RU2149187C1 - Vector for insertion of required gene in plant (variants), method of transgenic plant producing and method of insertion of at least two required genes in plant - Google Patents

Abstract

FIELD: plant selection, molecular biology, biotechnology. SUBSTANCE: vector has gene that to be inserted in plant and gene-marker. It is possible to avoid the presence of usual selective gene-marker in transgenic plant since this results often to undesirable effects for plant transformation as gene-marker is presented by gene inducing morphological anomality being easily removable from DNA after insertion of vector in plant. EFFECT: improved method of vector insertion. 29 cl, 31 dwg, 3 tbl, 10 ex

Description

 The present invention relates to a new vector for introducing a desired gene into a plant using genetic engineering methods to produce a transgenic plant; to a method for producing a transgenic plant not affected by a selectable marker gene using the specified vector; and a method for introducing at least two desired genes into a plant using said vector.

State of the art
The transformation of microorganisms and cultured cells using genetic engineering is currently used in the production of physiologically active substances used as drugs, thus making a great contribution to this industry. In the field of plant breeding, the industrial application of genetic engineering is lagging behind, because the plant life cycle is much longer than the microorganism cycle. However, due to the fact that this technology allows you to enter the desired gene directly into the breeding plants, it has the following advantages compared to classical selection, which requires multiple crosses.

 (a) Only the property to be improved is subject to change.

 (b) It is possible to impart properties to species other than plants (such as microorganisms, etc.).

 (c) A significant reduction in the selection period is possible.

 Thus, methods of genetic engineering for plant breeding were actively studied.

 Obtaining transgenic plants includes the following three stages.

 (1) Introduction of a desired gene into a plant cell (including introduction of a gene into chromosomes, nucleus, etc.).

 (2) Selection of plant tissue consisting only of cells into which the desired gene has been introduced.

 (3) Regenerating a plant from selected plant tissue.

 In order to select transgenic tissues into which the desired gene has been introduced, visual identification of the tissue in which the desired gene is expressed is necessary without regenerating a new plant. For this, the desired gene is usually introduced into the plant cell along with a selectable marker gene, the expression of which can be easily detected at the stage of cell cultivation. This means that expression of the selectable marker gene is used as an indicator of the introduction of the desired gene. Examples of conventional breeding marker genes include antibiotic resistance genes, such as the kanamycin resistance gene (i.e., the neomycin phosphotransferase gene); hygromycin resistance gene (i.e., hygromycin phosphotransferase gene); amino acid synthetase genes such as the nopaline synthetase gene, the octopine synthetase gene, and the sulfonylurea resistance gene (i.e., the aminoactate synthetase gene), which confers agricultural chemical resistance.

 However, the expression of a selectable marker gene can cause serious problems when using a transgenic plant for food. Those. it is difficult to state that a gene product produced by expressing a selective marker gene is safe for humans. Therefore, if a transgenic plant containing a selective marker gene is sold as a food product, a detailed study is necessary to determine the effect of the selective marker gene on the human body. For example, the neomycin phosphotransferase gene has been used as a selective marker gene at the laboratory level since the early 1980s. In 1994, the product of this gene was finally accepted as a nutritional supplement by the US Food and Drug Administration. Since that time, transgenic plants containing the neomycin phosphotransferase gene as a selective marker gene have been used for food purposes. However, some consumers of products containing the neomycin transferase gene are still concerned about the effects of this gene.

 Moreover, the selectable marker genes that are used in practice are the only genes, such as the neomycin phosphotransferase gene, that contribute to the detoxification of the plant cell growth inhibitory factor. Therefore, to select the transgenic tissue of the plant into which the desired gene was introduced, such tissue is cultivated in a culture medium containing growth suppression factor and expression of a selective marker gene, namely, the tissue resistance to growth suppression factor is determined and used as an indicator. However, even if the tissue has such resistance, cultivation in the presence of a suppression factor can lead to undesirable side effects on plant cells, such as decreased proliferation and transdifferentiation of transgenic tissue.

 Further, the expression of a selective marker gene in a plant cell after selection of transgenic tissue seriously interferes with plant selection by subsequent gene introduction. This means that when a different gene is introduced into a transgenic plant containing a selective marker gene, the introduction of such a gene should be controlled using another selective marker gene. However, the effectiveness of the selective marker gene depends on the type of plant, therefore, a preliminary test is necessary to determine the conditions for each selective marker gene (for example, it is reported that the hygromycin phosphotransferase gene more effectively affects rice plants than the neomycin phosphotransferase gene (K. Shimamoto et al., Nature (London), T. 338, p. 274, 1989) Moreover, since the species of the selective marker gene is limited, multiple gene introduction cannot be repeated indefinitely, simply by replacing of a selective marker gene, which means that the number of introductions of a gene into a particular plant is limited in itself by the type of selective marker gene that can be used for such a plant. In addition, the type of selective marker gene that can actually be used is limited, as indicated above Accordingly, it is desirable to find a way to remove the selective marker gene from the chromosome after selection of the transgenic tissue of the plant in order to exclude the influence of the selective marker gene on the cell, tissue and plant.

 Two methods are known for eliminating the effects of a selective marker gene. In accordance with the first method, a selective marker gene and a plant transposon are introduced into its chromosome and then removed from it (international application Laid-Open No. WO 92/01370). In accordance with the second method, a site-specific phage P1 recombination system is used instead of the transposon (International Application Laid-Open No. WO 93/01283). Using these methods, it is possible to obtain a cell in which a selective marker gene is removed from the plant chromosome in this respect after the introduction of the gene. Unfortunately, the likelihood that a selective marker gene is deleted is very low.

 Further, the cells of the plant from which the selective marker genes were removed using these methods are scattered among the cells in which the selective marker genes are still present and expressed. These two types of cells cannot be distinguished visually.

 Plant cells containing selective marker genes and desired genes can be selected based on their chemical resistance, nutritional requirements, etc. However, during selection, cells that do not contain selective marker genes exhibit strong growth inhibition and are often destroyed. Naturally, such selections cannot be used to obtain cells that do not contain selective marker genes.

 In order to obtain plants that do not contain a selective marker gene and contain the desired gene using the above methods, the tissue of a plant in which cells containing no selective marker gene are mixed and cells containing selective marker genes are proliferated, regenerated, and then analyzed for selection using methods such as Southern hybridization or polymerase chain reaction.

 This method is based on the premise that a regenerated cell is obtained from one cell and therefore all plant cells must have the same properties. Thus, a product obtained from a cell that does not contain a selective marker gene consists only of such cells. Unfortunately, the cells that make up such a regenerated product are not necessarily uniform. Cells that do not contain the chromosomes of the selective marker gene and cells containing the selective marker gene are present together and are rather unevenly distributed even in the same separate regenerated plant and in the same tissue. Thus, it is extremely difficult to obtain a product consisting only of cells that do not contain a selective marker gene at the stage when the cultured tissue is transdifferentiated to regenerate the product.

 In addition, well-known analytical screening methods use tissue, such as a sheet, as a test sample (not the entire product or a single cell). Therefore, only the general tendency is analyzed with respect to the state of the selectable marker gene present in one sheet. In this case, usually in the same product or tissue is present as a cell that does not contain a selective marker gene, and a cell containing the specified marker gene. So, even with the formation of a product consisting only of cells that do not contain a selective marker gene, it is difficult to isolate it. Even if the presence of a selective marker gene is not detected in this tissue, tissues at other sites of the same product may contain a selective marker gene, or it simply means that the amount of the selective marker gene is below the detection limit. Therefore, it is not possible to determine whether a test sample is completely free of cells containing a marker gene.

Using the above methods, it is possible to obtain a product that does not contain a selective marker gene only from an germ cell, such as pollen, an egg cell, and the like. Self-pollination using an egg cell that does not contain a selective marker gene results in a fertilized egg that does not contain a selective marker gene with a constant ratio in accordance with the classical hereditary law, and from this fertilized egg a product is obtained that has the same properties as and a fertilized egg. Using this product, conventional analytical methods such as Southern hybridization can be carried out. In particular, even if a cell that does not contain a selective marker gene is obtained using the method described in this link, a product made from such a cell is obtained for the first time by transdifferentiation of a plant from culture tissue containing such a cell by crossing the regenerated plants and receiving offspring F ₁ or later generations. The product thus obtained can be isolated as a product lacking a selective marker gene.

 In order to remove a selective marker gene from a transgenic plant, JP-A-6-276872 describes a method for introducing a gene according to which a selective marker gene is inserted into a separate plasmid vector other than a vector containing the desired gene. A plasmid containing a selective marker gene is removed from the cell after the introduction of the gene is complete (the term "JP-A" as used here means published Japanese patent application). However, this method requires a crossover step to remove the marker marker gene. In this regard, this method is the same as both of the above links.

The above methods are difficult to apply to woody plants having a long growth period, sterile products or a hybrid product in which F ₁ is valuable in itself. Further, when using deleted DNA elements such as transposon and the like, the ratio at which these elements are removed from the chromosomal DNA, viral DNA vector and the like where these elements are present and functioning is usually extremely low. Accordingly, it is necessary that the removal of these elements (in particular, the removal of the selective marker gene) is easily traceable in reality, at least at the stage of the cultured tissue. In the event that this cannot be traced to the transdifferentiation of the cultured tissue and the formation of a later generation by crossing the regenerated product, the method is not practical.

Disclosure of Invention
Accordingly, it is an object of the present invention to provide a vector containing a gene that is desired to be introduced into a plant and a selective marker gene, in which a plant containing said gene does not have a side effect on the human body when consumed, even when expressing a selective marker gene.

 Another objective of the present invention is the provision of a vector for introducing the desired gene into a plant, in which the vector contains a selective marker gene that allows the selection of transgenic tissue, without using a cell growth inhibitory factor that reduces plant cell activity.

 A further object of the present invention is to provide a vector for introducing a desired gene into a plant, wherein the vector contains a selective marker gene and acts so as to eliminate the effects of the selective marker gene by removing the selective marker gene from DNA in which the selective marker gene is present and functioning. Using this vector, the desired gene can be efficiently re-introduced.

Another objective of the present invention is the provision of a method for producing a transgenic plant using a vector that can eliminate the effects of a selective marker gene, not including the stage of production of F ₁ or later generations by crossing, and a method for the diversified introduction of genes into a plant by applying the method described above.

 These and other objectives of the present invention are achieved by using a vector comprising the desired gene and at least one gene with the induction of morphological abnormality as a selective marker gene.

 Further, these and other objectives of the present invention are achieved by using a vector in which a selective marker gene is removed from the DNA after its expression. The expression of the selective marker gene and the termination of its function is determined by morphological changes in the tissue into which the selective marker gene was introduced.

 Moreover, these and other objectives of the present invention are achieved by using a vector comprising the desired gene, at least one gene with the induction of morphological abnormality as a selective marker gene, and a deleted DNA element. A gene with the induction of morphological abnormality is placed in such a way that it behaves in the same way as a deleted DNA element. The desired gene is arranged in such a way that it does not behave the same as the deleted DNA element.

Further, these and other objectives of the present invention are achieved by providing a method for producing a transgenic plant free of the effects of a selective marker gene, comprising the following steps:
(A) introducing a vector into a plant cell, wherein said vector comprises a desired gene, at least one gene with induction of morphological abnormality and a deleted DNA element, wherein said gene with induction of morphological abnormality is placed in such a way that it behaves the same way as the deleted DNA element, and the desired gene is placed in such a way that it does not behave the same as the deleted DNA element;
(B) cultivating the plant cell obtained in (A), detecting the morphological abnormal plant tissue that appears during cultivation, and selecting morphologically abnormal tissue, and
(C) culturing said morphologically abnormal tissue selected according to subparagraph (B), detecting morphologically normal tissue that appears during cultivation, and selecting morphologically normal tissue.

Further, these and other objectives of the present invention are achieved by providing a method for introducing at least two desired genes into a plant, comprising at least twice the following steps:
(A) introducing a vector into a plant cell, wherein said vector comprises the desired gene, at least one gene with the induction of morphological abnormality as a selective marker gene, and a deleted DNA element, wherein said gene with the induction of morphological abnormality is placed in such a way that it behaves the same as the deleted DNA element, and the indicated desired gene is placed in such a way that it does not behave the same as the deleted DNA element,
(B) cultivating the plant cell obtained in (A), detecting morphologically normal plant tissue that appears during cultivation, and selecting said morphologically abnormal tissue, and
(C) culturing said morphologically abnormal tissue selected in (B), detecting morphologically normal tissue that appears during cultivation, and selecting said morphologically normal tissue.

 In addition, these and other objectives of the present invention are achieved by regenerating the plant by culturing the above morphologically normal tissue.

Brief Description of the Drawings
FIG. 1 is a Ti plasmid diagram and an endonuclease restriction map of the Pst I fragment in the T-DNA region of A. tumefaciens PO22 strain.

 FIG. 2 is a pIPT2 construction diagram.

 FIG. 3 is a diagram of the construction of pIPT3 from pIPT2.

 FIG. 4 is a construction diagram of pIPT4 from pIPT3.

 FIG. 5 is a restriction map of the endonuclease of a T-DNA region in the pIPT4 structure.

 FIG. Figure 6 shows the results of the analysis of the polymerase reaction of the synthesis of the extreme shoot of the tobacco phenotype into which the gene was introduced using pIPT4.

 FIG. 7 is a construction diagram of pNPI102.

 FIG. 8 is a construction diagram of pNPI103 from pIPT4 and pNPI102.

 FIG. 9 is a construction diagram of pNPI106 from pNPI103.

 FIG. 10 is a restriction map of the endonuclease of a T-DNA region in the structure of pNPI106.

 FIG. 11 is a photograph of shoot N 2 after a month of cultivation, as described in example 2.

 FIG. 12 is a photograph of shoot No. 8 after a month of cultivation, as described in Example 2.

 FIG. 13 shows the results of an analysis of the polymerase reaction of synthesis of the escape chain N 8, as described in example 2.

 FIG. 14 shows the results of the analysis of the polymerase reaction of chain synthesis of a normal product obtained from shoots NN 13-1 and 14-1, as described in example 3.

 FIG. 15 is a photograph of normal shoots differentiated from the extreme phenotype of a tobacco shoot, as described in Example 3.

 FIG. 16 shows the results of an analysis of the polymerase reaction of chain synthesis of a normal product obtained from a leaf formed from shoot No. 7, as described in Example 2.

 FIG. 17 is a construction diagram of pNPI128.

 FIG. 18 is a construction diagram of pNPI129 from pNPI128.

 FIG. 19 is a construction diagram of pNPI132 of pNPI101 and pNPI129.

 FIG. 20 is a restriction map of the endonuclease of a T-DNA region into the pNPI132 structure.

 FIG. 21 shows the results of the analysis of the polymerase reaction of chain synthesis of normal products obtained from shoots NN 15-21, as described in example 5, using seeds in which the presence of the ipt gene was detected.

 FIG. 22 shows the results of the analysis of the polymerase reaction of the synthesis of a chain of normal products obtained from shoots NN 15-21, as described in Example 5, using seeds, in which the elimination of the region retained by the Rs pair, including the ipt gene, was detected.

 FIG. 23 shows the results of the analysis of the polymerase reaction of chain synthesis of normal products obtained from shoots NN 15-21, as described in example 5, in which the presence of the GUS gene was detected.

 FIG. 24 shows the results of the analysis of the polymerase reaction of the synthesis of a chain of normal products obtained from a line that could not form an extreme shoot phenotype, as described in Example 5.

 FIG. 25 is a design diagram of pNPI702.

 FIG. 26 is a restriction map of the endonuclease of a T-DNA region in the structure of pNPI702.

 FIG. 27 is a photograph of normal shoots differentiated from the extreme phenotype of a hybrid aspen shoot, as described in Example 7.

 FIG. 28 is a restriction map of the endonuclease of a T-DNA region in the structure of pNPI140.

 FIG. 29 shows the results of the analysis of the polymerase reaction of the synthesis of a normal shoot chain, differentiated from the extreme shoot phenotype after various gene introductions, as described in example 8.

The best embodiment of the invention
In this description, a gene with the induction of morphological abnormality (MAI) means a gene that induces morphologically abnormal differentiation in plant tissue, such as dwarfism, impaired apex dominance, pigment changes, formation of gaps in the crown, formation of hairy roots, leaf waviness, etc. In addition to the preferred gene (MAI), genes isolated from bacteria of the genus Agrobacterium or the like, inducing tumors or teratomas (i.e., the formation of additional shoots and adnexa roots) in various plants can be used. Examples of these different MAI genes are cytokine synthesis genes (e.g. ipt (isopentenyl transferase) gene (AC Smigocki. LD Owens, Procl. Natl. Acad. Sci. USA, T. 85, p. 5131, 1988)), iaaM gene (tryptophan monooxygenase), (HJ Klee et al., GENES & DEVELOPMENT, vol. 1, p. 86, 1987), gene 5 (H. Korber et al., EMBO Journal, vol. 10, p. 3983, 1991), gene 6b (PJJ Hooykaas et al., Plant Mol. Biol., Vol. 11, p. 791, 1988), and rol genes such as rolA, rolB, rolC and rolD (FF White et al., J. Bacteriol., vol. 164, p. 33, 1985). Further examples are the iaaL gene (indoleacetic acid-lysine synthetase), such as Pseudomonas syringae subspecies savastanoi (A. Spena et al., Vol., Gen Genet., Vol. 227, p. 205, 1991), homeobox genes and phytochrome genes in various plants. Preferably, cytokine synthesis genes are used, such as the ipt gene or at least one gene selected from rol genes (more preferably rol genes containing rolA, rolB and rolC genes). The ipt gene is present in T-DNA of Agrobacterium tumefaciens and causes a violation of the apex dominance. Rol genes containing the rolA, rolB, and rolC genes are present in the T-DNA of Agrobacterium rhizogenes and at least one of them causes the formation of hairy roots, dwarfism, leaf waviness, etc. plants regenerated from hairy roots.

 Using the methods of the present invention, it is possible to program a combination of these selective marker genes in such a way that a specific structure, such as an extra shoot, subordinate root, etc., is transdifferentiated in a separate plant into which these selective marker genes are introduced. In accordance with the present invention, such a combination of MAI genes can be used in accordance with the conditions for obtaining a transgenic plant suitable for introducing genes.

 Morphologically abnormal tissue obtained by introducing the MAI gene into a cell consists only of cells containing this gene. Therefore, using this gene as a selective marker gene, a vector is constructed along with the desired gene. When this vector is introduced into a plant cell and the transgenic cell is cultured, a tissue consisting only of this cell, into which the selective marker gene and the desired gene are introduced, can be separated by visual separation of the resulting morphological abnormal tissue from this cell.

 Suitable vectors used in accordance with the present invention have a DNA sequence that embeds an extraneous gene into a host cell and which expresses an extraneous gene within the host cell.

 When a gene is introduced using the vector in accordance with the present invention, plant tissue consisting only of a transformed stand can be visually separated by simply culturing the cell after the operation of introducing the gene into a common culture medium, such as Murashige-Skoog culture medium, under normal culturing conditions . Since there is no need to use a special substance to isolate transformed tissue, such as a factor for suppressing the growth of plant cells, etc., the procedure is not only simplified, but also due to such a substance, the activity of plant cells does not decrease. In addition, the MAI gene is inherent in the plant or this gene is spontaneously introduced into the plant through infection with bacteria, etc. Therefore, the plant obtained using the vector in accordance with the present invention does not differ from natural plants having this morphological abnormality.

 Suitable vectors in accordance with the present invention include a vector in which the MAI gene is located so that it acts together with the deleted DNA, and the desired gene is placed in such a way that it does not work together with the deleted DNA element.

 As used herein, a deleted DNA element is an element of a DNA sequence that can itself be derived from the DNA in which the DNA element exists and functions. In plants, the transposon present on the chromosome is known as such an element. The structure, activity, and behavior of transposons have been established almost completely. In principle, for the functioning of the transposon, two components are necessary: (1) an enzyme that is expressed from the gene present in it and which catalyzes the excision (excision) and transposition of the transposon itself (transposase), and (2) the DNA binding sequences that are present at the terminal sites transposon and on which transposase acts. Using these elements, the transposon is excised from the chromosome in which it is contained, and then usually transferred to a new position in the DNA. However, at a certain ratio, the transposon also disappears without transfer. The present invention takes into account such a transposon transfer error.

 There are two types of transposon: autonomous transposon and non-autonomous transposon. Autonomous transposon supports two elements: transposase and DNA binding sequence. In an autonomous transposon, transposase is expressed and bound to the DNA binding sequence for action, while the transposon is autonomously excised from the chromosome. A non-autonomous transposon holds the terminal DNA binding sequence to which transposase is linked for action. In a non-autonomous transposon, a gene undergoes a mutation in which transposase is not expressed; Thus, the transposon cannot be excised from the chromosome autonomously. However, if a transposase enters a non-autonomous transposon from an autonomous transposon or from an independent transposase gene, the non-autonomous transposon behaves like an autonomous transposon.

 Examples of autonomous transposons are Ac and Smp isolated from maize (A. Gierl, H. Saedler, Plant Mol. Biol., Vol. 19, p. 39, 1992). Ac can be obtained by hydrolyzing the wx-m7 gene locus on the maize chromosome using the restriction endonuclease Sau3A (U. Behrens et al., Mol. Gen. Genet., Vol. 194, p. 346, 1984). This autonomous transposon is the most studied among plant transposons. In fact, the DNA sequence has already been determined (M. Muller-Neumann et al., Mol. Gen. Genet., Vol. 198, p. 19, 1984).

 Examples of non-autonomous transposons are Ds and dSpm obtained by deletion of the internal regions of Ac and Spm, respectively (H.-P. Doring, P. Starlinger, Ann. Rev. Genet., Vol. 20, p. 175, 1986), and transposons isolated from many plants (not corn), such as snapdragon, morning glory, etc. (e.g., Y. Inagaki et al., Plant Cell, vol. 6, p. 375, 1994).

 When these transposons are introduced into the chromosomes of exogenous plants, they are also excised from the chromosome and transferred, showing their activity (e.g. B. Baker et. Al., Proc. Natl. Acad. Sci. USA, vol. 83, p. 4844, 1986 )

 In accordance with the present invention, both autonomous and non-autonomous transposons can be used. Non-autonomous transposons can be used by insertion of a functioning transposase gene, too.

 Another mobile DNA gene that is not present in plants, but which can be used in accordance with the present invention, is an element derived from a site-specific recombination system. A site-specific recombination system consists of two elements: (1) a recombination site (corresponding to a movable DNA element in accordance with the present invention) having a characteristic DNA sequence, and (2) an enzyme that specifically binds a DNA sequence and catalyzes recombination between DNA sequences when the presence of two or more sequences (recombinase). When two DNA sequences are oriented in the same direction for a given interval of the same DNA molecule, the region held by these DNA sequences is excised from the DNA molecule, such as a plasmid, chromosome, etc. When two DNA sequences are oriented in opposite directions of the same DNA molecule, the portion held by these DNA sequences is inverted.

 The present invention preferably uses sequential excision. Both excision and inversion within the recombination site occur as a result of homologous recombination through a site-specific recombination system, which differs from the mechanism using transposon. It is known that the recombinase gene is not necessarily present in the same DNA molecule in which the recombination site is located. The recombinase gene only needs to be present in the same cell and expressed to excise or invert the region held by two DNA sequences (N.L. Craig, Annu. Rev. Genet., Vol. 22, p. 77, 1988).

 Currently, site-specific recombination systems have been identified in microorganisms such as phage, bacteria (e.g., E. coli), yeast, and the like. Examples are the Cre / lox system, the pSR1 system, the FLP system, the cer system, and the fim system (e.g., N.L. Craig, Annu. Rev. Genet., Vol. 22, p. 77, 1988). When a site-specific recombination system, separated from these microorganisms using the Cre / lox system obtained from phage 31 (WO 93/01283), is introduced into organisms (including plants) other than the organism from which this system was obtained, it behaves in the same way as in the original body. The site-specific yeast recombination system (Zygosaccharomycts rouxii) (pSR1 system (H. Matsuzaki et al., J. Bacteriology, vol. 172, p. 610, 1990)) can also be used in accordance with the present invention. This pSR1 system also (supports) retains its inherent function in higher plants (H. Onouchi et al., Nucleic Acid Res., Vol. 19, p. 6373, 1991).

 In accordance with the present invention, a gene with the induction of morphological abnormality (MAI) can be placed in such a position in which this gene is excised together with the deleted DNA element. For example, when a transposon is used as the deleted DNA element, the MAI gene can be placed in a position that does not affect the excision of the transposon and which is located above the promoter region of the transposase gene, but below the end region to which the transposase is associated. Using the pSR1 system, the MAI gene can be inserted at any position within the region held by the characteristic DNA sequences that inhibit recombinase expression.

 In accordance with the present invention, the MAI gene is preferably located within the deleted DNA element. On the other hand, the position of the desired gene is not particularly limited, however, it is preferable that the desired gene is located outside the deleted DNA element.

 Using a vector of this structure after introducing the desired gene, the MAI gene can be removed at a certain frequency along with the deleted DNA element from the DNA in which it is present and functioning. The desired gene, which does not act in conjunction with the selectable marker gene, remains in the same DNA. This vector can be used to repeatedly introduce the desired gene into a specific plant. In addition, since the loss of function of this MAI gene can be defined visually as a morphological change in the transgenic tissue during cultivation, a tissue consisting only of a cell containing the desired gene but not containing a selective marker gene can be easily separated without using a special procedure. Therefore, even if such a cell actually forms at a low ratio, this cell can be successfully isolated in order to make this procedure practically useful. Further, using this vector, you can not only repeatedly enter the gene, but also repeat this procedure until the regeneration of the adult plant. Thus, multiple administrations can be successfully carried out. In order to obtain a single transgenic plant consisting only of such cells, the plant can be regenerated from the tissue thus isolated without undergoing a crossover step. Thus obtained individual transgenic plant is completely free from side effects on the human body caused by the aforementioned selective marker gene. Moreover, the use of such a vector does not require the use of a factor for suppressing cell growth, which can reduce cell activity at the stage of isolation of transgenic tissue.

 The vector in accordance with the present invention can be used in any plants into which the gene can be introduced using genetic engineering methods. The desired gene in accordance with the present invention can be any gene that gives the necessary agricultural properties, and any gene that allows the study of the mechanisms of gene expression, etc., while not necessarily giving the necessary agricultural properties.

 To produce a protein, such as an enzyme, from a gene, a structural sequence of a gene encoding the information of a polypeptide and regulatory sequences of a structural gene, such as a promoter sequence (an expression initiating sequence), a termination sequence (an expression terminating sequence), etc., are usually required. Examples of a suitable promoter sequence that functions in a plant is the 35S promoter of cauliflower mosaic virus (JT Odell et al., Nature (London), vol. 313 , p. 810, 1985), a nopalysis synthetase promoter (WHR Langridge et al., Plant Cell Rep., vol. 4, p. 355, 1985) and a diphosphate carboxylase / oxygenase small subunit promoter (R. Fluhr et al., Proc. Natl. Acad. Sci. USA, vol. 83, p. 2358, 1986). Examples of suitable terminator sequences are the polyadenylation signal of nopaline synthetase (A. Depicker et al., J. Mol. Appl. Gen., vol. 1, p. 561, 1982) and the octapine synthetase polyadenylation signal (J. Gielen et al., EMBO J., vol. 3, p. 835, 1984). Accordingly, if necessary, the gene of the vector in accordance with the present invention includes a structural gene and its regulatory sequences expressing the gene. A given gene or gene and regulatory sequences can be obtained by chemical synthesis or by cloning cDNA or genomic DNA.

 The vector in accordance with the present invention can be indirectly introduced into the plant cell through viruses or bacteria that infected the plants (I. Potrycus, Annu. Rev. Plant Physiol. Plant Mol. Biol., Vol. 42, p. 205, 1991). Examples of suitable viruses are cauliflower mosaic virus, heminivirus, tobacco mosaic virus and mosaic bromine virus. Examples of suitable bacteria are Agrobacterium tumefaciens (hereinafter referred to as A. tumefaciens) and Agrobacterium rhizogenes (hereinafter referred to as A. rhizogenes). Dicotyledonous plants are generally known to be infected with bacteria of the genus Agrobacterium. Recently, the introduction of genes into monocotyledonous plants by infection of said plants with said bacteria has also been reported (for example, international patent application N WO 94/00977).

 The vector of the present invention can be directly introduced into the plant by physical and chemical methods such as microinjection, electroporation, polyethylene glycol method, fusion method and high-speed ballistic penetration (I. Potrycus, Annu. Rev. Plant Physiol. Plant Mol. Biol. , vol. 42, p. 205, 1991). Since the general method of indirect administration using the genus Agrobacterium cannot be applied to many monocotyledonous plants and dicotyledonous plants that are resistant to infection by Agrobacterium, the methods of the above direct administration are effective for these plants.

 The vector used in accordance with the present invention is not particularly limited as long as it meets the requirements of the present invention. For example, by indirectly introducing a vector into a plant cell, the vector may be a Ti vector or a viral vector. Examples of the Ti vector for use in accordance with the present invention are Bin19 (M. Bevan et al., Nucleic Acids Res., Vol. 12, p. 8711, 1984), pRAL3940 (A. Hoekema et al., Plant Mol. Biol ., vol. 5, p. 85, 1985), pGA492 and pGA482 (G. An, Plant Physiol., vol. 81, p. 86, 1986), pC22 (C. Simoens et al., Nucleic Acids Res., vol. 14, p. 8073, 1986), pAGS111 (P. van den Elzen et al., Plant Mol. Biol., vol. 5, p. 149, 1985), pEND4K (HJ Klee et al., Bio / Technology , vol. 3, p. 637, 1985), pGV831 (R. Delaere et al., Nicleic Acids Res., vol. 13, p. 4777, 1985) and pMCN200 (RT Fraley et al., Bio / Technology, vol . 3, p. 629, 1985). Examples of a viral vector for use in accordance with the present invention are the cauliflower mosaic viral vector (N. Brisson et al., Nature (London), vol. 310, p. 511, 1984), the heminivirus vector (RJ Hayes et al. , Nature (London), vol. 334, p. 179, 1988), vector of bromine mosaic virus (R. French et al., Science, vol. 231, p. 1294, 1986), vector of mosaic tobacco virus (N. Takamatsu et al., EMBO J., vol. 6, p. 307, 1987) and an agroinfectious vector (N. Grimsley et al., Nature (London), vol. 325, p. 177, 1987). However, vectors for use in accordance with the present invention are not limited to these vectors.

 Further, the desired gene for use in accordance with the present invention is not particularly limited. Examples of a desired gene for use in accordance with the present invention include disease resistance genes (e.g., the Bacillus thuringiensis endotoxin gene, WO 92/20802), herbicides (e.g., the mutant acetolactate synthase gene, WO 92/08794), storage protein grains (e.g., glutelin gene, WO 93/18643), to fatty acid synthesis (e.g., acyl-ACP thioexterase gene, WO 92/20236), to cell wall hydrolysis (e.g., polygalacturonase gene (D. Grierson et al., Nucleic Acids Res., Vol. 14, p. 8595, 1986)), to the biosynthesis of anthocyanin (e.g., the chalcone synthase gene (HJ Reif et al., Mol. Ge n. Genet., vol. 199, p. 208, 1985)), to ethylene biosynthesis (e.g., ACC oxidase gene (A. Slater et al., Plant Mol. Biol., vol. 5, p. 137, 1985) ), to an active oxygen utilization system (for example, the glutathione reductase gene (S. Greer & RN Perham, Biochemistry, vol. 25, p. 2736, 1986)) and to lignin biosynthesis (for example, the phenylalanine ammonia lyase gene, cinnamyl alcohol dehydrogenase gene , o-methyltransferase gene, cinnamate 4-hydroxylase gene, 4-coumarrate-CoA ligase gene, cinnamoyl CoA reductase gene (AM Boudet et al., New Phytol., Vol. 129, p. 203, 1995)). However, the desired genes for use in the present invention are not limited to these genes.

 Moreover, the host plant for use in accordance with the present invention is not particularly limited. Examples of herbaceous plants used as the host plant are tobacco (Tabacum), tomato (Lycopersicom), sweet potato (Impoea), potato (Solanum), carrot (Dacus), lettuce (Lactuca), cauliflower (Brassica), cabbage (Brassica), oilseed rape (Brassica), sunflower (Helianthus), sugar beets (Bela), asparagus (Asparagus), banana (Musa), cotton (Gossypium), arabidopsis (Arabidopsis), alfalfa (Medicago), peas (Pisum) ), soy (Glycine), rice (Oryza), corn (Zea) and rye (Secale). Examples of woody plants used as the host plant are poplar (Populus), eucalyptus (Eucalyptus), acacia (Acacia), pear (Pyrus), apple tree (Malus), grapes (Vitis), walnut (Juglans), plum (Prunus), rose (Rosa) and spruce (Picea). However, the host plants for use in accordance with the present invention are not limited to these plants.

In accordance with the present invention, the MAI gene is expressed in order to impart physiological abnormality to the interior of the cell. Physiological abnormalities mean the production of plant growth hormone in a plant cell, with the proliferation and differentiation of the cell containing the gene
MAIs are blended to cause various morphological abnormalities. For example, a collection of random shoots with a violation of apical dominance (extreme shoot phenotype), hairy roots, etc., can be obtained from a cell into which such an MAI gene is introduced. This phenotype is formed by abnormal proliferation and differentiation of the above cells. Thus, such morphologically abnormal tissue consists only of a cell containing this gene. Accordingly, when constructing a vector using such a gene as a selective marker gene together with the desired gene, introducing into the plant cell and cultivating the cell, tissue consisting only of the cell into which the selective marker gene and the desired gene were introduced can be simply isolated by visual selection of morphologically formed abnormal tissue from the plant cell. Thus, it is possible to conduct visual selection of transgenic tissue without the use of special procedures, such as the addition of a factor that inhibits the growth of plant cells, etc. to the culture medium.

 While traditional selective marker genes, such as the neomycin phosphotransferase gene, are not introduced into plants without genetic engineering, the MAI gene in accordance with the present invention is a gene inherent in plants or naturally introduced into plants by infection with bacteria, etc. For this reason, it is believed that the safety of this gene product for the human body is quite high.

 Further, in accordance with the present invention, the MAI gene is inserted into the position so that it acts inseparably with the movable DNA element. After the introduction of a vector having such a structure into the plant, the MAI gene, used as a selective marker gene, is removed from the DNA together with the deleted DNA element with a set frequency, leading to the loss of its function. Meanwhile, the desired gene, which acts differently from the mobile element of DNA, remains in the same DNA and performs its function. Thus, the expression of the same selective marker gene can be used as an index for introducing the desired gene again and again. Accordingly, this vector allows multiple gene introduction into a specific plant by simply changing the structure associated with the desired gene to be introduced without any changes in the structure of the selective marker gene, etc. For this reason, this vector can be reused an unlimited number of times.

 Since the loss of the function of the selective marker gene, i.e. loss of function of the MAI gene can be determined visually, tissue consisting of cells that do not contain a selective marker gene and contain the desired gene can be obtained easily and surely. This means that cultivation, visual selection and separation can be repeated without the need for any special procedure to obtain such tissue. Further, a plant consisting of the above cells can only be obtained by regenerating the plant from the resulting tissue without undergoing a crossover step. Moreover, despite the fact that it is difficult to completely remove the transposon from DNA because of its high transposability, this invention can be successfully applied in practice due to the high selection efficiency.

 The present invention is illustrated by the following examples, however, these examples should not be construed as limiting the invention.

 In the following examples, experiments were carried out in accordance with the instructions set forth in Molecular Cloning, 2nd Edition (Sambrook et al., Eds., Cold Spring Harbor Laboratory Press, New York, 1989) or at the discretion of the manufacturer, unless otherwise indicated.

Example 1
1. Vector construction
The ipt gene present in the T-DNA of the pathogenic A. tumefaciens PO22 strain (H. Wabiko, Chemical Regulation of Plants, v. 24, p. 35, 1989 (see FIG. 1)) was excised with the restriction endonuclease PstI, when this resulted in the plasmid pIPT1 by ligating the ipt gene to the restriction endonuclease PstI site of the plasmid pUC7 (Molecular Cloning, 2nd edition, volume 1, 4.10). The ipt gene containing the native promoter and the native polyadenylation signal is cut from the plasmid using the BamHI & PstI restriction endonucleases, and the pIPT2 plasmid is obtained by ligating the ipt gene to the BamHI-PstI restriction endonuclease sites of the pUC119 plasmid (obtained from Takarouz Ltd. ) Using the RsaI restriction endonuclease, a plasmid and a native ipt gene polyadenylation signal are cut from this plasmid, and the pIPT3 plasmid is obtained by ligating the ipt gene to the SmaI restriction endonuclease site of plasmid pUC119. Next, the ipt gene inserted into pIPT3 is excised using BamHI and SacI restriction endonucleases, whereby the pIPT4 plasmid is obtained by ligation of the fragment at the BamHI-SacI restriction endonuclease sites of the vector plasmid pBI121 (obtained from Clontech Co.), which can be used for introduction gene into the plant. When an A. tumefaciens plant having the pIPT4 plasmid is infected, the T-DNA region between the LB site and the RB site (here the approximately 5 kb portion having the neomycin phosphoransferase gene (NPTII) and the ipt gene) is integrated into the plant chromosome.

 This plasmid pIPT4 was introduced into E. coli (Escherichia coli), strain JM109, in accordance with the Budapest Treaty as E. coli JM109 (pIPT4) under Depository N FERM BP-5063.

 The procedure for constructing the plasmid pIPT4 is schematically shown in FIG. 2-4. A restriction endonuclease map of the T-DNA region of this endonuclease is shown in FIG. 5. In FIG. 2-4 and 5 circled by “P” and “T” indicate the native promoter and the native polyadenylation signal of the ipt gene itself, respectively. 35S-P means the 35S promoter of the cauliflower mosaic virus, and Nos-P means the promoter of the nopaline synthetase gene. T (FIG. 4) or Nos-T (FIG. 5) means the polyadenylation signal of the nopaline synthetase gene.

 In this example, as shown in FIG. 5, for the MAI gene, the ipt gene is used as a selective marker gene, which promotes the formation of an extreme shoot phenotype, causing breach of the apex dominance, while the NPTII gene is used as a model of the desired gene. The ipt gene belongs to oncogenes that hold pathogenic A. tumefaciens. The plant cell into which the ipt gene is introduced causes differentiation leading to the formation of an extreme shoot phenotype caused by excessive production of cytokinin, which is a plant hormone.

 In this example, the cauliflower mosaic virus 35S promoter is used for the promoter sequence of the ipt gene, and the native polyadenylation signal of the ipt gene itself is used for the terminator sequence.

II. Introduction of pIPT4 to Agrobacterium
A. tumefaciens, strain LBA4404 (obtained from Clonetech Co.), is inoculated in 10 ml of liquid culture medium YEB (containing 5 g / l of beef extract, 1 g / l of peptone, 5 g / l of sucrose and 2 mm MgSO ₄ , pH 7.2 at 22 ^o C (pH at 22 ^o C is used hereinafter, unless otherwise specified), and cultivated at 28 ^o C until then, until the level of OD ₆₃₀ reaches 0.4-0.6. Then the culture is exposed centrifuged at 6900 x g for 10 minutes at 4 ^° C to collect cells.The cells are suspended in 20 ml of 10 mM Tris-HCl (pH 8.0) and the suspension is again centrifuged at 6900 x g for 10 minutes at 4 ^o C. Next the collected cells are resuspended in 200 μl of YEB liquid culture medium, and this suspension is used as a cell solution for introducing the plasmid.

In a 15 ml tube (manufactured by Falcon), 200 μl of the cell solution for introducing the plasmid is mixed with 6 μl of the pIPT4 plasmid obtained in the above Step I, and the mixture is cooled by immersion for 5 minutes in ethanol cooled in liquid nitrogen for 30-40 minutes . The solution thus cooled, together with the tube, is kept in a water bath at 29 ^° C. for 25 minutes. Then, 750 μl of YEB liquid culture medium is added to it, and the mixed solution is cultured at 29 ^° C. for 1 hour with shaking. The cell solution is distributed on a YEB agar culture medium (containing 1.2% w / v agar and the same ingredients as the culture medium described above), to which 50 mg / L kanamycin is added and cultured at 28 ^° C. for 2 days . The cell colonies thus obtained are inoculated into the liquid culture medium of YEB and further cultured. The plasmid is then extracted from the cells using the alkaline method and cleaved using restriction endonucleases BamHI and EcoRI. Thus obtained fragments of the plasmid are analyzed by agarose gel electrophoresis, confirming the introduction of the plasmid pIPT4 into A. tumefaciens strain LBA4404.

III Introduction of pIPT4 from Agrobacterium to Tobacco
Formed tobacco leaves (Nicotiana tabacum cv. Xanthi, hereinafter tobacco means this variety, unless otherwise indicated), grown in a greenhouse, are immersed in a 1 vol./vol% aqueous solution of sodium hypochlorite for sterilization and washed 3 times with sterile water. Then the main leaf vein is removed, forming leaf blades with an area of approximately 8 mm square. The leaf blades thus obtained are immersed for approximately 1 minute in a cell suspension of A. tumefaciens strain LBA4404 with the introduction of pIPT4 as described above in stage II and infected with it (this suspension is diluted with sterilized water at OD ₆₃₀ = 0.25 after cultivation on overnight in liquid culture medium YEB). An infected leaf plate is placed on sterilized filter paper to remove excess cell suspension. Then it is placed on a hormone-free MS agar culture medium (T. Murashige and F. Skoog, Physiol, Plant., Vol. 15, p. 473, 1962 (provided that 0.8 wt./vol. Is added to it .% agar)) containing 50 mg / l of acetosyringone, the back of the sheet up, and cultivated for 3 days at 25 ^o C and full lighting (cultivation of ex-plants, plant tissue and plants was carried out under such temperature and light conditions, if not specified otherwise). The leaf blade thus cultivated is then transferred to a hormone-free MS agar culture medium containing only 500 mg / L carbenicillin, and cultivation continues. As a result, 22 additional shoots were transdifferentiated. These additional shoots were separated and then cultivated in a culture medium having the above composition in order to obtain 6 lines of extreme shoot phenotypes (EFP). These EPP lines are cross-cultivated every month in the same culture medium, and 3 months after infection, they are cross-cultured in MS agar culture medium free of hormones and carbenicillin. After the cessation of Agrobacterium proliferation, kanamycin resistance tests and analysis of the polymerase chain synthesis reaction are carried out.

IV. Analysis of tobacco into which the gene was introduced
A. Kanamycin Resistance Test
The 6 EPP lines obtained in the above described stage III were cultivated as such without reseeding. Leaves developed from these EPP lines were cut to form leaf blades having an area of approximately 3 mm square. The thus obtained leaf blades are placed on an MS agar culture medium (1 mg / L benzyl adenine and 0.2 mg / L α-naphthalene acetic acid are added to said medium) containing 200 mg / L kanamycin. After cultivation in this kanamycin containing the culture medium for 1 month on the leaf blades obtained from the EFP lines, the formation of these lines was also observed.

B. Analysis of the polymerase reaction of chain synthesis
Of all 6 EPP lines obtained in the above described stage III, chromosomal DNAs were extracted, and the genes introduced into them were analyzed using the polymerase chain reaction synthesis method.

 Chromosomal DNAs were extracted using the following modified CTAB method.

Initially, approximately 1 g of leaves obtained from EPP was ground in liquid nitrogen using a chilled mortar and pestle and suspended in 5 ml of a buffer solution (containing 2% w / v CTAB (hexadecyltrimethyl ammonium bromide), 1.4 M NaCl, 0 , 2 vol / vol% β-mercaptoethanol, 20 mM EDTA and 100 mM Tris-HCl (pH 8.0), which was heated to 60 ^° C. This suspension was heated at 60 ^° C. for 30-60 minutes with gentle shaking, and then cooled to room temperature.A mixture of chloroform and isoamyl alcohol (24: 1) was added to this suspension in equal proportions and the mixture was carefully mixed. Then the mixture was centrifuged at 1600 x g for 5 minutes to obtain the upper layer. Next, 2/3 volumes of isopropyl alcohol were added to the upper layer and the mixture was again carefully mixed. The mixture was kept on ice for 10 minutes to precipitate chromosomal DNA: This chromosomal DNA was collected by centrifugation at 1600 x g for 10 minutes, and the chromosomal DNA thus collected was washed with 70 v / v. ethanol, then dried in vacuum and dissolved in 300 μl TE (including 10 mM Tris-HCl and 1 mM EDTA).

In the meantime, in order to detect the ipt gene using the polymerase chain reaction method, several of the seeds used (oligonucleotide) were synthesized using a DNA synthesizer (manufactured by Applied Biosystems Co.). During binding to the ipt gene, the distance between the two primers became approximately 800 bp. In order to amplify the ipt gene, 1 μg of the extracted chromosomal DNA was dissolved in 50 μl of a mixed solution containing 0.2 μM of these seeds, 10 mM Tris-HCl (pH 8.8 at 25 ^° C), 50 mM KCl, 1, 5 mM MgCl ₂ , 1 w / v% Triton X-100, 0.1 mM dNTP and 1.25 units of Tag polymerase (obtained from CETUS Co). After heating the mixture at 94 ^° C for 1.5 minutes, a three-part heating cycle, namely: at 94 ^° C for 1 minute, at 55 ^° C for 2 minutes and at 72 ^° C for 3 minutes , repeated a total of 30 times in order to complete the reaction. The resulting agarose mixture was analyzed by agarose gel electrophoresis in order to detect the presence of the ipt gene in chromosomal DNA by amplifying the gene to approximately 800 bp.

 The results are shown in FIG. 6. As can be seen from FIG. 6, gene amplification to approximately 800 bp was observed in all 6 AFPs. In FIG. 6 values shown on the left side indicate the length of the bases, the strip ingredients of which were detected by electrophoresis of the DNA size marker.

Comparative Example 1
An analysis was made of 16 shoots that were not able to form EFP and were obtained from additional shoots transdifferentiated from a leaf infected with A. tumefaciens as described in stage III of Example 1. That is, 22 additional shoots were simultaneously cultivated, and 6 EPP lines were selected in stage III of Example 1. Morphologically normal shoots (hereinafter referred to as non-EPP) were released from A. tumafaciens and subjected to kanamycin resistance testing in the same manner as in stages III and IV of example 1. Next, 9 lines of non-EFP were subjected to analysis of the polymerase reaction of chain synthesis. However, after about 3 months, the leaf blades of these non-EFP lines laid on a culture medium containing kanamycin all turned brown and wilted. Further, as a result of the analysis of the polymerase reaction of chain synthesis, amplification of the DNA fragment to approximately 800 bp, proving the presence of the ipt gene, was not detected in any of the 9 analyzed lines.

 The results of the analysis of the polymerase chain synthesis reaction are shown in FIG. 6.

Example 2
I. Vector Design
Plasmid pHSG398 (obtained from Takara Shuzo Co., Ltd.) was hydrolyzed by restriction endonuclease BamHI. The cohesive ends obtained by hydrolysis were changed to blunt ends using T4DNA polymerase (large subunit), and plasmid pNPI100 was obtained by ligation of these ends. Those. pNPI100 is pHSG398 that has lost the restriction site of the BamHI endonuclease. Meanwhile, the plasmid pCKR97 (T. Izawa et al., Mol. Gen. Genet., Vol. 227, p. 391, 1991) was hydrolyzed using restriction endonuclease PstI. To obtain plasmid pNPI102, maize transposon Ac was excised and inserted into the restriction endonuclease PstI site of pNPI100 plasmid.

 Then, the 35S promoter of the cauliflower mosaic virus and the ipt gene connected to it using restriction endonucleases HindIII and SacI were cut from the pIPT4 plasmid constructed in accordance with Example 1. The cohesive ends of the fragment thus obtained were changed to blunt ends using T4DNA polymerase I, and the fragment was inserted into the HincII restriction endonuclease site of plasmid pUC119 to obtain plasmid pNPI101. The 35S promoter of cauliflower mosaic virus and the ipt gene were again cut from the plasmid pNPI101 using restriction endonucleases PstI and EcoRI, and the cohesive ends of the fragment were changed to blunt ends using polymerase I T4DNA. Next, the plasmid pNPI103 was obtained by ligation of the fragment at the "blunt" end of the BamHI endonuclease site of plasmid pNPI102. Those. in plasmid pNPI103, the 35S promoter and the associated ipt gene were present at the site of the old restriction endonuclease BamHI inside the Ac transposon.

 The desired vector was obtained by excising an Ac transposon containing the 35S promoter of cauliflower mosaic virus and the ipt gene from plasmid pNPI103 with the restriction endonuclease PstI and inserting this transposon Ac into the SseI restriction endonuclease site of the vector plasmid pBI121, which was designated as plasmid pNP102.

 This plasmid pNPI106 was also introduced into the E. coli strain JM109 and deposited in accordance with the Budapest treaty as E. coli JM109 (pNPI106) under deposit N FERM BP-5064.

 The procedure for constructing plasmid pNPI106 is schematically represented in FIG. 7-9. A restriction endonuclease map of the T-DNA region of this endonuclease is shown in FIG. 10. In FIG. 7 - 9 and 10, the terminal portion of the Ac transposon is shown by opposite black triangles, respectively. In FIG. 10 Ac-P represents a native promoter present within Ac. Other symbols have the same meaning as the symbols shown in FIG. 2 - 5.

 As seen in FIG. 10, this plasmid contains the ipt gene as a selective marker gene, and the NPTII gene and the GUS gene (β-galactosidase) as a model of the desired gene in the T-DNA region, namely, the region introduced into the plant chromosome. Further, the ipt gene is present when inserted into the transposon Ac. Since a cell containing the GUS gene metabolizes a special substrate that produces blue pigment, gene expression can be detected by detecting this pigment. Thus, the GUS gene is often used to analyze gene expression in a plant.

II. Introduction of pNPI106 into tobacco and analysis of tobacco into which the gene was introduced
A. Introduction of pNPI106 into tobacco and test for expression of the introduced gene
In the same manner as in Steps II and III of Example 1, pNPI106 was introduced into A. tumefaciens LBA4404 strain and tobacco leaf blades were infected with A. tumefaciens. The tobacco leaf thus infected was cultured in a hormone-free MS agar culture medium containing 50 mg / L acetosyringone, and then in a hormone-free MS agar culture medium to which 500 mg / L carbenicillin was added. Two months after such cultivation, 63 EFP lines were separated.

 These EPP lines were transplanted into a culture medium having the same composition (hormone-free MS agar culture medium containing 500 mg / L carbenicillin). A month later, 9 shoots were visually selected from slightly grown shoots of EPP (shoots grown from EPP, hereinafter referred to simply as “shoots”), which grew approximately two and three times in comparison with other shoots in which there was no increase in lateral shoots and in which the effect of the ipt gene looked reduced. The leaves of these shoots were subjected to the same kanamycin resistance test as described in step IV-A of Example 1 and the GUS gene expression test (GUS activity test) based on the method of Jefferson et al. Shoots obtained after leaf pruning were transplanted into hormone-free MS agar culture medium and cultivated. A month later, their shape was studied in order to identify the ability of formation on the shoots of AFP, thus detecting the expression of the ipt gene.

 The results are shown in table 1.

 As can be seen from table 1, although the shoot sheet N 8 is resistant to kanamycin and GUS activity, the EFP does not form, even if the shoot is cultivated for 1 month. Preferably, this is because the ipt gene, causing the formation of an AFP, is present in the introduced form inside the Ac transposon in plasmid pNPI106. This means that the ipt gene introduced into the tobacco chromosome by infection with A. tumefaciens, which contains this plasmid in the tissue and is expressed at the initial stage of tissue culture immediately after infection, is removed along with Ac due to the action of Ac during subsequent cultivation. Meanwhile, in the same vector, the NPTII gene and the GUS gene are introduced in a position in which they do not act in the same way as Ac, so these genes still remain on the plant chromosome and are expressed.

 In table 1, despite the fact that resistance to kanamycin and the ability to form EPPs are observed in shoots NN 3, 5, and 6, only GUS activity is negative. This means that in these shoots only the GUS gene from genes introduced using pNPI106 is not expressed. This may have been due to the erroneous integration that occurred during the integration of these genes into the plant chromosome. That is, when a gene is introduced through A. tumefaciens containing a plasmid having a pNPI106 structure or the like, the T-DNA region, namely the entire internal region between the RB site and the LB site, should integrate normally into the plant chromosome. However, this section is sometimes not fully integrated, but damaged, as a result of this, an inferior section is introduced, devoid of part of the ends of LB. In the T-DNA region of plasmid pNPI106, the gene is located at the closest position to the LB site. Accordingly, it is believed that due to erroneous integration during the introduction of the gene, the GUS gene is integrated into the chromosome in a torn form and having lost its own function, or the GUS gene is not inserted at all into it, thus, this gene is not expressed in these shoots and its activity not visible.

 A photograph of shoots N 2 and 8 after 1 month of cultivation is shown in FIG. 11 and 12.

 Given the growth of shoot leaves N 8 and the test for resistance to kanamycin, cultivation after the test continued further. Five additional shoots were obtained from this leaf, and all these shoots were non-EFP.

B. Analysis of the polymerase reaction of chain synthesis
The shoots 1 - 9 in table 1, after determining the ability to form EPPs, were subjected to the analysis of the polymerase chain synthesis reaction in the same manner as in stage IV-B of example 1 in order to further investigate the presence of the ipt gene in the chromosome, subject to the programmed binding of a pair of seeds with the NPTII gene and the GUS gene, respectively, in addition to the seeds used in step IV-B of Example 1. If these seeds are used to excise inserted Ac and ipt genes (Ac-ipt gene complex) from a T-DNA region of pNPI106, a DNA fragment having the approximate but 3 kd, amplifies in a polymer chain synthesis reaction. Accordingly, by using this amplification as an index, the excision of the Ac-ipt gene complex from DNA can be determined.

 The results of the analysis of the polymerase reaction of the synthesis of shoot chain N 8 are shown in FIG. 13, where the values indicated on the left have the same meanings as in FIG. 6. As follows from the results, amplification of the DNA fragment of approximately 3 kb is observed in the chromosomal DNA extracted from shoot N 8, which proves the excision of the Ac-ipt gene complex, while amplification of the DNA fragment of approximately 800 bp confirms the presence of the ipt gene is not observed. This means that the ipt gene is excised from the chromosomal DNA of this shoot along with Ac and disappears.

 On the other hand, with respect to shoots NN 1-7 and 9, amplification of a DNA fragment of approximately 3 kb was not detected in any of its chromosomal DNA samples. Accordingly, it is believed that in these shoots the ipt gene is still present in the chromosomal DNA along with Ac.

Reference Example 2
When cultivating leaves infected with A. tumefaciens, in stage II-A of example 2; 3 non-EPPs, transdifferentiated along with EPPs, were separated and subjected to a kanamycin resistance test, a GUS activity test, visual observation after a month of cultivation and analysis of the polymerase chain synthesis reaction in the same manner as in paragraph II of example 2.

 The results are shown in Table 1. The shoots obtained from these non-ESPs do not have any resistance to kanamycin, GUS activity, and the ability to form EFPs. In addition, as a result of the analysis of the polymerase chain synthesis reaction, amplification of DNA fragments of approximately 800 bp and approximately 3 kb was not detected.

Example 3
Further, the cultivation of 63 EPP lines separated in Example 2 was continued in hormone-free MS agar culture medium. Approximately two months after separation, a total of 7 shoots NN 13-1, 13-2, 13-3 and from 14-1 to 14-4, which were normal and could be visually identified, were obtained from two EFP lines, i.e. e. had apical dominant. These shoots were separated for transplantation into a culture medium having the above composition, they all had normal growth and roots. Of these, shoots 13-1 and 14-1 were subjected to analysis of the polymerase chain synthesis reaction, as described in stage II-B of Example 2. As a result, amplification of the DNA fragment of approximately 800 bp was not observed in any of the shoots NN 13-1 and 14-1. Meanwhile, amplification of a DNA fragment of approximately 3 kb was observed in both shoots. Thus, it was determined that the ipt gene was excised from the chromosomal DNA of these shoots along with Ac and disappeared. The results are shown in FIG. 14, where the values indicated on the left have the same meaning as in FIG. 6. Further, GUS gene expression was determined in all materials obtained from 7 shoots.

 FIG. 15 shows the state of a normal shoot differentiated from AFP.

 Example 4

 The leaf obtained from the shoot shown as shoot No. 7 in Table 1, among the 9 shoots selected in Example 2, was cultured in hormone-free MS agar culture medium for approximately 1 month. One normal shoot was visually selected and separated from 6 additional shoots, which transdifferentiated from the cultivated leaf. This normal shoot was transferred to a culture medium having the above composition, then a shoot having a normal length and roots was obtained. Further, this shoot was subjected to the analysis of the polymerase chain synthesis reaction in the same manner as described in stage II-B of Example 2, and based on the DNA fragment having approximately 800 bp and the amplification of the DNA fragment of approximately 3 kb, it was found that the ipt gene was excised from chromosomal DNA along with Ac and disappeared. The results are shown in FIG. 16, where the values indicated on the left have the same meaning as in FIG. 6. Then, expression of the GUS gene was also detected in the same materials.

Example 5
1. Separation of the site-specific recombinant system (pSR1 system) from yeast.

Yeast (Zygosaccharomyces rouxii) (obtained from the Institute of Fermentation) was inoculated in 5 ml of YPAD liquid culture medium (containing 10 g / l of yeast extract, 20 g / l of polypeptone, 0.4 g / l of adenine and 20 g / l of glucose) and cultured at 30 ^o C for 24 hours. The culture solution was centrifuged at 6900 x g for 3 minutes at 20 ^° C in order to collect cells (subsequently, cells were collected under the same conditions). The resulting cells were suspended in 2 ml of a solution containing 0.2 M Tris-HCl (pH 8.0) and 5 vol / vol% mercaptoethanol. The cell suspension was maintained at 25 ^° C. for 30 minutes with careful infrequent stirring, and then the cells were collected. Then, the collected cells were suspended in 1 ml of a solution (pH 6.8) containing 2.5 mg / ml Zaimolyeis-20T (obtained from SEIKAGAKU CORPORATION), 10% w / v% sorbitol and 5% w / v KPO ₄ . The suspension was maintained at 30 ^o C for 90 minutes, again subjected to centrifugation in order to again collect cells. The collected cells were resuspended in 1 ml of a solution containing 0.2 M NaCl, 0.1 M EDTA, 5% w / v sodium dodecyl sulfate, 50 mM Tris-HCl (pH 8.5), to which was added proteinase K up to 20 mg / ml. The stirred solution was left to stand at 60 ^° C. for 1 hour, then cooled to room temperature and extracted with a mixture of phenol and chloroform, and then purified with chloroform. An equal volume of isopropanol was added to the supernatant thus obtained in order to precipitate the chromosomal DNA and plasmid pSR1. The mixture was centrifuged at 6900 x g for 10 minutes at 4 ^° C to collect DNA, the collected DNA was washed with 70 vol. / vol.% ethanol, then dried in vacuum and dissolved in 100 μl TE.

 Using the DNA thus extracted (a mixture of chromosomal DNA and plasmid pSR1) as a template, only the site-specific recombination system present in plasmid pSR1 (hereinafter referred to as “pSR1 system”) was amplified using the polymerase chain reaction method. The pSR1 system consists of the R gene, which is the recombinase gene and the Rs recombination sequence, the DNA sequences of which have already been determined (H. Araki et al., J. Mol. Biol., Vol. 182, p. 191, 1985). In accordance with the present invention, in order to amplify the R gene, a seed was synthesized in which the XbaI restriction endonuclease site was added to position 5 ′ of 22 bases, namely bases 5595-5617 in the sequence of plasmid pSR1 (5′-CCTCTAGAATGCAATTGACCAAGGARACTG-3 ′) and the seed in which the SacI restriction endonuclease site was added to position 5 ′ of 22 bases, namely, bases 4126 to 4147 in the sequence of plasmid pSR1 (5′-CCGAGCTCTTAATCTTGTCAGGAGGTGTCA-3 ′). On the other hand, in order to amplify Rs, two pairs of seeds were synthesized, each of which included 30 bases (four types in total). Those. one pair included a seed in which 3 of the bases 287-316 of the pSR1 plasmid sequence were replaced and the Ssel restriction endonuclease site (5'-AGGATTGAGCTACTGGACGGGAATCCTGCA-3 ') was introduced, and a seed in which 4 of the bases 690-719 of the pSR1 plasmid sequence were introduced the HindIII restriction endonuclease site and the XhoI restriction endonuclease site (5'-CAACTCGAGCAATCAAAGCTTCTCGTAGTC-3 ') were replaced and introduced. The Rs amplified by this set of seeds was called "Rs1". The other pair included a seed in which 3 of the bases 287 - 316 of the pSR1 plasmid sequence were replaced and the restriction endonuclease XhoI site and a restriction endonuclease EcoRI site (5'-AGGATTGAGCTACTCGAGGGGAATTCTGGA-3 ') were introduced, and a seed in which 5 of the bases 688 717 sequences of plasmid pSR1 were replaced and the restriction endonuclease SseI site (5'-ACTGGACCAATCCCTGCAGGTCGTAGTCAA-3 ') was introduced. Rs amplified by this set of seeds was named "Rs2".

In order to amplify the R and Rs gene, 1 μl of the extracted DNA solution was added to each 50 μl of the mixed solution used in stage IV-B of Example 1 containing 0.2 μM of each set of seeds, respectively. The heating cycle, consisting of three parts, namely: at 95 ^o C for 30 seconds, at 55 ^o C for 30 seconds and at 72 ^o C for 1.5 minutes, was repeated with the mixture as a whole up to 30 times. The reaction mixture thus obtained was analyzed by agarose gel electrolysis to confirm amplification of the R and Rs gene.

II. Vector design
Rs1 amplified by the polymerase chain synthesis method was hydrolyzed by restriction endonucleases PstI and XhoI, resulting in plasmid pNPI126 by inserting this RS1 into the sites of restriction endonuclease PstI-XhoI pSL1180 (obtained from Pharmacia Biotech Co.).

 Then, in order to eliminate the EcoRI restriction endonuclease site and the HindIII restriction endonuclease site pHSG398, the hydrolysis of these restriction endonucleases was successively repeated by changing the hydrolyzed ends 2 to the blunt ends using T4 polymerase I (large subunit) and ligation of the blunt ends. Thus, plasmids pNPI121 with eliminated restriction endonuclease sites were obtained. Plasmid pNPI127 was obtained by hydrolysis of Rs2 amplified by the method of polymerase chain synthesis using restriction endonuclease XhoI and PstI, and incorporation of this Rs2 into the restriction endonuclease sites SalI-PstI of plasmid pNPI121.

 Plasmid pNPI128 was obtained by excising Rs1 from pNPI126 using restriction endonuclease SmaI and SpeI and incorporating this fragment into the restriction endonuclease sites of SmaI-XbaI plasmid pNPI127.

 The R gene amplified by the method of polymerase chain synthesis was hydrolyzed by restriction endonuclease XbaI and SacI and included in the restriction endonuclease sites of XbaI-SacI plasmid pHSG398. The plasmid thus obtained was designated as pNPI124.

 Then pBI221 (obtained from Clontech Co.) was hydrolyzed using restriction endonuclease PstI. The hydrolyzed ends were changed to blunt ends, and then were ligated in the above manner. Thus, the plasmid pNPI111 with the eliminated restriction endonuclease sites SseI and PstI was obtained. Then, the R gene, cut from pNPI125 using restriction endonuclease XbaI and SacI, was included in the restriction endonuclease sites XbaI-SacI of plasmid pNPI111, replacing the GUS gene to obtain plasmid pNPI125. Next, the 35S promoter of cauliflower mosaic virus, the R gene linked to the promoter, and the polyadenylation signal of nopaline synthetase were cut using HindIII and EcoRI restriction endonucleases and included in the HindIII-EcoRI restriction endonuclease sites of plasmid pNPI128 to obtain plasmid pNPI129.

 pNPI101 was hydrolyzed using SmaI restriction endonuclease, and the 5'-phospholylated HindIII linker (obtained from Takara Shuzo Co., Ltd) was included in the hydrolysis site to obtain plasmid pNPI122. Thus, this pNPI122 is the only one in which the SmaI restriction endonuclease site of plasmid pNPI101 is replaced by the HindIII restriction endonuclease site. Next, pNPI122 was hydrolyzed with the restriction endonuclease PstI, and the hydrolyzed ends were changed to blunt ends and ligated to obtain the plasmid pNPI123 with the eliminated restriction endonuclease sites SseI and PstI. The 35S promoter of cauliflower mosaic virus and the ipt gene were excised from this plasmid using the HindIII restriction endonuclease and included in the HindIII restriction endonuclease site of plasmid pNPI129 to obtain plasmid pNPI130.

 The desired vector was obtained by cutting out the fragment containing the ipt gene and the R gene, the 35S promoter of the cauliflower mosaic virus associated with them, respectively, and the Rs present on both terminals of these genes using the restriction endonuclease PstI and incorporating this fragment into the restriction endonuclease site SseI plasmids pBI121. The plasmid thus obtained was designated as pNPI132.

 Plasmid pNPI132 was also introduced into the E. coli strain JM109 and deposited in accordance with the Budapest treaty as E. coli JM109 (pNPI132) under deposit N FERM BP-5065.

 The procedure for constructing plasmid pNPI132 is schematically shown in FIG. 17-19. A map of the restriction endonuclease of its T-DNA region is shown in FIG. 20. In FIG. 17-19 and 20, the shaded triangle indicates the recombination sequence Rs obtained from the yeast plasmid pSR1 and the direction of its sequence. Other symbols have the meanings shown in FIG. 2-5.

 As follows from FIG. 20, the plasmid pNPI132 is the same as the plasmid pNPI106; it has the ipt gene as a selective marker gene, the NPTII gene, and the GUS gene as models of the desired gene in the T-DNA region. However, in this case, the region between the two Rs recombination sequences of the pSR1 system behaves as a removable DNA element. Therefore, the ipt gene is inserted in such a way that it is held by two recombination sequences in the same direction.

III. Introduction of pNPI132 into tobacco and analysis of tobacco into which the gene was introduced
In the same manner as in stages II and III of Example 1, the plasmid pNPI132 was introduced into the A. tumefaciens LBA4404 strain, and the tobacco leaf plate (Nicotiniana tabacum cv. SR1) was infected using this A. tumefaciens. The infected leaves were then cultured in a hormone-free MS agar culture medium containing 50 mg / L acetosyringone, and then in a hormone-free MS agar culture medium containing 500 mg / L carbenicillin.

 A month later, the cultivated leaves were transferred to a culture medium having the same composition, and cultivation continued for further 1 month. Then 48 lines of shoot extreme phenotypes (AFP) were separated.

 These EPP lines were again transplanted into a culture medium having the same composition, and cultivation continued further. About a month later (i.e., approximately 3 months after infection with A. tumefaciens), shoots, whose morphology was visually defined as normal, were generated from seven of the 48 EPP lines. These shoots were separated and transplanted into a culture medium having the same composition in order to obtain 10 normal individuals.

 These individuals were subjected to the analysis of the polymerase chain synthesis reaction in the same manner as in stage II-B of example 2, provided that a pair of seeds for determining the GUS gene was used in addition to the additives used in stage II-B of example 2. Carrying out the polymerase reaction chain synthesis using these seeds, a DNA fragment having approximately 800 bp, amplified in the presence of the ipt gene. A DNA fragment of approximately 3 kb was amplified when the ipt gene was excised in the T-region of the plasmid pNPI132 by excising the portion retained by Rs's (these results are the same as the results of the analysis of stage II-B of example 2). A DNA fragment having approximately 1.7 kb was amplified in the presence of the GUS gene. The results are shown in FIG. 21-23 and in table 2. In FIG. 21-23, the values indicated on the left have the same meanings as in FIG. 6.

 As follows from table 2, the presence of the ipt gene, which is a selective marker gene, was not detected in any chromosomal individuals that were selected by a simple visual study of their morphology, and on the contrary, amplification of the DNA fragment was detected, which means excision of the ipt gene. However, the presence of the GUS gene used as the desired gene was detected in all individuals.

 On the other hand, kanamycin resistance was determined using terminal kidneys of individuals that were derived from non-EPP, differentiating almost simultaneously with 48 EPP lines and showing normal length and roots when using hormone-free MS agar medium containing 200 mg / L kanamycin. As a result, 2 out of 16 individuals were found to be resistant to kanamycin.

 Further, these kanamycin-resistant individuals were further investigated by analyzing the polymerase chain synthesis reaction together with three individuals selected from 14 kanamycin-sensitive individuals in the same way as individuals derived from EPP. FIG. 24 shows the results, where the values indicated on the left have the same values as in FIG. 6.

 As clearly seen in FIG. 24, each of the two kanamycin-resistant individuals showed amplification of the DNA fragment, which means excision of the region containing the ipt gene and retained by Rs and the presence of the GUS gene. Thus, it was proved that the genes that arose in pNPI132 were integrated into these chromosomes. On the contrary, such amplification was not observed in three individuals sensitive to kanamycin. Further, any of these individuals (namely, neither individuals resistant to kanamycin, nor individuals sensitive to kanamycin) showed amplification of the DNA fragment, which means the presence of the ipt gene.

 It is assumed that these kanamycin-resistant individuals obtained from a strain incapable of forming an EPP, as well as kanamycin-sensitive individuals, arose in cells in the chromosomes of which pNPI132 was not introduced during infection with A. tumefaciens. Based on this assumption, it is unbelievable that the genes that arise in this sector are contained in the chromosomes. Moreover, it is impractical for individuals that do not contain the ipt gene but contain the NPTII gene (as evidenced by the fact that these individuals are resistant to kanamycin) and the GUS gene each in full form in the chromosomes to appear with such frequency, given that these vectors occur in the same vector.

 In particular, it is advisable to conclude that in these kanamycin-resistant individuals, pNPI132 was once introduced into chromosomes. In other words, it is believed that the T-DNA region of pNPI132 was once introduced into the chromosome due to infection with A. tumefaciens, but the pSR1 system function that was used for this vector was too effective and induced ipt excision before the formation of EFP after A.tumefaciens infection. As a result, the NPTII gene and the GUS gene exclusively remained on the chromosome. The fact that analysis of the polymerase reaction of chain synthesis showed individuals resistant to kanamycin, excision of the region containing the ipt gene and retained by Rs also confirms this assumption.

Example 6
I. Vector Design
The rol genes (S. Kiyokawa, Plant Physiol., Vol. 104, p. 801, 1994) containing rolA, rolB and rolC and having a total size of 7.6 kb that were included in the EcoRI restriction endonuclease site of plasmid pBluescriptII SK + (manufactured by Toyobo Co., Ltd), were cut using restriction endonuclease EcoRI. This fragment was included on the EcoRI restriction endonuclease site of plasmid pNPI129 to produce plasmid pNPI700.

 Using the restriction endonuclease, the 35S cauliflower virus promoter, the R gene associated with the promoter, and the Rs present at both terminals of these genes were excised from this plasmid pNPI700 and included in the restriction endonuclease SseI site of plasmid pBI131 to obtain the desired plasmid pNPI702.

 This plasmid pNPI702 was also introduced into the E. coli JM109 strain and was deposited in accordance with the Budapest agreement as E. coli JM109 (pNPI702) under depository N BP-5066.

 The procedure for constructing plasmid pNPI702 is schematically shown in FIG. 25. A map of the restriction endonuclease of its T-DNA region is shown in FIG. 26. The symbols in FIG. 25 and 26 have the same meaning as the symbols in FIG. 2 - 5.

 As follows from FIG. 26, plasmid pNPI702 is similar to plasmid pNPI132, except that the select marker marker ipt gene is replaced with rol genes. The rol genes used in this vector are present in A. rhisogenes T-DNA in nature. It is known that when rol genes are introduced into plant cells, hair roots are formed in plant tissue and that a plant regenerated from this hair root has morphological abnormalities such as dwarfism and the like.

II. Introduction of pNPI702 into tobacco and analysis of tobacco into which this gene was introduced
In the same manner as in stages II and III of Example 1, plasmid pNPI702 was introduced into A. tumefaciens strain LBA4404, and tobacco leaf blades were infected with A. tumefaciens.

 The tobacco leaf plate thus infected was cultured in an hormone-free MS agar culture medium containing 50 mg / L acetosyringone in a dark place for 3 days, and then in a hormone-free MS agar culture medium to which 400 mg / L tricarcillin was added. . Approximately 15 days after the start of cultivation, differentiation of hair roots was observed. The hair roots were separated one after another and laid on the induction culture medium of the shoot (MS agar culture medium containing 0.1 mg / L α-naphthalene acetic acid, 2.0 mg / L benzyladenine and 400 mg / L tricarcillin). Then, 18 shoots with visually normal morphology were selected from the transdifferentiated shoots and subjected to the polymerase chain synthesis analysis in the same manner as in stage II-B of example 2, using seeds to determine the excision region containing the rol gene and retained by Rs's through amplification of a DNA fragment having a size of approximately 3 kb (as well as the same seeds used in examples 2 to 5 and comparative example 2), and seeds for determining rol genes through the amplification of DNA fragments having size is approximately 1.1 kb. As a result, it was confirmed that the region containing the rol genes and retained by Rs's was excised from the chromosomes of 9 shoots.

Example 7
A gene was introduced into the hybrid aspen (Populus Sieboldii x Populus grandidentata) using the vector pNP1106 constructed above in Example 2.

 The hybrid aspen trunk, strain Y63 (grown in the Akita Jugo chemicals Co., Ltd. experimental forest), grown in a sterilized flask, was cut into 5 mm long knot-free parts. Then the part was cut vertically into two halves for use as a sample, and the sample was infected by introducing pNPI106 strain A. tumefaciens LBA4404 in the same manner as in Example 1. After infection, part of the stem was placed in a hormone-free modified agar culture medium MS (containing 0.2% w / v% sucrose and 0.8% w / v% agar) to which 40 mg / l acetosyringone was added and cultured therein for 3 days. Then it was transplanted into the same medium, but containing 500 mg of carbenicillin instead of acetosyringone, and cultivated in it further. The modified MS culture medium used is prepared by changing the concentrations of ammonia-nitrogen and nitrate-nitrogen of the usual MS culture medium by 10 mM and 30 mM, respectively.

 After about two months, the additional buds growing on this part were separated and cultivated further for 2 months. Thus, 6 EFP lines were obtained. These lines were reseeded and approximately 4 months after this (i.e., approximately 8 months after infection with A. tumefaciens), morphologically normal shoots growing from EPP were observed for the first time. Then, these shoots were transplanted into a 2/3 diluted MS medium (containing 2% w / v sucrose and 0.3% w / v gellan gum), to which 0.05 mg / l indolobutyric acid was added, and cultivated in her. Thus, 7 normal individuals with growing roots were obtained as a whole from two EPP lines within 10 months after infection with A. tumefaciens.

 These individuals were then analyzed for the polymerase chain synthesis reaction in the same manner as in Step III of Example 5. As a result, the ipt gene was not detected in any of them. On the other hand, among them, the presence of the GUS gene was detected in two individuals. Thus, it was confirmed that the vector in accordance with the present invention can be effectively used also in woody plants.

 By the way, in 5 of the remaining normal individuals, only a part of the GUS gene was detected. It seems that in individuals where the Ac transposon, introduced using the pNPI106 vector, excites their chromosomes together with the ipt gene, the GUS gene located in close proximity to it also undergoes excision and rupture.

 FIG. 27 shows the state of a normal shoot differentiated from AFP.

Example 8
A normal individual (obtained via EPP) containing genes introduced into it using pNPI132 in accordance with Example 5 was further subjected to gene introduction using a vector in accordance with the present invention.

 The GUS PNPI132 gene was replaced by the HPT gene (hygromycin resistant gene). Using the vector thus obtained (pNPI140, FIG. 28), these individuals underwent gene injection in the same manner as in Steps II and III of Example 1. Thus, 40 days after infection with A. tumefaciens with the vector introduced therein, 10 EFP lines were obtained. These EPP lines were separated, transplanted into a culture medium having the same composition (hormone-free MS agar culture medium containing 500 mg / L carbenicillin), and were further cultured in it. As a result, 20 days after this, shoot differentiation was observed in one of these lines with visually normal morphology (i.e., approximately two months after infection with A. tumefaciens).

 One of the thus differentiated shoots was subjected to the analysis of the polymer chain synthesis reaction in the same manner as in stages IV-B of Example 1. The results are shown in FIG. 29. It should be noted that in addition to the seeds used in stages IV-B of Example 1, in this case, a pair of seeds was used to bind to the NPTII gene and the HPT gene, respectively, in order to determine the excision of the region containing the ipt gene and retained by Rs's , from chromosomal DNA, as well as another pair of seeds to determine the presence of the HPT gene (determined by the amplification of DNA fragments of approximately 4 kb and approximately 1 kb, respectively). In FIG. 29, the values indicated on the left have the same values as in FIG. 6.

 As clearly seen in FIG. 29, as a result of the analysis of the polymer chain reaction, the chromosomal DNA extracted from the shoot showed an amplification of the DNA fragment of approximately 4 kb, which means that the ipt gene was excised through excision of the region retained by Rs's, as well as amplification of the DNA fragment of approximately 1 kb, which means the presence of the HPT gene. On the other hand, amplification of a DNA fragment of approximately 800 bp, which means the presence of the ipt gene in the same DNA, was not detected. These results suggest that the ipt gene, which was once introduced into the chromosomal DNA of this shoot, was excised from it through excision of the region held by Rs's, and thus disappeared, while the HPT gene remained in the DNA. In other words, an individual introduced with the desired gene (i.e., the NPTII gene and the GUS gene) using pNPI132 was further introduced with the new desired gene (i.e., the HPT gene) by using a vector in which the construct related to the desired gene , exclusively changed (i.e., the same ipt gene was used as a selective marker gene), while cultivation, visual selection, and separation were usually repeated. Moreover, the results showed the possibility of introducing a third, fourth or more desired genes using the same selective marker gene.

 As follows from the examples described above, the resulting EPP lines always contained the ipt gene inside their chromosomes, as shown in FIG. 6. Further, EPPs that showed a significant morphological abnormality, identified visually, without exception, showed resistance to kanamycin by expressing the NPRII gene, which was the desired gene as a model and was introduced with the ipt gene. This proves that such a MAI gene can fully serve as a selective marker gene for introducing a gene into a plant, and that a vector according to the present invention containing this MAI gene as a selective marker gene can also be used as a vector for introducing gene into the plant.

 After the gene has been introduced into the plant using the pNPI106 vector into which the ipt gene has been integrated inside the Ac transposon, the shoot or the like has lost its EPP-forming ability as a result of the disappearance of the ipt gene from the chromosome, while maintaining the properties provided by the desired gene (NPTII and / or the GUS genome), were obtained from tissue that once formed an AFP at the initial stage of cultivation, but after the gene insertion operation, as shown in Table 1 and FIG. 13, 14 and 16. In addition, the morphology of the tissue thus obtained (i.e., shoot or the like that has lost its EPP-forming ability) can be identified, as shown in FIG. 15 and 27. Further, after selection, separation and cultivation, an individual is obtained having a normal length, roots and normal morphology. Moreover, tissues that were differentiated from tissue obtained from shoots that lost their EPP-forming ability also showed normal morphology without an EPP-forming ability. This proves that such an escape or similar part consisted of uniform cells.

 The same results were observed when using the gene obtained from the site-specific recombination system as a mobile DNA element and when using the rol genes as the MAI gene. This means that when the gene was introduced into the plant using a vector in which the construct related to the transposon or transposon and ipt gene of the vectors used in examples 1-4 and 7 were replaced by the construct related to the above recombination system, and / or the rol gene, as described in examples 5 and 6, from a tissue showing an abnormal morphology immediately after the introduction of the gene, a morphologically normal tissue was obtained and a plant in which the MAI gene disappeared from its chromosome while maintaining the desired gene (Fig. 21-23, table 2). Further, it is also possible to introduce the desired genes into the same individual in various ways, repeating the steps of introducing the gene, culturing and visual selection by using a vector in which the design related to the desired gene is exclusively changed, while the same morphological gene causing abnormality, is used as a selective marker gene (example 8, Fig. 29).

 Accordingly, when using a vector in which the MAI gene is used as a selective marker gene placed in such a position that it acts together with the movable DNA element, a tissue consisting only of cells in which the desired gene remains on the chromosome, etc. . and performs its function, it is obtained only by performing the following stages: (1) culturing the cells immediately after the gene injection operation and visual selection of morphologically abnormal tissue that appears during cultivation, (2) further culturing the morphologically abnormal tissue and visual morphologically selection normal tissue that occurs during cultivation. Further, a plant consisting only of such cells can also be obtained from morphologically normal tissue.

 In addition, when using tissue derived from a site-specific recombination system as a movable DNA element, due to the fact that its excision occurred quickly and at a rather high frequency, morphologically normal tissue originating from morphologically abnormal tissue can also be detected quickly . Many normal individuals were obtained from it with good frequency.

 Table 3 shows the efficiency at which a normal individual was obtained from morphologically abnormal tissue when a transposon or tissue obtained from a site-specific recombination system was used as a mobile DNA element in a vector in accordance with the present invention.

 In examples 1-5, in the absence of hormones, the tissue containing transgenic cells proliferated, differentiated the additional shoot, and regenerated the plant. This is probably due to the action of the ipt gene, which was introduced into the chromosome of the transgenic cell as a selective marker gene. Those. through the expression of this gene inside the cell, the hormone of the plant is overproduced. Therefore, the plant hormone produced in a cell containing the ipt gene acted not only on the cell itself to differentiate tissue, such as EPP or similar tissue, but also to some extent on the tissue adjacent to the cells, thus causing the same effect. as well as the action caused by the artificial addition of plant hormone to the culture of the environment.

 In the vector of the invention, the MAI gene is used as a selective marker gene. Therefore, when a gene is introduced into a plant using this vector, the tissue into which the desired gene is introduced can be selected by culturing a cell that is being processed to introduce the gene in a normal culture medium under normal cultivation conditions without adding any chemicals for selection while the morphologically formed abnormal tissue can be identified visually. Accordingly, the procedure is simplified and the activity of plant cells during selection does not decrease.

 Further, such a MAI gene is inherent in a plant or is introduced into it by infection with bacteria or the like. in nature. For this reason, even with the expression of the MAI gene inside the cell of the plant into which the gene was introduced, its safety when it enters the human body is considered quite high.

 Moreover, when using the ipt gene as the MAI gene, the tissue containing the transgenic cell proliferated and differentiated the additional shoot under the action of this gene, thus eliminating the need to add plant hormones to the culture medium, which is generally considered necessary for proliferation and differentiation during cultivation of plant cells .

 In addition, in this vector, the MAI gene, used as a selective marker gene, is inserted into a position in which it behaves in the same way as the movable DNA element, while the selective marker gene is removed from the DNA where this gene is present and functions through excision of a DNA element at a given ratio after the gene has been introduced into the plant cell and loses its own function. Thus, only the desired gene occupying such a position of the vector that it does not behave the same as the mobile DNA element remains in the same DNA and retains the ability to perform its function. Accordingly, the vector in this structure causes multiple gene introductions into a specific plant, simply changing the portion of the desired gene being introduced, without changing the structure of the marker marker gene, etc. Thus, multiple introductions can be performed an unlimited number of times.

 Moreover, in this case, the loss of function of the MAI gene as a selective marker gene can be determined visually by the morphological change in transgenic tissue, as with the introduction of this gene. Therefore, a tissue consisting only of cells in which the desired gene remains on the chromosome and the like. one and retains its function, can be selected easily and correctly. As a result, multiple gene introduction can be carried out with high efficiency, and a transgenic plant consisting only of such cells, namely, an individual free from the effects of the selective marker gene and not presenting any health threat caused by the selective marker gene, can be obtained without using the crossover stage.

Notes:
Regarding the instructions for the deposition of the microorganism referred to in the description of the invention:
1) FERM BP-5063
2) FERM BP-5064
3) FERM BP-5065
4) FERM BP-5066
Four of the above microorganisms with depository numbers were simultaneously deposited in the following depositories.

Depository Name:
National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Osamu Suzuki, Dr., Director General)
Depository Address:
1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken 305 Japan
Date of deposit: April 5, 1995

Claims (29)

 1. A vector for introducing a desired gene into a plant, comprising said desired gene and at least one gene with induction of morphological abnormality as a selective marker gene, said morphological abnormality inducing gene being a cytokinin synthesis gene.  2. The vector according to claim 1, wherein said cytokinin synthesis gene is an ipt, isopentyl transferase gene present in T-DNA of Agrobacterium timefaciens.  3. A vector for introducing a desired gene into a plant, comprising said desired gene, at least one gene inducing morphological abnormality as a selective marker gene, and a removable DNA element in which said gene inducing morphological abnormality is located so that it is deleted along with the deleted DNA element, and in which the indicated desired gene is located so that it is not removed with the deleted DNA element.  4. The vector according to claim 3, in which the specified gene inducing morphological abnormality is present inside the specified deleted DNA element.  5. The vector according to claim 3, in which the specified deleted DNA element is a transposon.  6. The vector according to claim 3, in which the specified deleted element comes from a site-specific recombination system.  7. The vector according to claim 3, wherein said gene inducing morphological abnormality is obtained from a microorganism of the genus Agrobacterium.  8. The vector according to claim 3, wherein said gene inducing morphological abnormality is a cytokinin synthesis gene.  9. The vector of claim 8, wherein said cytokinin synthesis gene is an ipt, isopentyl transferase gene present in Agrobacterium timefaciens T-DNA.  10. The vector according to claim 3, wherein said morphological abnormality inducing gene is at least one gene selected from rol genes.  11. The vector of claim 10, wherein said rol genes include rolA, rolB and rolC present in T-DNA of Agrobacterium rhizogenes.  12. A method for producing a transgenic plant free of the effects of a selective marker gene, the method comprising the following steps: (A) introducing a vector into a plant cell, wherein said vector comprises the desired gene, at least one gene inducing morphological abnormality, as the marker gene and the deleted DNA element in which the indicated gene inducing morphological abnormality is located so that it is removed together with the deleted DNA element in which the indicated desired gene is located so that it is not together with the deleted DNA element, (B) culturing the plant cell obtained in (A), identifying the morphologically abnormal plant tissue that appears during cultivation, and selecting the indicated morphologically abnormal tissue, and (C) culturing the indicated morphologically abnormal tissue selected in (B), the identification of morphologically normal tissue that occurs during cultivation, and the selection of the specified morphologically normal tissue.  13. The method according to item 12, in which the specified gene inducing morphological abnormality is present inside the specified deleted DNA element.  14. The method according to item 12, in which the specified deleted DNA element is a transposon.  15. The method of claim 12, wherein said removable DNA element is derived from a site-specific recombination system.  16. The method of claim 12, wherein said gene inducing morphological abnormality is obtained from a microorganism of the genus Agrobacterium.  17. The method according to claim 12, wherein said gene inducing morphological abnormality is a cytokinin synthesis gene.  18. The method of claim 17, wherein said cytokinin synthesis gene is an ipt, isopentyl transferase gene present in Agrobacterium timefaciens T-DNA.  19. The method of claim 12, wherein said gene indicating morphological abnormality is at least one gene selected from rol genes.  20. The method according to claim 19, in which these rol genes are rol genes, including rolA, rolB and rolC present in the T-DNA of Agrobacterium rhizogenes.  21. A method for introducing at least two desired genes into a plant, comprising at least twice performing the following operations: (A) introducing a vector into a plant cell in which said vector comprises the desired gene, at least one gene, inducing morphological abnormality, as a selective marker gene and a deleted DNA element, in which the indicated gene inducing morphological abnormality is located so that it is removed along with the deleted DNA element, and in which the indicated desired gene is located to that it is not removed together with the deleted DNA element, (B) cultivating the plant cell obtained in (A), detecting the morphologically abnormal plant tissue that occurs during cultivation, and selecting the indicated morphologically abnormal tissue, and (C) culturing the morphologically indicated abnormal tissue selected in (B), the identification of morphologically normal tissue that occurs during cultivation, and the selection of the specified morphologically normal tissue.  22. The method according to item 21, in which the specified gene inducing morphological abnormality is present inside the specified deleted DNA element.  23. The method of claim 21, wherein said removable DNA element is a transposon.  24. The method of claim 21, wherein said removable DNA element is derived from a site-specific recombination system.  25. The method according to item 21, in which the specified gene inducing morphological abnormality is obtained from a microorganism of the genus Agrobacterium.  26. The method according to item 21, in which the specified gene inducing morphological abnormality, is a gene for the synthesis of cytokinin.  27. The method of claim 26, wherein said cytokinin synthesis gene is an ipt, isopentyl transferase gene present in Agrobacterium timefaciens T-DNA.  28. The method according to item 21, in which the specified gene, indicating morphological abnormality, is at least one gene selected from genes rol.  29. The method of claim 28, wherein said rol genes are rol genes comprising rolA, rolB and rolC present in Agrobacterium rhizogenes T-DNA. Priority on points:
11/09/1994, according to claims 1 - 3, 5 7 - 9, 12, 14, 16 - 18;
05/31/1995 according to claims 6, 10 - 11, 15, 19 - 21, 23 - 29;
10/04/1995 according to claims 4, 13 and 22.

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