DK173104B1 - Acceptor tumour inducing plasmid(s) - 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

DK 173104 B1

The present invention relates to a vector combination, an acceptor Ti plasmid, an intermediate cloning vector, a method for producing a hybrid Ti plasmid, a hybrid Ti plasmid vector, an Agrobacterium containing the vector combination or the hybrid Ti plasmid vector, and a a method of producing a transformed plant cell and a method of producing a plant using such an Agrobacterium method.

More particularly, the invention relates to DNA sequences which are to be copied into appropriate host plant cells. The recombinant DNA molecules in question are characterized by sequences encoding products, e.g. amino acids or polypeptides useful for plant growth or for improving its quality as a nutrient or for the formation of valuable metabolites, e.g. alkaloids or steroid precursors.

In the specification, the following terms are used: 20 25 30 2 DK 173104 B1

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boom area: DNA region where mob functions specifically stimulate and initiate autonomous DNA transfer.

Boundary sequence: DNA sequence containing the ends of the T-DNA.

Wide-host region replicon: A DNA molecule that can be transmitted and maintained in many different host cells.

Callus tissue: A mass of disorganized and undifferentiated cells.

Cloning: Providing a population of organisms or DNA sequences derived from such organism or sequence by unmarried reproduction, or better isolating a particular organism or part thereof and propagating this sub-fraction as a homogeneous population.

Cloning Agent: A plasmid, phage DNA, or other DNA sequences which can be copied into a host cell characterized by one or a small number of 20 endonuclease recognition sites at which such DNA sequences can be cleaved in a specific manner without consequent loss of an essential biological function of the DNA, e.g. copying, 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. tetracycline resistance or ampicillin resistance. A cloning agent 30 is often called a vector.

Coding sequence: DNA sequence which determines or attaches the amino acid sequence to a polypeptide.

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DK 173104 B1

Cointegrate: The structure that results from a single crossover between two circular DNA molecules.

Trans copying: A process in which a DNA molecule (replicon) not physically bound to another replicon can form or provide a diffusible substance that the second unbound replicon lacks and requires.

10 Conjugation: A method of transferring DNA from one type of baked cell to another type of bacteria during cell-cell contact.

Cross-over: Exchanging genetic material between homologous DNA sequences.

Deletion substitution: Removal and exchange of a DNA sequence with another DNA sequence.

differentiation; Process in which progeny of a cell achieve and maintain specialization in terms of structure and function.

DNA sequence or DNA segment: A linear sequence of nucleotides interconnected by phospho-diester linkages between the 3 'and 5' carbon atoms in adjacent pentose units.

Double crossover: Resolving a cointegrate structure into two circular DNA molecules. This process is used for the exchange of genetic information. One of the two circular DNA molecules has two homologous regions of the target DNA through which recombination can occur. These two regions surround a non-homologous DNA sequence to be replaced by the target DNA. If this second crossover occurs in the same DNA region as the first, the original circular DNA molecules will form. If this second crossover occurs in the second homologous region, then genetic exchange will occur between the two circular regions.

Copy: Copy or expression is the process that a structural gene undergoes to form a polypeptide. Copy is a combination of transcription and translation.

10 Copy Control Sequence: A nucleotide sequence that controls and regulates the copying of structural genes when operably linked to these genes.

F-type plasmid: Plasmid carrying the F (fertility) factor, which allows the transfer of a copy of the plasmid to a host that does not carry the F-factor.

Gene: A two-part DNA sequence, (1) the coding sequence for the gene product and (2) the sequences in the promoter region which control whether or not the gene is copied.

Genome: The entire DNA content of a cell or virus.

The genome includes the structural genes encoding the polypeptide or polypeptides, as well as operator, promoter, and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno sequences.

Genotype: The total amount of genetic information contained within an organism.

Homologous recombination: Recombination between two DNA regions containing 30 homologous sequences.

I-type plasmid: A group of self-transmitting plasmids of an incompatibility group different from F.

Incompatibility: The inability of two DNA molecules to coexist in the same cell in the absence of selective pressure.

Insertion: Addition of a DNA sequence to the DNA sequence of another molecule.

5 DK 173104 B1

Leader sequence: The region of an mRNA molecule extending from the 5 'end to the beginning of the first structural gene. It also includes sites that are important for initiating translation 5 of the coding sequence in the structural gene,

Meiosis: Two consecutive divisions which reduce the initial number of chromosomes from 4 n to 1 n in each of the four product cells. This process is important in gender formation.

mob (mobilization functions): A set of products that only in combination with tra functions promote DNA transfer. Mob can promote the transfer of plasmids containing a bomb site.

Mobilization: A process in which a DNA molecule which is unable to transfer to another cell is assisted for transfer by another DNA molecule.

Mobilization Helper Plasma: A plasmid capable of providing diffusible products that another plasmid lacks in order to be transferred to another host cell.

Nonconjugative Recombinant Plasmid: A DNA molecule which cannot itself be transferred from the host cell to another host cell during cell-cell contact.

In order to be transferred, it requires additional functions added by other DNA, e.g. of one or more helper plasmids.

Nucleotide: A monomeric DNA or RNA unit consisting of a sugar moiety (pentose), a phosphate and a nitrogen-containing heterocyclic base. The base is attached to the sugar moiety over a glycoside bond (the 1'-carbon atom of the pentose) and this combination of base and sugar is called a 35 nucleoside. The base characterizes 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").

DK 173104 B1 6

Phenotype: The observable characteristics of an individual due to the interaction between the genotype and the environment in which the development takes place.

Plasmid: A non-chromosomal double-stranded DNA sequence containing an intact "replicon" so that the plasmid is copied into a host cell. When the plasmid is placed in a single cell organism, the characteristics of that organism are altered or transformed as a result of the plasmid's DNA. For example, a plasmid carrying the tetracycline resistance gene transforms

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(Tc), a cell that was previously sensitive to tetracycline to a cell that is resistant to tetracycline. A cell that is trans-formed by a plasmid is called a "transformant".

Polypeptide: A linear sequence of amino acids interconnected by peptide bonds between the α-amino-20 and the carboxy groups of neighboring amino acids.

Promoter region: DNA sequences that are above the upstream beginning of the coding sequence that regulate the transcription of the gene.

Promoter Sequence: Sequence to which RNA polymerase binds and promotes the true transcription of "with current" sequences.

Recombinant DNA molecule or hybrid DNA: A hybrid DNA sequence containing at least two nucleotide sequences, the first sequence usually not found in nature with the second sequence.

Recombination: Forming a new link between DNA molecules or parts of DNA molecules.

Homology domain: A DNA region that shares the same DNA sequence as that found in another DNA region.

Replicon: A self-copying genetic element that contains a site for DNA copying and genes that specify the functions necessary to control copying.

7 DK 173104 B1

Restriction fragment: A DNA molecule produced by double-stranded cleavage by an enzyme that recognizes a specific target DNA sequence.

RNA polymerase. enzyme which causes transcription of DNA 5 to RNA.

Selectable marker gene: A DNA sequence which, after copying, gives this cell a growth advantage over cells which do not contain this DNA sequence when all the cells are in a growth medium capable of separating the two cell types. Commonly used selectable marker genes are those that encode antibiotic resistance.

Single crossover Recombination of 2 circular DNA molecules to form a cointegrate larger circle.

structural; A gene encoding a polypeptide.

T-DNA; Part of the Ti plasmid, as found stably integrated into the plant cell genome.

T region: Part of the Ti plasmid containing the DNA sequences transferred to the plant cell genome.

Ti plasmid: Large plasmids found in strains of Agrobacterium tumefaciens containing the genetic information for tumor (crown bile) induction on sensitive plants.

25 TL DNA and TR DNA; Octopin crown gall tumor cells may contain 2 T-DNA sequences, one left T-DNA, i.e.

TL DNA, and a right TR DNA. TL DNA contains sequences that are also found together with T-DNA from nopaline tumor cells, which, in contrast, is not the case with TR-DNA.

tra (transfer functions): Both plasmid-encoded diffusible products and sites of action used during DNA transfer between cells, e.g. products necessary to form a bridge 35 between two cells and the site where DNA transfer is initiated.

DK 173104 B1 8

Transcription: Formation of mRNA from a structural gene, or process involving base pairing, whereby the genetic information contained in DNA is used for a complementary sequence of bases in an RNA chain.

Transformation: Genetic modification induced by incorporation of exogenous DNA into the DNA complement in a cell.

Translation: The formation of a polypeptide from mRNA, or the process where the genetic information in an mRNA molecule controls the order 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 to transfer between different host cells.

20 The development of recombinant DNA technology has given the genetic engineering of microorganisms an incentive perspective. These techniques could be transmitted to multicellular eukaryotes if complete organisms could be recovered from single somatic cells. The cells of some higher plants exhibit 25 excellent regeneration properties and are therefore good materials for genetic engineering of higher organisms.

A major problem in plant genetic engineering is the availability of a system for introducing exogenous (foreign) DNA into the plant genome. Such a system is provided by the 30 tumor-inducing (Ti) plasmids carried by the Gram-negative soil bacterium Agrobacterium tumefaciens. This organism has been shown to induce a neoplastic transformation, called "crown gall", in the wound tissue of a very large number of two-gum leaf plants. The propagating neoplasms synthesize novel, Ti-specified metabolites called opines. Molecular basis for this transformation is the transfer and stable integration of a well-defined T-DNA (transferred DNA) fragment of the Ti plasmid into the plant cell genome. In other words, chromal gum tumors in the chromosomal DNA contain a DNA segment, termed the T-DNA, which is homologous to the DNA sequences in the Ti plasmid used to insert the tumor line. In all cases, this T-DNA corresponds to and is colinear with a continuous stretch of Ti plasmid DNA, which is therefore called the T region.

Ten plasmids are classified according to the type 10 opin which is formed in the crown gall cells. Agrobacterium strains which induce the synthesis of nopaline [Na- (1,3-dicarboxypropyl) -L-arginine] in chorionic cells are called nopaline strains, and strains which induce the synthesis of octopin [Na- (N1-carboxyethyl) -L-arginine ], called octopin strains. These are the 15 most commonly used Agrobacterium strains.

The use of T-DNA as a plant reflection vector was demonstrated by a model experiment in which the 14 kb bacterial transposon Tn7 is inserted in vivo near the right border of the T-DNA from the Ti plasmid from the strain Agrobacterium T37.

20 Nopaline synthesis is eliminated in the tumors provoked by agro-bacteria carrying this Ti plasmid. Furthermore, Southern blotting hybridization has shown that the entire Tn7 is present in the chromosomal DNA of these tumors as part of an otherwise normal T-DNA sequence (Hernalsteens et al., Nature 287 (1980), 654). 656, Holsters et al., Mol. Gen.

Genet. 185 (1982), 283-289). Thus, the insertion of this 14 kb DNA fragment into the 23 kb T DNA has not altered the latter's ability to transfer to the plant cell genome.

The boundaries of the T-DNA of the Ti plasmid from nopaline strain 30 but Agrobacterium T37 are certainly very accurate. It forms only a portion, approximately 23 kb, of the entire nopaline Ti plasmid. Furthermore, the boundaries of the T-DNA are known. The nucleotide sequences defining the boundaries of the T-DNA have been determined and compared with the same region of the nopaline Ti-35 plasmid (Zambryski et al. Science 209 (1980), 1385-1391, Zambryski et al. J. Mol. Appl. Genet. 1 (1982), 361-370).

DK 173104 B1 10

The boundaries of the T region are most likely involved in the integration of the T DNA into the plant cell genome.

Knowledge of the T-DNA sequences that determine the boundaries of the transferred DNA is a basic requirement for the use of the Ti plasmid as a vector for transferring DNA to plant cells. Thus, foreign DNA can be inserted within these boundaries to ensure its transmission to the plant cell genome. Furthermore, if one expects to use this system, it is important that the transformed 10 plant cells are normal and not tumor-like in terms of growth characteristics. The formation of normal cells after T-DNA transfer requires knowledge of the functions encoded by the T-DNA itself. Thus, the T region of the Ti plasmid has been subjected to intense genetic analysis to determine the regions responsible for the tumor phenotype.

The T-DNA encodes functions responsible for the crown-bile phenotype. The genes have been localized to specific regions of the T-DNA (Leemans et al., EMBO J. 1 (1982), 147-152 and Willmitzer et al., EMBO J. 1 (1982), 139-146).

In general, there are at least four genes controlling the undifferentiated phenotype for tumor callus tissue. Muntants in these genes can either lead to transformed tissues that appear bulky or rooty. The latter results are particularly important if one wishes to transfer DNA to 25 plants for copying in normal plant tissue and not in tumor tissue.

Recently, it has been found that a mutant Ti plasmid induces transformed shoots which are able to regenerate to completely normal plants. These plants are fertile and even transmit T-DNA specific sequences by meiosis, ie. that progeny plants still contain T-DNA specific sequences (Otten et al. Mol. Gen. Genet. 183 (1981), 209-213). However, the transformed plant tissue in the chromosomal DNA contains a T-DNA which is much smaller in size due to the formation of a large deletion which removed the region of the T-DNA which controlled the morphology. . It is not known whether the deletion occurred during the initial transformation or as a subsequent incident leading to shot formation.

The Ti plasmids are large (200 kb) and many genes located at different sites on the Ti plasmid are involved in the transformation of plant cells. Therefore, it is not possible to construct a small Ti plasmid-derived cloning vector with unique endonuclease recognition sites at appropriate 10 sites in the T region, and having all the functions essential for T-DNA transfer and stable incorporation into the plant cell genome. A known way of inserting a selected DNA fragment into specific restriction enzyme cleavage sites in the T region of a Ti plasmid is to generate cloning plasmids which can make copies in Agrobacterium as well as in Escherichia coli and which contain a selected restriction fragment of T -DNA'et. Such cloning plasmids are referred to as "intermediate vectors". Such intermediate vectors are used to analyze the functions encoded by the T region (Leemans et al., J. Mol. Appl.

Genet. 1 (1981), 149-164).

The present invention relates to the following: 1) A vector combination consisting of: (i) an acceptor Ti-plasmid which is essentially free of internal T-DNA sequences and unable to induce plant tumors comprising : (a) the two border sequences (1, 2) of the T region of a 30 wild-type Ti plasmid, (b) a DNA sequence (3) derived from cloning agent located between the two border sequences, and (c) a DNA segment (4) of a wild-type Ti plasmid containing DNA sequences essential for transmission by Agrobacterium of the T region of wild-type Ti plasmids in DK 173104 B1 12

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and (ii) an intermediate cloning vector, wherein this cloning vector comprises: (a) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants; and (b) a cloning agent segment (3 ') containing a DNA sequence which is homologous to the DNA sequence (b) of said acceptor Ti plasmid allowing a single crossover.

2) The acceptor Ti plasmid pGV2260, DSM 2799.

3) The intermediate cloning vector pGV750, DSM 2797.

A method for producing a hybrid Ti-20 plasmid, comprising preparing a vector combination of the invention in an Agrobacterium and performing a co-integration between the intermediate cloning vector and the acceptor Ti plasmid by a single crossover.

5) A hybrid Ti plasmid vector obtainable by co-integration between either an acceptor Ti plasmid which is unable to induce plant tumors and comprises: (a) the two border sequences (1, 2) of T the region of a wild-type Ti plasmid, (b) a DNA sequence (3) which is free of the oncogenic functions of the wild-type T-DNA, derived from a cloning agent located between the two boundary sequences, and containing a DNA sequence which is homologous to at least a portion of 3θ a DNA sequence in an intermediate cloning vector allowing a single crossover, and a wild-type DNA segment (4) -Ti plasmid containing DNA sequences essential for transfer by Agrobacterium of the T region of wild-type Ti plasmids into plant cell genomes, and an intermediate cloning vector comprising: (a) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants, and (b) a cloning agent segment (3 ') containing a DNA sequence which is homologous to the above DNA sequence (b) in the acceptor Ti plasmid, or between an acceptor Ti plasmid comprising: (A) a DNA segment (4) of a wild-type T1 plasmid without the T region and without the two boundary sequences of the T region and (B) a DNA sequence (3) derived from a cloning agent, and an intermediate cloning vector comprising: (A ') the left and right boundary sequence of a T 20 region of a wild-type Ti plasmid (1, 2), (B ') a cloning agent segment (3') located outside said two boundary sequences which is homologous to the DNA sequence (b) of said acceptor Ti plasmid enabling a single crossover, and (C ') a region substantially free of internal T-DNA sequences of a wild-type Ti plasmid and containing at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants, wherein said region is positioned between the two boundary sequences in a manner which allows its integration into the planar atom, said hybrid Ti plasmid vector at least comprising: (α) the two boundary sequences (1, 2) of the T region of a wild-type T plasmid, (β) not -oncogenic DNA sequences (3, 3 ') derived from a cloning agent, 5 (γ) a DNA segment (4) of the wild-type Ti plasmid containing the DNA sequences essential for transfer of the T region by wild type Ti plasmids with Agrobacterium for plant cell genomes, and ίο (δ) at least one gene of interest (5) under the control of promoter capable of directing gene expression in plants located between the two border sequences (1, 2).

6) An Agrobacterium containing a vector combination or a hybrid Ti plasmid vector of the invention.

7) A method of producing a transformed plant cell, comprising infecting plant cells with an Agrobacterium of the invention.

A process for preparing a plant comprising the following steps: (a) infecting a plant cell with an Agrobacterium of the invention, and (b) forming a plant from the transformed plant cells obtained in step (a).

30

The present invention relates to the introduction of expressable genes into plant cell genomes. Thereby, modified acceptor Ti plasmids are provided which allow the insertion of any 35 interesting genes into these plasmids. The gene or genes of interest to be introduced are contained in a novel intermediate cloning vector carrying a homology region with a corresponding region in the acceptor Ti plasmid.

The introduction of the gene or genes of interest into acceptor 5

The ten plasmids are obtained by a single crossover that takes place within the two homologous DNA segments of the acceptor Ti plasmid found in Agrobacterium and the intermediate cloning vector. The intermediate cloning vector is mobilized from Escherichia coli where it is propagated to Agrobacterium using helper plasmids. Such helper plasmids and their functions for mobilization are known (Finnegan et al., Mol. Gen. Genet. 185 (1982), 344-351).

The result of the single crossover in Agrobacterium is a hybrid Ti-piasmid vector.

15

The resulting hybrid plasmid vectors carried in Agrobacterium (hereinafter referred to as vector composition) can be used directly to infect plant cells which are then screened for the copy of the product or products of the gene or genes of interest. This method can be applied to any of the plant-transferable plasmids from Agrobacterium.

The vector combinations and the hybrid Ti plasmids differ from those previously described (Leemans et al., J. Mol.

Appl. Genet. 1, 149-164 (1981), and Leemans, in Molecular

Biology of Plant Tumors, Academic Press, 537-545) in that they enable simplified production of hybrid Ti plasmids at a single crossover and do not contain T-DNA genes that control neoplastic growth and are essentially free of internal T-DNA sequences. The use of the present vector combinations results in transformed plant cells that do not contain neoplastic growth T-DNA genes or are substantially free of internal T-DNA sequences. This is advantageous as these transgenic plants can be regenerated into morphologically normal transgenic plants. With the present invention, it is possible for the first time to omit all T-DNA genes controlling neoplastic growth, or even all internal T-DNA sequences, and still achieve successful transfer and integration of sequences located between the boundary sequences in the plant genome as well as expression of one or more transmitted genes of interest. Prior to the present invention, it could not be ruled out that some information necessary for transfer and / or integration into the plant genome was located in the T region.

The invention is explained in more detail below with reference to the drawings.

FIG. 1 shows an embodiment of an acceptor Ti plasmid according to the invention, which results from a deletion of the inner portion of the T region except for the boundary sequences (1) and (2). The boundary sequences are essential for the integration of the T region into a plant cell genome. The region (3) between the boundary sequences (1) and (2) is the DNA segment that will be transferred to the plant. The acceptor Ti plasmid contains a DNA segment (3) having a DNA sequence which is homologous to at least a portion of the DNA sequence in an intermediate cloning vector allowing integration of the intermediate cloning vector by a single crossover. .

35 DK 173104 B1 17

The ten-plasmid region (4) encodes functions that are essential for transmission by the T-region Agrobacterium to plant cell genomes. This area is referred to as the viral area.

FIG. 2 shows an intermediate cloning vector of the invention to be inserted by a single crossover into the acceptor Ti plasmid of FIG. It contains a cloning agent DNA segment (3 ') having a DNA sequence which is homologous to at least a portion of the DNA segment (3) in the acceptor Ti plasmid, which permits the desired single crossover. Furthermore, the intermediate cloning vector contains a gene or group of genes (5) that are of interest and which have the natural promoter sequence. Usually, plant genes can be used in this construction as they are very likely to be copied. However, in principle, any natural gene of interest can be inserted. The intermediate cloning vector may also contain a selectable marker gene (6).

This gene should contain a promoter sequence that allows copying of the gene into plant cells. Plant cells containing this marker gene should have a selective growth advantage over cells without this gene. In this way, plant cells that have been transformed by DNA containing this marker gene can be distinguished from non-transformed plant cells.

FIG. 3 illustrates another embodiment of an intermediate cloning vector of the invention similar to the intermediate cloning vector of FIG. 2, and which is to be inserted by a single crossover into the acceptor Ti plasmid of FIG. First

It contains the cloning agent DNA segment (3 '), a promoter sequence (8) which allows for controlled copying of the gene or genes (7) of interest and, if desired, a marker gene (6).

DK 173104 B1 18

FIG. 4 schematically illustrates the formation of hybrid Ti plasmid vectors of the invention from the acceptor Ti plasmid of FIG. 1 and the intermediate cloning vectors of FIG. 2 and 3 at a 5 single crossover.

FIG. 5 illustrates the steps involved in the genetic transfer of an intermediate cloning vector from Escherichia coli to Agrobacterium containing an acceptor Ti plasmid. The first step is the conjugation of the Escherichia coli strain (1) containing the intermediate cloning vector to a second Escherichia coli strain (2) containing two helper plasmids for the 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). When these helper plasmids are introduced by conjugation into Escherichia coli strain 20 (1), the intermediate cloning vector contained therein may be transferred to other bacterial strains. tra and mob helper plasmids contain, r also holds antibiotic resistant markers Ab r 3 and Ab, which are different from the markers that r1 25 exists in the intermediate cloning vector (Ab).

Thus, the presence of all the plasmids can be checked on selective media. A mobilization strain (3) is formed. This mobilization strain (3) is conjugated to an Agrobacterium tumacapiens strain (4) containing the acceptor Ti plasmid of FIG. 1, followed by selection for one or more antibiotic resistance markers from the intermediate cloning vector. Since the intermediate cloning vector cannot copy in Agro-bacterium, it can only be maintained if it has formed a cointegrate with the recipient acceptor Ti plasmid. This cointegrate structure of Agrobacterium (5) is the finished hybrid Ti plasmid, which is used in DK173104 B1 19 to transfer DNA to plant cell genomes.

FIG. 6 illustrates the formation of a model acceptor Ti plasmid (A) analogous to that of FIG. 1. Here, a double crossover 5 is made between a Ti plasmid and another plasmid which contains a DNA sequence which is intended to replace part of the original Ti plasmid. More specifically, the smaller plasmid contains the boundary sequences of the T region (1,2) in a cloning agent (3).

A double crossover results in the deletion of the inner portion T of the T region and exchange thereof with the cloning agent. The resulting acceptor Ti plasmid (A) can transfer DNA contained between the border sequences (1,2) to plant cell genomes. The 15 transformed plant DNA formed will not form tumorous coronary tissue as the genes controlling neoplastic growth are deleted in the Ti plasmid (A). Ti plasmid (A) is a highly generalized acceptor Ti plasmid for any intermediate cloning vector having homology to the cloning agent (3).

The cloning agent (3) may be a conventional plasmid such as pBR322 (or derivatives thereof).

FIG. 7 schematically illustrates the steps leading to the formation of an intermediate cloning vector in Esche-richia coli host cells. A gene (5) having interest * surrounded by restriction endonuclease site R1 and a selectable macer gene (6) surrounded by restriction endonuclease site R2 is inserted into a cloning agent (3 ') containing single restriction sites for the enzymes R1 and R2. All three molecules are degraded by restriction enzymes R1 and / or R2 and ligated or bound together using DNA ligase to form the intermediate cloning vector. The cloning agent 3 '35 should also contain additional DNA sequences encoding antibiotic resistance (Abr) for use as a selectable marker for bacterial genes. The gene (5) of interest may be under the control of its natural promoter or of an exogenous promoter as shown in FIG. 2 and FIG.

Third

FIG. 8 illustrates the formation of another embodiment of an acceptor Ti plasmid (B) of the invention.

Here, a double crossover is made between a Ti plasmid and a cloning agent (3) containing DNA sequences (9) and (10) which are homologous 10 to Ti sequences just outside the boundary sequences (1) and (2), respectively. The double crossing causes deletion of the entire T region T including the boundary sequences (1) and (2) and exchange thereof with the cloning agent (3). Ti plasmid (B) is an acceptor for intermediate cloning vectors containing the gene of interest cloned between the border sequences (1) and (2), cf. FIG. 9th

FIG. 9 illustrates an intermediate cloning vector of the invention to be inserted by a single crossover into the acceptor Ti plasmid (B) of FIG. 8. It contains the boundary sequences (1) and (2) which are located on the sides of the gene or genes (5) of interest. It also contains a cloning agent sequence (3 ') which is at least partially homologous to the cloning agent sequence (3) in the acceptor Ti plasmid (B) to allow homologous recombination between the two plasmids.

FIG. 10 schematically illustrates the formation of hybrid Ti plasmid vectors of the invention from the acceptor

The ten plasmid (B) of FIG. 8 and the corresponding intermediate cloning vector of FIG. 9. A single crossover introduces the intermediate cloning vector of FIG. 9 in the acceptor Ti plasmid (B) 35 of FIG. 8th

DK 173104 B1 21

The invention is explained in more detail with reference to FIG. 11-20 of the drawing.

FIG. 11 illustrates the insertion of the 5.2 kb HindIII fragment AcgB into pBR322 (see also Zambryski et al.,

5 Science 209 (1980), 1385-1391). This fragment AcgB

contains the right and left border regions of the nopaline Ti plasmid. This con pAcgB is used for the generation of acceptor plasmid pGV3850, which is an "A-like" acceptor plasmid as shown in FIG. 6. It will be appreciated by those skilled in the art that a clone analogous to clone pAcgB can be formed by using the cloned restriction fragments containing the left and right border regions of a wild-type Ti plasmid.

FIG. 12 illustrates the T region of nopaline Ti plasmid pGV3839.

The HindIII restriction endonuclease sites are designated (H). The mutated HindIII fragment 19 is designated (19 '). The acetylphosphotransferase gene, which provides camamycin or neomycin resistance, is designated as apt and is found in the black region.

The boundaries of the T range are indicated by arrows. The nopaline synthetase gene is denoted by nos.

The numbers refer to the size of the restriction fragments according to Depicker et al., 25 Plasmid 3 (1980), 193-211. The formation of Ti plasmid pGV3838 is described in Example 1 and in the two literature sites cited.

FIG. 13 illustrates the formation of acceptor Ti plasmid pGV 3850. The plasmid pBR322-pAcgB (Fig. 11) is shown in linear form. The pBR322 sequences are shown at the double-shaded region, and the ampicillin resistance gene of pBR322 at ApR. Part of the T region of pGV3839 shown in FIG. 12 is shown here. The HindIII fragments (10) and (23), which are part of homologous recombination with pAcgB and the apt gene, are shown. Double crossover leads to the formation of pGV3850 and a second replicon, which contains the lost lost apt gene.

FIG. 14 schematically shows the formation of the intermediate cloning vector pGV700, described in detail in Example 2. The following abbreviations are used to denote restriction endonuclease sites: B, BamHi, BG, Bglll, E, EcoRI, H, Hindlll, S, SalI, Sm , Narrow. Furthermore, the following abbreviations are used to designate antibiotic resistance: Aβ, ampicillin, 10 Cm, chloramphenocol, Sm, streptomycin, Tc, tetracycline. The numbers at the bottom of the figure referring to TL DNA indicate the RNA transcripts for this region (Willmitzer et al., EMBOJ. 1 (1982), 139-146).

FIG. 15 illustrates the structure of the intermediate cloning vector pGV750. Its preparation is described in Example 2. The restriction endonuclease sites are indicated by their relative locations listed in numbers as kilobase pairs (kb). Pstl sites are not specified, but there are three in the Km / Nm range and one in

D

20 Cb gene. The right and left boundaries are also indicated. The BglII / Bam HI and HpaI / SmaI sites used in the formation of pGV750 are listed but are not found in pGV750. The gray region corresponds to TL DNA, the black region to the Km / Nm region, and the white region to adjacent Ti plasmid sequences, and the line to the cloning agent pBR325. In addition, the following abbreviations are used: Ocs, octopin synthesis

R R

tase; Cm, chloramphenicol resistance; Cb, carbeni

R R

cillin resistance (analogous to ampicillin) and Km / Nm, 30 kanamycin resistance / neomycin resistance.

FIG. 16 illustrates the formation of the intermediate vector pGV745, which is described in detail in Example 3. pGV745 is used in the formation of the acceptor plasmid pGV2260, which is a "B-like" acceptor plasmid, shown in FIG. 8. The restriction endonuclease sites are designated as follows: B, BamHI; H, Hindlll, DK 173104 B1 23 and R, EcoRI. The ampicillin resistance gene is designated p as Ap. The shaded region denotes DNA homologous to the left side of the T-DNA region of the octopin Ti plasmid, and the white region denotes DNA which is homologous to the right side of the T-DNA region of octopine Ti-plasmid. The physical location and description of the starting plasmids pGV0219 and pGV0120 are described in De Vos et al. Plasmid 6 (1981), 249-253.

10 Pig. 17 illustrates the formation of the acceptor plasmid pGV2260.

The deletion substitution in pGV2217 is indicated as a black region containing the acetylphosphotranfera gene (designated apt) which provides neomycin and kanamycin resistance. The intermediate vector pGV745 15 (Fig. 16) is shown in linear form. It is opened on

The HindIII site of pGV745 shown in FIG. 16. The pBR322 se sequence is designated as a shaded region, and the ampicillin resistance gene is designated Ap. Double crossovers lead to the formation of pGV2260 and 20 loss of the apt gene. The restriction endonuclease sites are designated as follows: B, Bam HI; H, HindIII and R, EcoRI.

FIG. 18 illustrates the formation of a plasmid pLGV2381 for the copying of genes downstream or below the promoter of the nopaline synthetase gene (nos).

The numbers 5 'and 3' refer to the beginning and end of transcription, and ATG and TAA refer to the codons used to start and to interrupt translation. The thick black line 30 indicates the nos promoter region and the white region

D

indicates the nose coding range. Ap stands for ampicillin> resistance, and Km means kanamycin resistance.

FIG. 19 illustrates the formation of the plasmid pAGV10 containing the complete octopin synthetase coding sequence (ocs) and its insertion into plasmid pLGV2381 (see also Fig. 18) behind the nos promoter. The thick DK 173104 B1 24 black lines refer to the promoter regions, and the white areas refer to the ocs coding region.

The other names are as in FIG. 18th

FIG. 20 illustrates the nucleotide sequences around the promoter region of the nopaline synthetase gene (nos) and the nucleo time sequences around the same region after fusion with the coding region of the octopin synthetase gene. The fusion point is denoted by an asterisk (*). Several restriction endonuclease sites are listed / 10 BamHI, HindIII and SacII. The numbers 5 'and 3' refer to the beginning and end of transcription. ATG refers to the first codon used in translation, and TAA refers to the stop codon used in translation. The long arrows indicate the 15 coding regions of the nopaling gene in white and the octoping network in stripes.

In the following, the invention is further explained in detail with reference to the drawing.

FIG. 1 shows a simplified diagram of an acceptor Ti-20 plasmid. This acceptor Ti plasmid contains the two border sequences (1,2) or regions of the wild-type tumor-inducing (Ti) plasmid. The boundary sequences are important for the integration of the T region into Ti plasmids in a plant cell genome. In other words, they are important for the integration of any DNA sequence (3) or any T region into a plant cell genome located between these sequences.

The DNA sequence (3) of the acceptor Ti plasmid contains a DNA segment homologous to at least a portion of a DNA sequence (3 ') in an intermediate cloning vector illustrated in FIG. 2 and 3. This homology is necessary for cointegration by a single cross-over (homologous recombination) of the intermediate cloning vector by the acceptor Ti plasmid. The frequency of obtaining cointegrates is essentially determined by the length of the homology range.

In order to induce a homologous recombination with high frequency B1, the ranges from 1-4 kb are usually used (Leemanns et al., J. Mol. Appl. Genet. 1 (1981), 149-164).

The acceptor Ti plasmid further contains sequences (4), 5 which are important for the transmission by Agrobacterium of the T region of the Ti plasmids to plant cell genomes.

The formation of such acceptor Ti plasmids and their co-integration with intermediate cloning vectors illustrated in FIG. 2 and 3 are described in greater detail below in connection with the discussion of FIG. 4th

In FIG. 2 and 3, simplified diagrams for intermediate cloning vectors are shown for the cloning of any prokaryotic or eukaryotic genes of interest and to be propagated or copied, i.e. are transcribed under the control of a promoter and translated into plant cells. These intermediate cloning vectors contain a DNA segment (3 ') of a cloning agent containing a DNA sequence which is homologous to at least a portion of the DNA segment (3) of the acceptor Ti plasmid, allowing a single cross-over. Furthermore, the intermediate cloning vectors contain at least one gene (5; 7) which is of interest and which contains the natural promoter sequence. The promoter sequence allows for copying of the inserted gene sequence (s).

Examples of various forms of regulation include the following: (1) tissue-specific copying; leaves, roots, stems, flowers, (2) level of copy, viz. high or 3Q low, and (3) inducible copying, i.e. using temperature, light or added chemical factors.

Examples of genes of interest for the intermediate cloning vectors are DNA fragments or sequences of the genetic information that control the synthesis of products, such as amino acids or sugars, for modifying a plant's nutritional or growth potential, or products that provide protection against external pathogenic pathogens, such as resistance to disease organisms or stressful environmental factors, or products that provide information on basic plant processes that need to be modified by remediation techniques.

FIG. 2 and 3 both show intermediate cloning vectors which may contain selectable marker genes (6). Examples of selectable marker genes include those which encode resistance to antibiotics or toxic analogues (e.g., amino acid analogs), or genes that can remedy a defect in the recipient host cell.

FIG. 4 illustrates the structures involved in the formation of hybrid Ti plasmid vectors; and FIG. Figure 5 illustrates the actual conjugation steps involved in the isolation of Agrobacterium carrying the hybrid Ti plasmid vectors. Since the intermediate cloning vector is formed in Escherichia coli, these steps are necessary to transfer the intermediate cloning vector to the acceptor Ti plasmid in Agro-bacterium.

The known transfer process used to produce modified Ti plasmids in which a portion of the T region is interchanged with an altered sequence comprises many steps. Usually, most DNA recombinant manipulations are performed in specially designed cloning agents such as pBR322 (Bolivar, Gene 2 (1977), 75-93). However, these cloning agents cannot themselves be transferred to Agrobacterium.

In the known methods, this problem is solved as follows: a) The pBR cloning agent sequence is exchanged with another broad-host region cloning agent such as the mini-Sa plasmid (Leemanns et al., Gene 19 (1982), 361). 364), which can also multiply in Agrobacterium. These manipulations are performed in Escherichia coli. An intermediate cloning vector is formed.

B) Conjugation of the Escherichia coli strain carrying the intermediate cloning vector containing the DNA of interest to another Escherichia coli strain carrying a helper plasmid which cannot copy in Agrobacterium but which can mediates transfer of itself and other DNAs to Agrobacterium.

C) Conjugation of Escherichia coli formed in step b) with Agrobacterium containing a Ti plasmid. The helper plasmid is lost.

d) Since the interradial cloning vector is capable of copying and existing in Agrobacterium as a separate replicon, the conjugants formed in step c) are a mixture of cells containing either the cointegrate between the intermediate cloning vector and the Ti plasmid or other cells containing the intermediate cloning vector and the Ti plasmid where no cointegration has occurred. In order to isolate the cointegrates exclusively, it is necessary to perform a second conjugation to another Agrobacterium strain without a Ti plasmid. This transfer is mediated by functions, scan encoded by the Ti plasmid itself. Transfer of the intermediate cloning vector to the second Agrobacterium strain is carried out only in the form of a cointegrate with the Ti plasmid.

e) To generate the final modified Ti plasmid with the desired exchange, another crossover is made (Leemanns et al., J. Mol. Appl. Genet. 1 (1981), 149-164).

The only other known method is broadly similar to that described above, however, in step d), another plasmid incompatible with the intermediate cloning vector is introduced into Agrobacterium. In this case, the cointegration (single crossover) can be selected, as all the intermediate cloning vectors remaining as independent replicons will be lost. (Matzke et al., J. Mol. Appl. Genet.

1 (1981), 39-49).

The present invention relates to a novel and extremely simple method for introducing intermediate cloning vectors into acceptor Ti plasmids into Agrobacterium. In short, this method is based on the recognition that helper plasmids from Escherichia coli allow direct transfer of many of the commonly used cloning plasmids, such as pBR322, into Agrobacterium. Since none of these plasmids can copy in Agrobacterium, only those plasmids capable of cointegrating with the acceptor Ti plasmid will remain. Furthermore, according to the invention, this cointegrate in Agrobacterium is used directly as a vector composition for infection of plant cells. In this way, it is possible to eliminate the steps d) and e) described above, which greatly increases the ability 10 to use acceptor Ti plasmids as vectors for DNA transfer to plant cell genomes by both reducing the time required for formation of modified hybrid Ti plasmids, and by increasing the flexibility of the possible constructs.

15 As shown in FIG. 5, the insertion of the intermediate cloning vector into the acceptor Ti plasmid is thus carried out in two steps. First, an Escherichia coli strain (1) carrying the intermediate cloning vector is conjugated to another Escherichia coli strain (2) carrying two plasmids which help to mobilize the intermediate cloning vector for Agrobacterium. Typical and preferred examples of these helper plasmids are R64drdll containing mob functions and pGJ28 containing tra functions (Finnegan et al., Mol. Gen. Genet. 185 (1982), 344-351).

A bomb site (Warren et al., Nature 274 (1978), 259-261) on the cloning agent of the intermediate cloning vector is recognized by the functions encoded by the other two plasmids so that transfer can take place. .

Preferably, all plasmids should contain antibiotic resistance markers to detect their presence.

Second, the Escherichia coli strain formed, i.e. the mobilized strain (3) carrying all three plasmids into Agrobacterium containing an acceptor Ti plasmid having a homology region with the intermediate cloning vector. A single crossover between the intermediate cloning vector and the acceptor Ti plasmid can be detected by selection of the antibiotic resistance marker in the intermediate cloning vector.

DK 173104 B1 29

FIG. 6 schematically illustrates the DNA molecules used to form the acceptor Ti plasmid shown in FIG.

1. This acceptor ^ Ti plasmid is designated (A) to distinguish it from the second acceptor Ti plasmid (B) (Fig. 8). The formation requires a double crossover between a Ti plasmid and another plasmid, which carries the boundary sequences (1) and (2) of a cloning agent (3). Scan shown in the figure, the cloning agent sequence (3) lies between the boundary sequence (1) on the left and (2) on the right. To show the correct 10 polarity of the DNA strands, this one can be drawn on a circle. However, in order to show the homology ranges used in the double crossing, it is necessary to break this circle as shown. This is an important feature to understand and it is also used in the generation of acceptor Ti Ti plasmid (B) in FIG. 8, i.e. if the boundary sequences (1) and (2) are merely inserted into the cloning agent sequence (3), a double crossover would form a Ti plasmid with a deleted T region but without the cloning agent sequence between the boundary sequences (1) and (2). As also shown in FIG. 6, the double crossover also produces a circular DNA molecule containing the original T region between the boundary sequences (1) and (2). Since this is not a replicon, it will be lost. Genetic selection for these events can be achieved e.g. by selecting for the loss of an antibiotic resistance marker contained in the T region of the Ti plasmid and selecting for an antibiotic resistance marker contained in the cloning agent sequence (3).

FIG. 7 shows schematically the formation of an intermediate cloning vector according to FIG. 2 and FIG. 3. A gene (5) of interest and a selectable marker gene (6), both surrounded by a restriction endonuclease site R 1 or R 2, respectively, are inserted into a cloning agent sequence (3 ') which contains unambiguous restriction sites for enzymes R 1 and R 2, by degradation and ligation or binding of all molecules.

The recombinant DNA molecule formed is used to transform Escherichia coli host cells and the transformants p i are selected for the antibiotic resistance marker (Ab) in the cloning agent sequence (3 ') · 30 DK 173104 B1

FIG. 8 schematically shows the DNA molecules used to form another embodiment of the invention, i.e. acceptor Ti plasmid (B). Here, a double crossover is made between a Ti plasmid and a plasmid which contains the cloning agent sequence (3) between the DNA sequences (9) and (10), which are located just outside the boundary sequences (1) and (2). To illustrate the homologous regions used at the crossover, the small plasmid has been broken (as in Fig. 6). The product of the double 10 crossover is acceptor Ti plasmid (B) and another lost circular DNA molecule containing the T region of the original Ti plasmid plus the DNA sequences (2), (10), (9) and (1). Genetic selection can be achieved as described in connection with FIG. 6th

FIG. 9 schematically illustrates an intermediate cloning vector to be used in combination with the acceptor Ti plasmid B of FIG. 8. Here, the gene (5) of interest is inserted between the boundary sequences (1) and (2) contained in a cloning agent sequence (31).

FIG. 10 schematically shows how a single crossover introduces the intermediate cloning vector of FIG. 9 to the acceptor Ti plasmid (B). In this case, selection of the antibiotic resistance marker in the cloning agent sequence (3 ') of the intermediate cloning vector ensures that a hybrid Ti plasmid can be found which results from a co-integration between the two plasmids. A hybrid Ti plasmid is formed with the gene of interest located between the boundary sequences (1) and (2). The hybrid plasmid thus formed does not contain in its T region a sequence which is a direct repeat, e.g. sequences (3) and (3 '), in the hybrid Ti plasmid of FIG. 4, avoiding any instability problems with the hybrid vector or with the transformed DNA in the plant cell genome as a result of intramolecular recombination.

35 Laboratory tests further show that for the formation of the intermediate vector of FIG. 9 is not essential to have both boundary sequences 1 and 2. It is sufficient but essential to have at least the right boundary sequence (2) (see Fig. 1 and Fig. 9) to achieve integration of the desired DNA sequence. sequence in the plant genome.

Knowledge of the restriction endonuclease plans of the Ti plasmids in Agrobacterium, e.g. nopaline or octopin Tiplasmids (Depicker et al., Plasmid 3 (1980), 193-211, and De Vos et al., Plasmid 6 (1981), 249-253), and the knowledge of the restriction fragments containing T-DNA boundary. 10 (Zambryski et al., J. Mol. Appl. Genet. 1 (1982), 361-370, and De Beuckeleer et al., Mol. Gen. Genet. 183 (1981), 283-288), do so now. it is possible to generate acceptor Ti plasmids according to the method of the invention.

It is only required that conventional 15 recombinant DNA operations and basic bacterial genetic manipulations be performed. The present invention is unique in that it specifically provides the described acceptor Ti plasmids which can be used for the formation of hybrid Ti plasmid vectors. Furthermore, these 20 acceptor Ti plasmids are designed to form part of a method for introducing genes into the plant cell genome.

The following examples serve to further illustrate the acceptor Ti plasmids, intermediate cloning vectors, hybrid Ti plasmid vectors and vector compositions, and to demonstrate the efficacy of the vector compositions in providing transformed plant cells and plants exhibiting copy of the foreign gene (s) , which is integrated into the plant cell genome.

Example 1

Formation of acceptor Ti plasmid PGV3850 (type A)

Starting strains and plasmids:

Agrobacterium tumefaciens (rifampicin resistant strain C58C1 and chloramphenicol erythromycin resistant strain C58C1 derived from wild type Agrobacterium).

DK 173104 B1 32

Ti plasmid = pGV3839

Plasmid of FIG. 11 = pAcgB

The ten plasmid pGV3839 is generated from a nopaline plasmid pTiC58trac (pGV3100, Holsters et al., Plasmid 3 (1980), 5,212-230). It contains a deletion substitution mutation near the center of the T region. SmaI fragment 24 within HindIII fragment 19 (Depicker et al., Plasmid 3 (1980), 192-211) has been exchanged for a HindIII fragment of pKC7 (Rao et al., Gene 7 (1979), 79- 82) which contains the apt (acetyl-10 £ hosphotranferase) gene of Tn5. This gene encodes resistance to the aminoglycosides neomycin and kanamycin.

In FIG. 12 is a restriction map for the T region of PGV3839.

The plasmid pAcgB is an insertion portion of AcgB in pBR322, which contains only the boundaries of the T-DNA (see Fig. 11). The boundaries are defined as the ends of the T-DNA, and these regions play a role in the stable integration of the T-DNA into the plant cell genome. The origin and analysis of this clone is described in detail by Zambryski et al., 20 Science 209 (1980), 1385-1391. This clone is formed by isolating portions of the transformed tobacco DNA from the T-DNA. pAcgB contains the junction site of two consecutive T-DNA copies so as to contain the left and right boundaries of the T-DNA. In addition, pAcgB contains the nopaline synthetase gene, as these genetic information maps are very close to the right T-DNA border. Plasmid pAcgB is used to generate an "A-like" acceptor Ti plasmid, pGV3850. FIG. 6 shows the structures involved, and FIG. Fig. 13 shows more precisely 30 the DNA regions involved in the double junctions leading to pGV3850.

The described plasmid pAcgB carries a ColE1-specific barrier region in the pBR322 moiety and can be mobilized from Escherichia coli to Agrobacterium using the helper plasmids 35 R64drdll and pGJ28. The plasmids R64drdll and pGJ28 contained in Escherichia coli are introduced into the Escherichia coli strain, DK 173104 B1 33 carrying pAcgB, by conjugation. Transconjugants are selected as ampicillin-resistant colonies (from the pBR322 sequences in pAcgB), as streptomycin-resistant colonies (from R64drdll) and as kanamycin-resistant colonies (from 5 pGJ28).

The Escherichia coli strain, which carries all three plasmids, is conjugated to the Agrobacterium strain C58C1, which is rifam-picin resistant and which contains the Ti plasmid PGV3839.

The ampicillin resistance of pBR322 is used to select 10 for the first single crossover with the nopaline Ti plasmid. The only way in which the ampicillin resistance can be stabilized in Agrobacterium is a crossover after homologous recombination with pGV3839 through one of the homology regions near the T-region boundaries. At a second over-crossing through the second homology domain, the central portion of the T region of pGV3839 including the apt gene (kanamycin resistance) is interchanged with the pBR322 sequences in the clone pAcgB.

Thus, other recombinants are ampicillin resistant and kanamycin sensitive. To increase the probability of isolation of a second recombinant, the rifampin-resistant Agrobacterium carrying the first recombinant (pAcgB: pGV3839) is conjugated with a second chloramphenicol / erythromycin-resistant Agrobacterium strain without a Ti plasmid. In this way, a chloramphenicol / erythromycin-resistant Agrobacterium pGV3850, which is ampicillin-resistant and kanamycin-sensitive, can be obtained, with a frequency of approx. one in 600 colonies.

Of course, there are other Ti plasmids that can be used for acceptor Ti plasmids of the pGV3850 type. Any Ti-30 plasmid carrying a selectable marker gene near the center of the T region can be used as a recipient. Furthermore, a pAcgB-like plasmid can be generated by inserting the T-region boundary fragments into pBR322 in such a way that the pBR322 sequences lie between the left border fragment and the right border fragment in the orientation left border fragment pBR322 right border fragment. . For example, the left and right border fragments of the nopaline Ti plasmid are 34 and 173104 B1, respectively, HindIII fragments 10 and 23 (Depicker et al.

Plasmid 3 (1980), 193-211).

A single crossover will introduce an intermediate cloning vector containing any gene of interest which is inserted into pBR322 (or derivatives thereof) into the modified T-DNA region of pGV3850. The only requirement is that the DNA to be inserted contains an additional resistance marker gene in addition to those already found in pBR322 for use in selecting for the transfer of the intermediate cloning vector from Escherichia coli to Agrobacterium. This resistance marker may be contained either in the pBR sequences (such as Cm for pBR325 p or Km for pKC7) or in the DNA to be tested in the plant cell. In the acceptor Ti plasmid pGV3850 can

ISLAND

Furthermore, the Aβ gene pBR322 is exchanged for another resistance gene.

D

the marker gene, such as Km. In this way, even pBR322-containing intermediate cloning vectors which are Aβ can be directly mobilized to this pGV3850-like acceptor Ti plasmid.

A further advantage of the pGV3850-20 type acceptor Ti plasmid is that it does not generate tumors in transformed plant cells. Cells that have been "transformed" by pGV3850 can be readily screened from untransformed cells by assaying for the presence of nopaline, since the truncated T-DNA region of pGV3850 still contains the gene. which encode nopaline synthase. Of course, if the intermediate cloning vector co-integrated with the acceptor Ti plasmid pGV3850 contains a marker gene, it can also be directly screened or selected.

In addition to inserting defined intermediate cloning vectors containing a pBR322 sequence at a single cross-over into the acceptor Ti plasmid pGV3850, this acceptor Ti plasmid may also be used as a recipient of cloned groups of DNA in pBR322 or derivatives thereof in an experiment of " shotgun "type. The total population of hybrid-35 plasmid vectors in Agrobacterium can be used to infect plant cells and then screened for copying DK173104 B1 35 of one or more selectable genes of interest.

One can, for example. easily select for genes encoding amino acid synthesis by transferring the entire group to plant cells that do not form the selected amino acid.

The acceptor Ti plasmid pGV3850 has two phenotypic characteristics; (1) It cannot form tumors and (2) it is capable of synthesizing nopaline if T-DNA transfer to the plant cell genome occurs. Thus, various tests are conducted to check these characteristics in different plant tissues infected with Agrobacterium-containing pGV3850.

a) Experiment with potato and carrot slices.

Inoculation of potato and carrot slices with the acceptor Ti plasmid pGV3850 causes formation of a small amount of callus tissue. This wall is tested for the presence of nopaline and the test is positive. Interestingly, this mutant is capable of forming a little callus tissue. However, it can only be formed if the slices are grown in media containing low concentrations of both auxins and 20 cytoquinins.

b) Inoculation of whole plants with acceptor Ti plasmid pGV3850.

Tobacco and petunia seedlings grown on sterile agar medium (without hormones) are inoculated with pGV3850. Slight tissue growth can only be detected after several months (usually "wild-type" tumors are detected after 2 weeks). This tissue does not grow on hormone-free media but can be further propagated as sterile tissue culture on media containing both auxin and cytoquinin. This tissue is also found to be nopa-30 lin positive.

c) Furthermore, since pGV3850 "transformed" cells are not tumor-like, these cells can regenerate in normal plants, which in the genome still contain the transferred DNA segment. Normal plants can be obtained by growing the transformed cells on conventional regeneration media (see also Example 5).

DK 173104 B1 36

To demonstrate the utility of pGV3850 as an acceptor plasmid, the following experiments are performed. An intermediate cloning vector containing oncogenic functions of the octopin 5 T-DNA of pBR325 is recombined into Agrobacterium containing pGV3850. The hybrid Ti plasmid formed in Agrobacterium formed by a single crossover is inoculated into wounded tobacco plants. After approx. two weeks later, tumor tissue develops. This shows that the tumor-inducing DNA has been re-introduced into pGV3850 and copied correctly in transformed plant cells.

Example 2

Formation of intermediate cloning vectors pGV700 and pGV7 5 0_15 An overview of the constructs is shown schematically in FIG. 14. HindiIl fragment 1, which represents the right portion of the TL DNA of the octopin Ti plasmid B6S3, found in pGV0201 (De Vos et al., Plasmid 6 (1981), 249-253), is first inserted into HindIII site of the broad-host region vector pGVH22 (Leemanns et al., Gene 19 (1982), 361-364). The recombinant plasmid pGV0201 contains the HindIII fragment 1 inserted into the unique 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., 25 Fed. Proc. 35 (1976), 2037-2043. 2 µg of pGV0201 DNA is completely degraded by 2 units of HindIII (all restriction enzymes are from Boehringer Mannheim) for 1 hour at 37 ° C in a final volume of 20 µl. The incubation buffer is described by O'Farrell et al., Mol. Gen. Genet 179 (1980), 421-435. 2 µg of pGVH22 DNA is completely digested with HindIII under the same conditions.

0.1 µg HindIII digested pGV0201 binds to 0.05 µg HindIII digested pGV1122 with 0.02 units of T4 ligase (Boehringer Mannheim) at a final volume of 20 μΐ. The incubation buffer used and the conditions used are recommended by the manufacturer (brochure "T4 ligase", Boehringer Mannheim, August 1980, DK 173104 B1 37 no. 10.M.880.486). The ligation mixture is transformed into competent Escherichia coli K514 hsr ”hsm + cells (Colson et al., Genetics 52 (1965), 1043-1050) as described by Dagert and Ehrlich, Gene 6 (1980), 23-28.

Cells were placed on LB medium plates (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York) added to strptomycin (20 µg / ml), and spectinomycin (50 µg / ml). Transformants containing recombinant plasmids are screened for tetracycline sensitivity 10 (10 µg / ml) as a result of the inserted inactivation of the gene encoding tetracycline resistance (Leemanns et al., Gene 19 (1982), 361-364). Streptomycin-resistant and spectimycin-resistant as well as tetracycline-sensitive colonies are physically characterized. Microscale 15 DNA preparations are made using the method of Klein et al.

Plasmid 3 (1980), 88-91 The orientation of the HindIII fragment 1 at the HindIII site of pGV1122 is determined by digestion with SalI. Degradation (conditions used by O'Farrell et al., Mol. Gen. Genet. 179 (1980), 421-435) 20 of recombinant plasmids yield 2 fragments after agarose gel electrophoresis. In the α orientation, there are fragments of 0.77 kb and 22.76 kb, whereas in the β orientation there are fragments of 10.33 kb and 13.20 kb. A recombinant plasmid having the α orientation is used for subsequent cloning and is designated pGV1168.

A BglII-SalI fragment containing the left portion of the TL DNA (including the left border sequence) is introduced into pGVII68, digested with BglII-SalI. This fragment is formed from the recombinant plasmid pGV0153 (De Vos et al., Plasmid 6 (1981), 249-253), which contains Bam HI fragment 8, from the T region of pTiB6S3 inserted into the vector pBR322. pGV0153 and pGVH68 DNA are prepared using the method of Betlach et al., (Fed. Proc. 35 (, 976), 2037-2043). 10 µg of pGV0153 DNA is completely degraded by 10 units of BglII and 35 units of SalI for 1 hour at 37 ° C in a final volume of 100 µl.

The degradation mixture is applied to a preparative 0.8% agarose gel. The 2.14 kb BlgXI-SalI fragment is recovered from this gel by electroelution as described by Allington et al., Anal.

DK 173104 B1 38

Biochim. 85 (1978), 188-196. 2 µg pGV1168 DNA is completely degraded by 2 units BgflI and 2 units SalI. 0.1 µg BglII-SalI fragment DNA binds to 0.02 µg BglII-SalI digested pGV1168 with 0.02 units of T4 DNA ligase in a final volume of 20 µl. The ligation mixture is transformed into competent Escherichia coli K514 hsr hsm + cells (Dagert and Ehrlich, Gene 6 (1980) 23-28). The cells are placed on LB medium plates (Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, 10 New York) to which streptomycin (20 µg / ml) and specin-tinomycin (50 µg / ml) are added.

Microscale DNA preparations (Klein et al., Plasmid 3 (1980), 88-91) are prepared from streptomycin-resistant and spectinomycin-resistant transformants. Recombinant-15 plasmids in which the 2.14-kb BglII kb. A plasmid with a degradation pattern corresponding to these molecular weights (2.14 kb and 21.82 kb) is used for 20 additional cloning and is designated pGV1171. A 12.65-kb fragment from pGV1171 contains the right and left TL DNA boundary sequences (De Beuckeleer et al., In Proceedings IVth International Conference on Plant Pathogenic Bacteria, Μ.

Ridé (ed.) (1978), I.N.R.A., Angers, 115-126), and genes that allow oncogenic proliferation (Leemanns et al., EMBO J.

(1982), 147-152). This HindIII fragment is inserted into the plasmid pBR325 (Bolivat, Gene 4 (1978), 121-136). pGV1171 and pBR325 are prepared using the method of Betlach et al., Fed. Proc. 35 (1976), 2037-2043. Two µg of 30 each DNA is completely digested with two units of HindIII for 1 hour at 37 ° C (incubation buffer described by O'Farrell et al., Mol. Gen. Genet. 179 (1980), 421-435). 0.1 µg HindIII degraded pGV1171 binds to 0.05 µg pBR32% linearized with HindIII with 0.02 units of T4 DNA ligase. Transforming the ligation mixture into competent Escherichia coli K514 hsr ”hsm cells is performed as described by Dagert and Ehrlich,

Gene 6 (1980), 23-28. The cells are placed on plates with LB medium (Miller, Experiments in Molecular Genetics (1972), DK 173104 B1 39

Cold Spring Harbor Laboratory, New York), to which is added carbenicillin (100 µg / ml). Carbenicillin-resistant clones are screened for sensitivity to tetracycline (10 µg / ml), as a result of inserted inactivation of the gene encoding tetracycline resistance (Bolivar, Gene 4 (1978) 121-136). Carbenicillin-resistant and tetracycline-sensitive clones are typically characterized by restriction enzyme degradation of DNA prepared from these clones using the microscale technique (Klein et al., Plasmid 3 (1980), 88-91). Bam HI-10 degradation yields 4 DNA fragments: in the α orientation, fragments of 0.98 kb, 4.71 kb, 5.98 kb and 7.02 kb are formed, whereas the β orientation yields 0.98 kb, 4 fragments. 71 kb, 1.71 kb and 11.20 kb. A recombinant plasmid designated pGV700 is obtained, corresponding to the α orientation, and this plasmid is used for further experiments.

PGV750 is derived from pGV700 by exchanging a 2.81 kb BamHI-HpaI fragment encoding kanamycin resistance with a 3.49 kb BglII-Sma I fragment encoding functions important for oncogenicity. The TL area inserted 20 into the pGV700. The Bam HI-Hpa I fragment encoding kanamycin resistance is obtained from 1 :: Tn5 (Berg et al., Proc.Natl. Acad.

Sci USA 72 (1975), 3628-3632). Preparation of λ :: Tn5 is done as described by Hiller, Experiments in Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York-25 pGV700 DNA is prepared using the method of Betlach et al. , (Fed. Proc. 35 (1976), 2037-2043).

2 µg pGV700 DNA is completely degraded by 2 units Bgll and 2 units Narrow. 2 µg Xs * Tn5 DNA is completely degraded by 2 units of BamHI and 2 units of Hpal. 1 µg Bam HI-Hpal digested X :: Tn5 binds to 0.2 µg BgIII-Smal digested pGV700 with 0.5 units of T4 DNA ligase in a final volume of 10 µl (using the conditions recommended by the manufacturer). The ligation mixture is transformed into competent Escherichia coli K514 hsr hsm + cells (Dagert and Ehrlich, Gene 35 6 (1980) 23-28). The cells are placed on plates with LB medium (Miller, Experiments in Molecular Genetics (1972), Cold

Spring Harbor Laboratory, New York)

R R

cillin (100 ug / ml) and kanamycin (25 ug / ml). Cb and Km clones are physically characterized by restriction enzyme analysis of DNA prepared according to the microscale technique (Klein et al., Plasmid 3 (1980), 88-91). BglII /

Bam HI double digests of this DNA yield 3 fragments 5 of 3.94 kb, 5.89 kb and 8.09 kb, whereas HindIII digests yield 3 fragments of 2.68 kb, 5.99 kb and 9.25 kb.

A plasmid showing these degradation patterns is designated pGV750 and is schematically illustrated in FIG. 17th

pGV700 and pGV750 are two different intermediate cloning vectors containing the left and right border sequences of TL DNA of the octopin Ti plasmid pTIB6S3.

Furthermore, the inner T region is deleted to different extents in each of the two plasmids. pGV700 contains a truncated T-region of genetic information for octo-15 pin synthetase (transcript 3) and 3 other products, 4, 6a and 6b (see Willmitzer et al., EMBO J. 1 (1982), 139-146, for a description of T-range products). The combination of these three products (4, 6a and 6b) will induce shoot formation in transformed plants. The pGV750 contains 20 even less of the T region, i. only the octopin synthetase gene. The information for products 4, 6a and 6b has been exchanged with the antibiotic resistance marker gene encoding kanamycin (neomycin) resistance.

pGV700 and pGV750 are examples of intermediate cloning vectors which can be used for B-type acceptor Ti plasmids (see Figure 8 and Example 3 below). These vectors are partially analogous to the vector shown in FIG. 9, except that they do not contain a gene of interest. A gene of interest can be readily inserted into these vectors, as they contain single restriction endonuclease sites for cloning within the modified T regions (see Figures 14 and 15).

41 DK 173104 B1

Example 3

Formation of acceptor Ti plasmid pGV2260 (type B)

Starting 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

The formation of the Ti plasmid pGV2217 is described in detail by Leemans: et al., EMBO J 1 (1982), 147-152. It contains a deletion substitution mutation of the entire TL region of the octopin Ti plasmid BamHI fragments 8, 30b, 28, 17a and the left 3.76 kb BamHI EcoRI fragment of BamHI fragment 2 (De Vos et al., Plasmid 6 (1981), 249-253) 15 have been exchanged with an EcoRI-BamHI fragment of pKC7 (Rao & Rogers, Gene 7 (1979), 79-82), which contains the apt (acetylphosphotransferase) gene of Tn5. This gene encodes resistance to the aminoglycosides neomycin and kanamycin.

The formation of the intermediate vector pGV745 is shown schematically in FIG. 16 and is described as follows. The recombinant plasmid pGV713 is derived from the octopin Ti plasmid subclone PGV0219 (De Vos et al., Plasmid 6 (1981), 249-253), which contains HindIII fragments 14, 18c, 22e and 38c in α-25 orientation. pGV0219 DNA is completely degraded with BamHI and then bound under conditions that promote self-binding of the plasmid (final concentration of DNA in ligation mixture <1 µg DNA / ml). Transformants are selected for ampicillin resistance and physically characterized by restriction enzyme degradation. A clone that no longer contains the 6.5 kb Bam HI fragment in pGV0219 is isolated and designated pGV713. This clone pGV713 is used for subsequent cloning (see below). The recombinant plasmid pGV738 is derived from pGV0120 (De Vos et al., 35 Plasmid 6 (1981), 249-253), which contains the BamHI fragment. PGV0120 DNA is digested with E.coRI and self-bound (as above by the formation of pGV713). Transformants are selected for ampicillin resistance and analyzed by restriction enzyme degradation. A clone in which EcoRI fragments 20, 12 and a 2.95 kb EcoRI fragment containing parts of EcoRI fragment 19a and parts of pBR322 are deleted are used for further cloning and are designated pGV738. This plasmid still contains a 5.65 kb Eco RI-BamHI fragment from the right portion of BamHI fragment 2 (De Vos et al.,

Plasmid 6 (1981), 249-253).

Then, pGV713 DNA is digested with HindIII and BamHI and the degradation mixture is placed on a preparative agarose gel. Following electrophoresis, the 2.30 kb HindIII-Bam HI fragment contained in pGV713 is purified by electroelution (as described by Allington et al., Anal. Biochem. 85 (1975), 188-196). This fragment binds to pGV738, which is completely degraded by HindIII and BamHI. Effter transformation, ampicillin-resistant clones are physically characterized by restriction enzyme degradation. For example, EcoRI-BamHI degradation should yield 2 fragments of 3.98 kb (= vector portion) and 7.95 kb (= inserted portion), respectively. A recombinant plasmid having these properties is designated pGV745 and used as an intermediate vector for the formation of the acceptor Ti plasmid pGV2260.

Plasmid pGV745 contains a ColE1-specific bom site in the pBR322 moiety and can be mobilized from Escherichia coli 25 to Agrobacterium using the helper plasmids R64drdll and pGJ28, as described in Example 1 (formation of acceptor Ti plasmid pGV3850).

pGV745 is mobilized to Agrobacterium strain C58C1, which is rifampicin resistant and contains the Ti plasmid pGV2217.

The first crossover is selected using the ampicillin resistance of pBR322 in the same manner as described in Example 1 (formation of the acceptor Ti plasmid pGV3850). A second crossover deletes the deletion substitution mutation in pGV2217 with the pBR322 sequences 35 of plasmid pGV2217. Other recombinants are selected by direct screening for the ampicillin-resistant transconjugants, formed by co-integration of pGV745 with pGV2217, for loss of kanamycin resistance. In this way, a rifampicin Agrobacterium strain C58C1 containing pGV2260 (ampicillin resistant, kanamycin sensitive) is obtained.

This Ti plasmid pGV2260 will be used as an acceptor plasmid (type B) for intermediate cloning vectors of the pGV700 or pGV750 type. They are composed of (1) a DNA fragment carrying the ampicillin resistance gene, copy region and barrier site of pBR322, (2) a DNA fragment containing the left and right border sequences of the TL DNA and an additional resistance marker. in addition to those already present in pBR322, which will allow for genetic selection for the transfer of the intermediate cloning vector from Escherichia coli to Agrobacterium as well as for its co-integration into the acceptor Ti plasmid pGV2260.

For example, it has been possible to show that Agrobacterium, which contains a cointegrate between pGV2260 and pGV700, can transfer the expected DNA sequences (the sequences found between the T-DNA boundaries) to plant cell genomes. The transformed plant cells exhibit the expected phenotype, viz. tumors that produce shoots, provided that pGV700 contains the genetic information for three products (4, 6a, 6b; cf. Willmitzer et al., EMBO J. 1 (1982), 139-146). In this way, it has been shown that a B-type acceptor Ti plasmid is capable of transferring DNA to plant cells when used as a cointegrate with an intermediate cloning vector of the type illustrated in FIG. 9 and described in Example 2.

Example 4 Formation of an intermediate cloning vector containing a gene to be copied into plants

Thus far, the insertion of whole genes in more or less cases positions in the T region of Ti plasmids has not caused copying of the foreign sequence after transfer to the plant genome. In the method of the invention, the coding region of one or more foreign foreign genes of interest can be bound to transcription initiation and termination signals known to be functional in the plant cell. The utility of this method 5 is demonstrated by the present invention in experiments involving the DNA sequences encoding the nopaline synthase gene. The entire sequence of this gene and the exact onset and interruption of the transcription are known (Depicker et al., J. Mol. Appl. Genet. 1 (1982), 561-574).

.10 According to the present invention, the protein coding region of any foreign gene can be inserted in a neighboring position to the nos promoter. As an example of a foreign gene sequence, the coding region of the octopin synthase gene (De Greve et al., J. Mol. Appl. Genet. 1 15 (1982), 499-512) is inserted in a neighboring position to the nos promoter. This construct is mobilized to an acceptor Ti plasmid and used to infect plants. The tumor tissue formed is tested for the presence of octopin and the sample is positive.

The formation of the intermediate cloning vector containing the unreal nopaline promoter: the octopin synthase structure gene is shown and described in FIG. 18-20.

Briefly, the restriction fragment HindIII-23 containing the nos gene is constructed in vitro to remove most of the nos coding sequence and while maintaining the 25 nos promoter in the neighboring position to the restriction endonuclease site BamHI (Fig. 18). 10 µg of pGVQ422 (a pBR322 derivative carrying the HindIII-23 fragment containing the complete nos gene, (Depicker et al., Plasmid (1980), 193-211) is digested with Sau3A, and the 350 bp fragment carrying The nos-30 promoter is isolated from a preparative 5% polyacrylamide gel. The promoter fragment binds to BglII-digested pKC7 (Rao et al., Gene 7 (1979), 79-82), previously treated with bacterial base phosphatase (BAP). ) to remove 5'-terminal phosphate groups. 20 µg of the 35 plasmid formed (pLGV13) is digested with BglII and treated with 7 units of Bal31 exonuclease (Biolabs, New England) for 4-10 minutes in 400 μΐ 12 mM MgCl CalCl2, 0.6 M NaCl, 45 µl of EDV and 20 xnM Tris-HCl, pH 8.0, at 30 ° C.

During this time, approx. 20-50 bp DNA. The Bal31-treated molecules are digested with BamHI and incubated with the Klenow fragment of DNA polymerase and all four des-oxynucleoside triphosphates (in 10 mM each) for filling at the ends. The plasmids are screened for a regenerated BamHI site derived from the binding of a filled BamHI end and the end of the Bal31 deletion. The size of the BamHI-SacII fragment of several candidates is evaluated in a 6% 10 urea polyacrylamide gel and the nucleotide sequences of the candidates of sizes between 200-280 nucleotides are determined. The clone pLGV81 containing the SacII-BamHI fragment of 203 bp containing the promoter is used to exchange the SacII-BamHI fragment of the nos gene in 15 pGV0422. The final promoter vector is designated pLGV2381.

All the recombinant plasmids are selected by transformation of Escherichia coli strain HB101.

The plasmid vector containing the formed nos promoter is digested with BamHI and the coding sequence for ocs contained in a BamHI fragment is inserted at this site. The ocs coding sequence is also prepared in vitro for the environment with the BamHI restriction endonuclease site as described in FIG. 19. 10 µg of BamHI fragment 17a of the octopin Ti plasmid B6S3 (De Vos et al., Plasmid 6 (1981), 249-253) 25 is digested with BamHI and SmaI and the fragment containing the ocs coding sequence is isolated from a 1% agarose gel and binds to the large Bam HI-PvuII fragment of pBR322. 20 µg of the resulting plasmid pAGV828 is digested with BamHI, treated with the exonuclease Bal31 as described in 18, after which it is degraded with HindIII and the ends are filled and bonded to themselves. The sizes of the Bal31 deletions are determined in a 6% polyacrylamide gel. The multiple candidate nucleotide sequences are determined and a candidate with only 7 bp remaining of the 5 'nontranslated leader sequence is selected for further use (pOCSa). To surround the OCS sequence with BamHI sites, the Clal-Rsal fragment is filled in and subcloned to the Ball site by pLC236 (Remaut et al., Gene 15 (1981), 81-93). The resulting plasmid pAGV40 DK 173104 B1 46 is digested with BamHI and the fragment carrying the ocs sequence is isolated by electroelution from a preparative 1% agarose gel and bound to pLGV2381 previously digested with BamHI and treated with BAP (bacterial base phosphatase). The insertion of the ocs sequence into pLGV2381 is achieved in both orientations (pNO-1 and pNO-2).

The nucleotide sequences showing the exact junction site in the nosrocs fusion are shown in FIG. 20th

Furthermore, the plasmid vector containing the formed nos promoter is used to introduce DNA from plasmid R67 which encodes the enzyme dihydrofolate reductase. The coding sequence containing the dihydrofolate reductase gene is found on a BamHI fragment as described by O'Hare et al., Proc.

Natl. Acad. Sci. USA 78 (1981), 1527-1531, and thus can be readily inserted into the nos promoter vector containing the BamHI site adjacent to the promoter region as described above. This gene is an example of a selectable marker gene (see, for example, Figures 2, 3, 4, 5 and 7) as it provides copy resistance to the antibiotic 20 methotrexate upon copying. When this intermediate cloning vector is mobilized to an Agrobacterium containing a wild-type nopaline acceptor Ti plasmid, a single crossover occurs and a hybrid Ti plasmid vector is formed. The vector composition is used to infect plants. The tumor tissue formed is capable of sustained growth in the presence of 0.5 µg / ml methotrexate.

The formation of the intermediate cloning vectors containing the ox and dihydrofolate reductase coding regions behind the aforementioned nosprorotor and their transfer and copying into transformed plant cells after co-integration with the Ti plasmid of Agrobacterium is evidence that foreign genes can be transmitted and copied in the plant cells according to the method. .

47 DK 173104 B1

Example 5

Isolation of plant cells and plants Containing the desired gene (s) inserted into the chromosomes Plant cells and whole plants transformed with non-oncogenic acceptor Ti plasmid derivatives are prepared (e.g.

pGV3850) using one of the following three methods (1) In vivo inoculation of whole plants, followed by in vitro cultivation on media permitting shoot regeneration.

(2) In vivo coinfection of whole plants in the presence of other Agrobacteria strains that directly induce shoots at the wound site.

(3) In vitro cocultivation of single plant cell protoplasts.

Each of these methods is described in more detail below.

The first method is based on a modification of the methods commonly used to induce infection of whole plant tissue with wild-type Agrobacterium strains, which results in the formation of coronary tissue. Since pGV3850 is a 20 non-tumor producing (nononcogenous) Agrobacterium derivative, no tumor growth is detected at the site of infection.

However, if the infected tissue is removed and grown in tissue cultures, transformed tissue can be readily obtained. After an initial culture period (to simply increase the mass of the tissue), wound site tissue is grown under conditions that allow the formation of shoots. Both untransformed and pGV3850 transformed cells will form shoots. The transformed shoots can be easily separated by simple analysis for the presence of nopaline.

30 pGV3850 transformed calli and shoots are derived from cut tobacco seedlings of Nicotioina tabacum Wisconsin 38 using the following procedure (all steps are performed under sterile conditions in a laminar flow extractor).

DK 173104 B1 48 (1) 6-week-old tobacco seedlings grown in small jars (diameter 10 cm, height 10 cm) are used on solid Murashige & Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15 (1962 ) 473-497) containing 0.8% agar.

5 (2) The youngest top leaves are removed with a scalpel and discarded.

(3) Wound surfaces are inoculated with a spatula or toothpick containing Agrobacterium derived from a fresh plate culture grown under selective conditions (e.g. to the rifampicin-resistant, ampicillin-resistant Agrobacterium strain containing Ti plasmid pGV3850, YEB medium containing 100 YEB medium: 5 g / l Bacto meat extract, 1 g / l Bacto yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 x 10 5 MgSO value 7.2 and 15 g / l agar At least 8 plants are inoculated for each pGV3850 construct.

(4) Incubate for 2 weeks, there should be no response or only weak response at the inoculation site. Sometimes very small calli occur.

(5) A thin section with a thickness of less than 1 mm is removed from the wound surface. This section of the injured assay is incubated on a plate containing Linsmaier & Skoog (LS) agar medium (Linsmaier and Skoog, Physiol. Plant.

18 (1965) 100-127) with auxins and cytoquinines (1 mg / 1 NAA, 0.2 mg / 1 BAP) and 1% sucrose.

(6) After approx. 6 weeks the callus must be sufficiently large, diameter at least approx. 5 mm to allow a portion to be tested for the presence of nopaline. Not all wound calli form nopaline. Ca. one in four plants forms nopaline positive wound callus.

(7) Nopaline-positive calli is transferred to agar plates containing the regeneration medium: LS medium as above + 1% sucrose and 1 mg / 1 BAP cytoquinine.

(8) Shoots of good size (1 cm high) appear after 35 approx. 4-6 weeks. The shoots are transferred to fresh agar plates containing LS medium + 1% sucrose without hormones for further growth and rooting.

(9) The shoots are grown for 1 or 2 weeks so that part (1 or 2 small leaves) can be removed for testing for nopaline in situ.

(10) Nopaline positive shoots are transferred to larger containers (10 cm containers as above) containing MS medium as in (1) for further growth.

5 N.B. All plant culture media for infected tissues contain 500 µg / ml of the antibiotic cefotaxime ("Claforan®") as a selection against Agrobacterium containing pGV3850.

This drug works well to prevent growth of all Agrobacteria (including those which are carbenicillin-resistant).

Another method of providing transformed and shot tissue is developed by the applicants. This method is based on the recognition that certain mutant Ti plasmid strains of Agrobacterium induce crown gall tumors which produce shoots. Such shoot-inducing (shi) mutants map a particular region of the T-DNA (transferred DNA segment) into the Ti plasmids of Agrobacterium tumefaciens (Leemanns et al., EMBO J. 1 (1982) 147-152 and Joos et al. .

Cell 32 (1983) 1057-1067). Often, the induced shots are 20. composed of perfectly normal non-transformed cells. Therefore, it has been attempted to inoculate plants with a mixture of two different Agrobacteria, one carrying an octopin Ti plasmid shoot mutant and another carrying pGV3850.

In this way, there is a good chance that the octopin shot mutant can induce shoots that have been transformed down pGV3850. Plants have been inoculated with Agro bacterium containing Ti plasmid pGV3850 and an octopin shot inducing Ti plasmid in a 5: 1 ratio. In this way, we have succeeded in producing pGV3850 transforated shoots.

These shots can be easily screened by analyzing for the presence of nopaline. This approach avoids all labor-intensive tissue culture methods. The nopaline-positive shoots are transferred to media containing simple salts and sugars with arbitrary growth regulating hormones which allow further growth. Once the shoots have reached a sufficient size, they can easily be transferred to soil and grown. This co-infection method is 5 ° DK 173104 B1 particularly useful for transforming plant species that cannot be easily grown in tissue cultures. Thus, a whole range of agronomically and economically significant plants, such as legumes, medicinal plants and ornamental plants, can be formed by Agrobacterium.

The third method allows the isolation of Nicotiana tabacum protoplasts and the selection of hormone-independent T-DNA transformed cell clones after cocultivation of protoplast-derived cells with oncogenic Agrobacterium strains. An analogous technique can be used for the selection of transformed cells when using other dominant selective markers such as antibiotic resistant genes formed in such a way that they are copied into higher plant cells (see Example 3).

In this case, however, the selection conditions must be optimized in each case (concentration of the selection agent, time between transformation and selection, concentration of the protoplast-derived cells or cell colonies in the selection medium). If it is not possible to make selection for the transformed cells, e.g. due to the use of avirulent T-DNA mutants, such as e.g. pGV3850 or pGV2217 (Leemans et al., EMBO J. 1 (1982), 147-152), it is possible to grow the cells after genetic transformation on auxin- and cytoquinin-containing medium, e.g. Murashige and

Skoog medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473-497) with 2 mg / l NAA (α-naphthalene acetic acid) and 0.3 ml / l kinetin, and to identify the transformed colonies by their opinindhold. In this way, after 30 electrophoretic analysis for agropin and mannopin synthesis (method see Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164) can be found. 660 colonies formed following infection with pGV2217, a Nicotiana tabacum SR1 cell line that synthesizes the TR-encoded open mannopin (N2- (1-mannityl) -glutamine). A large number of shots are formed after incubation of callus pieces of this cell line on regeneration medium (Murashige and Skoog medium with BAP (6-benzylaminopurine) (1 mg / 1) as the only plant growth regulator). All of the 20 shots analyzed are still capable of synthesizing mannopin.

After transfer to hormone-free Murashige and Skogg medium, shoots grow as morphologically normal tobacco-5 plants that still contain mannopin.

The protoplast isolation and transformation described here for Nicotiana tabacum can also be used for Nicotiana plum-baginifolia.

2. Experimental Procedures 10 2.1 Shooting Cultivation Conditions

Cultures of Nicotiana tabacum shoots are kept in 250 ml glass containers on hormone-free Murashige and Skoog medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473-497) under sterile conditions in a culture room (16 to 15 days, 1500 lux white fluorescent light ("ACEC LF 58 W / 2 4300 ° K Economy"), 24 ° C, 70% relative humidity). Five-week-old shoot cultures are used for protoplast isolation.

2.2 Protoplast Insulation 20 Acceptic technique is used for all the steps during protoplast insulation and cultivation. The protoplasts are isolated by a mixed enzyme procedure.

All leaves, except very young leaves less than 2 cm, can be used for protoplast insulation. The leaves 25 are cut into strips with a width of approx. 2-3 mm with a sharp knife. 2-3 g of leaf material is incubated for 18 hours at 24 ° C in 50 ml of enzyme blending in the dark without stirring. The enzyme mixture consists of 0.5% cellulase Ono-zuka R-10 and 0.2% macerozyme Onozuka R-10 in hormone-free K3 medium (Nagy and Maliga, Z. Pflanzenphysiol.

78 (1976), 453-455). The mixture is sterile filtered through a 0.22 µm pore membrane and the mixture can be stored for at least 6 months at -20 ° C without appreciable loss of activity.

DK 173104 B1 52 2.3 Protoplast culture

After 18 hours of incubation, mixtures are gently stirred to release the protoplasts. Then the mixture is filtered through a 50 µm sieve and the filtrate is transferred to 10 ml centrifuge tubes. After centrifugation for 6 minutes at 60-80 g in a rotating curve, the protoplasts form a dark green liquid band. The liquid under the protoplasts and residues forming a pellet is removed using a capillary tube connected to a peristaltic pump. The protoplasts are poured into a tube and washed twice with culture medium. The culture medium is K3-mediated (Nagy and Maliga, Z.

Pflanzenphysiol. 78 (1976), 453-455) with NAA (0.1 mg / l) and kinetin (0.2 mg / 1) as growth regulators. The medium is adjusted to pH 5.6 and sterilized through a 0.22 µm filter membrane. After the second wash, the protoplasts are counted using a Thoma hemacytometer (from the company "Assistant", France), 20 and the cysteoplasts are resuspended in culture medium at a final concentration of 10. protoplasts / ml. The protoplasts are grown in a volume of 10 ml per ml. tissue culture quality petri dish with. a diameter of 9 cm. The dishes are sealed with "Parafilm" ® and incubated for 25 hours in the dark and then in subdued light (500-1000 1uj $ at 24 ° C.

2.4 Transformation by co-culture The protoplast cultures are infected 5 days after isolation. Agrobacterium cultures are grown for 18 hours in liquid LB medium (Miller, Experiments in Molecular

Genetics (1972), Cold Spring Harbor Laboratory, New

York), and resuspended in K3 culture medium at a density of 2 x 10 celler cells / ml. 50 µl of this suspension is added to the plant protoplast cultures and after 35 sealing with "Parafilm" ® the cultures are incubated under the same conditions as described above under 2.3. ·

After 48 hours, the cultures are transferred to 10 ml centrifuge tubes and centrifuged in a rotor with swine basket at 60-80 g for 6 minutes. The liquid tape and pellet are combined and resuspended in 10 ml K3 medium (Nagy and Maliga, Z. Pflanzenphysiol.

78 (1976), 453-455) added an antibiotic (carbenicillin 1000 µg / ml or cefotaxime 500 µg / ml).

After two weeks of incubation, the protoplast-derived microcalli are centrifuged and resuspended in K3 medium (Nagy and Haliga, Z, Pflanzenphysiol. 78 (1976), 453-455) with the same growth regulator and 10 antibiotic concentrations as before but with a sucrose concentration. of 0.3 M instead of 0.4 M. The cell density in this medium is set to approx. 25 x 103 micro-calli per ml.

After two weeks of further incubation under the same conditions, calli is transferred to K3 medium with the same antibiotic concentrations as before, but with a reduced sucrose content (0.2 M) and a reduced content of growth regulators (NAA 0.01 ml / l and kinetin 0.02 mg / l).

20 After incubation for another 2 or 3 weeks, the putative transformants can be recognized by their light green and compact appearance and better growth. These colonies are then transferred to hormone-free Linsmaier and Skoog medium (Linsmaier and Skoog, Physiol. Plant. 18 (1965), 25 100-127), which are fortified with 0.6% agar and contain reduced antibiotic concentrations (carbenicillin 500 µg / ml or cefotaxime 250 µg / ml).

Opin tests can be performed on the putative transformants that grow on this hormone-free medium when they have reached a diameter of 3-4 mm. Half of each colony is used for the detection of octopin 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). This test 35 allows confirmation of the transformed nature of these colonies selected on hormone-free medium.

Then, the selected colonies can be grown on antibiotic-free medium.

2.5 Co-cultivation without selection on hormone-free medium 5 When it is not possible to select for trans-formed cells (for example, because avirulent T-DNA mutants are used) or when it is not necessary because a dominant one exists selectable marker, such as an antibiotic-resistant gene, in the T-DNA, the treatment of the protoplast-derived cells can be simplified (the hormone reduction steps are no longer needed). The protoplasts are treated as described above up to the stage of infection. 48 hours after the addition of the bacteria, the protoplastic-derived cells (6 minutes, 60-80 g) are centrifuged and resuspended in medium AG (Caboche, Planta 149 (1980), 7-18) capable of supporting cell growth at very low density. The cells are counted using a Fuchs-Rosenthal counting chamber (from 20 "Assistant", France), and the cells are resuspended at the density required for subsequent work.

If the colonies are to be treated individually for open-label testing, low cell density plates (100 proto-plastic-derived cells and cell colonies per ml) yield large-cell colonies 25 after 1 month of incubation. If it is possible to do drug selection for the transformed cells, the cells are incubated at a higher density (103-104 / ml), and the selection agent used is added to the medium at a concentration and at a time point that must be optimized for each type of selection.

2.6 Regeneration of whole plants from callus tissue

Normal plants are readily obtained from callus tissue (for example, either derived from protoplate transformation or from whole-plane inoculation (see 2.7). Callus tissue is grown on Murashige and Skoog medium containing 1 mg / ml BAP. This medium induces shoot formation after 1 These shoots can be transferred to a medium without hormones to form roots and a complete plant.

2.7 Tumor Infection on Tobacco Climbs

Tobacco seeds (e.g. cultivar Wisconsin 38) are surface sterilized by treatment with 70% denatured ethanol / f 2 O for 2 minutes followed by 10% commercial bleach and 0.1% sodium dodecyl sulfate (SDS), then further rinse five times with sterile water. The sterile seeds were seeded in large test tubes 10 (25 mm wide) containing the salts of Murashige and

Skoog medium solidified with 0.7% agar and covered with polycarbonate peaks. Then the tubes are incubated in a culture room (12,000 lux, 16 hours light / 8 hours dark, 70% relative humidity, 24 ° C) - After 4-6 15 weeks the plants are ready for use. They remain optimal for at least another month.

The seedlings must have a height of at least 3 cm and have 4 or more leaves. Then the plants are transversely cut through the youngest internode with a new 20 sterile scalpel. The upper part of the plant is removed from the tube and bacteria from an agar plate culture are placed on the wound surface with a flamed microspatula.

Tumors appear after 2 weeks for the onset of the wild type and after a prolonged period for some of the attenuated mutant strains. This method is used to inoculate tobacco (Nicotiana tabacum), Nicotians plumbaginifolia and Petunia hybrida.

Concluding remarks.

The present invention enables for the first time transformation of plants with Agrobacterium containing a hybrid Ti plasmid vector lacking the oncogenic functions of the T region in wild-type Ti plasmids. Since the influence of DK 173104 B1 56 on the oncogenic functions of the T region on the transfer of DNA from the Ti plasmid to plant cells was not known, it is surprising that, nevertheless, transfer of the modified T region containing 5 or more interesting genes for plant cells. There is co-integration and stable maintenance of this transferred DNA in the plant cell genome. Furthermore, copying of the selected gene (s) of interest may be obtained, provided that the gene or genes either contain - or are designed to contain - suitable promoter sequences. The implementation of a single crossover between an intermediate cloning vector containing the selected gene (s) with a specially designed acceptor Ti plasmid greatly facilitates the formation of an arbitrary hybrid Ti plasmid vector useful for the transformation of plant cells. The specially designed acceptor Ti plasmids contain the DNA segment of a conventional cloning agent, so that one or more random genes (which have been inserted into the same or a related cloning agent as part of an intermediate cloning vector) can form a cointegrate at a single crossover. The two segments of the cloning agent (s) provide the necessary homology regions for recombination.

Microorganisms and intermediate cloning vectors, acceptor

Ten plasmids and hybrid plasmid vectors prepared by the method of the invention are exemplified by cultures deposited in the German Collection of Microorganisms (DSM), Gottingen, December 21, 1983 and identified therein as follows: (1) Intermediate vector plasmid pAcgB in Escherichia coli K12 HB101, (2) Agrobacterium tumefaciens C58C1 rifampicin-resistant strain carrying carbenicillin-resistant acceptor Ti plasmid 35 pGV3850, (3) Intermediate vector plasmid pGV700 in Escherichia coli K12 strain K514 (thr leu th1 lac

ISLAND

(4) Intermediate vector plasmid pGV750 in Escherichia coli K12 strain K514 (as above under (3)).

(5) Agrobacterium tumefaciens C58C1 rifampicin-resistant strain carrying carbenicillin-resistant acceptor Ti plasmid pGV2260, (6) Intermediate vector plasmid pNO-1 bearing the octopin synthase coding region under control of the nopaline promoter in Escherichia coli K12 HB1.

(7) Strain used to immobilize intermediate vectors for Agrobacterium = GJ23-bearing mobilization plasmids pGJ28 and R64drdll (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).

15

These cultures have been given the following deposit numbers: 2792 (1), 2798 (2), 2796 (3), 2797 (4), 2799 (5), 2833 (6) and 2793 (7).

The deposits were made in accordance with the Budapest-20 Treaty.

25 30 35

Claims (42)

A vector combination consisting of: (i) an acceptor Ti plasmid which is substantially free of internal T-DNA sequences and unable to induce plant tumors, comprising: (a) the two border sequences (1, 2) of the T region of a wild-type Ti plasmid, (b) a DNA sequence (3) derived from cloning agent located between the two boundary sequences, and (c) a DNA segment (4) of a wild-type Ti plasmid containing DNA sequences essential for transmission by Agrobacterium of the T region of wild-type Ti plasmids into plant cell genomes, and (ii) an intermediate cloning vector comprising: (a) ) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants, and (b) a cloning agent segment (3 ') containing a DNA sequence homologous to the DNA sequence. (b) in said acceptor Ti plasmid allowing a single over-1 junction. Vector combination according to claim 1, characterized in that the intermediate cloning vector further contains at least one selectable marker gene adjacent to the gene or genes of interest (5). Vector combination according to claim 1, characterized in that the gene or genes of interest (5) contained in the intermediate cloning vector are under the control of its (their) natural promoter. DK 173104 B1 Vector combination according to claim 1, characterized in that the gene or genes of interest (5) contained in the intermediate cloning vector are under the control of a promoter which is exogenous to the gene or genes of interest. 5. A vector combination consisting of (i) an acceptor Ti plasmid which is unable to induce tumors in plants and is free of boundary sequences and internal T-DNA sequences of a wild-type Ti plasmid (a) a DNA segment (4) of a wild-type Ti plasmid without the T region and without the two boundary sequences of the T region and (b) a DNA sequence (3) derived from a cloning agent, and (ii) ) an intermediate cloning vector comprising: (c) the left and right boundary sequence of a T region of a wild-type Ti plasmid (1, 2), (d) a cloning agent segment (3 ') located outside the two boundary sequences which is homologous to the DNA sequence (b) of said acceptor Ti plasmid which allows for a single crossover, and (e) a region substantially free of internal T-DNA sequences of a wild-type T1 plasmid and contains at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants thereby placing said region one between the two boundary sequences in a way that enables its integration into the planar atom. 30 The vector combination of claim 5, wherein the acceptor Ti plasmid is pGV2260, DSM 2799. DK 173104 B1 The vector combination of claim 5 or 6, wherein the intermediate cloning vector further contains at least one selectable marker gene adjacent to the gene or genes of interest (5). The vector combination of claim 9, wherein the selectable marker gene encodes resistance to an antibiotic. The vector combination of claim 5 or 6, wherein the intermediate cloning vector is pGV750, DSM 2797. The vector combination of claim 5 or 6, wherein the intermediate cloning vector is further characterized in that the gene (s) of interest (5) is under the control of its (their) natural promoter. The vector combination of claim 5 or 6, wherein the intermediate cloning vector is further characterized in that the gene or genes of interest (5) are under the control of promoter exogenous to the gene or genes of interest. The vector combination of claim 11, wherein the promoter sequence provides tissue-specific regulation of transcription of said downstream sequences, preferably in leaves, roots, stems or flowers of a plant containing the cell. The vector combination of claim 11, wherein the promoter sequence provides inducible regulation of transcription of said downstream sequences, preferably by temperature, light or added chemical factors. DK 173104 B1 The vector combination of claim 11, wherein the promoter sequence is the promoter sequence of an Opin synthase gene of Agrobacterium T-DNA, preferably the promoter sequence of the nopaline synthase gene. The vector combination of any one of claims 5-14, wherein the gene of interest controls the synthesis of products such as amino acids or sugars. 10 The vector combination of any of claims 5-14, wherein the gene of interest is a structural gene. The vector combination of any of claims 5-14, wherein the gene of interest controls the synthesis of products which confer protection against external pathogenic agents, such as resistance to disease-causing organisms or stress-causing environmental factors. 18. Acceptor Ti plasmid pGV2260, DSM 2799. 19. The intermediate cloning vector pGV750, DSM 2797. A method of producing a hybrid Ti plasmid, comprising producing a vector combination according to any one of claims 1-4 in an Agrobacterium and performing a co-integration between the intermediate cloning vector and the acceptor Ti plasmid by a single crossover. 30 A method for producing a hybrid Ti plasmid, comprising producing a vector combination DK 173104 B1 according to any of claims 5-17 in an Agrobacterium and performing a cointegration between the intermediate cloning vector and the acceptor Ti plasmid at a single crossover. 5 A hybrid Ti plasmid vector obtainable by co-integration between either an acceptor Ti plasmid which is unable to induce plant tumors and comprises: (a) the two border sequences (1, 2) of the T region of a wild-type Ti plasmid, (b) a DNA sequence (3) which is free of the oncogenic functions of the wild-type T-DNA, derived from a cloning agent located between the two boundary sequences, and containing phen DNA sequence which is homologous to at least a portion of a DNA sequence in an intermediate cloning vector allowing a single crossover, and (c) a DNA segment (4) of a wild-type Ti plasmid containing DNA sequences essential for transfer by Agrobacterium of the T region of wild-type Ti plasmids into 20 µ plant cell genomes, and an intermediate cloning vector comprising: (a) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants, and (b) a cloning agent segment (3 ') containing a D NA sequence which is homologous to the above DNA sequence (b) in the acceptor Ti plasmid, or between an acceptor Ti plasmid comprising: (A) a DNA segment (4) of a wild-type Ti plasmid without The hybrid Ti plasmid vector of claim 22, further comprising at least one selectable marker gene adjacent to the gene or genes of interest (5). DK 173104 B1 The hybrid Ti plasmid vector of claim 23, wherein the selectable marker gene encodes resistance to an antibiotic. The hybrid Ti plasmid vector of claim 24, wherein the selectable marker gene encodes resistance to methotrexate. The hybrid Ti plasmid vector according to claim 23, wherein the selectable marker gene is an Opin synthase gene of Agrobacterium T DNA, preferably the nopaline synthase gene or the octo-pin synthase gene. Hybrid Ti plasmid vector according to any one of claims 22-26, characterized in that the gene (s) of interest (5) is under the control of its (their) natural promoter. Hybrid Ti plasmid vector according to any one of claims 22-26, characterized in that the gene or genes of interest (5) are under the control of a promoter which is exogenous to the gene or genes of interest. The hybrid Ti plasmid vector of claim 28, wherein the promoter sequence provides tissue-specific regulation of transcription of said downstream sequences, preferably in leaves, roots, stems or flowers of a plant containing the cell. The hybrid Ti plasmid vector of claim 28, wherein the promoter sequence provides inducible regulation of transcription of said downstream sequences, preferably by temperature, light or added chemical factors. DK 173104 B1 The T region and without the two boundary sequences of the T region and (B) a DNA sequence (3) derived from a cloning agent, and an intermediate cloning vector comprising: DK 173104 B1 (A ') the left and right boundary sequence of a Τ 'region of a wild-type Ti plasmid (1, 2), (Bf> a cloning agent segment (3') located outside the two boundary sequences which are homologous to DNA in the sequence (b) of said acceptor -Ti plasmid allowing a single crossover, and (C ') a region essentially free of internal S T DNA sequences of a wild-type Ti plasmid and containing at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants, whereby said region is located between the two boundary sequences in a manner which enables its integration into the plant atom, said hybrid Ti plasmid vector at least comprising: (a) the two border sequences (1, 2) of the T region of a wild-type T plasmid, (β) keon oncogenic DNA sequences (3, 3 ') derived from a cloning agent, 20 (γ) a DNA segment (4) of the wild-type Ti plasmid containing the DNA sequences essential for transfer of T the region of wild-type Ti plasmids with Agrobacterium for plant cell genomes, and (δ) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants located between the two boundary sequences (l, 2). The hybrid Ti plasmid vector of claim 28, wherein the promoter sequence is the promoter sequence of an open synthase gene of Agrobacterium T-DNA, preferably the promoter sequence 5 of the nopaline synthase gene. The Hybrid Ti plasmid vector according to any of claims 22-31, wherein the gene of interest controls the synthesis of products such as amino acids or sugars. 10 The Hybrid Ti plasmid vector of any of claims 22-31, wherein the gene of interest is a structural gene, A hybrid Ti plasmid vector according to any one of claims 22-31, wherein the gene of interest controls the synthesis of products that confer protection against external pathogenic agents, such as resistance to disease-causing organisms or stress-causing environmental factors. The hybrid Ti plasmid vector of any one of claims 22-31, wherein the gene of interest is a selectable marker gene. The hybrid Ti plasmid vector of claim 35, wherein the gene of interest is an antibiotic resistance gene. An agrobacterium containing a vector combination according to any one of claims 1-17. 38. Agrobacterium containing a hybrid Ti plasmid vector according to any one of claims 22-36. DK 173104 B1 A method of producing a transformed plant cell, comprising infecting plant cells with an Agrobacterium containing a vector combination according to claim 37. 5 A method of producing a transformed plant cell, comprising infecting plant cells with an Agrobacterium containing a hybrid Ti plasmid vector according to claim 38. A method of producing a plant, comprising the following steps: (a) infecting a plant cell with an Agrobacterium according to claim 37, and (b) forming a plant from the transformed plant cells obtained in step (a). A method of producing a plant, comprising the steps of: (a) infecting a plant cell with an Agrobacterium according to claim 38, and (b) forming a plant from the transformed plant cells obtained in step (a). 25