MXPA97003440A - Vector and methods to introduce at least dosgenes in a pla - Google Patents

Abstract

A vector for introducing a desired gene into a plant, which comprises the desired gene and, at least, a morphological abnormality induction (AMI) gene as a marker gene, or comprising the desired gene, at least one gene of IAM and a removable element. A method to produce a transgenic plant free of the influence of a marker gene. A method to introduce many desired genes into a plan

Description

VECTOR AND METHODS FOR ENTERING AT LEAST TWO GENES ON A PLANT Technical Field The present invention relates to a novel vector for introducing a desired gene into a plant using genetic engineering methods to obtain a transgenic plant; a method for producing a transgenic plant free of the influence of a selectable marker gene using this vector; and a method for introducing at least two desired genes into a plant using this vector. Background Art The transformation of microorganisms and cells grown using genetic engineering is currently applied to the production of physiologically active substances useful as medicines and, therefore, contributes largely to the industry. In the field of plant breeding, the industrial application of engineering lags behind because plant life cycles are much longer than those of microorganisms. However, since this technology allows a desired gene to be introduced directly into plants to be reproduced, it has the following advantages compared to classical reproduction which requires multiple crosses. (a) It is possible to enter only one characteristic to be improved. (b) It is possible to introduce characteristics of species other than plants (such as microorganisms and the like). (c) It is possible to shorten the reproduction period to a large extent. Therefore, genetic engineering methods for plant cultivation have been vigorously researched. The production of transgenic plants requires the following three steps. (1) Enter the desired gene into the plant cell (including introduction of the same into the chromosomes, nucleus and the like). (2) Select the tissue of plants formed only of cells in which the gene has been introduced. (3) Regenerate a plant from the desired tissue of the plants. In order to select transgenic tissues in which a desired gene has been introduced, it has been convenient to visualize the tissue in which the desired gene is expressed without regenerating the new plant. To achieve this, the desired gene is normally introduced into the plant cell together with a selectable marker gene from which expression in the culture stage of the cell can be easily detected. That is, the expression of the selectable marker gene is used as an index of introduction of the desired gene. Examples of conventional selectable marker genes include antibiotic-resistant genes, such as kanamycin-resistant gene (i.e., NPTII, neomycin phosphotransferase gene), a hygromycin-resistant gene (i.e., HPT, hygromycin phosphotransferase gene) , amino acid synthetase genes, such as a nopaline synthetase gene (NOS), an octopine synthetase gene (OCS), and a sulfonylurea-resistant gene (ie, ALS; acetolactate synthetase gene) that imparts chemical resistance agricultural. However, the expression of a selectable marker gene can cause serious problems when said transgenic plant is used for food. That is, it is very difficult to ensure that a gene product produced by expressing a selectable marker gene is safe for the human body. Consequently, if a transgenic plant containing a selectable marker gene is to be sold as a food, a detailed investigation must be carried out to determine the influence of the selectable marker gene on the human body. For example, the NPTII gene has been used as a selectable marker gene at the laboratory level since the early 1980s. In 1994, the product of that gene was finally accepted as a food additive by the U.S. Food and Drug Administration (FDA). Since then, transgenic plants containing the NPTII gene as a selectable marker gene have been used for food.

However, some consumers of products that contain the NPTII gene are still anxious about the effect of this gene. further, the selectable marker genes that are practically used, are only genes, such as the NPTII gene, which contributes to the detoxification of a growth inhibitory substance in plant cells. Therefore, in order to select transgenic tissue from plants into which the desired gene has been introduced, the tissue is cultured in a growth medium containing the growth inhibitory substance, and the expression of the selectable marker gene, viz. Resistance in the tissue to the growth inhibitory substance is evaluated and used as an index. However, even when a tissue has such resistance, culture in the presence of an inhibitory substance can result in undesirable side effects on plant cells, such as a decrease in proliferation and redifferentiation of the transgenic tissue. In addition, the expression of a selectable marker gene in a plant cell after the selection of transgenic tissue seriously obstructs the reproduction of plants by the subsequent introduction of genes. That is, when another gene is introduced into a transgenic plant containing a selectable marker gene, the introduction of the gene should be monitored using a different selectable marker gene. However, the effectiveness of a selectable marker gene varies with plant species. Therefore, a preliminary test is required to establish the conditions for each selectable marker gene (for example, it is reported that the HPT gene is more effective in rice plants than the NPTII gene (K. Shimamoto et al., Nature (London ), volume 338, page 274, 1989)). In addition, since the selectable marker gene varieties are limited, the multiple introduction of a gene can not be repeated indefinitely only by changing the selectable marker gene. That is, the number of gene introductions in a certain plant is limited by itself by the variety of selectable marker genes that can be used in that plant. In addition, the class of selectable marker gene that can be used is actually limited as mentioned above. Consequently, it is convenient to find a method for removing the selectable marker gene from the chromosome after the selection of transgenic plant tissue to exclude the influence of the selectable marker gene of the cell, tissue and plant. To eliminate the influence of a selectable marker gene, two methods have been reported. In one method, selectable marker ungen and a transposon of the plant is introduced into a plant chromosome and subsequently removed following the transposon (International Application Open to the Public No. WO 92/01370). In a second method, the site-specific recombination system of phage P1 is used in place of the transposon (International Application Open to the Public No. WO / 01283). Using these methods, it is possible to obtain a cell in which the selectable marker gene has been removed from the plant chromosome in a given ratio after the introduction of the gene. Unfortunately, the likelihood of the selectable marker gene being removed is very low. In addition, plant cells in which selectable marker genes have been removed from chromosomes using these methods, diffuse into cells in which selectable marker genes are still present and expressed. These two kinds of cells can not be visually distinguished. Plant cells containing selectable marker genes and a desired gene can be selected based on their chemical resistance, nutritional requirements and the like. However, at the time of selection, cells lacking selectable marker genes exhibit serious growth inhibition and are destroyed in many cases. Consequently, these selections can not be applied to obtain cells that lack selectable marker genes. In order to obtain plants lacking a selectable marker gene and containing the desired gene using the methods mentioned above, the plant tissue, in which the cells lacking the selectable marker gene and the cells containing the selectable marker genes are mixed, proliferated. they are 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 individual is derived from a single cell and therefore all the cells of the plant must have the same characteristics. Therefore, an individual derived from a cell lacking the selectable marker gene is made only from said cells. Unfortunately, the cells that constitute said regenerated individual are not necessarily uniform. Cells lacking the chromosome of the selectable marker gene and cells containing the selectable marker gene are co-existent and very irregularly distributed even in the same individual regenerated plant and in the same tissue thereof. Therefore, it is extremely difficult to obtain an individual made only of cells lacking the selectable marker gene at the stage at which the cultured tissue was re-branched to regenerate the individual. In addition, known analytical methods of selection, use a tissue, such as a sheet, as a test sample (not a complete individual or a single cell). Consequently, only the overall trend is analyzed with respect to the state of the selectable marker gene present in a leaf. Furthermore, in this case, it is common for the cell free of the selectable marker gene and the cell containing the selectable marker gene to be both present in the same individual or tissue. Therefore, even if only an individual made only of cells lacking the selectable marker gene is formed, it is difficult to select it. Even if the presence of the selectable marker gene is not detected in this tissue, tissues in other sites of the same individual may contain the selectable marker gene or simply show that the amount of selectable marker gene is below the detected limit. Therefore, it is impossible to determine whether the test sample is completely free of the cells that contain the marker gene. Using the methods mentioned above, an individual lacking the selectable marker gene is obtained only from an embryonic cell, such as a pollen, an egg cell and the like. When the self-pollination is carried out using the egg cell lacking the selectable marker gene, a fertilized egg lacking the selectable marker gene is obtained in a fixed ratio according to a classical hereditary law and from this fertilized egg, an individual is produced formed only of cells that have the same characteristics as the fertilized egg. Conventional analytical methods such as Southern hybridization can be carried out using this individual. Namely, even if the cell lacking the selectable marker gene is produced by the method described in the report referred to herein, the individual made only from said cell is obtained for the first time of redifferentiation of the plant of the plant. cultured tissue containing said cell, carrying out crossings of the regenerated plant and obtaining progeny of F or later generations. The individual thus obtained can be selected as an individual lacking the selectable marker gene. In order to remove the selectable marker gene from the transgenic plant, JP-A-6-276872 reports a technique for introducing genes into which a selectable marker gene is inserted into a separate plasmid vector different from the vector containing the gene. wanted. The plasmid containing the selectable marker gene is removed from the cell after completion of the introduction of the gene (the term "JP-A" as used herein means a published Japanese patent application). However, this technique requires a crossing step for the removal of the selectable marker gene. In this regard, the technique is the same as that of the two reports mentioned above. The above methods are difficult to apply to woody plants that have a long growth period, to sterile individuals and hybrid individuals in which it is thought to have F? Value. Further, when the removable DNA elements, such as a transposon and the like, are used, the relationship in which these chromosomal DNA elements, virus vector DNA and the like are removed, where these elements are present and functioning, is usually extremely low Consequently, it is necessary that the removal of these elements (namely, the removal of the selectable marker gene) can be easily detected in a real way, at least, at the stage of the cultured tissue. When this can not be detected before the redifferentiation of the cultured tissue and the formation of a later generation via the crossing of the regenerated individual, the method is not practical. Description of the Invention Accordingly, an object of the present invention is to provide a vector containing a gene that is desired to be introduced into a plant and a selectable marker gene, wherein a plant containing the same has no adverse effect on the human body when it is ingested, even if the selectable marker gene is expressed. Another objective of the present invention is to provide a vector for introducing a desired gene into a plant, wherein the vector contains a selectable marker gene that allows the selection of a transgenic tissue without the use of a decreasing plant cell growth inhibiting substance. the activity of the plant cell. Yet another objective of the present invention is to provide a vector for introducing a desired gene into a plant, wherein the vector contains a selectable marker gene and functions to exclude the influence of the selectable marker gene by removing the selectable marker gene from the DNA, wherein the selectable marker gene is present and works. Using this vector, a desired gene can be introduced repeatedly and efficiently. A further objective of the present invention is to provide a method for producing a transgenic plant using said vector, which can exclude the influence of the selectable marker gene without undergoing the step of producing Fi or late generations by crossing and a method for introducing massively genes in a plant applying the method described above.

These and other objects of the present invention have been achieved by using a vector, which comprises a desired gene and, at least, a morphological abnormality induction gene (hereinafter referred to as "IAM") as a selectable marker gene. . In addition, these and other objects of the present invention have been achieved using said vector, wherein the selectable marker gene is removed from the DNA after its expression. The expression of the selectable marker gene and the disappearance of the function thereof are detectable by morphological changes in the tissue in which the selectable marker gene has been introduced. In addition, these and other objects of the present invention have been achieved by using a vector which comprises a desired gene, at least one IAM gene is a selectable marker gene and a removable DNA element. The IAM gene is positioned so that it behaves integrally with the removable DNA element. The desired gene is positioned so that it does not behave integrally with the removable DNA element. In addition, these and other objects of the present invention have been achieved by a method for producing a transgenic plant free of the influence of a selectable marker gene, which comprises the following steps: (A) introducing a vector into a plant cell, wherein said vector comprises a desired gene, at least one IAM gene as a selectable marker gene, and a removable DNA element, wherein said IAM gene is positioned so that it behaves integrally with the removable DNA element and wherein said desired gene is positioned so that it does not behave integrally with the removable DNA element, (B) cultivate the plant cell obtained in (A), detecting a morphologically abnormal plant tissue that appears during cultivation, and select said morphologically abnormal tissue, and (C) culturing said morphologically abnormal tissue selected in (B), detecting a morphologically normal tissue that appears during culture and selecting said morphologically normal tissue. further, these and other objects of the present invention have been achieved by a method for introducing at least two desired genes in a plant, which comprises carrying out the following steps, at least twice: (A) introducing a vector in a plant cell, wherein said vector comprises a desired gene, at least one IAM gene as a selectable marker gene and a removable DNA element, wherein said IAM gene is positioned so that it behaves integrally with the removable DNA element and wherein said desired gene is positioned so that it does not behave integrally with the removable DNA element, (B) cultivate the plant cell obtained in (A), detecting a morphologically abnormal plant tissue that appears during culture, and selecting said morphologically abnormal tissue, and (C) culturing said morphologically abnormal tissue selected in (B), detecting a morphologically abnormal tissue appearing during culture, and selecting said morphologically normal tissue. Additionally, these and other objects of the present invention have been achieved by a regenerated plant by growing the morphologically normal tissue described above. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of the Ti plasmid and a restriction endonuclease map of Pst I fragment in a T-DNA region of A. tumefaciens strain P022. Figure 2 is a diagram of the construction of plPT2. Figure 3 is a diagram of the plPT3 construction of plPT2. Figure 4 is a diagram of the plPT4 construction of plPT3. Figure 5 is a restriction endonuclease map of a T-DNA region of the plPT4 structure. Figure 6 is the result of the PCR analysis of a phenotype of extreme buds of a tobacco in which a gene has been introduced using plPT4. Figure 7 is a diagram of the construction of pNPI102. Figure 8 is a diagram of the construction of pNPI103 of plPT4 and pNPI102.

Figure 9 is a diagram of the construction of pNPI106 of pNPI103. Figure 10 is the restriction endonuclease map of a T-DNA region in the structure of pNPMOT. Figure 11 is a photograph of outbreak No. 2 after one month of culture in Example 2. Figure 12 is a photograph of outbreak No. 8 after one month of culture in Example 2. Figure 13, is the result of PCR analysis of outbreak No. 8 in Example 2. Figure 14 is the result of PCR analysis of normal individuals obtained from shoots Nos. 13-1 and 14-1 in Example 3. Figure 15, is a photograph of normal outbreaks differentiated from a phenotype of extreme buds of a tobacco in Example 3. Figure 16 is the result of the PCR analysis of a normal individual obtained from a leaf formed from bud No. 7 in Example 2. Figure 17 is a diagram of the construction of pNPI128. Figure 18 is a diagram of the construction of pNPI129 of pNPI128. Figure 19 is a construction diagram of pNPM32 of pNPI101 and pNPI129. Figure 20 is the restriction endonuclease map of the T-DNA region in the structure of pNPI132.

Figure 21 is the result of PCR analysis of normal individuals obtained from shoots Nos. 15 to 21 in Example 5 using primers in which the existence of an ipt gene was detected. Figure 22 is the result of PCR analysis of normal individuals obtained from shoots Nos. 15 to 21 in Example 5 using primers in which the removal of a region maintained by a pair of Rs including an ipt gene was detected . Figure 23 is the result of PCR analysis of normal individuals obtained from shoots Nos. 15 to 21 in Example 5 using primers in which the existence of a GUS gene was detected.

Figure 24 is the result of PCR analysis of normal individuals obtained from the line that could not form an extreme bud phenotype in Example 5. Figure 25 is a diagram of the construction of pNPI702. Figure 26 is the restriction endonuclease map of the T-DNA region in the structure of pNP1702. Figure 27 is a normal shoot photograph differentiated from an extreme poplar shoot phenotype in Example 7. Figure 28 is the restriction endonuclease map of the T-DNA region in the structure of pNPI140. Figure 29 is the result of PCR analysis of a differentiated normal outbreak phenotype after the multitudinous introduction of genes in Example 8. Best Mode for Practicing the Invention As used herein, the gene for AMI is a gene that is induced in a tissue of a morphologically abnormal differentiation of the plant such as a dwarf, destruction of apical dominance, change in pigments, formation of a roughness in the crown, formation of hair roots, rippling of the leaves or Similar. With respect to the preferred AMI gene, genes isolated from bacteria of the genus Agrobacterium or the like that induce tumor or teratoma (e.g., formation of adventitious shoots and adventitious roots) can be found in several plants. Examples of these different IAM genes include cytokinin synthesis genes (e.g., ipt (isopentenyltransferase) gene (AC Smigocki, LD Owens, Proc. Nati, Acad. Sci. USA, vol 85, p.5131, 1988)), iaaM gene (tryptophan mono-oxygenase) (HJ Klee et al., GENES & DEVELOPMENT, vol. 1 p. 86, 1987), gene 5 (H. Kórber et al., EMBO Journal, vol.10, p. 3983, 1991), gene 6b (PJJ Hooykaas et al., Plant Mol. Biol., Vol.11, p.791, 1988) and role genes such as ro1A, ro1B, ro1C, and ro1D (FF White et al, J. Baceriol., Vol 164, p.33, 1985). In addition, examples thereof include a gene for iaaL (indoleacetic acid synthase-lysine) such as Pseudomonas syringae subsp. Savastanoi (A. Spena et al., Mol.Gen. Genet., Vol.227, page 205, 1991), genes. of homogeneous compartments and phytochrome genes in several plants. Preferably, cytokinin synthesis genes are used such as the ipt gene or at least one gene selecting from the role genes (more preferably, role genes containing genes ro1A, ro1B and ro1C). The ipt gene is present in the T-DNA of Agrobacterium tumefaciens and induces the destruction of apical dominance. Role genes containing the genes ro1A, ro1B and ro1C are present in the T-DNA of Agrobacterium rhizogenes and, at least, one of these induces the formation of hairy roots, dwarfs, ripple of the similar leaves of a regenerated plant of the hairy root. Using the techniques of the present invention, a combination of these selectable marker genes can be designed so that a specific structure, such as an adventitious bud, an adventitious root or the like, is redifferentiated in a specific plant into which these genes are introduced. selectable markers In the present invention, said combination of IAM genes can be used, according to the conditions for producing the transgenic plant, such as the class of a plant in which the genes are to be introduced. The morphologically abnormal tissue produced by the introduction of the IAM gene into the cell is formed only from the cells that contain this gene. Therefore, using this gene as the selectable marker gene, a vector is constructed together with the desired gene. When this vector is introduced into the cell of the plant and the transgenic cell is cultured, the tissue formed solely from this cell in which the selectable marker gene is introduced and the desired gene can be selected by visually choosing the morphologically abnormal tissue formed from this cell. Suitable vectors useful in accordance with the present invention have a DNA sequence that introduces a foreign gene into a host cell and that expresses the foreign gene within a host cell. When the gene is introduced using the vector of the present invention, the plant tissue formed solely from the transformed cell can be visually selected by culturing only the cell after the operation for the introduction of the gene into a culture medium such as culture medium. MS (Murashige-Skoog) under normal culture conditions. Since there is no need to use a special substance to select the transformed tissue, such as a plant growth inhibiting substance from plants or the like, not only is the procedure simplified, but also the activity of the plant cell is not diminished through said substance. In addition, the plant inherently has the lAM gene, or the lAM gene is spontaneously introduced into the plant by infection with bacteria or the like. Consequently, a plant obtained using the vector of the present invention, is not different from the plants that are present in nature that have this morphological abnormality. Suitable vectors according to the present invention include a vector wherein the lAM gene is positioned so that it behaves integrally with a removable DNA element and the desired gene is positioned so that it does not behave integrally with the element of Removable DNA As used herein, a removable DNA element is an element of a DNA sequence that by itself is removable from the DNA where the DNA element exists and functions. In plants, the transposon present in a chromosome is known as this element. The structure, activity and behavior of transposons has almost been completely identified. For the transposon to work, in principle two components are required, (1) an enzyme that is expressed from the gene present in it and that catalyzes the transposition excision of the transposon itself (transposase) (2) DNA binding sequences that are present in the terminal region of the transposon on which the transposase acts. Through these elements, the transposon is removed from the chromosome in which it exists, and then it is usually transposed to a new position in the DNA. However, in a certain proportion, the transposon also disappears without being transposed. The present invention makes use of said transposon transposition error. The transposon can be one of two types, either an autonomous transposon or a non-autonomous transposon. The autonomous transposon maintains the two elements, the transposase and the DNA binding sequence. In the autonomous transposon, the transposase is expressed and binds to the DNA binding sequence for action, whereby the transposon is autonomously excised from the chromosome. The non-autonomous transposon retains the terminal DNA binding sequence to which the transposase binds to act. In the non-autonomous transposon, the transposase gene undergoes mutation so that the transposase is not expressed; therefore, the transposon can not be removed autonomously from the chromosome. However, when the transposase is supplied to the autonomous transposon of the autonomous transposon or of an independent transposase gene, the non-autonomous transposon behaves similar to the autonomous transposon. Examples of autonomous transposons include Ac and Spm isolated from maize (A. Gierl and H. Saedler, Plant Mol. Biol., Vol.19, page 39, 1992). Ac can be obtained by digesting sites on the wx-m7 gene in the chromosome of the maize with restriction endonuclease Sau3A (U. Behrens et al., Mol.Gen.Genet., Vol 194, p.346, 1984). This autonomous transposon is the most analyzed among plant transposons. In fact, the DNA sequence has already been determined (M. M? Ller-Newman et al., Mol. Gen. Genet., Vol 198, p.19, 1984). Examples of non-autonomous transposons include Ds and sSpm obtained by deleting the internal regions of Ac and Spm respectively (H.-P. Doring and P. Starlinger, Ann. Rev. Genet .. Voter 20, P. 175, 1986) and those isolated from many plants, other than corn, such as snapdragon, bell vine, and the like, (eg, Y. Inagaki et al., Plant Cell, vol.6, p.375, 1994). When these transposons are introduced into chromosomes of exogenous plants, these transposons are also excised from a chromosome and transposed upon display of the activity (eg, B. Baker et al., Proc. Nati, Acad. Sci. USA, vol. P. 4844, 1986). In the present invention, both autonomous and non-autonomous transposons can be used. Non-autonomous transposons can be used by inserting a transposase gene into the same transposase. Another removable DNA element, which is not present in plants, but which can be used according to 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 the removable DNA element of the present invention) having a characteristic DNA sequence, and (2) an enzyme that it binds specifically to the DNA sequence and catalyzes the recombination between the DNA sequences if two or more of the sequences exist (recombinase). When the two DNA sequences are oriented in the same direction at a given interval in the same DNA molecule, the region maintained by these DNA sequences are excised from the DNA molecule, such as a plasmid, chromosome or the like. When the two DNA sequences are oriented in opposite directions in the same DNA molecule, the region maintained by these DNA sequences is inverted. The present invention preferably utilizes the first excision. Both extirpation and inversion within the recombination site occur as a result of homologous recombination through the site-specific recombination system, which is different from the mechanism used by the transposon. It is known that the recombinase gene is not necessarily present in the same DNA molecule, in which the recombination site exists. The recombinase gene only needs to be present in the same cell and is expressed to excise or reverse the region maintained by the 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 (eg, E. coli), yeast and the like. Examples thereof include a Cre / lox system, a pSR 1 system, an FLP system, a cer system, and a fim system (eg, N. L. Craig, Annu. Rev. Genet., vol.22, p.77, 1988). when the site-specific recombination system separated from these microorganisms with the use of a Cre / lox system derived from phage P1 (WO 93/01283) is introduced into organisms (including plants) different from the organism from which this system has been derived, behaves in the same way as in the original organism. The specific recombination system for a yeast site (Zygosaccharomyces rouxii) (pSR 1 system (H. Matsuzaki et al., J. Bacteriology, vol 172, page 610, 1990)) can also be used according to the present invention. invention. This pSR 1 system also maintains its inherent function in higher plants (H. Onouchi et al., Nucleic Acid Res., Vol 19, p 6373, 1991). In the present invention, the morphological abnormality induction gene (lAM) can be inserted into a position where this gene is removed together with the removable DNA element. For example, when the transposon is used as the removable DNA element, the IAM gene can be inserted in a position that does not influence the removal of the transposon and that is upstream of the promoter region of the transposase gene but downstream of the transposon gene. terminal region to which the transposase is attached. When the pSR1 system is used, the IAM gene can be inserted at any position within the region maintained by the characteristic DNA sequences that does not inhibit recombinase expression. In the present invention, the lAM gene is preferably present within the removable DNA element. On the other hand, the position of the desired gene is not particularly limited; however, preferably, the desired gene is present in the removable DNA element. Using the vector of said structure after the introduction of the desired gene, the IAM gene can be removed at a certain frequency, together with the removable DNA element, from the DNA in which it is introduced and functions. The desired gene that does not behave integrally with the selectable marker gene remains in the same DNA. The vector can be used to multiply a desired gene in a certain plant. In addition, since the loss of function of this lAM gene can be visually detected as a morphological change of the transgenic tissue during culture, the tissue formed only from the cell with the desired gene but without the selectable marker gene, can be selected with ease and without needing a special procedure, consequently, even when said cell is actually formed at a low ratio, the cell can be selected sufficiently to make the procedure practically useful. Furthermore, not only the multiple introduction of the gene using this vector can be repeated many times, but it can be repeated before a mature plant is generated. Therefore, multiple introduction can be carried out efficiently. In order to obtain the individual transgenic plant formed only of said cells, the plant can be regenerated from the selected tissue, without having to endure the crossing step. The individual transgenic plant thus obtained is completely free of any adverse effects on the human body caused by the selectable marker gene as mentioned above. In addition, the use of this vector does not require a cell growth inhibiting substance that can decrease the activity of the cell in the step of selecting the transgenic tissue.

The vector of the present invention can be used in any plant in which the gene can be introduced by genetic engineering methods. The desired gene according to the present invention can be any gene by which agriculturally excellent characteristics can be imparted and any gene that allows studies of gene expression mechanisms and the like, although agriculturally excellent characteristics are not necessarily imparted. To produce a protein such as an enzyme of a gene, a structural gene sequence encoding polypeptide information and regulatory sequences of the structural gene, such as a promoter sequence (expression initiation sequence), a sequence sequence (sequence), is generally required. terminating expression) and the like. Examples of suitable promoter sequence that functions in the plant include the 35S promoter of a cauliflower mosaic virus (JT Odell et al., Nature (London), vol.3133, p.810, 1985), the promoter of a syntetase nopaline (WHR Langridge et al., Plant Cell Rep., vol.4, p.355, 1985) and the promoter of the small carboxylase / oxygenase subunit of ribulose diphosphate (R. Fluhr et al., Proc. Nati. Acad. Sci. USA, vol 83, p 2358, 1986). Examples of the suitable terminator sequence include the polyadenylation signal of a nopaline synthetase (A. Depicker et al., J. Mol. Appl. Gen., vol.1, p.561, 1982) and the polyadenylation signal of a Octopine synthetase (J. Gielen et al., EMBO J., Vol.3, p.835, 1984). Consequently, when necessary, a gene in the vector of the present invention comprises a structural gene and the regulatory sequences of expression of the gene thereof. The gene, or gene and regulatory sequences, can be obtained by chemical synthesis or by cloning cDNA or genomic DNA. The vector of the present invention can be introduced indirectly into the plant cell through viruses or bacteria with which the plants are infected (I. Potrykus, Annu, Rev. Plant Physiol. Plant Mol. Biol., Vol 42, p. 205, 1991). Examples of suitable viruses include cauliflower mosaic virus, geminivirus, tobacco mosaic virus and brominated mosaic virus. Examples of suitable bacteria include 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 the bacterium of the genus Agrobacterium. Recently, the introduction of genes into monocotyledonous plants has also been reported by the infection of these plants with them (for example, in International Open to the Public No. WO 94/00977). The vector of the present invention can be introduced directly into the plant cell by physical and chemical methods such as microinjection, electroporation, a polyethylene glycol method, a melting method and high velocity ballistic penetration ( I. Potrykus, Annu, Rev. Plant Physiol. Plant Mol. Biol., Vol 42, p 205, 1991). since the general indirect introduction method using the genus Agrobacterium can not be applied to many of the monocotyledonous and dicotyledonous plants that are resistant to infection with Agrobacterium, the direct introduction methods mentioned above are effective for these plants. The vector to be used in the present invention is not particularly limited as long as the requirements of the present invention are met. For example, if the vector is introduced indirectly into the cell of the plant, the vector can be a Ti vector or a virus vector. Examples of the vector Ti for use in the present invention include 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 p.GA482 (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., Nucleic Acids Res., Vol.13, p.4777, 1985), and pMON200 (RT Fraley et al., Bio / Technology, vol.3, p.629, 1985) . Examples of the virus vector for use in the present invention include cauliflower mosaic virus vector (N. Brisson et al., Nature (London), vol 310, p.511, 1984), geminivirus vector (RJ Hayes et al. , Nature (London), Vot 334, p.799, 1988), vector of brominated mosaic virus (R. French et al., Science vol.231, p.1294, 1986), vector of tobacco mosaic virus ( N. Takamatsu et al., EMBO J .. vol.6, p.307, 1987), and agroinfection vector (N. Grimsley et al., Nature (London), vol.325, p.177, 1987). However, the vectors for use in the present invention are not limited to the same.

In addition, the desired gene for use in the present invention is not particularly limited. The nature of the desired gene by itself is not critical to the present invention. Examples of the desired gene for use in the present invention include genes for disease resistance (e.g., Bacillus thuringiensis endotoxin gene, WO 92/20802), herbicide resistance (e.g., mutant acetolactate synthase gene). , WO 92/08794), seed storage protein (e.g., glutelin gene, WO 93/18643), fatty acid synthesis (e.g., acyl thioesterase gene-ACP, WO 92/20236 ), cell wall hydrolysis (eg, polygalacturonase gene (D. Grierson et al., Nucleic Acids Res., vol 14, P. 8595, 1986)), anthocyanin biosynthesis (e.g. Chalcone synthase (HJ Reif et al., Mol.Gen.Genet., vol 199, p. 208, 1985)), ethylene biosynthesis (e.g., ACC oxidase gene (A. Slater et al., Plant Mol. Biol., Vol.5, p.137, 1985), active oxygen scavenging system (e.g., glutathione reductase gene (S. Greer &RN Perham, Biochemistry, vol.25, p. 2736, 1986), and lignin biosynthesis (e.g., ammonium gene from phenylalanine lyase, cinnamyl alcohol dehydrogenase gene, o-methyltransferase gene, cinnamate 4-hydroxylase gene, 4-coumarate-CoA ligase gene, CoA cinnamoyl reductase gene (A. M. Boudet et al., New Phytol., Vol. 129, p. 203, 1995)). However, the genes desired for use in the present invention are not limited thereto. In addition, the host plant for use in the present invention is not particularly limited. Examples of herbaceous plant used as the host plant include tobacco (Tabacum), tomato (Lycopersícom), sweet potato (Impoea), potato (So / ar? Í / m), carrot (Dacus), lettuce (Lactuca), cauliflower (Brassica) ), cabbage (Brassica), oilseed rape (Brassica), sunflower (Helianthus), beet (Bela), asparagus (Asparagus), banana (Musa), cotton (Gossypium), arabidopsis (Arabidopsis), alfalfa (Medicago), pea (Pisum), soy (Glycine), rice (Oryza), corn (Zea), and rye (Sécale). Examples of the arboreal plant used as the host plant include white poplar (Populus), eucalyptus (Eucalyptus), acacia (Acacia), pear (Pyrus), apple (Malus), grape (Vitis), walnut (Juglans), plum ( Prunus), rose (Rosa), and fir (Picea). However, host plants for use in the present invention are not limited thereto. In the present invention, the lAM gene is expressed to render abnormal the internal physiological part of the cell. Physiological abnormalities include the production of plant growth hormone in a plant cell, with the result that the proliferation and differentiation of the cell containing the IAM gene are confused to induce various morphological abnormalities. For example, an aggregate of disordered shoots with destroyed apical dominance (extreme bud phenotype: FBE), hair roots or the like, can be derived from a cell in which a lAM gene is introduced. This phenotype is formed by proliferation and abnormal differentiation of the cell mentioned above. Therefore, this morphologically abnormal tissue is formed solely from the cell that contains this gene. Consequently, if the vector is constructed using this gene as the selectable marker gene together with the desired gene and is introduced into the cell of the plant and the cell is cultured, the tissue formed only of the cell in which the selectable marker gene and The desired gene has been introduced, it can be chosen by visually selecting only the morphologically abnormal tissue formed from the plant cell. Therefore, it is possible to visually select the transgenic tissue without carrying out any special procedure such that the addition of the growth inhibitory substance of the plant cell and the like to a culture medium. While conventional selectable marker genes such as NPTII gene, are not introduced into plants without genetic engineering; the lAM gene of the present invention is a gene whose plants inherently retain or which are naturally introduced into plants by infection with bacteria or the like. For this reason, it is considered that the safety of the product of the gene for the human body is very high. In addition, in the present invention, the lAM gene is inserted in a position so that it behaves integrally with the removable DNA element. After the vector having said structure is introduced into the plant, the lAM gene used as the selectable marker gene is removed from DNA together with the removable DNA element at a fixed frequency resulting in the loss of this function. Meanwhile, the desired gene that does not behave integrally with the removable DNA element remains in the same DNA and maintains its function. Therefore, expression of the same selectable marker gene can be used as an index for the introduction of a desired gene over and over again. Consequently, this vector causes the multiple introduction of the gene into a certain plant merely by changing the structure related to the desired gene that will be introduced without imposing any changes in the structures of the selectable marker gene and the others. For this reason, the vector can be used repetitively for an unlimited number of times. Since the loss of the function of the selectable marker gene, ie the loss of the function of the lAM gene, can be detected visually, the tissue formed of cells lacking the selectable marker gene and containing the desired gene can be obtained from safe and easy way. That is, the culture, visual selection and separation can be repeated without the need for any special procedure to obtain said tissue. In addition, the plant formed only of the cell mentioned above can be obtained only by the regeneration of the obtained tissue plant, without having to undergo the crossing step. Still further, even if a transposon is hard to remove completely from the DNA since this element has a high transposon capacity, the invention can be placed sufficiently in practical use since the selection is highly efficient. EXAMPLE The present invention will be illustrated with reference to the following Examples, but the present invention should not be construed to be limited thereto. In the following Examples, the experiments were carried out according to the instructions of Molecular Cloning, 2nd edition (Sambrook et al., Eds., Cold Spring Harbor Laboratory Press, New York, 1989) or by a manufacturer unless otherwise instructed., EXAMPLE 1 I. Construction of a vector An ipt gene present in R-DNA of the P022 strain of pathogenic A. tumefaciens (H. Wabiko, Chemical Regulation of Plants, vol.24, p.35, 1989 (see Fig. 1)) was separated with the restriction endonuclease Pst, and the plasmid plPT1 was obtained by ligating the ipt gene at the endonuclease site of Psti restriction of plasmid pUC7 (Molecular Cloning, 2nd edition, col 1, 4.10). From this plasmid, an ipt gene containing a native promoter and a native polyadenylation signal was separated from the restriction endonucleases BamH1 and Pst1, and the plasmid pIPT2 was obtained by ligating the ipt gene at the restriction endonuclease sites SamHI-Psfl from plasmid pUC119 (obtained from Takara Shuzo Co., Ltd.). From this plasmid, the structural gene and the native polyadenylation signal of the ipt gene were separated with the restriction endonuclease Rsal and the plasmid pIPT3 was obtained by ligating the ipt gene at the Smal restriction endonuclease site of the plasmid pUC119. In addition, the ipt gene inserted into plPT3 was separated with the restriction endonucleases BamHl and Sacl, and the plasmid plPT4 was obtained by ligating the fragment at the restriction endonuclease sites ßamHI-Sací of the plasmid pBM21 of the vector (obtained from Clontech Co. ) which is useful for the introduction of the gene in a plant. When a plant is infected with A. tumefaciens having the plasmid plPT4, a T-DNA region that exists between a LB site and an RB site, a region of approximately 5 kb having the NPTII gene and the ipt, enter to integrate into the chromosome of the plant. This plasmid pIPT4 was introduced into strain JM109 of E. coli (Escherichia coli), and was deposited according to the Budapest Treaty as E. coli JM109 (plPT4) under Deposit No. FERM BP-5063. The strategy for constructing the plasmid pIPT4 is shown schematically in Figures 2 to 4. The restriction endonuclease map of the T-DNA region thereof is shown in Figure 5. In Figures 2 to 4 and 5, the " P "and" T "enclosed in a circle indicate a native promoter and a native polyadenylation signal of the ipt gene itself, respectively. 35S-P indicates a 35S promoter from a cauliflower mosaic virus and Nos-P indicates a promoter from a nopaline synthetase gene. T (Fig. 4) or Nos-T (Fig. 5) indicates a polyadenylation signal of the nopaline synthetase gene. In this Example, as shown in Figure 5, for the lAM gene as the selectable marker gene, the ipt gene was used that contributes to the formation of an ESP by inducing the destruction of apical dominance, and the NPTII gene was used as a model of the desired gene. The ipt gene is a member of oncogenes that retains pathogenic A. tumefaciens. A plant cell in which this ipt gene is introduced, causes differentiation that leads to the formation of an ESP by the suppression of cytokinin, which is a plant hormone. In this Example, the 35S promoter from a cauliflower mosaic virus was used for an ipt gene promoter sequence, and the native polyadenylation signal from the ipt gene itself was used for a terminator sequence. II. Introduction of pIPT4 in Agrobacterium The strain LBA4404 of A. tumefaciens (obtained from Clontech Co.) was inoculated in 10 ml of YEB liquid culture medium (containing 5 g / liter of meat extract, 1 g / liter of yeast extract, 1 g / liter of peptone, 5 g / liter of sucrose, and 2 mM of MgSO4, pH of 7.2 at 22 ° C (the pH at 22 ° C applies to the following unless indicated otherwise)), and it was grown at 28 ° C until OD63o was within the range of 04 to 0.6. Then, the culture was centrifuged at 6,900 x g for 10 minutes at 4 ° C to collect the cells. The cells were suspended in 20 ml 10-mM TrisHCI (pH 8.0) and the suspension was recentrifuged at 6,900 x g for 10 minutes at 4 ° C. Subsequently, the collected cells were resuspended in 200 μl of YEB liquid culture medium., and this suspension was used as a cell solution to introduce a plasmid. In a 15 milliliter tube (made by Falcon), 200 μl of the cell solution to introduce the plasmid was mixed with 6 μg of plasmid pIPT4 obtained in step 1 described above the mixture was cooled by immersing it for 5 minutes in ethanol which was It had cooled in liquid nitrogen for 30 to 40 minutes. The solution thus cooled, together with the tube, was allowed to stand in a water bath of 29 ° C for 25 minutes. Then, 750 μl of YEB liquid culture medium was added thereto and the mixed solution was cultured at 29 ° C for 1 hour while stirring. This cell solution was dispersed in YEB agar culture medium (containing 1.2% w / w of agar and the same ingredients as those of the aforementioned culture medium) to which 50 mg / l of kanamycin was added, and were cultured at 28 ° C for 2 days. The colonies of cells thus obtained were inoculated in liquid culture medium YEB and further cultured. Then, the plasmid was extracted from the cells by an alkaline method and divided with restriction endonucleases Psfl, SamHl and EcoRl. The fragments thus obtained from the plasmid were analyzed by agarose gel electrophoresis and it was confirmed that the plasmid pIPT4 was introduced into a strain of A. tumefaciens LBA4404. lll. Introduction of Aqrobacterium pIPT4 into a tobacco The mature leaves of a tobacco (Nicotina tabacun cv. Xanthi, hereinafter a tobacco means this variety unless otherwise indicated) developed in a greenhouse, were immersed in an aqueous solution of sodium hypochlorite at 7% v / v for sterilization and washed three times with sterile water. Then, the central rib of the sheet was removed to form sheet disks of approximately 8 mm square. The leaf discs thus obtained were immersed for approximately 1 minute in a suspension of cells of pIPT4 introduced in the strain LBA4404 of A. tumefaciens in step II described above, and were infected therewith (the suspension of which was diluted with a water sterilized at OD63o = 0.25 after overnight culture in liquid culture medium YEB). The infected leaf disc was placed on a sterilized filter paper to remove any extra cell suspension. Then, it was placed on a culture medium of MS agar without hormones (T. Murashige and F. Skoog, Physiol. Plant., Vol.15, p.473, 1962 (with the proviso that 0.8p / v% agar has been added to it)) containing 50 mg / liter of acetosyringone with the back of the leaf facing upwards and cultivated for 3 days, at 25 ° C in full light (the culture in an explant, a plant tissue and a plant was carried out under these temperature and lighting conditions unless otherwise indicated). The leaf disk thus cultivated, was then transplanted into growth medium of MS agar without hormone containing only 500 mg / liter of carbenicillin, and the culture was continued. As a result, 22 adventitious shoots were redifferentiated. These adventitious shoots were further separated and cultured in a culture medium having the composition mentioned above to obtain 6 ESP lines. These ESP lines were subcultured in the same culture medium each month, and were subcultured in MS agar culture medium without hormones that several times did not contain carbenicillin 3 months after infection. After the proliferation of Agrobacterium was not observed, a test for kanamycin resistance and PCR analysis was carried out. IV. Analysis of a tobacco in which gene A has been introduced. Test for resistance to kanamycin. The 6 lines of ESP obtained in step Ul described above, were cultivated as such without subcultures. The developed leaves of these ESP lines were cut to form sheet discs of approximately 3 square mm. The leaf discs thus obtained were placed on the MS agar culture medium (1 mg / liter of benzyl adenine and 0.2 mg / liter of α-naphthalene acetic acid) containing 200 mg / l of kanamycin was added thereto. After cultivation in this culture medium containing kanamycin for 1 month, the formation of ESP lines of the leaf disks obtained from these ESP lines was also observed. B. PCR analysis Chromosomal DNs were extracted for all 6 lines of ESP obtained in step III described above, and the genes introduced thereto, were analyzed by the PCR method. The chromosomal DNA was extracted using the following modified CTAB method. First, about 1 g of the developed leaves of the ESP were milled in liquid nitrogen using a hardened mortar and grinder and suspended in 5 ml of a buffer solution (containing 2% w / v of C (hexadecyltrimethylammonium bromide), 1.4 M NaCl, 0.2% v. / v of β-mercaptoethanol, 20 mM EDTA and 100 M Tris-HCl (pH 8.0)) that has been heated to 60 ° C. This suspension was heated to 60 ° C for 30 to 60 minutes while stirring gently, and then cooled to room temperature. To this suspension was added a mixture of chloroform and isoamyl alcohol (24: 1) at an equal volume and these were mixed gently. Then, the mixture was centrifuged at 1.600 x g for 5 minutes to recover a supernatant. Subsequently, 2/3 volume of isopropyl alcohol was added to the supernatant and mixed again gently. The mixture was allowed to stand on ice for 10 minutes to precipitate the chromosomal A DN. This chromosomal DNA was collected by centrifugation at 1, 6000 x g for 10 minutes. The chromosomal DNA thus collected was washed with 70% v / v ethanol, then dried under vacuum and dissolved in 300 μl of TE (comprising 10 mM Tris-HCl and 1 mM EDTA). Meanwhile, in order to detect the ipt gene by the PCR method, a pair of primers (oligonucleotides) were synthesized by a DNA synthesizer (manufactured by Applied Biosystems Co.). When they joined the ipt gene, the distance between the two primers was approximately 800 bp. 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 primers, 10 mM Tris-HCl (pH of 8.8 at 25 ° C), 50 mM KCl, MgCl 2 1.5 mM, 1% w / v of Triton X-100, 0.1 mM dNTP and 1.25 units of Taq polymerase (obtained from CETUS CO.). After the mixture was heated at 94 ° C for 1.5 minutes, a heating cycle was repeated in three parts, namely at 94 ° C for 1 minute, at 55 ° C for 2 minutes and at 72 ° C for 3 minutes for a total of 30 times to complete the reaction. The obtained reaction mixture was analyzed by agarose gel electrophoresis to detect the presence of the ipt gene in the chromosomal DNA by the amplification of the approximately 800 bp gene. The results are shown in Figure 6. As is clear from Figure 6, the amplification of the approximately 800 bp gene was observed in all 6 ESPs. In Figure 6, the values shown on the left side indicate the base length, which was of the detected band ingredients (hereinafter referred to as the "band") in the electrophoresis in the DNA size marker. COMPARATIVE EXAMPLE 1 The analysis was carried out with respect to 16 shoots that did not have the capacity to form an ESP and were obtained from the redifferentiated adventitious shoots of the leaf infected with A. tumefaciens in step III of Example 1. That is, at the time when 22 adventitious shoots were cultivated and the lines of ESP in step 111 of Example 1, those showing morphologically normal shoots (hereinafter referred to as "sin-ESP") were also released from A. tumefaciens and subjected to the kanamycin resistance test in the same manner as in steps III and IV of Example 1. In addition, the 9 lines without ESP were subjected to analysis of PCR. However, with respect to these non-ESP lines, the leaf discs placed on the culture medium containing kanamycin turned brown and became white after 3 months. In addition, in the analysis of PCR, in none of the nine lines analyzed, no amplification of a DNA fragment of approximately 800 bp was detected, which proves the presence of the ipt gene. The PCR results are shown in Figure 6. EXAMPLE 2 I. Construction of a vector Plasmid pHSG398 (obtained from Takara Shuzo Co., Ltd.) was digested with the restriction endonuclease SamHI. The cohesive endings produced by the digestion were changed at the blunt end terminations with DNA polymerase I4 (large subunit), and the plasmid pNPHOO was obtained by ligating these endings. That is, the pNPHOO was the pHSG398 that loses the restriction endonuclease site of SamHl. Meanwhile, plasmid pCKR97 (T. Izawa et al., Mol.Gen. Genet., Vol.227, p.391, 1991) was digested with restriction endonuclease Pst. Ac transposon from a corn was cut and inserted into the Psfl restriction endonuclease site of pNPHOO to obtain plasmid pNPH02. Subsequently, of the plasmid pIPT4 constructed in Example 1, a 35S cauliflower mosaic virus promoter and an ipt gene attached thereto were cut with the restriction endonucleases HindU and Sacl. The cohesive terminations of the fragment thus obtained were changed to the blunt end terminations with the T4 DNA polymerase I and the fragment was inserted into the Hincll restriction endonuclease site of the plasmid pUC119 to obtain the plasmid pNPHOI. From this plasmid pNP16101, the 35S cauliflower mosaic promoter virus and the ipt gene were again cut with restriction endonucleases Psfl and EcoRI and the cohesive ends of the fragment were changed into blunt end terminations with DNA polymerase 16 T4 In addition, plasmid pNP16103 was obtained by ligating the blunt-ended endonuclease BamH1 site fragment of pNPI102. That is, in the plasmid pNPI103, the 35S promoter and the ipt gene linked thereto existed at the old restriction endonuclease site of Sa HI within the Ac transposon. The desired vector was obtained by cutting the Ac transposon containing the 35S cauliflower mosaic virus promoter and the ipt gene of the plasmid pNPI103 with the restriction endonuclease Psfl and inserting this transposon Ac into the restriction endonuclease site Ssel of the plasmid of vector pBI121. This was designated as plasmid pNPI106. This pNP106 plasmid was also introduced into E. coli strain JM109 and was deposited according to the Budapest Treaty as E. coli JM109 (pNPI106) under No., Depository FERM BP-5064. The strategy for constructing the plasmid pNPHOd was shown schematically in Figures 7 to 9. A restriction endonuclease map of the T-DNA region thereof is shown in Figure 10. In Figures 7 to 9 and 10, the region Transposon terminal Ac is shown by the black opposite triangles, respectively. In Figure 10, Ac-P is a native promoter present within Ac. Other symbols are the same as those shown in Figures 2 to 5. As is clear from Figure 10, this plasmid has the ipt gene as the selectable 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 in the region that will be integrated into the plant chromosome. In addition, the ipt gene is present as it is inserted into the Ac transposon. Since the cell that has the GUS gene metabolizes a special substrate to produce a blue pigment, the expression of the gene can be identified by detecting this pigment. Therefore, the GUS gene is often used for the analysis of gene expression in the plant. II. Introduction of pNPH06 in a tobacco and analysis of a tobacco in which gene A was introduced. Introduction of pNPH06 in a tobacco and test for expression of the introduced gene. In the same manner as in steps II and Ul of Example 1, pNPI106 was introduced into the strain LBA4404 of A. tumefaciens and the leaf discs of the tobacco were infected with this A. tumefaciens. The tobacco leaf, thus infected, was cultivated in culture medium of MS agar without hormone containing 50 mg / liter of acetosyringone. and then in the culture medium of MS agar without hormone to which 500 mg / liter of carbenicillin was added. After two months of said cultivation, 63 lines of ESP were separated. The ESP lines were transplanted into the culture medium of the same composition (growth medium of MS agar without hormone containing 500 mg / liter of carbenicillin). One month later, among the outbreaks of ESPs that grew slightly, 9 shoots (those that were generated from the ESPs are called from here on, simply "buds") were visually selected which had a growth of approximately two or more times in comparison with the other shoots, in which the growth of the lateral shoots was not observed and that the influence of the ipt gene seemed to decrease. The leaves of the shoots were subjected to the same test for resistance to kanamycin that was carried out in step IV-A of Example 1 and the test for the expression of the GUS gene (test for GUS activity) based on the Method of Jefferson and others. The shoots obtained after the leaves were cut, were transplanted in culture medium of MS agar without hormones and were cultured. One month later, the configurations were observed to detect the ability to form the ESPs of the shoots, thus exhibiting the expression of the ipt gene. The results are shown in Table 1. TABLE 1 Results for the expression of the gene introduced in a tobacco by the vector pNPH06 Outbreak Resistance Morphology Activity to GUS after kanamycin 1 month of culture Example 2 1 ESP 2 ESP 3 ESP 4 ESP 5 ESP 6 ESP 7 ESP 8 normal 9 ESP Example 10 normal Comparative 11 normal 2 12 normal Notes: 1. In resistance to kanamycin, + is "resistant", and - is "non-resistant". 2. In the GUS activity, + is "active", and - is "inactive". 3. "normal" means an individual that causes the dominant growth of an apical bud and the formation of roots.

As is evident from Table 1, although root leaf No. 8 has resistance to kanamycin and GUS activity, an ESP is not formed even if the root is grown for 1 month. This is presumably because the ipt gene that causes the formation of ESP is present in the form inserted within the Ac transposon in the plasmid pNPI106. That is, the ipt gene, which is introduced into the tobacco chromosome by infection with A. tumefaciens that contains this plasmid for tissue and expressed in the initial stage of tissue culture just after infection, is removed together with AC by the action of Ac during the subsequent culture. Meanwhile, in the same vector, the NPTII gene and the GUS gene are inserted in a position where they do not behave integrally with Ac, so that these genes still remain in the plant chromosome and are expressed. In Table 1, although resistance to kanamycin and ESP formation capacity is observed in shoots Nos. 3, 5, and 6, only GUS activity is negative. This means that in these outbreaks only the GUS gene of the genes introduced using pNPI106 is not expressed. It is considered that this is due to the erroneous integration that occurs when these genes were integrated into the plant chromosomes. That is, when the gene was introduced via A. tumefaciens containing the plasmid having the structure of pNPHOd or the like, the T-DNA region, namely, the overall integral region between the RB site and the LB site, they should normally be integrated into the plant chromosome. However, this region is sometimes not fully integrated, but the defective part that lacks some portion of the LB terminals is broken and inserted. In the T-DNA region of pNPI106, the GUS gene is present in the position closest to the LB site. Consequently, it is considered that due to the wrong integration in the introduction of genes, the GUS gene is integrated into the chromosome in a condition in which the GUS gene is broken into pieces and its own function is lost, or the gene of GUS is not completely inserted in it, so the GUS gene is not expressed in these outbreaks and the activity of the same is not observed. The photograph of outbreak No. 2 and No. 8 after one month of cultivation, is shown in Figures 11 and 12. With respect to the growth of bud leaf No. 8 and subjected to the test for resistance to kanamycin present , the culture was further continued after the test. Five adventitious buds were obtained from this leaf and all were without ESPs. B. PCR analysis. In shoots Nos. 1 to 9 in Table 1, after the ESP formation capacity was observed, the PCR analysis was carried out in the same manner as in step IV-B of Example 1 to further examine the presence of the ipt gene in the chromosome, as long as the pair of primers that were designed are linked to the NPTII gene and the GUS gene were respectively used in addition to the primers used in step IV-B of Example 1. In the In case these primers were used, when the Ac and the ipt gene inserted therein (γ-c-gene complex) were excised from the T-DNA region of pNPI106, a DNA fragment of approximately 3 kb in the PCR. Consequently, the removal of the ipt-Ac-gene complex from the DNA can be detected using eta amplification as an index. The results of the PCR analysis in the outbreak No. 8, are shown in Fig. 13, in which the values indicated on the left side are the same as those shown in Figure 6. As is evident from the results, in the chromosomal DNA extracted from shoot No. 8, observed the amplification of the approximately 3 kb DNA fragment that proves the removal of the ipt Ac-gene complex, whereas the amplification of a DNA fragment of approximately 800 bp that proves the presence of the ipt gene is not observed. This means that the ipt gene is excised from the chromosomal DNA of this outbreak along with the Ac and disappears. On the other hand, with respect to outbreaks Nos. 1 to 7 and 9, the amplification of the DNA fragment of approximately 3 kb was not detected in any chromosomal DNA sample thereof, while the amplification of the DNA fragment was detected. approximately 800 bp in all chromosomal DNA samples thereof. Consequently, it is considered that in these outbreaks, the ipt gene is still present in the chromosomal DNA together with the Ac. COMPARATIVE EXAMPLE 2 In the leaf culture infected with A. tumefaciens in step II-A of Example 2, the 3 were separated without redifferentiated ESPs together with the ESPs, and were subjected to the test for kanamycin resistance, the test for GUS activity, visual observation after 1 month of culture and PCR analysis in the same way as in II of Example 2. The results are shown in Table 1. The outbreaks obtained from this without-ESPs have no resistance to kanamycin, GUS activity and ability to form ESP. In addition, the amplification of the DNA fragments of approximately 800 bp and approximately 3 kb were not detected in the PCR analysis. EXAMPLE 3 In addition, the culture of the 63 separate ESP lines in Example 2 were continuous in culture medium of MS agar without hormone. Approximately two months after separation, a total of seven shoots Nos. 13-1 to 13-3 and 14-1 to 14-4 were normal shoots capable of being visually identified, ie, that exhibited the apical domain were obtained from the 2 lines of ESP. These cans were separated for transplantation into the culture medium having the aforementioned composition, then extended and formed roots normally. Of these, the individuals obtained from sprouts Nos. 13-1 and 14-1 were subjected to PCR analysis in the same manner as in step 1I-B of Example 2. As a result, amplification of a fragment was not observed. of DNA of approximately 800 bp in none of the outbreaks Nos. 13-1 nor 14-1. Meanwhile, amplification of a DNA fragment of approximately 3 kb was observed in both of these outbreaks. Therefore, it was determined that the ipt gene was excised from the chromosomal DNA of these individuals together with the Ac and disappearance. The results are shown in Figure 14, in which the values indicated on the left side are the same as those shown in Figure 6. In addition, expression of the GUS gene was detected in all individuals obtained from the seven outbreaks. Figure 15 shows the status of a normal outbreak differentiated from an ESP. EXAMPLE 4 The leaf obtained from an outbreak that is shown as outbreak No. 7 in Table 1 among the 9 roots selected in Example 2 was grown on MS growth medium without hormones for about 1 month. A normal shoot was visually selected and separated from 6 adventitious shoots that were redifferentiated from the cultivated leaf. This normal shoot was transplanted into a culture medium of the composition mentioned above, then an extended normal individual with roots formed was obtained. In addition, this individual was subjected to PCR analysis in the same manner as in step II-B of Example 2, and on the basis of the disappearance of the approximately 800 bp DNA fragment and the amplification of the approximately 3 DNA fragment. kb, it was determined that the ipt gene has been excised from the chromosomal DNA together with the Ac and disappeared. The results are shown in Figure 16, in which the values indicated on the left side are the same as those shown in Figure 6. In addition, in the same individuals, the expression of the GUS gene was also detected. EJ EM PLO 5 1. Separation of a site-specific recombination system (yeast pSR D system) The yeast (Zygosaccharomyces rouxii (obtained from I n stitute for Fermentation)) was inoculated into 5 ml of YPAD liquid culture medium (containing 10 g / liter of yeast extract, 20 g / liter of polypeptone, 0.4 g / liter of adenine and 20 g / liter of glucose), and cultured at 30 ° C for 24 hours.The culture solution was centrifuged at 6,900 xg for 3 minutes at 20 ° C to collect the cells (hereafter, the cells were harvested under the same conditions) The cells obtained were suspended in 2 ml of a solution comprising 0.2M Tris-HCl (pH 8.0). and 5% v / v of mercaptoethanol The cell suspension was allowed to stand at 25 ° C for 30 minutes while gently shaking several times and the cells were collected.In addition, the collected cells were suspended in 1 ml of a solution ( pH 6.8) containing 2.5 mg / ml of Zaimolyeis-20 T (obtained from SEIKAGAKU CORPORATION), 10% w / v of sorbitol and 5% w / v of KPO4. The suspension was allowed to stand at 30 ° C for 90 minutes and recentrifuged to collect the cells again. The collected cells were resuspended in 1 ml of a solution containing 0.2 M NaCl, 0.1 M EDTA, 5% w / v SDS, 50 mM Tris-HCl (pH 8.5) and Proteinase K were added to reach 20 mg / ml Of the same. The mixed solution was allowed to stand at 60 ° C for 1 hour and was returned to room temperature and extracted with a mixture of phenol and chloroform and then with chloroform to purify it. The supernatant thus obtained was added in an equal volume of isopropanol to precipitate chromosomal DNA and plasmid pSR1. The mixture was centrifuged at 6,900 x g for 10 minutes at 4 ° C to collect the DNA and the collected DNA was washed with 70% v / v ethanol, then dried under vacuum and dissolved in 100 μl of TE. Using the DNA thus extracted (the mixture of the chromosomal DNA and the plasmid pSR1) as a template, only one specific recombination system for a site that was present in the plasmid pSR1 (hereinafter referred to as the "pSR1 system") was amplified by the PCR method. The pSR1 system consists of a R gene which is a recombinase gene and a Rs recombination sequence and its DNA sequences have already been determined (H. Araki et al., J. Mol. Biol., Vol.182, p. 191, 1985). In the present invention, in order to amplify the R gene, an initiator in which an Xbal restriction endonuclease site was added to a 5 'position of 22 bases, namely, 5,596a - 5,617, was used for use. to bases in the sequence of the plasmid pSR1 (5'-CCTCTAGAATGCAATTGACCAAGGATACTG-3 ') and an initiator in which the Sacl restriction endonuclease site was added to the 5' position of 22 bases, namely, 4.126a - 4.147a bases in the sequences of the plasmid pSR1 (5'-CCGAGCTCTTAATCTTGTCAGGAGGTGTCA-3 '). On the other hand, in order to amplify Rs, two pairs of primers were synthesized to be used, each comprising 30 bases (a total of four types). That is, a pair was composed of an initiator in which the three bases 287a-316a of the sequence of plasmid pSR1. were replaced and a restriction endonuclease site Ssel (5'-AGGATTGAGCTATGGACGGGAATCCTGCA-3 ') and an initiator in which the four bases 690a-719a of the plasmid pSR1 sequence were replaced and the restriction endonuclease site was introduced Hindlll and the Xhol restriction endonuciease site (5'-CAACTCGAGCAATCAAAGCTTCTCGTAGTC-3 '). The Rs that will be amplified with this established initiator was called "Rs1". Another pair was constituted by an initiator in which the three bases 287a-316a of the plasmid pSR1 sequence were replaced and an Xhol restriction endonuclease site and an EcoRI restriction endonuclease site (5'-AGGATTGAGCTACTCGAGGGGAATTCTGGA-3 'were introduced. ) and an primer in which the five bases 688a to 717a of the plasmid pSR1 sequence were replaced and introduced into the Ssel restriction endonuclease site (5'-ACTGGACCAATCCCTGCAGGTCGTAGTCAA-3 '). The Rs to be amplified with this established initiator was called "RS2". In order to amplify the R gene and the Rs's, 1 μl of the extracted DNA solution was added to each 50 μl of the mixed solution used in step IV-B of Example 1 containing 0.2 μM of each established primer respectively. A heating cycle in three parts, namely at 95 ° C for 30 seconds, at 55 ° C for 30 seconds and at 72 ° C for 1.5 minutes was repeated in the mixture for a total of 30 times. The reaction mixture thus obtained was analyzed by agarose gel electrolysis to confirm the amplification of the R gene and the Rs's. II. Construction of a vector The Rs1 amplified by the PCR method was digested with restriction endonucleases Psfl and Xhol, and a plasmid pNPI126 was obtained by inserting this Rs1 into the restriction endonuclease sites Psfl-Xftol of pSL1180 (obtained from Pharmacia Biotech Co .). Subsequently, in order to remove the EcoRI restriction endonuclease site and the Hindlll restriction endonuclease site of pHSG398, digestion of these restriction endonucleases, by changing the digested endings in blunt end terminations with polymerase I T4 (large subunit) and the ligatures of the ends of blunt ends were repeated in sequence. In this form, the plasmid pNPI121 was obtained with these deleted restriction endonuclease sites. The plasmid pnPI127 was produced by digesting the amplified Rs2 by the PCR method with the restriction endonucleases X? OI or Psfl and inserting this Rs2 into the Sall-PstI restriction endonuclease sites of the plasmid pNPI121. The plasmid pNPH28 was obtained by separating Rs1 from pnPI126 with the restriction endonucleases Smal and Spel and inserting this fragment at the sites of restriction endonucleases Smal-X £ >to the? NPI127. The R gene amplified by the PCR method was digested with the restriction endonucleases Xbal and Sacl and inserted into the Xbal-Sacl restriction endonuclease sites of pHSG398. The plasmid thus obtained was designated pNPI124. Then pBI221 (obtained from Clontech Co.) was digested with the restriction endonuclease Psfl. The digested termini were changed to blunt ended ends and then ligated in the manner described above. Therefore, plasmid pNPI111 was obtained with the restriction endonuclease sites Ssel and Psfl removed. Then, the R gene separated from pNPI124 with the restriction endonucleases Xbal and Sacl were inserted into the restriction endonuclease sites Xbal-Sacl of pNP1111 replacing the GUS gene to produce the plasmid pNPM25. In addition, a 35S promoter from cauliflower mosaic virus, the R gene linked to the promoter and a polyadenylation signal from nopaline synthetase, were separated with the restriction endonucleases Hindlll and EcoR] and inserted into the restriction endonuclease sites Hindlll -EcoRl of pNPI128 to obtain the plasmid pNPI129. pNPI101 was digested with the restriction endonuclease Smal and a 5'-phosphorylated Hind linker (obtained from Takara Shuzo Co., Ltd.) was inserted into the digestion site to obtain the plasmid pNPI122. That is, pNPI122 is one in which the Smal restriction endonuclease site of pNPI101 was replaced with the HindIII restriction endonuclease site. In addition, pNPI122 was digested with the restriction endonuclease Psfl and the digested terminations were changed to blunt ended ends and ligated to produce the plasmid pNPH23 with the restriction endonuclease sites Ssel and Psfl removed. From this plasmid pNPI123, a 35S promoter from cauliflower mosaic virus and ipt gene were ligated to the promoter where they were cut with the restriction endonuclease HindIII and inserted into the HindIII restriction endonuclease site of pNPI129 to obtain the plasmid pNPI130. The desired vector was obtained by separating the fragment containing the ipt gene and the R gene, the 35S promoters of the cauliflower mosaic virus bound to them respectively and the Rs' present at both terminals of these genes with the restriction endonuclease Psfl and inserting this fragment at the Ssel restriction endonuclease site of pBI121. The plasmid thus obtained was designated pNPI132. This plasmid pNPI132 was also introduced into the E. coli strain JM109, and was deposited according to the Budapest Treaty as E. coli JM109 (pNPH32) under Deposit No. FERM BP-5065. The strategy for constructing plasmid pNPM32 is shown schematically in Figures 17-19. The restriction endonuclease map of the T-DNA region thereof is shown in Figure 20. In Figures 17 to 19 and 20, a dotted triangle indicates the recombination sequence Rs derived from the yeast pSR1 pyramid and the direction of this sequence. Other symbols are the same as those shown in Figures 2 to 5. As is evident from Figure 20, the plasmid pNPI132, like the plasmid pNPI106, has the ipt gene as the selectable marker gene and 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 recombination sequences Rs's of the pSR1 system behaves like the removable DNA element. Therefore, the ipt gene is inserted so that it is maintained by the same two directed recombination sequences. III. Introduction of pNPH32 into a tobacco and tobacco analysis in which the gene has been introduced In the same manner as in steps II and III of Example 1, the plasmid pNPI132 is introduced into the strain of A. tumefaciens LBA4404 and the disks of leaf of a tobacco (Nicotina tabacum cv. SR1) were infected with this A. tumefaciens. The infected leaves were then cultured in a culture medium of MS agar without hormone containing 50 mg / liter of acetosyringone and then in culture medium of MS agar without hormone containing 500 mg / liter of carbenicillin. One month after, the cultivated leaves were transplanted in the culture medium of the same composition and the culture was further continued for 1 month. Then, 48 lines of ESP were separated. The ESP lines were again transplanted into the culture medium in the same composition and the culture was further continued. Approximately one month later (ie, approximately 3 months after infection with A. tumefaciens), outbreaks that were visually detectable by having a normal morphology were generated from seven of the 40 ESP lines. These shoots were separated and transplanted into the culture medium of the same composition and then 10 normal individuals could be obtained. These individuals were subjected to the PCT analysis in the same manner as in step II-B of Example 2, as long as a pair of primers are used for detection of the GUS gene in addition to the primers used in step II-B of the Example 2. Conducting PCR using these primers, a DNA fragment of approximately 800 bp was amplified when the ipt gene was present; a fragment of DNA of approximately 3 kb was amplified when the ipt gene was excised from the T-DNA region of plasmid pNPH32 by removing the portion maintained by Rs (these results are the same as in the analysis of step TT) B in Example 2); and a DNA fragment of approximately 1.7 kb was amplified when the GUS gene was present. The results are shown in Figures 21-23 and Table 2. In Figures 21-23, the values indicated on the left side are the same as those shown in Figure 6. TABLE 2 Results of the analysis of a transgenic gene in a tobacco in which the gene was introduced with the pNPI132 vector Line ESP Plant re ipt re GUS Individual No. 800 bp 3 kb 1.7 kb No. 15 1 2 16 1 17 1 18 1 19 1 2 20 1 2 21 1 Notes: + indicates that the corresponding DNA fragment is amplified and - indicates that it is not amplified. As is evident from Table 2, the presence of the pt gene that was the selectable marker gene was not detected in any of the chromosomes of the individuals that were selected simply by visual observation of their morphology and instead detected the amplification of the DNA fragment that indicated the removal of the ipt gene in all individuals. On the other hand, resistance to kanamycin was examined using the terminal buttons of individuals that were obtained from sin-ESPs differentiating almost simultaneously with the 48 ESP lines and showed normal elongation and root formation with the use of an MS agar medium without hormone containing 200 mg / l of kanamycin. As a result, it was found that two out of 16 individuals have resistance to kanamycin. Subsequently, these kanamycin-resistant individuals were further examined by subjecting them to PCR analysis together with three individuals selected from 14 kanamycin sensitive individuals in the same manner as was used for individuals obtained from ESPs. Figure 24 shows the results where the values indicated on the left side are the same as those shown in Figure 6. As shown clearly in Figure 24, each of the two individuals resistant to kanamycin exhibited the amplification of a DNA fragment. , which indicates the extirpation of a region that contains the ipt gene and was maintained by Rs and the presence of the GUS gene. Therefore, it was proved that the genes originated in pNP ± 132 has been integrated into these chromosomes. In contrast, no amplification was observed in the three individuals sensitive to kanamycin. In addition, none of these individuals (ie, individuals resistant to kanamycin and individuals sensitive to kanamycin) showed amplification of a DNA fragment indicating the presence of the ipt gene. It was assumed that these kanamycin-resistant individuals obtained from a strain lacking the ability to form ESPs as well as the kanamycin-sensitive individuals targeted on cells in the chromosomes of which pNPI132 has not been introduced into the infection with A tumefaciens Based on this assumption, it is possible that the genes that originate in this vector are contained in the chromosomes. Furthermore, it is unreasonable for individuals who lacked the ipt gene but were contained in the NPTII gene (as indicated by the fact that these individuals were resistant to kanamycin) and the GUS gene each in the full form in the chromosomes, they appeared in this frequency considering all these genes originated in the same vector. Namely, it is reasonable to conclude that, in these individuals resistant to kanamycin, pNPI132 was once introduced into the chromosomes. That is, it is calculated that the T-DNA region of pNPI132 was once introduced into the chromosome due to infection with A. tumefaciens, but the excessively efficient function of the pSR1 system used for this vector induced the excision of the ipt gene before of the formation of ESPs following infection with A. tumefaciens. As a result, the NPT1I gene and the GUS gene remained exclusively on the chromosome. The fact that the kanamycin-resistant individuals shown in the PCR analysis, the removal of the region containing the ipt gene and maintained by Rs's, also support this calculation. EXAMPLE 6 I. Construction of a vector Role genes (S. Kiyokawa, Plant Physiol., Vol.104, p.801, 1994) containing ro1A, ro1B and ro1C and having a total size of 7.6 kb, whose genes have been inserted at the EcoRI restriction endonuclease site of pBluescriptII SK + (made by Toyobo Co., Ltd.), an EcoR1 restriction endonuclease was removed. This fragment was inserted into the EcoRI restriction endonuclease site of pNPI129 to produce plasmid pNPI700. Of this plasmid pNPI700, the role genes, the 35S promoter of the cauliflower mosaic virus, the R gene linked to the promoter and the Rs' present in both terminals of these genes, were separated with the restriction endonuclease Ssel and inserted in the endonuclease site. Ssel restriction of pB1121 to obtain the desired plasmid pNPI702. Plasmid pNP! 702 was then introduced into the E. coli strain JM109 and was deposited in accordance with the Budapest Treaty as E. coli JM109 (NP1702) under the Deposit No.. FERM BP-5066. The strategy for constructing the plasmid pNPI702 is shown schematically in Figure 25. The restriction endonuclease map of the T-DNA region thereof is shown in the figure 26. The symbols in Figures 25 and 26 are the same as those in Figures 2 to 5. As is evident from Figure 26, the plasmid pNPl702 is similar to pNPH32, but only the selectable marker gene was changed from the ipt gene to the role genes. Role genes used for this vector are present in the T-DNA of A. rhizogenes in nature. It is known that when role genes are introduced into plant cells, hairy roots are generally found in the tissue of the plant and that the regenerated plant of these hair roots shows morphological abnormality such as dwarfism or the like. II. Introduction of pNPI702 into a tobacco and tobacco analysis in which the gene was introduced In the same manner as in steps II and III of Example 1, the plasmid pNPI702 was introduced into the strain of A. tumefaciens LBA4404, and disks of tobacco leaf were infected with this A. tumefaciens. The leaf disc of tobacco thus infected was cultured in culture medium of MS agar without hormone containing 50 mg / liter of acetosyringone in a dark place for 3 days and then in culture medium of MS agar without hormone to which 400 mg was added. / liter of ticarcillin. Approximately 15 days after starting the culture, differentiation of the hair roots was observed. The hair roots were separated one after the other, placed on an induction culture medium (MS agar culture medium containing 0.1 mg / liter of a-naphthaleneacetic acid, 2.0 mg / liter of benzyladenine and 400 mg / liter of ticarcillin). Then, among the redifferentiated shoots, 18 shoots were selected which were considered to have normal morphology, and were subjected to PCR analysis in the same manner as in step II-B of Example 2, using the primers for detection in order of removing the region containing the role genes and maintained by Rs' by amplifying a DNA fragment of approximately 3 kb (same as the primers used in Examples 2 to 5 and Comparative Example 2) and primers for detecting the presence of the role genes by amplifying a DNA fragment of approximately 1.1 kb. As a result, it was confirmed that the region containing the RP genes and maintained by Rs was excised from the chromosomes of the 9 outbreaks. EXAMPLE 7 Using the pNPI106 vector constructed in Example 2 above, a hybrid poplar (Populus sieboldii x Populus grandidentata, a woody plant) was subjected to gene introduction. The stem of strain Y63 of the hybrid poplar (grown in the experimental forest of Akita Jujo Chemicals Co., Ltd.) developed in a sterilized flask was cut into a section without nodes of 5 mm in length. It was then cut vertically into two pieces to be used as a sample and the sample was infected with the strain LBA4404 of A. tumefaciens introduced in pNPI106 in the same manner as in the step of Example 1. After infection, the section of the stem placed in a culture medium of modified MS agar without hormone (containing 2% w / v of sucrose and? 8% w / v of agar) to which 40 mg / liter of acetosyringone was added and in the same was cultivated during 3 days. Subsequently, it was transplanted into the same medium but containing 500 mg of carbenicillin instead of acetosyringone and further cultured therein. The modified MS culture medium employed herein, is one prepared by combining the ammonium-nitrogen and nitrate-nitrogen concentrations of the usual MS culture medium respectively at 10 mM and 30 mM respectively. After approximately two months, the adventitious shoots developing in this section were separated and further cultured for 2 months. In this way, 6 lines of ESP were obtained. These lines were further subcultured and approximately 4 months later, (that is to say, approximately 8 months after the infection with A. tumefaciens), morphologically normal shoots were observed growing from the ESPs from the first moment. These shoots were then transplanted in a gellan gum medium (containing 2% w / v of sucrose and 0.3% w / v of gellan gum) to which 0.05 mg / liter of IBA (indole butyric acid) was added and they cultivated in it. Therefore, several roots in development of normal individuals were obtained in total of 2 EPS lines within ten months after the invention with A. tumefaciens. These individuals were then subjected to PCR analysis in the same manner as in step III of Example 5. As a result, the ipt gene was not detected from any of them. On the other hand, the presence of the GUS gene was detected in two individuals among them. Therefore, it was confirmed that the vector of the present invention would also work efficiently in a woody plant. Incidentally, in the remaining 5 normal individuals, only part of the GUS gene was detected. It appears that, in said individual, when the Ac transposon introduced by the pNPI106 vector was excised from the chromosome together with the ipt gene, the GUS gene located in the vicinity thereof was involved in the excision and thus broke. Figure 27 shows the state of a normal outbreak differentiated from an ESP. EXAMPLE 8 The normal individual (obtained via ESP) having the genes introduced therein by pNPI132 in Example 5, was further subjected to the introduction of the gene with the use of the vector of the present invention. The GUS gene of pNPI132 was replaced by an HPT gene (hygromycin resistance gene). Using the vector thus obtained (pNPI140, Figure 28), the normal individuals mentioned above were subjected to the introduction of the gene in the same manner as in steps II and III of Example 1. Therefore, 10 lines of ESP 40 were obtained. days after infection with >;TO. tumefaciens having this vector introduced in it. These ESP lines were separated, transplanted in a culture medium of the same composition (culture medium of MS agar without hormone containing 500 mg / liter of carbenicillin) and further cultured therein. As a result, differentiation of shoots, with visually normal morphology, was observed in one of these lines 20 days later (namely, approximately 2 months after infection with A. tumefaciens). One of these normal sprouts, thus differentiated, was subjected to PCR analysis in the same manner as in the steps of IV-B of Example 1. Figure 29 shows the result. It should be noted that a pair of primers designed to bind in the NPTII gene and the HPT gene respectively, to detect the excision of the region containing the ipt gene and maintained by Rs's of the chromosomal DNA and another pair of primers to detect the presence of the HPT gene (detected through the amplification of DNA fragments of about 4 kb and about 1 kb, respectively) were employed herein in addition to the primers used in step IV-B of Example 1. In Figure 29, the values indicated on the left side are the same as those shown in Figure 6. As shown clearly in Figure 29, in the PCR analysis, the chromosomal DNA extracted from the shoot showed the amplification of the DNA fragment of approximately 4 kb, which indicated that the ipt gene has been excised by excising the region maintained by Rs's and amplifying the DNA fragment by approximately 1 kb, which indicated the presence of the HPT gene. On the other hand, the amplification of the approximately 800 bp DNA fragment, which indicated the presence of the ipt gene, was not detected in the same DNA. These results suggest that the ipt gene, which has been introduced once into the chromosomal DNA of this outbreak, was excised from it by removing the region maintained by Rs and thus disappearing, while the HPT gene still remained. in the DNA. That is, an individual introduced with the desired genes (ie, the NPTII gene and the GUS gene) by pNPI132 was also introduced with a desired novel gene (ie, the HPT gene) using a vector, wherein the construct that it refers to the desired gene altered exclusively (ie, the same ipt gene used as the selectable marker gene) repeating culture, visual selection and usually separation. In addition, the results showed that it is quite possible that the third, fourth or more desired genes could be introduced with the use of the same selectable marker gene. As is evident from the Examples described above, the ESP lines obtained always from ESP had the ipt gene within their chromosomes as shown in Figure 6. In addition, the ESPs, which showed remarkable morphological abnormality that could be identified visually, it exhibits resistance to kanamycin, without exception by the expression of the NPTII gene which was the desired gene as a model and was introduced together with the ipt gene. This provides that said lAM gene is completely available as a selectable marker gene for introducing a gene into a plant and that the vector of the present invention containing this lAM gene as the selectable marker gene is also available as a vector for introducing a gene. gene in a plant. When a gene was introduced into a plant using the pNPII06 vector in which the ipt gene was integrated into the transposon Ac, an outbreak or the like that loses its ability to form ESP as a result of the disappearance of the ipt gene from the chromosome , while retaining characteristics provided by the desired gene (NPTII gene and / or GUS gene), was obtained from the tissue that once formed the ESP in the initial stage of the culture just after the gene introduction operation, as shown in Table 1 and Figures 13, 14 and 16. In addition, the morphology of this tissue obtained (ie, the outbreak or similar ones that lose their ability to form esp) could be visually identified as shown in Figures 15 and 27. In addition, when this was selected, separated and cultivated, an extended and rooted individual was obtained having a normal morphology. In addition, the tissues that were redifferentiated from the tissue obtained from the outbreak that loses its ability to form ESP also showed normal morphologies without having the capacity to form ESP. This proves that said outbreak or the like consisted of uniform cells. The same results were also observed when one derived from the site-specific recombination system was used as the removable DNA element and when the role genes were used as the lAM element. That is, when a gene was introduced into a plant using a vector, in which the construct that refers to the transposon or the transposon and the ipt gene of the vectors used in Examples 1 to 4 and 7, were replaced with the which refers to the recombination system mentioned above and / or role genes as described in Examples 5 and 6, the morphologically normal tissue and plant in which disappearance of the IAM gene of this chromosome, while maintaining the desired gene, is obtained from the tissue that showed the abnormal morphology immediately after the introduction of the gene (Figures 21-23, Table 2). In addition, it is also possible to introduce the desired genes in the same individual, repeating the steps of gene introduction, culture and visual selection using the vector in which the construction that refers to the desired gene is exclusively altered while the same gene is used. induces the morphological abnormality as the selectable marker gene (Example 8, Figure 29). Accordingly, when said vector is used, in which the lAM gene is used as the selectable marker gene and inserted into the position such that it behaves integrally with the removable DNA element, a tissue made only of cells in which the The desired gene only remains in the chromosome or similar and maintains its function, it is obtained only by carrying out the following steps: (1) culturing the cells just after the gene introduction operation and visually selecting a morphologically abnormal tissue that appears during the culture, (2) further culturing the morphologically abnormal tissue and visually selecting a morphologically normal tissue that appears during culture. In addition, a plant formed only of said cells can also be obtained from said morphologically normal tissue. In addition, when one derived from the site-specific recombination system was used as the removable DNA element, since excision of it occurred rapidly and at a very high frequency, the morphologically normal tissue appearing from morphologically normal tissue could also be detected rapidly and many normal individuals were obtained with good efficiency. Table 3 shows the efficiency in which the normal individual was obtained from morphologically abnormal tissue when the transposon or a site-specific recombination system derivative was used as the removable DNA element in the vector of the present invention. TABLE 3 Difference in efficiency to obtain normal individuals depending on a type of a removable DNA element.

Vector Removable DNA Number Number Element lines of ESP lines in which normal individuals are regenerated Example 3 pNPH06 Ac 63 2 (7 (transposon) individuals) Example 5 pNPI132 region 48 7 (10 maintained by Rs's of the system individuals) pSR1 (site-specific recombination system) Notes: 1. ESP separated after two months of culture. 2. The normal shoot can be detected after four months of culture in Example 3 and after three months of culture in Example 5. 3. Each of the normal individuals mentioned above contained a GUS gene as a model of the desired gene . In Examples 1 to 5, under the conditions without hormone, the tissue containing the transgenic cells proliferated, differentiated the adventitious bud and regenerated the plant. This is presumably attributed to the action of the ipt gene that was introduced into the chromosome in the transgenic cell as the selectable marker gene. That is, by the expression of this gene, the hormone of the plant was overproduced inside the cell. Consequently, the plant hormone produced in the cell containing the ipt gene not only influenced the cell itself to differentiate tissue such as ESP or the like, but also the tissue adjacent to the cell to some degree, so that the same state was created as that given by the artificial addition of the hormone of the plant to the culture medium. In the vector of the present invention, the lAM gene was used as the selectable marker gene. Therefore, when the introduction of the gene was carried out in the plant using this vector, the tissue in which the desired gene was introduced can be selected by culturing the cell subjected to the treatment for the introduction of the gene, in a medium of common culture under common culture conditions without adding any chemical substance for selection and visually identifying the morphologically abnormal resultant tissue. Consequently, the procedure was simplified and the activity of the plant cell did not decrease during the selection. In addition, said lAM gene is inherent in the plant or introduced into the plant by infection with bacteria or the like in nature. For this reason, even if the IAM gene was expressed within the cell of the plant into which the gene was introduced, its safety is considerably high when it was ingested in the human body. Furthermore, when the ipt gene was used as the lAM gene, the tissue containing the transgenic cell proliferated and differentiated the adventitious outbreak by the action of this gene, making it possible to eliminate the need for the addition of plant hormones to the culture medium that was generally considered indispensable for the proliferation and differentiation in the cultivation of the cell of the plant. In addition, in this vector, the lAM gene was used as the selectable marker gene was inserted into the position so that it behaves integrally with the removable DNA element, whereby the selectable marker gene was removed from the DNA where This gene exists and works through the excision of the DNA element in a given proportion after the gene was introduced into the cell of the plant, and it loses its own function. Therefore, only the desired gene present in a position of the vector so that it does not behave integrally with the removable DNA element, remains in the same DNA and maintains the ability to express its function. Consequently, in this structure, the vector causes multiple introduction that refers to the gene in a certain plant merely by changing the portion of the desired gene to be introduced without any change of structures of the selectable marker gene and the others. Therefore, the multiple introduction can be carried out a number or a limited number of times. Furthermore, in this case, the loss of the function of the lAM gene as the selectable marker gene can be detected visually through the morphological change of the transgenic tissue as in the introduction of this gene. Thus, the tissue formed only of cells in which the desired gene only remains in the chromosome or the like and maintains its function, can be selected safely and easily. As a result, the multiple introduction of the gene can be carried out with high efficiency, and the transgenic plant formed only of said cells, namely, the individual free from the influence of the selectable marker gene and completely free of any health risk presented by the product of the selectable marker gene can be obtained without having to undergo the crossing step . *** Notes *** Regarding INDICATIONS REFERRING TO A DEPOSITED MICROORGANISM listed in the specification as follows: 1) FERM BP-5063 on page 31, lines 22 2) FERM BP-5064 on page 40, line 4 3) FERM BP-5065 on page 53, line 16-17 4) FERM BP-5066 on page 59, line 25-26 The four identified above with the Access Numbers were simultaneously deposited with the next depository institution.

Name of the depository institution: National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Osamu Suzuki, Dr., DIRECTOR GENERAL) Address of the depository institution: 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki ken 305 JAPAN Deposit date: April 5, 1995.

Claims (32)

1. CLAIMS 1. A vector for introducing a desired gene into a plant, which comprises said desired gene, and at least one morphological abnormality induction gene as a selectable marker, wherein said morphological abnormality induction gene is a cytokinin synthesis.
2. 2. The vector of claim 1, wherein said cytokinin synthesis gene is an ipt, isopentenyltransferase gene, which is present in the T-DNA of Agrobacterium tumefaciens.
3. 3. A vector for introducing a desired gene into a plant cell together with a selectable marker gene, wherein said selectable marker gene expresses its function once in the plant cell, then is removed from the DNA where it exists in accordance with a behavior of a removable DNA element and the function of said selectable marker gene disappears and the expression of said selectable marker gene and the disappearance of the function thereof are detected by morphological change of the tissue derived from the cell of the plant in which said selectable marker gene is introduced.
4. 4. A vector for introducing a desired gene into a plant, comprising said desired gene, at least one morphological abnormality inducing gene such as a selectable marker gene and a removable DNA element, wherein said gene induction morphological abnormality it is positioned so that it is removed together with the removable DNA element and where said desired gene is placed so that it is not removed together with the removable DNA element.
5. The vector of claim 4, wherein said morphological abnormality induction gene is present within said removable DNA element.
6. 6. The vector of claim 4, wherein said removable DNA element is a transposon.
7. The vector of claim 4, wherein said removable DNA element is derived from a site-specific recombination system.
8. The vector of claim 4, wherein said morphological abnormality induction gene is obtained from a microorganism of the genus Agrobacterium.
9. 9. The vector of claim 4, wherein said morphological abnormality induction gene is a cytokinin synthesis gene.
10. 10. The vector of claim 9, wherein said cytokinin synthesis gene is an ipt, isopentenyltransferase gene, which is present in the T-DNA of Agrobacterium tumefaciens.
11. 11. The vector of claim 4, wherein said morphological abnormality induction gene is at least one gene selected from role genes.
12. 12. The vector of claim 11, wherein said role genes are role genes containing ro1A, ro1B and ro1C, which are present in the T-DNA of Agrobacterium rhizogenes.
13. 13. A method for producing a transgenic plant free of the influence of a selectable marker gene, which comprises the following steps: (A) introducing a vector into a plant cell, wherein said vector comprises a desired gene, at least a gene for induction of morphological abnormality as a selectable marker gene, and a removable DNA element, wherein said morphological abnormality induction gene is positioned so that it is removed together with the removable DNA element and wherein said desired gene is placed so that it is not removed along with the removable DNA element, (B) cultivate the cell of the plant obtained in A (), detecting a morphologically abnormal plant tissue which appears during the culture and selecting said morphologically abnormal tissue, and (C) culturing said morphologically abnormal tissue selected in (B), detecting a morphologically normal tissue that appears during cultivation and selecting said morphologically normal tissue.
14. The method of claim 13, wherein said morphological abnormality induction gene is present within said removable DNA element.
15. 15. The method of claim 13, wherein said removable DNA element is a transposon.
16. The method of claim 13, wherein said removable DNA element is derived from a site-specific recombination system.
17. The method of claim 13, wherein said morphological abnormality induction gene is obtained from a microorganism of the genus Agrobacterium.
18. 18. The method of claim 13, wherein said morphological abnormality induction gene is a cytokinin synthesis gene.
19. 19. The method of claim 18, wherein said cytokinin synthesis gene is an ipt, Slyntenyltransferase gene. which is present in the T-DNA of Agrobacterium tumefaciens.
20. The method of claim 13, wherein said morphological abnormality induction gene is at least one gene selected from role genes.
21. The method of claim 20, wherein said role genes are role genes that contain ro1A, ro1B and ro7C genes. in which the T-DNA of Agrobacterium rhizogenes is present.
22. 22. A method for introducing at least two desired genes is a plant, which comprises carrying out the following steps at least twice: (A) introducing a vector into a plant cell, wherein said vector comprises a gene desired, at least one gene for induction of morphological abnormality as a selectable marker gene, and a removable DNA element, wherein said morphological abnormality induction gene is positioned so that it is removed together with the removable DNA element and in wherein said desired gene is placed so that it is not removed together with the removable DNA element, (B) cultivating the cell of the plant obtained in A (), detecting a morphologically abnormal plant tissue which appears during cultivation and selecting said morphologically abnormal tissue, and (C) culturing said morphologically abnormal tissue selected in (B), detecting a morphologically normal tissue that appears during cultivation and selecting d icho morphologically normal tissue.
23. 23. The method of claim 22, wherein said morphological abnormality induction gene is present within said removable DNA element.
24. The method of claim 22, wherein said removable DNA element is a transposon.
25. 25. The method of claim 22, wherein said removable DNA element is derived from a site-specific recombination system.
26. 26. The method of claim 22, wherein said induction gene of morphological abnormality is obtained from a microorganism of the genus Agrobacterium.
27. 27. The method of claim 22, wherein said morphological abnormality induction gene is a cytokinin synthesis gene.
28. The method of claim 27, wherein said cytokinin synthesis gene is an ipt, isopentenyltransferase gene, which is present in the T-DNA of Agrobacterium tumefaciens.
29. 29. The method of claim 22, wherein said morphological abnormality inducing gene is at least one gene selected from ro l genes.
30. 30. The method of claim 29, wherein said ro genes are ro genes that contain ro 1A, ro 1 B and ro 1 C genes, in which the T-DNA of Agrobacterium rhizogenes is present.
31. 31 A transgenic plant free from the influence of a selectable marker gene, produced by a method, which comprises the following steps: (A) introducing a vector into a plant cell, wherein said vector comprises a desired gene, at least one morphological abnormality inducing gene as a selectable marker gene, and a removable DNA element, wherein said morphological abnormality inducing gene is positioned so that it is removed together with the removable DNA element and wherein said desired gene is placed so that it is not removed together with the removable DNA element, (B) cultivate the cell of the plant obtained in A (), detecting a morphologically abnormal plant tissue which appears during the culture and selecting said morphologically abnormal tissue, ( C) culturing said morphologically abnormal tissue selected in (B), detecting a morphologically normal tissue that appears during cultivation and selecting said tissue morphologically normal; and (D) cultivating said morphologically normal tissue to regenerate a plant.
32. 32. A plant that contains two or more desired genes produced by a method which comprises the following steps: (A) introducing a vector into a plant cell, wherein said vector comprises a desired gene, at least one induction gene of morphological abnormality as a selectable marker gene, and a removable DNA element, wherein said morphological abnormality inducing gene is positioned so that it is removed together with the removable DNA element and wherein said desired gene is placed in such a manner that it is not removed together with the removable DNA element, (B) cultivate the cell of the plant obtained in (A), detecting a morphologically abnormal plant tissue which appears during the culture and selecting said morphologically abnormal tissue, (C) cultivate said morphologically abnormal tissue selected in (B), detecting a morphologically normal tissue that appears during the culture and selecting said morphologically normal tissue wrong; (D) carrying out steps (A) to (C) at least once; and (E) cultivating said morphologically normal tissue to regenerate a plant. RESU MEN A vector for introducing a desired gene into a plant, which comprises the desired gene and, at least, a morphological abnormality inducing gene (IAM) as a marker gene, or comprising the desired gene, at least a lAM gene and a removable element. A method to produce a transgenic plant free of the influence of a marker gene. A method for introducing many desired genes into a plant.