between the intermediate cloning vector and the acceptor Ti plasmid can be detected by selecting for the marker for antibiotic resistance of the intermediate cloning vector.

6 explains schematically the DNA molecules that are used to construct the in 1 shown acceptor Ti plasmid can be used. We call this acceptor Ti plasmid (A) to distinguish it from the other acceptor Ti plasmid (B) ( 8th ) to distinguish. The construction requires a double crossover event between a Ti plasmid and another plasmid, which the edge sequences ( 1 ) and ( 2 ) in a cloning vehicle ( 3 ) wearing. As shown in the figure, the cloning vehicle sequence ( 3 ) between the edge sequence ( 1 ) on the left and ( 2 ) On the right side; to show the correct polarity of the DNA strands, they can be drawn on a circle. However, to show the homology areas used in the double crossover event, this circle has been broken up as indicated. This is an important subtlety to be observed, which is also important in the construction of acceptor Ti plasmid (B) 8th is used, i.e. if the border sequences ( 1 ) and ( 2 ) simply within the cloning vehicle sequence ( 3 ), a double crossover event would result in a Ti plasmid with a deleted T region, but without the cloning vehicle sequence between the edge sequences ( 1 ) and ( 2 ) produce. Like here in 6 is also shown, the double crossover event also creates a circular DNA molecule that retains the original T region between the edge sequences ( 1 ) and ( 2 ) contains; but since this is not a replicon, it will be lost. Genetic selection for these events can be achieved, for example, by the loss of an antibiotic resistance marker contained in the T region of the Ti plasmid and an antibiotic resistance marker contained in the cloning vehicle sequence ( 3 ) is selected.

7 explains schematically the construction of an intermediate cloning vector of the 2 and 3 , A desired gene ( 5 ) and a gene for a selectable marker ( 6 ), each of which is bracketed by a restriction endonuclease site R1 or R2, are inserted into a cloning vehicle sequence ( 3 ' ), each containing a single restriction site for the enzymes R1 and R2, by cleavage and ligation of all molecules. The recombinant DNA molecule obtained is used to transform E. coli host cells and the transformants are placed on the antibiotic resistance marker (Ab ^R1 ) of the cloning vehicle sequence ( 3 ' ) selected.

8th explains schematically the DNA molecules used in the construction of the acceptor Ti plasmid (B). There is a double crossover event between a Ti plasmid and a plasmid that encodes the cloning vehicle sequence ( 3 ) between the DNA sequences ( 9 ) and ( 10 ) that contains just outside the border sequences ( 1 ) and ( 2 ) lie; in order to illustrate the homologous regions used in the crossover event, the small plasmid has been broken up (as in 6 ). The product of the double crossover event is acceptor Ti plasmid (B) and another ring-shaped DNA molecule that precedes the T region from the original Ti plasmid along with the DNA sequences ( 2 ), ( 10 ), ( 9 ) and ( 1 ) contains. The genetic selection can, as for 6 described can be achieved.

9 schematically illustrates an intermediate cloning vector for use together with the acceptor Ti plasmid B of B , Here is the gene you want ( 5 ) between those in a cloning vehicle sequence ( 3 ' ) included edge sequences ( 1 ) and ( 2 ) inserted.

10 explains schematically how a simple crossover event. the intermediate cloning vector of 9 into the acceptor Ti plasmid (B). In this case, the selection for the antibiotic resistance marker of the cloning vehicle sequence ( 3 ' ) of the intermediate cloning vector ensures that a hybrid Ti plasmid can be found which is the result of a cointegration between the two plasmids. A hybrid Ti plasmid is produced which contains the desired gene within the border sequences ( 1 ) and ( 2 ) contains. The hybrid plasmid constructed in this way does not contain a sequence in its T region that represents a direct repetition (such as the sequences ( 3 ) and ( 3 ' ) in the hybrid Ti plasmid of 4 ), which raises potential problems of instability of the hybrid vector or the transformed DNA in the plant cell genome. Consequence of intramolecular recombination can be avoided.

Unpublished results from our laboratory also show that it is essential for the construction of the intermediate vector in 9 is not essential, both marginal sequences 1 and 2 to own; it is sufficient, but it is essential that at least the right margin sequence ( 2 ) is present (see 1 and 9 ) to achieve the integration of the desired DNA sequence into the plant genome.

Knowledge of the restriction endonuclease maps of the Agrobacterium Ti plasmids, e.g. B. the Nopalin or Octopin-Ti plasmids (Depicker et al., Plasmid 3 (1980), 193-211; De Vos et al., Plasmid 6 (1981), 249-253), together with the knowledge of the restriction fragments containing the T-DNA fringe areas (Zambryski et al., J. Mol. Appl. Genet. 1 (1982), 361-370; De Beuckeleer et al., Mol. Gen. Genet. 183 (1981), 283 –288) now enables each researcher to construct acceptor Ti plasmids as described here. All that is required is the ability to use common DNA recombination techniques and perform basic genetic manipulations on bacteria.

The following examples are provided around the disclosed acceptor Ti plasmids, intermediate cloning vectors, Hybrid Ti plasmid vectors and vector compositions continue to increase explain and the effectiveness of the vector compositions in providing of transformed plant cells and plants to show the Expression of the foreign integrated in the plant cell genome Show gens.

EXAMPLE 1

Construction of acceptor Ti plasmid pGV3850 (type A) parent strains and plasmids:

Agrobacterium tumefaciens (rifampicin-resistant strain C58C1 and chloramphenicol-erythromycin-resistant strain C58C1, derived from wild-type Agrobacterium) Ti-plasmid pGV3839 Plasmid of Fig. 11 pAcgB

The Ti plasmid pGV3839 is constructed from a nopaline plasmid pTiC58tra ^c (pGV3100; Holsters et al., Plasmid 3 (1980), 212-230). It contains a deletion substitution mutation near the center of the T region; the SmaI fragment 24, which is internal to HindIII fragment 19 (Depicker et al., Plasmid 3 (1980), 193-211), has been replaced by a HindII fragment of pKC7 (Rao et al., Gene 7 ( 1979), 79-82), which contains the apt (acetylphosphotransferase) gene of Tn5. This gene encodes resistance to the aminoglycosides neomycin and kanamycin. In 12 a restriction map of the T region of pGV3839 is shown.

The plasmid pAcgB represents an incorporation of AcgB in pBR322, in which only the edges of the T-DNA are contained (see 11 ). The edges are defined as the ends of the T-DNA, and these areas play a role in the stable integration of the T-DNA into the plant cell genome. The origin and analysis of this clone have been described in detail (Zambryski et al., Science 209 (1980), 1385-1391). This clone was obtained by re-isolating sections of the T-DNA from transformed tobacco DNA. pAcgB contains the connection of two copies of T-DNA, which are arranged one behind the other so that the left and right edges of the T-DNA are contained. In addition, pAcgB contains the nopaline synthase gene, since this genetic information is very close to the right edge of the T-DNA. The plasmid pAcgB is used to construct an "A-like" acceptor Ti plasmid, pGV3850. 6 shows the structures involved and 13 reflects the regions of DNA that are involved in the double crossover events leading to pGV3850.

The plasmid pAcgB described carries a ColEi-specific bom site in the pBR322 section and can be obtained by using the Helper plasmids R64drd11 and pGJ28 from E. coli on Agrobacterium be mobilized. The plasmids R64drd11 contained in E. coli and pGJ28 are conjugated into the pAcgB-bearing E. coli strain introduced. Transconjugants are considered ampicillin-resistant (from the pBR322 sequences from pAcgB), streptomycin-resistant (from R64drd11) and kanamycin-resistant (from pGJ28) Colonies selected.

The E. coli strain, all three Carries plasmids, is conjugated to Agrobacterium strain C58C1, the rifampicin-resistant and contains the Ti plasmid pGV3839. Ampicillin resistance from pBR322 is used to point to the first simple crossover event with the Nopalin-Ti plasmid. The only way how ampicillin resistance can be stabilized in Agrobacterium, is a crossover event over homologous recombination with pGV3839 through one of the homology areas in nearby the edges the T region. A second crossover event by the other Homology area becomes the central portion of the T region of pGV3839, comprising the apt gene (kanamycin resistance), through the pBR322 sequences of the clone pAcgB replaced. The second recombinants are therefore ampicillin resistant and kanamycin sensitive. To the possibility the isolation of a second recombinant the rifampicin-resistant Agrobacterium, which is the first recombinant (pAcgB :: pGV3839) wearing, with a second chloramphenicol / erythromycin resistant Agrobacterium strain conjugated without a Ti plasmid. This way, a chloramphenicol / erythromycin resistant Agrobacterium pGV3850 can be obtained with one frequency of about one in 600 colonies is ampicillin resistant and kanamycin sensitive.

Of course there are other Ti plasmids for the construction of acceptor Ti plasmids of the pGV3850 type can be used. Any Ti plasmid that has a gene for a selectable marker near the center of the T region carries, can as receiver be used. Furthermore, a pAcgB-like plasmid can be constructed by inserting the edge fragments of the T region into pBR322 that the pBR322 sequences between the left margin fragment and the right one Edge fragment in the alignment of the left edge fragment - pBR322 - right Marginal fragment. The left and right edge fragments of the Nopalin Ti plasmid are, for example, HindIII fragments 10 and 23 (Depicker et al., Plasmid 3 (1980), 193-211).

A simple crossover event will result in an intermediate cloning vector containing any desired gene incorporated into pBR322 (or its derivatives) into the modified one Introduce pGV3850 T-DNA region. It is only necessary that the DNA to be introduced contains a gene for a resistance marker in addition to those already found in pBR322 so that it can be used as a means to select the transfer of the intermediate cloning vector from E. coli to Agrobacterium. This resistance marker can either be contained in the pBR sequences (such as Cm ^R for pBR325 or Km ^R for pKC7) or in the DNA to be tested in the plant cell. In addition, in the acceptor Ti plasmid pGV3850, the Ap ^R gene of pBR322 can be replaced by another resistance marker gene such as e.g. B. Km ^R , to be replaced; in this way even pBR322-containing intermediate cloning vectors that are Ap ^R can be directly mobilized to this pGV3850-like acceptor Ti plasmid.

An additional advantage of the acceptor Ti plasmid of the pGV3850 type is the fact that it is in transformed plant cells no tumors generated. Cells that have been "transformed" by pGV3850 can easily be transformed from untransformed cells can be distinguished by examining for the presence of nopaline be, because the shortened Contains T-DNA region of pGV3850 nor the gene encoding nopaline synthase. If the in the Acceptor Ti plasmid pGV3850 cointegrated intermediate cloning vector contains a marker gene can of course can also be searched directly for him; or it can be selected directly become.

In addition to the insertion of defined intermediate Cloning vectors containing a pBR322 sequence by a simple crossover event into the acceptor Ti plasmid pGV3850 this acceptor Ti plasmid can also be used as a receiver for cloned DNA banks in pBR322 or its derivatives can be used in a "shotgun" experiment. The entire population of hybrid plasmid vectors in Agrobacterium can for. Infection from plant cells can and will be used subsequently for expression of any desired selectable gene (s) searched. For example, one can easily select genes that Amino acid synthesis encode by applying the entire bank to plant cells which the selected one amino acid is missing.

The acceptor Ti plasmid pGV3850 has two phenotypic Features: (i) the inability to To produce tumors, and (ii) the ability to synthesize nopaline, provided the transfer of T-DNA takes place in the plant cell genome. Therefore, several Conducted experiments to in various plant tissues containing Agrobacterium containing pGV3850 are infected to look for these characteristics.

• a) Tests with potato and carrot slices The Inoculation of potato and carrot slices with the acceptor Ti plasmid pGV3850 leads to Generation of a small mass of callus tissue. This tissue is on the presence of nopaline has been tested and found positive. It's interesting, that these Mutant capable is to create small callus tissue; however, it can only get if you let the disks grow in media that low concentrations of both auxins and cytokinins contain.
• b) Inoculating whole plants with acceptor Ti plasmid pGV3850 The growing on sterile agar media (without hormones) Tobacco and Pentunia plants will be inoculated with pGV3850. A small amount of tissue growth can only be observed after several months (usually "wild-type" tumors become after two Weeks proven). This tissue does not grow on hormone-free Media, but can be viewed as sterile tissue culture on media that both Contain auxin as well as cytokinin, can be propagated further. It was also shown that this Tissue is nopalin positive.
• c) Furthermore, since the cells "transformed" with pGV3850 do not behave like tumor cells, are these cells capable of to regenerate normal plants that still have the transferred in their genome DNA segment included. The breeding of transformed cells on usual Regeneration media will lead to the maintenance of normal plants (see also example 5).

To the benefit of pGV3850 as one To demonstrate acceptor plasmid, the following experiment is performed. On intermediate Cloning vector, the oncogenic functions of the Octopin-T-DNA in pBR325 contains is recombined in Agrobacterium harboring pGV3850. The resulting hybrid Ti plasmid in Actrobacterium, obtained through a simple crossover event vaccinated on wounded tobacco plants. After about two weeks tumor tissue develops. This is proof that the tumor-inducing DNA reintroduced into pGV3850 and is accurately expressed in transformed plant cells.

EXAMPLE 2

Construction of the intermediate cloning vectors pGV700 and pGV750

An overview of the constructions is in 14 shown schematically. HindIII fragment 1, which is the right part of the TL-DNA of the octopine-Ti plasmid B6S3 and is present in pGV0201 (De Vos et al., Plasmid 6 (1981), 249-253), is first in the HindIII site of the broad host range vector pGV1122 (Leemans et al., Gene 19 (1982), 361-364). The recombinant plasmid pGV0201 contains the HindIII fragment 1 inserted into the only HindIII site of the multicopy vector pBR322 (Bolivar et al., Gene 2 (1977), 95-113). pGV0201 and pGV1122 DNA are prepared as described by Betlach et al., Fed. Proc. 35 (1976), 2037-2043). 2 μg of pGV0201-DNA are completely cleaved with 2 units of HindIII (all restriction enzymes are obtained from Boehringer Mannheim) for 1 hour at 37 ° C. in a final volume of 20 μl. The incubation buffer is from O'Farrell et al., Mol. Gen. Genet. 179: 421-435 (1980). 2 μg of pGV1122-DNA are completely digested with HindIII under the same conditions.

A tenth of a μg of HindIII-cleaved pGV0201 are ligated to 0.05 μg of HindIII-cleaved pGV1122 with 0.02 units of T4 ligase (Boehringer Mannheim) in a final volume of 20 μl. Incubation buffers and conditions are as recommended by the manufacturer (brochure "T4 Ligase", Boehringer Mannheim, August 1980, # 10.M.880.486). The transformation of the ligation mixture into competent E. coli K514 hsr ^- hsm ⁺ cells (Colson et al., Genetics 52 (1965), 1043-1050) is carried out as described by Dagert and Ehrlich, Gene 6 (1980), 23-28 , described. The cells are plated on LB medium (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York), to which streptomycin (20 μg / ml) and spectinomycin (50 μg / ml) are added. The transformants containing recombinant plasmids are screened for tetracycline sensitivity (10 μg / ml) based on inactivation by insertion of the gene coding for tetracycline resistance (Leemans et al., Gene 19 (1982), 361-364 ). Streptomycin and spectinomycin resistant and tetracycline sensitive clones are physically characterized. Micro-scale DNA preparations are according to Klein et al. (Plasmid 3 (1980), 88-91). The orientation of HindIII fragment 1 in the HindIII site of pGV1122 is determined by SalI cleavage. The cleavage (conditions according to O'Farrell et al., Mol. Gen. Genet. 179 (1980), 421-435) of recombinant plasmids gives two fragments after agarose gel electrophoresis. Fragments of 0.77 kb and 22.76 kb are present in the α alignment, while fragments of 10.33 kb and 13.20 kb are present in the β alignment. In the subsequent cloning, a recombinant plasmid with α orientation is used and called pGV1168.

A BglII-SalI fragment containing the left part of the TL-DNA (including the left margin) is inserted into pGV1168, which is digested with BglII-SalI. This fragment is obtained from the recombinant plasmid pGV0153 (De Vos et al., Plasmid 6 (1981), 249-253), containing BamHI fragment 8, from the T region of pTiB6S3, inserted into the vector pBR322. pGV0153 and pGV1168 DNA are according to Betlach et al. (Fed. Proc. 35 (1976), 2037-2043). 10 μg of pGV0153-DNA are completely cleaved with 10 units of BalII and 10 units of SalI in 1 hour at 37 ° C. in a final volume of 100 μl. The fission mixture opens up. a preparative 0.8% agarose gel was applied. The 2.14 kb BalII-SalI fragment is electroeluted from this gel as described by Allington et al., Anal. Biochem. 85 (1978), 188-196). 2 μg pGV1168-DNA are completely cleaved by 2 units of BalII and 2 units of SalI. A tenth of a µg of BalII-SalI fragment DNA is ligated to 0.02 µg of BglIII-SalI-cleaved pGV1168 with 0.02 units of T4 DNA ligase in a final volume of 20 µl. The ligation mixture is transformed into competent E. coli K514 hsr ^- hsm ⁺ cells (Dagert and Ehrlich, Gene 6 (1980), 23-28). The cells are plated on LB medium (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York), to which streptomycin (20 μg / ml) and spectinomycin (50 μg / ml) are added.

Micro-scale DNA preparations (Klein et al., Plasmid 3 (1980), 88-91) are carried out with streptomycin and spectinomycin-resistant transformants. Recombinant plasmids in which the 2.14 kb BalII-SalI fragment is inserted into BalII-SalI-cleaved pGV1168 are identified by BalII-SalI cleavage, giving two fragments of 2.14 kb and 21.82 kb become. A plasmid with a cleavage pattern corresponding to these molecular weights (2.14 kb and 21.82 kb) is used for the further cloning and is called pGV1171. A 12.65 kb fragment of pGV1171 contains the right and left TL-DNA edge sequences (De Beuckeleer et al., In Proceedings IVth International Conference on Plant Pathogenic Bacteria, M. Ridé (ed.) (1978), INRA, Angers, 115-126) and also genes that enable oncogenic growth (Leemans et al., EMBO J. (1982), 147-152). This HindIII fragment is inserted into plasmid pBR325 (Bolivar, Gene 4 (1978), 121-136). pGV1171 and pBR325 are according to Betlach et al., Fed. Proc. 35: 2037-2043 (1976). 2 μg of each DNA are completely cleaved with 2 units of HindIII in 1 hour at 37 ° C. (incubation buffer is described by O'Farrell et al., Mol. Gen. Genet. 179 (1980), 421-435). One-tenth of a µg of HindIII-digested pGV1171 is ligated to 0.05 µg pBR325 linearized with HindIII with 0.02 units of T4 DNA ligase. The transformation of the ligation mixture into competent E. coli K514 hsr ^- hsm ⁺ is carried out as described by Dagert and Ehrlich, Gene 6 (1980), 23-28. The cells are plated on LB medium (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York), to which carbenicillin (100 μg / ml) has been added. Carbenicillin-resistant clones are screened for sensitivity to tetracycline (10 μg / ml), which is based on inactivation by insertion of the gene coding for tetracycline resistance (Bolivar, Gene 4 (1978), 121-136). Carbenicillin-resistant and tetracycline-sensitive clones are characterized physically by using the micro-scale technique (Klein et al., Plasmid 3 (1980), 88-91) produced DNA is cleaved by a restriction enzyme. Cleavage with BamHI yields 4 DNA fragments: fragments of 0.98 kb, 4.71 kb, 5.98 kb and 7.02 kb are found in the α-orientation, while the β-orientation fragments of 0.98 kb , 4.71 kb, 1.71 kb and 11.20 kb. A recombinant plasmid corresponding to the α orientation is obtained, called pGV700 and used for further experiments.

pGV750 is derived from pGV700 by adding a 2.81 kb BamHI-HpaI fragment coding for kanamycin resistance to a 3.49 kb BglII SmaI fragment coding for functions relevant to oncogenicity internally to that in pGV700 inserted TL region is inserted. The BamHI-HpaI fragment encoding a kanamycin resistance is obtained from λ :: Tn5 (Berg et al., Proc. Natl. Acad. Sci. USA 72 (1975), 3628-3632). λ :: Tn5 is produced as described (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York), pGV700-DNA is according to Betlach et al. (Fed. Proc. 35 (1976), 2037-2043). 2 μg pGV700 DNA are completely digested with 2 units BglII and 2 units SmaI. 2 μg of λ :: Tn5-DNA are completely digested with 2 units of BamHI and 2 units of HpaI. One μg of BamHI-HpaI-cleaved λ :: Tn5 is ligated to 0.2 μg of BglII-SmaI-cleaved pGV700 with 0.5 units of T4-DNA ligase in a final volume of 10 μl (conditions as recommended by the manufacturer). The ligation mixture is transformed into competent E. coli K514 hsr ^- hsm ⁺ cells (Dagert and Ehrlich, Gene 6 (1980), 23-28). The cells are plated on LB medium (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York), to which carbenicillin (100 μg / ml) and kanamycin (25 μg / ml) are added. Cb ^R and Km ^R clones are physically characterized by restriction enzyme analysis of the micro-scale DNA (Klein et al., Plasmid 3 (1980), 88-91). Double cleavage with BalII / BamHI of this DNA gives 3 fragments of 3.94 kb, 5.89 kb and 8.09 kb, while cleavage with HindIII 3 fragments of 2.68 kb, 5.99 kb and 9.25 kb results. A plasmid that shows these cleavage patterns is called pGV750 and is shown in 17 schematically explained.

pGV700 and pGV750 are two different intermediate Cloning vectors, which are the left and right edge sequences the TL DNA of the octopine Ti plasmid pTiB6S3 is also included the internal T region in each of the two plasmids is different Size dropped out. pGV700 contains a shortened one T region with genetic information for octopine synthase (transcript 3) and 3 other products, 4, 6a and 6b (see Willmitzer et al., EMBO J. 1 (1982), 139-146, for a description of the products of the T region). The combination of these three products (4, 6a, 6b) the shoot formation in transformed plants cause. pGV750 contains even less of the T region, i. H. only the octopine synthase gene. The information for the products 4, 6a and 6b is due to the gene for the antibiotic resistance marker which encodes kanamycin (neomycin) resistance.

pGV700 and pGV750 are examples of intermediate cloning vectors that are compatible with B-type acceptor Ti plasmids (see 8th and Example 3) below can be used; some of these vectors are analogous to those in 9 with the exception that they do not contain a desired gene. A desired gene can be easily inserted into these vectors since they each contain individually present restriction endonuclease sites for cloning in their modified T regions (see FIGS 14 and 15 ).

EXAMPLE 3

Construction of acceptor Ti plasmid pGV2260 (type B) parent strains and plasmids

Agrobacterium tumefaciens (rifampicin-resistant strain C58C1 and erythromycin-chloramphenicol-resistant strain C58C1, derived from wild-type Agrobacterium) Ti-plasmid pGV2217 Intermediate vector (Fig. 16) pGV745

Construction of Ti plasmid pGV2217 is detailed (Leemans et al., EMBO J. 1 (1982), 147-152). It contains one Deletion substitution mutation of the entire TL region of the octopine-Ti plasmid: the BamHI fragments 8, 30b, 28, 17a and the left 3.76 kb BamHI-EcoRI fragment BamHI fragment 2 (De Vos. et al., Plasmid 6 (1981), 249-253) by an EcoRI-BamHI fragment of pKC7 (Rao & Rogers, Gene 7 (1979), 79-82), the containing the apt (acetyl phosphotransferase) gene of Tn5 Service. This gene encodes resistance to the aminoglycosides Neomycin and kanamycin.

The construction of the intermediate vector pGV745 is shown in 16 is shown schematically and is described as follows. The recombinant plasmid pGV713 was derived from the octopin-Ti plasmid subclone pGV0219 (De Vos et al., Plasmid 6 (1981), 249-253), which contains the HindIII fragments 14, 18c, 22e and 38c in the α-orientation contains. pGV0219 DNA was completely digested with BamHI and then ligated under conditions which favor self-ligation of the plasmid (final concentration of DNA in the ligation mixture <1 μg DNA / ml). Transformants were selected for ampicillin resistance and physically characterized by restriction enzyme cleavage. A clone that in pGV0219 no longer contains 6.5 kb BamHI fragment was isolated and named pGV713. This clone pGV713 was used in the subsequent cloning (see below). The recombinant plasmid pGV738 was derived from pGV0120 (De Vos et al., Plasmid 6 (1981), 249-253) and contains BamHI fragment 2. pGV0120 DNA was digested with EcoRI and self-ligated (as in the construction of pGV713). Transformants were selected for ampicillin resistance and analyzed by restriction enzyme cleavage. A clone in which the EcoRI fragments 20, 12 and a 2.95 kb EcoRI fragment, containing part of EcoRI fragment 19a and part of pBR322, were deleted, was used for further cloning and was designated pGV738. This plasmid also contains a 5.65 kb EcoRI-BamHI fragment from the right part of BamHI fragment 2 (De Vos et al., Plasmid 6 (1981), 249-253).

Next was the pGV713 DNA cleaved with HindIII and BamHI and the cleavage product on preparative Agarose gel applied. After electrophoresis, this was described in pGV713 contained 2.30 kb HindIII-BamHI fragment purified by electroelution (as described by Allington et al., Anal. Biochem. 85 (1975), 188-196), This fragment was ligated to pGV738 and HindIII and BamHI Completely split. After the transformation, ampicillin became resistant Physically characterized clones by restriction enzyme cleavage. The cleavage with EcoRI and BamHI should, for example, 2 fragments of 3.98 kb (= vector part) and 7.95 kb (= insert part). A recombinant plasmid with these characteristics was made with pGV745 referred to and as intermediate Vector used in the construction of the acceptor Ti plasmid pGV2260.

The plasmid pGV745 contains a ColE1 specific bom position in the pBR322 area and can be mobilized from E. coli on Agrobacterium using the helper plasmids R64drd1l and pGJ28, as described in Example 1 (construction of the acceptor Ti plasmid pGV3850).

pGV745 was grown on Agrobacterium strain C58C1 mobilized, the rifampicin resistant and the Ti plassmid pGV2217 contains. The first crossover event was selected by the ampicillin resistance of pBR322 was used, just as described in Example 1 (construction of the acceptor Ti piasmid pGV3850). By a second The crossover event becomes the deletion substitution mutation present in pGV2217 replaced by the pBR322 sequences from plasmid pGV745. The second Recombinants were removed by using the ampcillin-resistant transconjugants, that came from the co-integration of pGV745 with pGV2217, directly were screened for loss of kanamycin resistance. To this A rifampicin Agrobacterium strain C58C1 was obtained, which pGV2260 (ampicillin resistant, kanamycin sensitive) contained.

This Ti plasmid pGV2260 will be used in the future as a Acceptor plasmid (Type B) for intermediate Cloning vectors of the pGV700 or pGV750 type used. This are composed of (i) a DNA fragment which contains the ampicillin resistance gene, carries the origin of replication and the bom site of pBR322; (Ii) a DNA fragment that encompasses the left and right edge sequences of the Contains TL-DNA and also an additional Resistance markers to those already on pBR322, the both the genetic selection for the transfer of the intermediate cloning vector of E. coli on Agrobacterium as well as on its cointegration into the acceptor Ti plasmid pGV2260 enables.

For example, we were able to show that Agrobacterium, which has a cointegrated structure between pGV2260 and pGV700, is capable of transferring the expected DNA sequences (those contained between the edges of the T-DNA) to plant cell genomes. The transformed plant cells show the expected phenotype, ie tumors that produce shoots if it is assumed that pGV700 contains the genetic information for three products (4, 6a, 6b; see Willmitzer et al., EMBO J. 1 (1982), 139-146). In this way, we have shown that a B-type acceptor Ti plasmid is capable of transferring DNA to plant cells if it is a cointegrated structure with an intermediate cloning vector of the in 9 explained and used in Example 2 is used.

EXAMPLE 4

Construction of an intermediate cloning vector, that contains a gene that is to be expressed in plants

Before the present invention became known, the insertion of whole genes into more or less random positions in the T region of Ti plasmids following the transfer to the plant genome did not lead to the expression of the foreign sequence. As described here is
the coding region of (any) desired foreign gene (s) is bound to transcriptional initiation and termination signals which are known to be functional in the plant cell. The benefit of this approach is
shown by experiments comprising the DNA sequences encoding the nopaline synthase gene. The entire sequence of this gene and the exact start and end of the transcription are known: (Depicker et al., J. Mol. Appl. Genet. 1 (1982), 561-574). The protein coding region of any foreign gene can be inserted adjacent to the nos promoter. As an example foreign gene sequence, the coding region of the octopine synthase gene (De Greve et al., J. Mol. Appl. Genet. 1 (982), 499-512) is inserted adjacent to the nos promoter. This construction is mobilized in an acceptor Ti plasmid and used to infect plants. The resulting tumor tissue is examined for the presence of octopine and found to be positive.

The construction of the intermediate cloning vector containing the chimeric nopaline promoter: octopine synthase structural gene is shown in US Pat 18 to 20 shown and described.

In brief, the restriction fragment HindIII-23, which contains the nos gene, is genetically engineered in vitro in such a way that most of the nos coding sequence is removed and the nos promoter adjacent to the BamHI restriction endonuclease site is retained ( 18 ). 10 μg of pGV0422 (a pBR322 derivative which carries the HindIII-23 fragment which contains the complete nos gene; Depicker et al., Plasmid 3 (1980), 193-211.) Are digested with Sau3A, and that 350 bp fragment carrying the nos promoter is isolated from a preparative 5% polyacrylamide gel. The promoter fragment is ligated to BalII-cleaved pKC7 (Rao et al., Gene 7 (1979), 79-82) which has previously been treated with bacterial alkaline phosphatase (BAP) to remove 5'-terminal phosphate groups. 20 μg of the resulting plasmid (pLGV13) are cleaved with BglII and with 7 units of the Bal31 exonuclease (Biolabs, New England) for 4 to 10 minutes in 400 μl 12 mM MgCl ₂ , 12 mM CaCl ₂ , 0.6 M NaCl, 1 mM EDTA and 20 mM Tris-HCl, pH 8.0, treated at 30 ° C. During this time, approximately 20 to 50 bp of the DNA is removed. The Bal31 treated molecules are digested with BamHI and incubated with the Klenow fragment of DNA polymerase and all four deoxynucleoside triphosphates (10 mM each) to fill in the ends. The plasmids are regenerated on a. BamHI site searched, which comes from the ligation of a filled BamHI end and the end of the Bal31 deletion. The respective size of the BamHI-SacII fragment from different candidates is determined in a 6% urea-polyacrylamide gel, and the nucleotide sequences of the candidates, whose sizes are in the range between 200 and 280 nucleotides, are determined. Clone pLGV81, containing the 203 bp SacII-BamHI fragment carrying the promoter, is used to replace the SacII-BamHI fragment in the nos gene in pGV0422; the final promoter vector is named pLGV2381. All recombinant plasmids are selected by transforming E. coli strain HB101.

The plasmid vector containing the genetically modified nos promoter is digested with BamHI and the sequence coding for ocs contained on a BamHI fragment is inserted at this point. The ocs coding sequence is also genetically modified in vitro in such a way that it is bracketed by the BamHI restriction endonuclease site, as described in 19 is described. 10 μg of BamHI fragment 17a of the octopine Ti plasmid B6S3 (De Vos et al., Plasmid 6 (1981), 249-253) are digested with BamHI and SmaI, the fragment containing the ocs coding sequence is obtained from a 1st % Agarose gel isolated and ligated to the large BamHI-PvuII fragment of pBR322; 20 μg of the resulting plasmid pAGV828 are cleaved with BamHI, with the exonuclease Bal31, as in 18 treated, then cleaved with HindIII, and the ends are filled in and self-ligated. The sizes of the Bal31 deletions are determined in a 6% polyacrylamide gel. The nucleotide sequences of several candidates are determined, and a candidate that has only 7 bp remaining of the untranslated 5 'leader sequence is selected for further work (pOCSΔ). To bracket the ocs sequence with BamHI sites, the ClaI-RsaI fragment is filled in and subcloned into the BalI site of pLC236 (Remaut et al., Gene 15 (1981), 81-93). The resulting plasmid pAGV40 is digested with BamHI, the fragment bearing the ocs sequence is isolated by electroelution from a preparative 1% agarose gel and ligated to pLGV2381, which had previously been digested with BamHI and treated with BAP (bacterial alkaline phosphatase). The insertion of the ocs sequence into pLGV2381 is obtained in both orientations (pNO-1 and pNO-2).

The nucleotide sequences which show the exact connection point in the nos: ocs fusion are shown in 20 shown.

Furthermore, the plasmid vector, which contains the genetically modified nos promoter, is used to insert DNA from the plasmid R67, which encodes the enzyme dihydrofolate reductase. The coding sequence containing the dihydrofolate reductase gene is, as described, contained on a BamHI fragment (O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527-1531) and is therefore simply described in insert the nos promoter vector containing the BamHI site adjacent to the promoter region as described above. This gene is an example of a gene for a selectable marker (see e.g. the 2 . 3 . 4 . 5 and 7 ) because, when expressed, it provides resistance to the antibiotic methotrexate. When this intermediate cloning vector is mobilized into an Agrobacterium containing a wild-type nopaline acceptor Ti plasmid, a simple crossover event occurs and a hybrid Ti plasmid vector is obtained. The vector composition is used to infect plants. It is shown that the resulting tumor tissue is able to grow sustainably in the presence of 0.5 μg / ml methotrexate.

The construction of the intermediate clony tion vectors which contain the ocs- and dihydrofolate reductase-coding regions behind the nos promoter described above, and their transfer and expression in transformed plant cells subsequent to the cointegration with the Ti plasmid from Agrobacterium provides the evidence that foreign genes on plant cells can be transmitted and expressed in it.

EXAMPLE 5

Isolation of plant cells and plants that produce the desired Gene (s) inserted contained in their chromosomes

• (1) Beimpfung in vivo von ganzen Pflanzen, worauf anschließend die Kultur in vitro auf Medien folgt, welche die Regeneration von Sprossen ermöglichen;
• (2) Coinfektion in vivo von ganzen Pflanzen in Gegenwart von anderen Agrobacterium-Stämmen, welche an der verwundeten Stelle direkt Sprosse induzieren;
• (3) Cokultivierung in vitro von einzelnen Pflanzenzellen-Protoplasten.

We have received plant cells and whole plants transformed with non-oncogenic acceptor Ti plasmid derivatives (e.g. pGV3850) using one of the following three methods:

• (1) In vivo inoculation of whole plants, followed by in vitro culture following media that allow sprout regeneration;
• (2) in vivo co-infection of whole plants in the presence of other Agrobacterium strains which directly induce shoots at the wounded site;
• (3) In vitro cocultivation of single plant cell protoplasts.

We'll follow each of these Describe the procedure.

The first method is based on one Modification of procedures that are normally used to infect whole plant tissues with wild-type Agrobacterium strains preserved, which leads to the production of root-gall gall tissues. There pGV3850 is a non-tumor-producing (non-oncogenic) Agrobacterium derivative no tumor growth is observed at the infected site. Unless, however, the infected tissue is removed and in tissue culture can be increased, transformed Tissues can be easily obtained. After an initial cultivation period (simply to increase the mass of the tissue) becomes the tissue of the wound site grown under conditions that allow the formation of sprouts. Both untransformed and pGV3850 transformed cells will Create rung. The transformed rung can easily be replaced by a simple one Test for the presence of nopaline can be distinguished.

We have pGV3850 transformed Calli and rung, decapitated by tobacco plants from Nicotiana tabacum Wisconsin 38, using the following protocol (all manipulations are under sterile conditions in a laminar flow hood).

• (1) 6-week-old tobacco seedlings are used, those in little pots (10 cm diameter × 10 in cm height) on firm Murashige & Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15 (1962) 473-497) 0.8% agar grown were.
• (2) The youngest top leaves are removed with a scalpel and thrown away.
• (3) The wound surface is inoculated with a spatula or toothpick containing Agrobacterium, which is derived from a fresh plate culture grown under selective conditions (e.g. for the rifampicin-resistant, ampicillin-resistant Agrobacterium strain which contains the Ti Plasmid pGV3850 contains, YEB medium containing 100 μg / ml rifampicin and 100 μg / ml carbenicillin; YEB medium: 5 g / l Bacto beef extract, 1 g / l Bacto yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 x 10 ^-3 M MgSO ₄ , pH 7.2, and 15 g / l agar). At least 8 plantlets are inoculated for each pGV3850 construction.
• (4) incubate for 2 weeks; there should be little or no response occur at the site of inoculation; sometimes appear very little calli.
• (5) A thin one Section less than 1 mm thick is removed from the wound surface. The wound surface is incubated on a plate, the Linsmaier & Skoog (LS) agar medium (Linsmaier and Skoog, Physiol. Plant. 18 (1965) 100-127) with auxins and cytokinins (1 mg / l NAA, 0.2 mg / l BAP) and 1% sucrose.
• (6) After about 6 weeks, the callus should be big enough, at least about 5 mm in diameter, to be part of the presence of nopaline testing. Not all wound calli produce nopaline; about one in four Planting creates a nopaline-positive wound callus.
• (7) Nopalin positive calli are transferred to agar plates, the regeneration medium contain: LS medium as above + 1% Sucrose and 1 mg / l BAP cytokinin.
• (8) Rungs of acceptable size (1 cm high) appear after about 4 up to 6 weeks. The sprouts are transferred to fresh agar plates, the LS medium + 1%. Sucrose without hormones included for more Allow growth and rooting.
• (9) The rung is left Grow 1 or 2 weeks so that a Part (one or two small leaflets) for testing the presence of nopaline can be removed.
• (10) Nopaline-positive shoots are used for further breeding larger vessels (10 cm Pots as above), the MS medium as contained in (1).

Note: All plant culture media for infected tissues contain 500 μg / ml of the antibiotic cefotaxim (Claforan ^® , Hoechst) as a selection against the pGV3850-containing Agrobacterium. This drug works so that it is the growth of all agro prevents bacteria (including those that are carbenicillin resistant).

Another method of obtaining of transformed and sprouting tissues is recently in ours Laboratory. This process is based on the observation that certain mutated Ti plasmid strains of Agrobacterium induce tumors that sprout produce. Such shoot-inducing (shi) mutants can be found in a specific area of T-DNA (Transmitted DNA segment) of the Ti plasmids map from A. tumefaciens (Leemans et al., EMBO J. 1 (1982) 147-152; Joos et al., Cell 32 (1983) 1057-1067), The induced rungs are often from completely normal untransformed Cells assembled. We therefore tried to plant with one Inoculate mixture of two different Agrobacteria, whereby one an octopine-Ti plasmid shoot mutant and the other wore pGV3850. In this way there is a good one Chance that Octopin sprout mutation rung which can be transformed with pGV3850. We have inoculated plants with Agrobacterium, which is the Ti plasmid pGV3850 and a shoot-inducing Octopin-Ti plasmid in a ratio of 5: 1 contained. This way we have pGV3850 transformed Preserved rung; these rungs can be easily picked out by is tested for the presence of nopaline. Through this procedure complicated tissue culture procedures are no longer necessary. The nopaline positive Rungs are transferred to media, the simple salts and sugars with some growth regulators Contain hormones, which enables further growth. After the rung has reached a sufficient size, you can easily transferred to earth for propagation become. This method of coinfection should be particularly useful for the transformation of plant species that are not easily accessible to tissue cultures. In this way it will be possible a whole range of agronomically and economically important plants, such as B. legumes, medicinal plants and ornamental plants to be genetically engineered by Agrobacterium.

The third method enables Isolation of Nicotiana tabacum protoplasts and selection of hormone independent T-DNA transformed cell clones after coculturing of protoplasts derived cells with oncogenic Agrobacterium strains. A analog technology can for that Selection of transformed cells to be used when others dominant selective markers are used, such as. B. genes for antibiotic resistance, which are constructed so that they in higher plant cells can be expressed (see example 3).

In this case, however, the conditions for the selection must be optimized in any case (concentration of the selective agent, time between transformation and selection, concentration of the cells or cell colonies derived from protoplasts in the selective medium), provided that no selection for the transformed cells is possible , e.g. B. because avirulent T-DNA mutants are used, such as. B. pGV3850 or pGV2217 (Leemans et al., EMBO J. 1 (1982), 147-152), it is possible, after genetic transformation, on medium containing auxin and cytokinin (e.g. Murashige-und- Cultivate Skoog medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473-497) with 2 mg / l NAA (α-naphthalene acetic acid) and 0.3 mg / l ki netin) and transform the colonies by their opine Identify content. In this way, after electrophoretic analysis for agropine and mannopine synthesis (method see Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164), about 660 colonies obtained after infection with pGV2217 , a Nicotiana tabacum SR1 cell line, which synthesizes the TR-coded opin mannopin (N ² - (1-mannityl) glutamine). Numerous shoots are formed after incubation of callus pieces from this cell line on regeneration medium (Murashige and Skoog medium with BAP (6-benzylaminopurine) (1 mg / l) as the only growth regulator of the plant cells). All 20 shoots analyzed can still synthesize mannopine. After transfer to hormone-free Murashige-and-Skoog medium, the shoots grow as morphologically normal tobacco plants that still contain mannopin.

The isolation described here for N. tabacum and transformation of protoplasts can also be used for N. plumbaginifolia become.

2. Experimental method

2.1. Culture conditions for rung

Shoot cultures of Nicotiana tabacum are in 250 ml glass pots on hormone-free Murashige-and-Skoog medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473-497) under sterile conditions in a culture room held (16 hours, 1500 lux white fluorescent light ("ACEC LF 58 W / 2 4300 ° K Economy "), 24 ° C, 70% relative humidity). Five weeks old shoot cultures be for used the isolation of protoplasts.

2.2. Protoplast isolation

Germ-free techniques are used for all stages in the isolation and culture of protoplasts. The protoplasts are isolated by an enzyme mixture method. All leaves, except very young leaves, which are smaller than 2 cm, can be used for the isolation of protoplasts. The leaves are cut into strips with a sharp scalpel knife, which are about 2 to 3 mm wide. 2 to 3 g of the leaf material are in a 50 ml enzyme mixture in one at 18 ° C incubated dark room without movement. The enzyme mixture consists of 0.5% Onozuka R-10 cellulase and 0.2% Onozuka R-10 macerozyme in hormone-free K3 medium (Nagy and Maliga, Z. Pflanzenphysiol. 78 (1976), 453-455). The mixture is sterile filtered through a membrane with 0.22 μm pores and can be stored for at least 6 months at -20 ° C without noticeable loss of activity.

2.3. Protoplast culture

After 18 hours of incubation, the mixture is gently agitated to release the protoplasts. The mixture is then filtered through a 50 μm sieve and the filtrate is transferred to 10 ml centrifugation tubes. After centrifugation at 60 to 80 g for 6 minutes in a swinging bucket rotor, the protoplasts form a dark green floating band. The liquid under the protoplasts and the debris forming the pellet are removed using a capillary tube connected to a peristaltic pump. The protoplasts are collected in a tube and washed twice with culture medium. The culture medium consists of K3 medium (Nagy and Maliga, Z. Pflanzenphysiol. 78 (1976), 453-455) with NAA (0.1 mg / l) and kinetin (0.2 mg / l) as growth regulators. The medium is adjusted to a pH of 5.6 and sterilized through a 0.22 μm filter membrane. After the second wash, the protoplasts are counted using a Thoma hemacytometer (obtained from "Assistant", FRG) and resuspended in culture medium with a final density of 10 ⁵ protoplasts / ml. The protoplasts are grown in a volume of 10 ml per Petri dish in tissue culture quality with a diameter of 9 cm. The dishes are sealed with Parafilm ^® and incubated for 24 hours in the dark and then in dim light (500 to 1000 lux) at 24 ° C.

2.4. Transformation through Co-cultivation

The protoplast cultures are grown 5 days after isolation. infected. Agrobacterium cultures are grown for 18 hours in liquid LB medium (Miller, Experiments in Molecular Geneticics (1972), Cold Spring Harbor Laboratory, New York) and resuspended in K3 culture medium with a density of 2 × 10 ⁹ cells / ml. 50 .mu.l of this suspension are added to the plant protoplast cultures, and after sealing with Parafilm ^®, the cultures are incubated under the same conditions as described under 2.3. After 48 hours, the cultures are transferred to 10 ml centrifuge tubes and centrifuged in a vibrating beaker rotor at 60 to 80 g for 6 minutes. The floating band and the pellet are combined and in 10 ml of K3 medium (Nagy and Maliga, Z. Pflanzenphysiol. 78 (1976), 453-455), which with an antibiotic (carbenicillin 1000 μg / ml or cefotaxime 500 μg / ml ) is supplemented, resuspended.

After a 2-week incubation, the protoplast-derived microcalli are centrifuged and resuspended in K3 medium (Nagy and Maliga, Z. Vegetable Physiol. 78 (1976), 453-455), with the same concentrations of growth regulator and antibiotics as above, but 0 , 3 M sucrose instead of 0.4 M can be used. The cell density in this medium is adjusted to approximately 2 5 × 10 ³ micro calli per ml.

After an incubation of 2 weeks under the same conditions, the calli are transferred to K3 medium, where the same antibiotic concentrations as above, but less Sucrose (0.2 M) and growth regulators (NAA 0.01 mg / l and kinetin 0.02 mg / l) can be used.

After an incubation of 2 to 3 Weeks can the suspected Transformants by their bright green and compact appearance and be recognized by better growth. These colonies then become transferred to hormone-free Linsmaier and Skoog medium (Linsmaier and Skoog, Physiol. Plant. 18 (1965), 100-127), which is solidified with 0.6% agar and reduced antibiotic concentrations contains (Carbenicillin 500 μg / ml or cefotaxim 250 μg / ml).

With the putative transformants on growing in this hormone-free medium, opine tests can be carried out when they have a diameter of 3 to 4 mm. Half everyone Colony is used to detect octopine and nopaline (Aerts et al., Plant Sci. Lett. 17 (1979), 43-50) or agropin and mannopin (Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164) used. With this test, the transformed nature of the hormone-free Medium selected colonies can be confirmed. Then you can selected colonies are grown on antibiotic-free medium.

2.5. Co-cultivation without Selection on hormone-free media

If selection for transformed cells is not possible (e.g. because avirulent T-DNA mutants are used) or is not required because a dominant selectable marker, e.g. For example, a gene for antibiotic resistance in which T-DNA is present, the treatment of cells derived from protoplasts can be simplified (the stages with hormone reduction are then no longer necessary). The protoplasts are treated up to the infection stage as previously described. 48 hours after the addition of the bacteria, the cells derived from protoplasts are centrifuged (6 minutes, 60 to 80 g) and resuspended in AG medium (Caboche, Planta 149 (1980), 7-18), which promotes cell growth at very low levels Density is suitable. The cells are counted using a Fuchs-Rosenthal count mer (obtained from "Assistant", FRG) is used, and resuspended with a density required for the subsequent work. If the colonies have to be manipulated individually for opine tests, plating with low cell density (100 cells derived from protoplasts and cell colonies per ml) results in large cell colonies after one month of incubation. If selection with drugs for the transformed cells is possible, the cells are incubated at a higher density (10 ³ to 10 ⁴ / ml) and the selective agent used is added to the medium in a concentration and at a time appropriate for each Selection type must be optimized.

2.6 regeneration of whole Plants made from callus tissue

It's simple, normal plants from callus tissue (for example, either from the Protoplast transformation or comes from the inoculation of whole plants (see 2.7)). The Callus tissue is placed on Murashigeund-Skoog medium containing 1 mg / ml BAP contains, bred; this Medium induces sprout formation 1 to 2 months. These rungs can transferred to medium without hormones, so that roots form and complete Plant arises.

2.7. Tumor induction on Tobacco seedlings

Tobacco seeds (e.g. Kultur-Wisconsin-38) are surface sterilized by treatment with: 70% denatured ethanol / H ₂ O, 2 minutes; then 10% commercial bleach and 0.1% sodium dodecyl sulfate (SDS); further rinsing five times with sterile H ₂ O.

The sterile seeds are placed in large test tubes (25 mm wide) sown, containing the salts of the Murashige and Skoog medium, solidified with 0.7% agar and covered with polycarbonate lid. Then the tubes in the Culture room incubated (12,000 lux, 16 hours light / 8 hours dark; 70% relative humidity; 24 ° C). After 4 to 6 weeks you can the plants are used. You stay at least one more Optimally usable for months.

The plants should be at least 3 cm high and have four or more leaves. The plants then diagonally with a new, sterile scalpel blade through the youngest Internode decapitated; the upper part of the plant is removed from the tube and bacteria from a culture on agar plate with a flamed micropatula on the wound surface brought.

Tumors appear in the wild type 2 weeks and for some of the weakened mutant strains a longer one Time. This procedure is used to inoculate tobacco (Nicotiana tabacum), Nicotiana plumbaginifolia and Petunia hybrida used.

Final remarks

The present invention provides for the first time dicotyledonous plant cells and plants ready with Transformed Agrobacterium harboring a hybrid Ti plasmid vector, that does not contain T-DNA genes, that control neoplastic growth, and essentially free of internal T-DNA sequences of a wild-type Ti plasmid with the exception of promoter sequences is. Because the influence of oncogenic functions of the T region. on the transfer of DNA from the Ti plasmid was not known on plant cells, it is surprising that anyway the transfer the modified T region containing the desired gene (s) Plant cells takes place. There is cointegration and stable Preserving this transferred DNA in the plant cell genome. Expression can also of the selected one desired Gens (genes) can be reached, provided that the gene or genes are suitable Promoter sequences either contain or are constructed to be them contain. The concept of a simple crossover event between an intermediate Cloning vector containing the selected gene (s) with Executing a specially designed acceptor Ti plasmid simplifies construction from any for the Transformation of plant cells useful hybrid Ti plasmid vector considerably. The specially designed acceptor Ti plasmids contain the DNA segment of a usual Cloning vehicle so that any (any) desired (desired) Gene e) (which is in the same or a related cloning vehicle as Part of an intermediate Cloning vector inserted a cointegrated structure through a simple crossover event can form. The two segments of the cloning vehicle (s) provide the necessary Homology areas for a recombination ready.

• (1) intermediäres Vektorplasmid pAcgB in Escherichia coli K12, HB101;
• (2) Agrobacterium tumefaciens C58C1, Rifampicin-resistenter Stamm, der das Carbenicillin-resistente Akzeptor-Ti-Plasmid pGV3850 trägt;
• (3) intermediäres. Vektorplasmid pGV700 in Escherichia coli K12, Stamm K514 (thr leu thi lac hsdR);
• (4) intermediäres Vektorplasmid pGV750 in Escherichia coli K12 Stamm K514 (wie vorstehend in (3));
• (5) Agrobacterium tumefaciens C58C1, Rifampicin-resistenter Stamm, der das Carbenicillin-resistente Akzeptor-Ti-Plasmid pGV2260 trägt;
• (6) intermediärer Plasmidvektor pNO-1, der den Octopin-Synthase codierenden Bereich unter der Kontrolle des Nopalin-Promotors trägt, in Escherichia coli K12 HB101;
• (7) Stamm, der bei der Mobilisierung von intermediären Vektoren auf Agrobacterium verwendet wird = GJ23, trägt mobilisierende Plasmide pGJ28 und R64drd11 (Van Haute et al., EMBO J. 2 (1983), 411–418); GJ23 ist Escherichia coli K12, JC2926, ein recA-Derivat von AB1157 (Howard-Flanders et al., Genetics 49 (1964), 237–246).

For the microorganisms and intermediate cloning vectors, acceptor Ti plasmids and hybrid plasmid vectors described hereinabove; cultures serve as examples, which were deposited in the German Collection of Microorganisms (DSM), Göttingen, on December 21, 1983, and are identified there as follows:

• (1) intermediate vector plasmid pAcgB in Escherichia coli K12, HB101;
• (2) Agrobacterium tumefaciens C58C1, rifampicin-resistant strain, which carries the carbenicillin-resistant acceptor Ti plasmid pGV3850;
• (3) intermediate. Vector plasmid pGV700 in Escherichia coli K12, strain K514 (thr leu thi lac hsdR);
• (4) intermediate vector plasmid pGV750 in Escherichia coli K12 strain K514 (as above starting in (3));
• (5) Agrobacterium tumefaciens C58C1, rifampicin-resistant strain, which carries the carbenicillin-resistant acceptor Ti plasmid pGV2260;
• (6) intermediate plasmid vector pNO-1, carrying the octopine synthase coding region under the control of the nopalin promoter, in Escherichia coli K12 HB101;
• (7) strain used in the mobilization of intermediate vectors on Agrobacterium = GJ23 carries mobilizing plasmids pGJ28 and R64drd11 (Van Haute et al., EMBO J. 2 (1983), 411-418); GJ23 is Escherichia coli K12, JC2926, a recA derivative of AB1157 (Howard-Flanders et al., Genetics 49 (1964), 237-246).

The following deposit numbers have been assigned to these cultures:
2792 (1), 2798 (2), 2796 (3), 2797 (4), 2799 (5), 2833 (6) and 2793 (7).

Claims (4)

Cell of a dicotyledonous plant, obtainable by agrobacterial transformation, which contains a foreign DNA stably integrated into its genome, which is characterized in that: (a) it does not contain T-DNA genes which control neoplastic growth, and essentially is free from internal T-DNA sequences of a wild-type Ti plasmid except for promoter sequences; and (b) it comprises at least one gene of interest comprising: (i) a coding sequence; and (ii) a promoter region that contains a different promoter sequence than the coding sequence, and wherein the promoter sequence regulates the transcription of downstream sequences containing the coding sequence to generate an RNA in the cell. The cell of claim 1 in which the gene of interest is a structural gene and the RNA is an mRNA which contains a "leader" sequence. Plant composed of cells according to claim 1 or 2. A plant seed according to claim 3, which is built up is made of cells according to claim 1 or 2.