JP5186683B2 - Method for producing transformed plant body and use thereof - Google Patents

Description

  The present invention relates to a technique for producing a transformed plant, and more particularly to a technique for producing a transformant useful for exploring the gene function of a plant or producing a useful individual, and use thereof.

  Conventionally, mating and grafting have been used as breeding methods for producing useful plants. In recent years, molecular breeding using genetic engineering techniques such as introduction of a foreign gene that expresses a useful trait has been performed. Further, in the search for gene functions in plants, genetic engineering techniques such as suppressing or enhancing the expression of specific endogenous genes are useful.

  In plants, multiple genes are often involved in the expression or deletion of specific traits. For this reason, modification of a plurality of genes related to the traits is important for expression of useful traits and improvement of traits. In addition, it is important to modify the expression of a plurality of genes in gene function and expression analysis.

  Conventionally, various methods are used to introduce or suppress a plurality of genes in a plant body. For example, there is a method of obtaining a transformed plant in which a single gene is modified, and crossing such a transformant with a transformant in which another gene is modified. In addition, a method using the Agrobacterium method has been tried as a transformation method often used for plants. For example, a method is also disclosed in which expression suppression gene cassettes, overexpression gene cassettes, and the like are ligated onto one binary vector in any combination, and a plurality of genes are modified by one transformation using this binary vector. (Patent Document 1).

In addition, regarding the Agrobacterium method, it has also been disclosed that two or more foreign genes were introduced in tandem when Agrobacterium carrying a single foreign gene on T-DNA was infected with Arabidopsis thaliana. (Non-Patent Documents 1 and 2).
International Publication No. WO2004 / 056993 Marks et al., The Plant Cell., Vol. 1,104-1050 Feldman e tal., Mol. Gen. Genet. 208, 1-9

  However, the method of sequentially modifying genes through mating requires a period of more than half a year even if only two genes are modified. Further, in order to determine a combination of genes to be introduced into a plant body, it has been necessary to try to obtain plant tissues or plant individuals for all possible combinations. Therefore, to obtain a target gene combination having the expected trait. Numerous combinations of gene inserted tissues or individuals had to be created and selected.

Moreover, as described in Patent Document 1, a plurality of genes may be introduced by linking target genes. However, even in this case, an expression construct must be constructed for each combination of a plurality of genes to be combined. Therefore, an enormous amount of experimentation is required to comprehensively search for a preferable combination of genes or obtain an optimal combination of genes.

  As described above, when introducing a plurality of genes into a plant body, the introduction method itself is still inefficient, and a method for efficiently determining a plurality of genes to be introduced has not been studied. .

  Furthermore, although the Agrobacterium method disclosed that multiple single genes were introduced into plants, the mechanism of integration of foreign genes into host chromosomes by Agrobacterium has become completely clear. I don't mean. Therefore, it was not known at all what caused the aneuploidy of the number of introduced foreign genes. In other words, whether the result of introducing a single foreign gene into a single plant is the result of infection with one type of Agrobacterium or the result of infection with multiple Agrobacterium. Was not known at all.

  Accordingly, an object of the present invention is to provide a method for producing a transformed plant that can efficiently introduce and transform a plurality of genes into a plant, and a method for introducing a gene into a plant suitable for the production method. And In addition, the present invention provides a screening method useful for a gene function analysis method, and also provides a transformed plant library into which a foreign gene is introduced in various combinations of a plurality of foreign genes, and a method for producing the same. Is another purpose. Furthermore, another object of the present invention is to provide a composition for gene transfer into a plant body.

  In the genetic modification of a plant body via Agrobacterium, the present inventors infect the plant body with two or more types of Agrobacterium each having a foreign gene for genetic modification on T-DNA, The present inventors have obtained the knowledge that a modification exogenous gene possessed by each of these Agrobacterium is introduced into a plant body, and as a result, a transformant newly retaining a plurality of genes can be efficiently obtained. In addition, the present inventors have found that using a plurality of types of Agrobacterium each holding a plurality of types of foreign genes, these plurality of types of genes are introduced into the plant body in a possible combination. The present inventors have obtained the knowledge that it is possible to easily create a transformed plant body set having a foreign gene in various combinations. The present inventors have completed the present invention based on these findings. According to the present invention, the following means are provided.

  According to the present invention, there is provided a method for producing a transformed plant, the step of introducing two or more kinds of constructs each holding a different foreign gene into a host plant chromosome so as to be incorporated into the host plant chromosome, and the introduction step. And a step of regenerating a plant individual from the plant body that has passed through. In this method, the introduction step can be a step of infecting the plant with two or more types of Agrobacterium each holding the construct.

Furthermore, the infection step in the production method can be a step of supplying the plant body with a mixture of the two or more types of Agrobacterium. In addition, the foreign gene can include a polynucleotide region that suppresses the expression of the endogenous gene in the plant body.

In addition, according to the present invention, there is provided a method for introducing a gene into a plant, the method comprising introducing two or more types of constructs each holding a different foreign gene so as to be capable of being incorporated into a host plant chromosome, into the plant. Is provided.

  According to the present invention, there is provided a method for screening a gene in a plant, the step of infecting a plant with two or more types of Agrobacterium each having a different foreign gene incorporated therein into a host plant chromosome, and the infection step The method comprises: regenerating a plant individual from the plant body that has undergone the process; and selecting or classifying the transformed plant body using the phenotype of the regenerated plant individual or its progeny as an index. This screening method can further comprise a step of determining the foreign gene incorporated in the selected or classified chromosome of the plant body. Furthermore, in the present screening method, the foreign gene can include a polynucleotide region that suppresses the expression of the endogenous gene of the plant body.

  According to the present invention, there is provided a transformed plant library comprising a transformed plant body into which the foreign gene has been introduced in a combination of selecting two or more types from two or more types of foreign genes. The library can include a transformed plant into which one kind of the foreign gene has been introduced.

  According to the present invention, there is provided a method for producing a transformed plant library, the step of introducing two or more constructs each holding different foreign genes so as to be capable of being incorporated into the host plant chromosome, and the introduction step. There are provided a step of regenerating a plant individual from the plant body having undergone the above and a preparation method comprising the same.

  According to the present invention, there is provided a composition for introducing a foreign gene into a plant, comprising two or more types of Agrobacterium each having a different foreign gene. In addition, the foreign gene can include a polynucleotide region that suppresses the expression of the endogenous gene in the plant body.

  The method for producing a transformed plant of the present invention comprises a step of introducing two or more kinds of constructs each holding a different foreign gene into a host plant chromosome so as to be able to be incorporated into the plant, and a plant from the plant after the introduction step. And regenerating the individual. By introducing two or more types of constructs having different foreign genes into one plant body, a plant individual in which two or more types of these foreign genes are introduced into one plant body can be obtained. By introducing n types of constructs (n is an integer of 2 or more) into a plant and transforming it, it is possible to regenerate a plant into which one or more n types of foreign genes have been introduced. In addition, the possible combinations of foreign genes introduced into the plant body are all combinations that select one or more of n genes. Therefore, according to this production method, n types of foreign species can be obtained by infecting plants with n types of Agrobacterium each holding n types of foreign genes to obtain transformed plants and transformed plant individuals. A recombinant set (library) into which a foreign gene has been introduced can be obtained by a combination of selecting two or more genes. That is, it is possible to obtain transformed plants and individual plants by introducing genes in various combinations selected from n types of foreign genes at once without passing through crossing. It is possible to efficiently obtain transformants and plant individuals.

  Furthermore, according to this production method, transformed plants into which genes have been introduced in various combinations can be obtained. For this reason, by classifying or selecting plants using the phenotype of these transformant plants or their progeny as indicators, and determining the introduction form of the foreign gene for the classified plants and plant individuals, You can easily associate phenotypes. That is, it is only necessary to analyze the introduced form and expression of a foreign gene only for a transformed plant expressing the intended phenotype. Therefore, the time and labor required for gene function search and gene screening for gene modification can be significantly reduced. This introduction method is particularly useful when two or more types of genes are involved and the trait changes. Moreover, the intended useful transformed plant body can also be produced easily.

  The method for introducing a foreign gene of the present invention can comprise a step of introducing two or more kinds of constructs each holding different foreign genes so that they can be incorporated into the host plant chromosome, into the plant body. In particular, it is preferable to infect the plant body with Agrobacterium each holding such a construct. By infecting one plant body with these two or more types of Agrobacterium, two or more types selected from these foreign genes can be easily introduced into one plant body. Conventionally, many attempts have been made to transform plants by infection with Agrobacterium, but infecting two or more types of Agrobacterium and transgenes held by each Agrobacterium to infect plants. It was not attempted. In addition, the mechanism by which Agrobacterium is infected has not been clarified. When two or more types of Agrobacterium are infected, it is predicted whether the genes held by each Agrobacterium will be introduced into the host DNA. It was also difficult to do. The introduction method of the present invention is a gene introduction method suitable for the method for producing a transformed plant of the present invention.

  FIG. 1 shows an example in which the production method of the present invention is applied to a function search of an in-family gene by producing a transformed plant knocked down with respect to three endogenous genes constituting the family.

  As shown in FIG. 1A, according to this production method, a family gene is infected by infecting a plant with three types of Agrobacterium each holding three types of foreign genes that knock down the three family genes, respectively. Transformed plants that are suppressed by various combinations can be obtained. And the knowledge about functional duplication of a family gene can be obtained by carrying out gene analysis and expression analysis only about the transformed plant body in which the change was recognized in the character among them. On the other hand, as shown in FIG. 1B, according to the conventional method, Agrobacterium holding a knockdown construct for knocking down individual genes is prepared, and these Agrobacterium are individually infected to the plant body. Thus, three types of transformed plants or individual plants in which each gene is knocked down are obtained. When two genes in a family have overlapping functions, there is no change in traits when one gene is knocked down. Next, the obtained three types of transformed plants are crossed in three combinations to obtain transformed plants in which two family genes are knocked down. In the conventional method, knowledge about gene duplication can be obtained for the first time by creating a transformed plant in which genes are knocked down in a stepwise manner. FIG. 1C shows the merit of the gene function analysis method to which the present invention is applied in comparison with the conventional method.

  Hereinafter, a method for producing a transformed plant, a gene screening method, a gene introduction method, a transformed plant library, a method for producing the library, and the like, which are embodiments of the present invention, will be described in detail.

(Method for producing transformed plant individual)
This production method can include a step of introducing two or more kinds of constructs each holding a different foreign gene into a host plant chromosome so as to be able to be integrated into a plant body.

  In the present invention, the term “gene” generally refers to a polynucleotide associated with the production of a polypeptide and various polynucleotides (genomic DNA, mRNA, cDNA, etc. typically associated with suppression of the production of a polypeptide, cDNA) region. This region may contain regulatory regions and intervening sequences that precede or follow the coding region. The “foreign gene” means a gene introduced from the outside into the plant body to be introduced. Therefore, "foreign gene" refers to a plant that is a target of introduction in addition to a gene that is different from the plant that is the target of introduction, a heterogeneous plant, and a plant of the same type but that is present in other varieties of plants. It may be a gene inherent in The “foreign gene” does not include genes that are not essential for plant modification, such as marker genes such as commonly used selectable marker genes and expression marker genes, or that are associated with genes for modification.

  The foreign gene can include a promoter that overexpresses the encoded polypeptide. By introducing such an overexpressed foreign gene into a plant body, expression profiling of the polypeptide encoded by the foreign gene in the plant body and other genes related to the polypeptide is performed, and the like. Can be analyzed. Examples of the overexpression include a case where an endogenous gene is expressed at a higher level than a normal expression level and a case where an endogenous gene is expressed. Promoters suitable for such overexpression are not particularly limited and promoters known in the art can be used. Examples thereof include viral promoters such as 35S promoter derived from cauliflower mosaic virus (CaMV), and plant-derived constitutive promoters such as nopaline synthase gene (NOS) and plant actin promoter.

  The foreign gene can include a region that suppresses the expression of the endogenous gene of the plant to be introduced. Suppressing the expression of an endogenous gene refers to suppressing the destruction of the gene encoding the endogenous polypeptide and the transcription and translation of the gene, as well as introducing mutations and expressing it as an incomplete protein. It includes reducing or deactivating, or expressing a substance that interacts with the protein and reduces the action of the protein. For example, as a method for suppressing the expression of a specific gene, in addition to knocking out a specific gene, it is possible to suppress the expression of mRNA using an antisense method, a cosuppression method, an RNA interference method, a ribozyme method, or the like. Introducing and retaining a nucleic acid construct. A method for suppressing the expression of a specific endogenous gene is not particularly limited, and a conventionally known method can be applied. For example, by introducing mutations such as substitution / deletion / addition (insertion) into the gene sequence to inhibit complete protein synthesis, by inhibiting transcription promoters or activating transcriptional repressors, Examples thereof include a method of inhibiting gene transcription, a method of inhibiting protein synthesis using RNA interference, and a method of overexpressing a dominant negative mutant of the gene product. Examples of the RNA interference method include methods using antisense RNA, short inhibitory RNA (siRNA), micro RNA (miRNA) and the like.

In addition, as the foreign gene, an organ or tissue-specific promoter, various environment-responsive promoters, and the like can be used. Furthermore, a cis sequence such as an enhancer, a 5 'translation leader sequence, and a terminator sequence can also be provided.

The selection marker gene that functions in a plant body for selecting a transformant is appropriately selected according to the plant species and variety. For example, a kanamycin resistance gene, a G418 resistance gene, a hygromycin resistance gene and the like are used.

Although it does not specifically limit as 2 or more types of foreign genes, It can select from the gene group estimated to be concerned with the expression or defect | deletion of a predetermined character. For example, by selecting a foreign gene that suppresses the expression of two or more endogenous genes that are related to or similar to each other, which endogenous gene combination is involved in the expression, deletion, or change of a given trait Can be detected. In addition, for example, it is possible to detect which combination of endogenous genes is involved in the expression, deletion, or change of a predetermined trait by using a foreign gene that overexpresses a gene that is related or similar to each other. .

As gene groups related to each other, for example, a gene group constituting a family, a multigene family, a gene group having an autoregulation mechanism, a gene group that is a quantitative trait locus, or any of these relationships is presumed. The gene group etc. which are made are mentioned.

In addition, the combination of foreign genes includes the production amount, production period, production site, composition, seed and fruit maturation of humans such as proteins, starches, oils and fats, improved nutritional properties of animals such as humans, pharmaceutical peptides Productivity improvement, yield, hatching, taste, heading time, petal color, herbicide resistance, nematode and other pest resistance, fungi resistance, virus resistance, bacterial resistance, environmental stress resistance, digestibility improvement And combinations of genes considered to be related to enhancement, deletion or modification of expression of various traits including useful traits such as industrial enzyme production ability and nitrogen fixing ability.

Furthermore, such combinations include, for example, a combination of a foreign gene that simultaneously suppresses two or more types of endogenous genes, a combination of a foreign gene that enhances the expression of the endogenous gene and a foreign gene that suppresses the expression of the endogenous gene, 2 It includes a combination of foreign genes that simultaneously suppress the expression of more than one endogenous gene.

  When it is desired to obtain a plant individual transformed by introducing two or more foreign genes in a possible combination with respect to n types (n is an integer of 2 or more), n holding each of the n types of foreign genes By introducing a kind of construct into a plant body, a transformed plant body into which two or more kinds of foreign genes selected from n kinds of foreign genes are introduced can be obtained. In addition, transformed plants into which a single foreign gene has been introduced can also be obtained. That is, a transformant can be obtained with a possible combination of n types of foreign genes. For example, when introducing foreign genes of A, B, and C, if three types of constructs each holding them are introduced into a plant, a transformed plant that retains only A, and a transformed plant that retains only B , A total of seven types of plants: a transformed plant holding only C, a transformed plant holding only AB, a transformed plant holding only AC, a transformed plant holding only BC, and a transformed plant holding ABC Transformants may be obtained. When n is large, it is unlikely that a transformed plant body having n or a number of foreign genes close to it can be obtained, but various combinations of foreign genes (two types, three types, etc.) can be combined. Can be obtained.

  The construct used in this introduction step can take a form according to the introduction method. Typically, it takes the form of a vector such as a DNA fragment or a plasmid, but is not particularly limited.

In this production method, it is preferable to use a construct that holds different foreign genes so that they can be introduced into the host plant chromosome. That is, each construct may hold two or more foreign genes. Preferably, a foreign gene for modification related to the function and usefulness of the plant other than the marker gene is a single foreign gene. It is preferable to provide a gene.

  Moreover, the number of foreign genes for modification to be introduced is preferably 25 or less. This is because if the number exceeds 25, it is necessary to search for 1000 or more transformants in order to search for transformants into which two foreign genes have been introduced. More preferably, the number is 10 or less.

(Plant)
There are no particular restrictions on the plant species or varieties of the plants to which foreign genes are to be introduced. In the present invention, the “plant” is not particularly limited, and for example, a plant used for food, feed, ornamental use, environment use, experiment use and resource use can be used. Further, the plant is, for example, a seed plant, which may be a gymnosperm or an angiosperm. The plant may be a monocotyledonous plant or a dicotyledonous plant. Examples of plants for food and feed include, for example, monocotyledonous plants such as wheat, barley, sorghum, millet, rye, triticale, corn, rice, oats, sugarcane, rapeseed such as rapeseed, pepper, Arabidopsis, cabbage or canola Legumes such as soybean, alfalfa, peas, kidney beans or peanuts, potatoes, tobacco, tomatoes, eggplants such as eggplant or pepper, asteraceae such as sunflower, lettuce or calendula, cucurbitaceae such as melon, pumpkin or zucchini, And dicotyledonous plants such as celery family such as carrot. Ornamental plants include roses such as roses, rhododendrons such as rhododendrons and azaleas, euphorbiaceae such as drosophila and croton, radisaceae such as citrus, solanaceae such as petunia, genus genus such as african violet, genus genus , Such as orchidaceae such as orchid, orchidaceae such as gladiolus, iris, freesia, crocus, asteraceae such as marigold, auraceae such as geranium, lily such as dracaena, mulberry such as fig. Examples of environmental and resource plants include various trees, and examples of experimental plants include Arabidopsis thaliana.

In the present invention, the “plant body” includes plant cells such as seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, plant tissues and organs in addition to individual plants.

(Introduction of foreign genes)
The method for introducing a foreign gene that can be used in this introduction step is not particularly limited. For example, protoplasts are isolated from plant cells by treatment with cell wall degrading enzymes such as cellulase and hemicellulase, and polyethylene glycol is added to a suspension of the protoplasts and an expression vector containing an expression cassette for a foreign gene to cause endocytosis A method of incorporating the expression vector into a protoplast in the process of (PEG method), a method of inserting an expression vector into a lipid membrane vesicle such as phosphatidylcholine by sonication, etc., and fusing the vesicle and protoplast in the presence of PEG (Liposome method), a method of fusing in the same process using minicells, a method of applying an electric pulse to a suspension of protoplast and expression vector to incorporate the vector in the external solution into the protoplast (electroporation method) Is mentioned. However, these methods are complicated in that they require a culture technique for redifferentiation from protoplasts to plants. As a means for gene introduction into intact cells with cell walls, a micropipette is inserted into the cell, and vector DNA in the pipette is injected into the cell by hydraulic pressure or gas pressure, and micro gold particles coated with DNA There are a direct introduction method such as a particle gun method that accelerates the explosives using explosives and gas pressure, and introduces them into cells, and a method that uses infection by Agrobacterium. Microinjection requires the skill of operation and has the disadvantage that the number of cells that can be handled is small. Therefore, considering the ease of operation, it is preferable to transform plants by the Agrobacterium method and the particle gun method. The particle gun method is further useful in that a gene can be directly introduced into the apical meristem of a plant under cultivation. Further, in the Agrobacterium method, a plant vector, for example, a genomic DNA of a gemini virus such as tomato golden mosaic virus (TGMV) is simultaneously inserted into a binary vector between border sequences, so that an arbitrary site of a plant being cultivated can be detected. By simply inoculating cells with a bacterial suspension using a syringe or the like, viral infection spreads throughout the plant body, and the target gene is also introduced into the entire plant body. These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by the person skilled in the art.

(Infecting Agrobacterium plants)
In this production method, it is preferable to introduce the construct by Agrobacterium infection. That is, the introduction step is preferably a step of infecting a plant with two or more types of Agrobacterium each holding a construct capable of integrating different transgenes into the host plant chromosome. Agrobacterium-mediated gene transfer differs in efficiency depending on plant species and cultivars, but by adopting appropriate methods for Agrobacterium types, infected tissues, infection methods, pretreatment and marker genes, etc. Efficient gene transfer is possible.

Agrobacterium is not particularly limited and can be selected and used according to the type of plant. In general, Agrobacterium tumefaciens and Agrobacteriumu
rhizogenes) and other Agrobacterium bacteria having a transferable plasmid such as a Ti plasmid or Ri plasmid having a DNA region that can be transferred to the host plant chromosome, and mutants thereof can be used. Typically, Agrobacterium tumefaciens is used.

  When an Agrobacterium infection method is used as a method for introducing a construct, such a foreign gene is retained in a construct that can be introduced into a host plant chromosome in an Agrobacterium. Specifically, the foreign gene is provided on a transfer DNA on a transferable plasmid such as a Ti plasmid or Ri plasmid possessed by an Agrobacterium. In order to introduce a foreign gene into Ti plasmid or the like, a known method such as an intermediate vector method or a binary vector method can be used. For example, in the intermediate vector method, a pBR322-based intermediate vector holding a desired gene is introduced into an Agrobacterium holding a Ti plasmid into which an arbitrary sequence such as pBR322 is inserted using a helper plasmid or the like. In Agrobacterium into which an intermediate vector has been introduced, a desired gene can be incorporated on the Ti plasmid by homologous recombination at the pBR322 portion. By attaching an appropriate selection marker to the foreign gene, Agrobacterium having the foreign gene on the Ti plasmid can be easily selected. In addition, a binary vector generally has a selection marker gene that functions in Agrobacterium, a selection marker gene that functions in a plant body on T-DNA, and a multicloning site into which a desired foreign gene can be introduced. I have. This binary vector introduced with a desired foreign gene is introduced into Agrobacterium having a Ti plasmid lacking T-DNA and cultured in an appropriate selective medium to obtain Agrobacterium having the desired foreign gene. Can do. As such binary vectors, for example, pBI101 and pBI121 (both CLONTECH) are commercially available.

  Plants that infect Agrobacterium are whole plants; organs such as leaves, stems, sleeping, flower vases, growth and growth points, seeds, germs; tissues such as epidermis, phloem, soft tissue, xylem, vascular bundles; Any of plant culture tissues such as callus may be used. In addition, if necessary, pretreatment such as scratching or culturing under a predetermined condition can be applied to these infection targets.

  The host plant chromosome into which a foreign gene is integrated by infection with Agrobacterium may be a chromosomal chromosome as well as a nuclear chromosome.

As a technique for infecting a plant with Agrobacterium having a foreign gene, an appropriate technique is adopted depending on the type of the plant. For example, leaf disc method (Horsch, R. & B., et al .: A simple and general method for trams-ferring
cloned genes into plants. Science, 227, 1229-1231, 1985), protoplast co-culture method (Marton, L., et al .: In vitro transformation of cultured cell from
Nicotiana tabacum by Agrobactevium tumefaciens. Nature, 277, 129-131, 1981), callus regeneration method (Plant Cell Reports, 12, 7-11,
1992), reduced pressure infiltration method (The Plant Journal, 19 (3), 249-257, 1999) and the like can be employed.

  In the case of infection with Agrobacterium, it is preferable to use two or more types of Agrobacterium that hold different foreign genes so that they can be incorporated into the chromosome of the host plant body. Two or more types of Agrobacterium may be simultaneously supplied to the subject of infection as a mixed bacterial solution in which these are mixed, or may be supplied simultaneously as separate bacterial solutions. Further, two or more types of Agrobacterium may be sequentially supplied to the infection target. From the viewpoint of operational efficiency and transformation efficiency, it is preferable to supply two or more types of Agrobacterium at the same time, and it is more preferable to supply them as a mixed bacterial solution.

  There is no particular limitation on the preparation of a mixed bacterial solution containing two or more types of Agrobacterium. Two or more types of Agrobacterium separately seeded and cultured may be mixed, or two or more types of Agrobacterium may be seeded together and cultured to prepare a mixed bacterial solution. . Preferably, the bacterial solution obtained by individually seeding and culturing is mixed as necessary to obtain a mixed bacterial solution. The concentration of Agrobacterium used for infection is not particularly limited. It can set suitably according to the infection method to be used.

  Each Agrobacterium holds a selection marker, an expression marker, etc., and may hold two or more foreign genes, but the plant modification gene is a single foreign gene. Is preferably maintained. Since one type of Agrobacterium has a single foreign gene, the number of types of Agrobacterium can be matched with the number of types of foreign genes for modification. It becomes possible to obtain a converter.

This production method can comprise a step of selecting a transformed plant body under selection conditions according to the type of selection marker gene. The selection process of the transformed plant body is appropriately selected according to the technique employed in the infection process. In the T0 generation, the selection step may be performed at the tissue or cell level, or in the T1 generation, the selection step may be performed at the stage of growing from T1 seeds.

  For example, after infecting a plant with Agrobacterium, a transformant (T0 plant) is selected by culturing in a selective medium according to the type of the selectable marker gene, if necessary, and seeds from the T0 plant. (T1 seed) can be obtained, and the T1 seed can be germinated to obtain a T1 plant. Moreover, a transformed plant body (T1 plant body) can also be obtained by sowing seeds obtained without selection and germinating them under selective conditions. Transformed plant individuals that are homozygotes for foreign genes can be obtained by self-pollinating such transformed plants (T1 plants) to obtain T2 seeds and T3 seeds.

  Moreover, in this production method, regardless of whether or not the selection process based on the selection marker gene is performed, a plant body of T1 generation or its progeny is obtained, and the plant body is selected using an intended phenotype such as a useful trait as an index. May be. By performing the selection process using the trait of the transformed plant individual as an index, a transformed plant individual having a good trait can be easily created. In this production method, transformed plant individuals by combinations of various foreign genes are obtained as a set, so that it is effective to carry out a selection process based on the phenotype. In other words, since transgenic plant individuals transformed with various combinations of exogenous genes are created and then selected according to the phenotype of the individual, conventional trial and error can be eliminated and transformed plant individuals that retain useful traits The production process can be saved. When performing such a phenotypic selection process, a selection process based on a selectable marker gene is not necessarily required.

  DNA can be extracted from the obtained transformed plant individual according to a conventional method, and the presence or absence of a foreign gene can be confirmed by Southern hybridization or the like. In addition, RNA is extracted in accordance with conventional methods, Northern hybridization using an appropriate probe, hybridization on a microarray using cDNA prepared from RNA, or real-time RT-PCR, etc. It is also possible to confirm the expression state of the foreign gene and other genes.

  In addition, the obtained transformed individuals are classified or selected using desired traits, for example, phenotypes such as yield, storage substance composition, etc. as indicators, and the presence or absence, types and genes of foreign genes in the transformed plant individuals classified or extracted The expression state may be confirmed. In addition, classification and selection using phenotype as an index can be performed using an index of any trait change including expression, enhancement, deficiency, and suppression of the trait.

  The transformed individual obtained by the production method can be provided with two or more foreign genes selected from two or more foreign genes. At maximum, it can have all foreign genes of the type used. Furthermore, the transformed individual obtained by this method can also have only a single foreign gene selected from two or more types of foreign genes. Therefore, according to this production method, it can be said that a set of transformed individuals into which a foreign gene has been introduced can be obtained with a combination selected from two or more types of foreign genes.

  Therefore, according to this production method, a transformed plant individual holding two or more kinds of genes can be obtained easily and efficiently. Compared with the conventional method which acquired the plant individual which crossed the plant individual provided with a different gene and introduce | transduced multiple types of foreign genes, labor and time are reduced significantly. Moreover, according to this production method, transformed plant individuals that retain two or more genes in various combinations can be obtained all at once. Compared to the law, labor and time are drastically reduced. Furthermore, according to this production method, since a transformed plant individual into which a single foreign gene has been introduced can also be obtained, the phenotypic comparison between a single introduced transformed plant individual and a plurality of introduced transformed plant individuals is facilitated, This is also preferable.

  In addition, according to this production method, a set of transformed plant individuals carrying two or more types of foreign genes can be obtained, so that interaction or cooperation between two or more types of foreign genes and related endogenous genes can be achieved. It can be easily grasped. In addition, when a transformant having a single foreign gene can be obtained at the same time, it is useful for exploring the functions of individual foreign genes and corresponding endogenous genes, and two or more types of foreign genes and endogenous genes. It is also a useful information source for the interaction of

  According to this production method, for such a set, a transformed plant is classified or selected using the desired trait of the plant as an index, and the presence or type of a foreign gene is confirmed only for the transformed plant that has been classified or extracted, or By confirming the expression states of the foreign gene and other genes, genes suitable for expression of useful characters and combinations thereof can be efficiently screened. At the same time, it is possible to obtain knowledge about the function and cooperative action of these genes.

(Methods for introducing foreign genes into plants)
The method of introducing a foreign gene into a plant body of the present invention can comprise a step of introducing two or more types of constructs each holding a different foreign gene so that it can be incorporated into a host plant chromosome, into the plant body. This introduction step can be a step of infecting a plant with two or more types of Agrobacterium each holding a construct capable of integrating different foreign genes into the host plant chromosome. According to this introduction method, a foreign gene can be introduced into a plant by a combination selected from two or more kinds of foreign genes. As a result, a transformed plant body (set) in which two or more kinds of foreign genes are introduced in various combinations and transformed can be easily obtained. Therefore, this introduction method is useful for searching gene functions, searching for combinations of genes that can express a desired trait by control with two or more genes, and useful traits by modifying two or more genes. Useful for the production of plants. In addition, in this introduction method, the various aspects about a foreign gene, a plant body, a construct, and the introduction method in the production method of the transformed plant body of this invention can be applied as it is.

(Gene screening method)
In addition to the introduction step and the regeneration step, the gene screening method of the present invention further comprises a step of selecting or classifying transformed plants using the phenotypes of the regenerated plant individual and its progeny plants as indicators. A genetic screening method for a plant body comprising the same is also provided. According to the screening method of the present invention, transformed plants that retain foreign genes in various combinations can be obtained at once by the introduction step. For this reason, it is possible to easily identify a useful foreign gene of a transformed plant individual having a good trait by selecting or classifying the obtained transformed plant individual or its progeny plant using a phenotype as an index. it can. Up to now, a large number of trials and errors such as individual trial production of transformed plant individuals with various genes and combinations and subsequent analysis of traits or expression have been necessary. In order to obtain a set, select a transformed plant individual from these or its progeny plants using the phenotype as an index, and determine a foreign gene of the transformed plant individual. Combinations can be easily detected. That is, in order to create a transformed plant body at once, select by trait, and then determine a foreign gene, the determination of a gene for a useful transformed plant body is labor-saving.

  In addition, since the present screening method can obtain a transformed plant body of a combination of two or more kinds of foreign genes, it is possible to easily screen the function and cooperative action of genes whose functions are unknown. For example, in the past, even if the expression of an endogenous gene alone was suppressed, the phenotype did not change and functional analysis was difficult. However, according to this screening method, it is easy to suppress the expression of related genes. Therefore, phenotypic changes can be easily detected. For this reason, the function and cooperation of the endogenous gene can be easily detected. In addition, in this screening method, the various aspects about the foreign gene in the production method of the transformed plant body of this invention, a plant body, a construct, and the introduction method can be applied as it is.

(Transformed plant library and method for producing the same)
The transformed plant body library of the present invention can contain a transformed plant individual introduced by a combination of two or more foreign genes selected from two or more foreign genes. A combination of a phenotype found in a transformed plant contained in this library and a foreign gene contributing to the phenotype is useful for molecular breeding and gene function search. This library can include not only transformed plants by a combination of two or more foreign genes, but also transformed plants into which a foreign gene has been introduced alone. According to such a library, it is useful for the interaction and cooperation of foreign genes and functional analysis of endogenous genes.

This library can also be composed only of transformed plants into which foreign genes have been introduced in combinations of two or more foreign genes. In this library, various aspects of the foreign gene, plant, construct and introduction method in the method for producing a transformed plant of the present invention can be applied as they are.

This library can be easily prepared as a set of transformed plants by the method for producing transformed plants of the present invention. That is, using the production method, including a transformed plant individual into which the foreign gene has been introduced in a combination of two or more types selected from the introduction step, the regeneration step, and two or more types of the foreign genes This can be carried out as a method for producing a transformed plant library, comprising a step of producing a library.

(Composition for introducing foreign genes into plants)
The composition for introducing a foreign gene of the present invention can contain two or more types of Agrobacterium that hold different foreign genes so that they can be introduced into the host plant chromosome. According to the present composition, a group of transformed plants can be easily obtained by efficiently carrying out the foreign gene introduction step of the production method and the introduction method. Therefore, the present composition can be used for producing a transformed plant body or for genetic screening of a plant body.

This composition is usually provided as an Agrobacterium solution, but may be a lyophilized product as necessary. Moreover, this composition may be comprised as kits, such as an individual Agrobacterium liquid or powder which should be mixed at the time of use. That is, a kit for plant transformation is also provided as another embodiment of the present composition. About foreign genes, Agrobacterium, etc. held by these Agrobacterium, overexpression or suppression of expression of foreign genes or endogenous genes that may be related to useful traits in plants, or endogenous genes with unknown functions Therefore, various embodiments can be applied to the present invention as already described in the production method of the present invention.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

(For RNAi (Gate
Way (trade name) specification) Construction of binary vector pYU501)
DNA containing two Gate Way cassettes (attR1, Cm ^R , ccdB and attR2 arranged in tandem) arranged in reverse orientation across the first intron of the Arabidopsis WRKY33 gene and the 35S terminator sequence of cauliflower mosaic virus The fragment was excised from pJawoh8 with XhoI and PmeI and recombined into the XhoI-EcoRV site of vector pYU502 in which the nos terminator was incorporated into the XbaI site of pBluescript (trade name) II SK (-) (Stratagene). The obtained plasmid was designated as pYU505. The larger fragment obtained by digesting pYU505 with PspOMI and KpnI and pSK1 (Kojima, S., Banno, H., Yoshioka, Y., Oka, A., Machida, C., and
Machida, Y. (1999). A binary plasmid for gene expression in plant cells that is
stably maintained in Agrobacterium cells. DNA Res. 6, 407-410.) was digested with NotI and KpnI, and the larger fragment was joined together. The obtained plasmid was designated as pYU501 (FIG. 2). For the construction of pYU505 and pYU501, E. coli DB3.1 strain (Invitrogen) was used. pJawhl8 is Dr. Beir Ulker and Imre E. E. of Max Planck Institute. Granted by Dr. Somsschich.

(Cloning of Arabidopsis gene encoding histone deacetylase (HDAC) belonging to HD2 family)
Amplification by PCR was performed from cDNA derived from the shoot apex of Arabidopsis thaliana using specific primers (Table 1) for amplifying each gene. Each fragment was cloned into pENTR-D-TOPO (trade name) (Invitrogen).

(Construction of a binary plasmid to suppress the expression of each gene)
Each cloned cDNA was recombined into pYU501 using LR clonase (trade name) (Invitrogen). For Escherichia coli, Stbl2 strain (Invitrogen) was used. Individual plasmids were independently Agrobacterium GV3101 / pMP90 (Koncz, C., and Schell, J.
(1986) .The promoter of T _L -DNA gene 5 controls the tissue-specific
expression of chimeric genes carried by a novel type of Agrobacterium binary
vector. Mol. Gen. Genet. 204, 383-396).

(Plant transformation and construction of HDT-RNAi library)
Each of the four types of Agrobacterium transformed lines prepared in Example 3 was cultured in 5 mL of LB medium containing 50 μg / mL carbenicillin for 3 nights (full growth, OD ₆₀₀ > 2). A glycerol stock was prepared by mixing equal amounts of these four types of Agrobacterium cultures, and used as an HDT-RNAi Agrobacterium library (a composition for introducing a foreign gene).

The HDT-RNAi Agrobacterium library was subjected to the floral dip method (Ye, G.-N., Stone, D., Pang, S.-Z.,
Creely, W., Gonzalez, K., and Hinchee, M. (1999). Arabidopsis ovule is the
target for Agrobacterium in planta vacuum infiltration transformation.Plant J.
19, 249-257) by Arabidopsis as2-1 mutant (Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C., and
Machida, Y. (2001). The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana
regulates formation of a symmetric lamina, establishment of venation and
repression of meristem-related homeobox genes in leaves.Development 128,
1771-1783.). The obtained seed was added to Murashige-Skoog (MS) medium containing 15 μg / mL hygromycin [1 × strength MS salt, 3 mg / L thiamine, 5 mg / L nicotinic acid, 0.5 mg / L pyridoxine, 0.5 g / L-Mes-KOH (pH 5.7), 0.5% (w / v) gellan gum (Wako Pure Chemical Industries)] was used as a primary transformant (HDT-RNAi Arabidopsis thaliana library).

(Screening by phenotype)
From the obtained transformants, individuals that formed leaves that did not develop leaf blades were searched. In addition, when an HDAC inhibitor is administered to the as2 mutant, the leaf blades do not expand (having a property on the dorsal axis side) to form rod-shaped leaves. As a result of searching the HDT-RNAi Arabidopsis thaliana library using the individual's phenotype (leaf morphology) as an indicator, individuals exhibiting the desired traits (the leaf blades did not develop) could be isolated. The results are shown in Table 2. Table 2 shows the results of analyzing 11 of the obtained 20 systems. An example of the blade of the transformant (transformed lines # 1 and # 3) is shown in FIG.

(Confirmation of genes introduced into transformants)
Further, genomic DNA was extracted from the obtained transformant, and confirmed by PCR using primers specific to the HDAC gene and the vector (HDAC gene: primer GSP) (vector: primer Rv59 or pU104) ( (See the upper part of FIG. 4). In addition, pU508, pU510, pU512 and pU514 (all described in Table 1) were used for the HDT1 gene, HDT2 gene, HDT3 gene and HDT4 gene, respectively. In addition, nested PCR was performed using the following primers for identification of RNAi T-DNA of HDT3. The primers used are shown in Table 3. GSP is a primer specific for each HDT gene. The lower part of FIG. 4 shows the results of confirming genes in transformed lines # 1 (left) and # 301 (right). It was confirmed that T-DNA for RNAi of HDT1 and HDT2 was introduced into transformed line # 1, and that of HDT2 was introduced into # 301. Lanes 1 to 4 show amplified fragments using GSP specific to HDT 1 to 4, respectively. The two left lanes indicate size markers.

As shown in Table 2, when the introduced T-DNA was analyzed from 11 plants out of the 20 strains obtained as transformants, 1 type, 2 types and 3 types of T-DNA were introduced. The individuals that had been made were 7 individuals (64%), 3 individuals (27%) and 1 individual (9%), respectively. This indicates that the introduction of a plurality of T-DNAs into one host plant cell is due to the infection of a plurality of Agrobacterium. Actually, this result shows that the number of loci of T-DNA loci of transformed plants obtained when Agrobacterium introduced one kind of T-DNA into Arabidopsis thaliana (1 locus, 2 loci, 3 genes). The probability of introducing a locus is 53%, 25% and 8%, respectively; Feldmann, KA (1991). T-DNA insertion
mutagenesis in Arabidopsis: mutational spectrum.Plant J.1,71-82). This kind of report is unprecedented and can be said to contribute to the basic understanding of plant transformation via Agrobacterium.

  Further, as shown in Table 2, the identified T-DNAs were 3, 3, 5 and 5 in total for HDT1, HDT2, HDT3 and HDT4, respectively. Assuming that each Agrobacterium cell exists equally and genes are introduced to the same extent, the average number is 4, so it does not deviate significantly from the expected value, and an HDT-RNAi Agrobacterium library can be produced as expected. It shows that.

(Gene expression analysis in transformed line # 1)
The expression level of HDT gene in transformed line # 1 was determined by RT-PCR (Semiarti, E., Ueno,
Y., Tsukaya, H., Iwakawa, H., Machida, C., and Machida, Y. (2001). The
ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a
symmetric lamina, establishment of venation and repression of meristem-related
homeobox genes in leaves. Development 128, 1771-1783). RNA was extracted from the individual on the 10th day after sowing and RT-PCR was performed. In addition to HDT1, HDT2, HDT3 and HDT4, transcripts of genes for inactivating hygromycin (HPT; drug marker gene on T-DNA), PHABULOSA (PHB), KANADI2 (KAN2) and ETIN (ETT) genes The accumulation of was also investigated. PHB, KAN2 and ETT are genes related to the phenotype of this plant body. HPT was examined to determine the presence or absence of T-DNA. The primers used were as follows. The results are shown in FIG.

HDT1, HDT2,
HDT3 and HDT4: see Table 1

HPT
: HPT1
(5'-AGCCTGAACTCACCGCGACGT-3 ') (SEQ ID NO: 13)
HPT2
(5'- TGCCCTCGGACGAGTGCTGGG-3 ') (SEQ ID NO: 14)

PHB
: pU579
(5'-ATGATGATGGTCCATTCGATGAGCAGA-3 ') (SEQ ID NO: 15)
pU580
(5'-GGGCCTCCTCTGCTATAGAAAGGAGTCCT-3 ') (SEQ ID NO: 16)

KAN2:
pU593
(5'- ATGGAGCTGTTTCCTGCTCAGCCCGAT-3 ') (SEQ ID NO: 17)
pU594
(5'-GTGAGATCGACCCAGAGTAAACTCAAG-3 ') (SEQ ID NO: 18)

ETT:
pU223
(5'-ATGGGTGGTTTAATCGATCTGAACGTGATGG-3 ') (SEQ ID NO: 19)
pU224
(5'-GGAGGGAAGCAATCCTCAGCAGC-3 ') (SEQ ID NO: 20)

TUBA:
pU51 and pU52 Semiarti, E., Ueno, Y.,
Tsukaya, H., Iwakawa, H., Machida, C., and Machida, Y. (2001). The ASYMMETRIC
LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina,
establishment of venation and repression of meristem-related homeobox genes in
leaves. Development 128, 1771-1783.

As shown in FIG. 5, the accumulation of transcripts of HDT1 and HDT2 was certainly suppressed in the transformed line # 1, but HDT3 and HDT4 had no effect. Although no significant difference was observed between KAN2 and ETT, the accumulated amount of PHB transcripts was significantly reduced compared to non-transformants. These results indicate that HDT1 and HDT2 RNAi suppress their expression as expected, and the effect appears to affect the expression of PHB, which is a downstream gene. TUBA was used as an internal standard.

The trait and the presence or absence of T-DNA in the F2 generation obtained by crossing the transformed strain # 1 back to the wild type were examined in the same manner as in Example 4. The results are shown in Table 4.

  As shown in Table 4, the t-DNA introduced into transformed line # 1 was linked to the phenotype.

  According to the above example, as a result of examining T-DNA introduced into the transformed lines # 1 and # 301 that markedly exhibit the desired trait, two types of HDT1 and HDT2 and one type of RNAi T-DNA T- Each DNA was confirmed (Table 2 and FIG. 4). In fact, in the transformed line # 1, accumulation of transcripts of HDT1 and HDT2 was suppressed (FIG. 5), and these T-DNAs were linked to the phenotype (Table 4). The traits used as a search index here are those observed when an HDAC inhibitor (TSA or the like) is administered to the as2 mutant. As in the HD2 family, the same search was performed for the RPD3 / HDA1 family, which is an HDAC family that is sensitive to TSA, but an individual exhibiting a characteristic trait could not be obtained. From these results, HDT1 and HDT2 could be identified as genes involved in leaf formation.

  In addition, according to the above embodiment, Agrobacterium introduced with different T-DNAs is mixed, and these Agrobacterium are infected with plant cells, whereby heterologous T-DNA is simultaneously introduced into the same host plant cell. It was found that it could be introduced. It was also found that this method can greatly reduce the time and labor required to create multiple transformed plants. Furthermore, it has been found that the use of such a method makes it possible to identify genes from the traits of various combinations of multiple transformed plants. In fact, the RNAi library in this example showed that gene identification was possible using changes in plant traits associated with functional defects as an index. In the above examples, screening was performed using the appearance of the individual as an index. However, various applications such as tracking the composition and activity of the extract are possible, and various applications such as function-deficient transformants are possible. It was also found that the function-acquisition type transformant can be similarly applied.

SEQ ID NOs: 1-20: synthetic primers

The figure which shows an example of the gene-analysis method to which this invention is applied. The figure which shows an example of the conventional gene-analysis method. The table | surface figure which shows the merit of the gene-analysis method to which this invention is applied. The figure which shows the structure of pYU501. However, Riori, origin of replication in Agrobacterium; Amp ^R , ampicillin resistance gene (β-lactamase, drug marker of E. coli and Agrobacterium); pUC ori, origin of replication in E. coli; tnos, nos terminator; t35S , Cauliflower mosaic virus 35S terminator; recombination sites of attR1 and attR2, LR clonase ™; Cm ^R , chloramphenicol resistance gene (chloramphenicol acetyltransferase, Escherichia coli and Agrobacterium drug markers); ccdB, Lethal gene in E. coli; WRKY33intron, first intron of Arabidopsis WRKY33 gene; p35S, cauliflower mosaic virus 35S promoter; HPT, hygromycin resistance gene (hygromycin phosphotransferase, plant, Agrobacterium and E. coli drug markers -). The figure which shows the example of a transformed individual obtained from the HDT-RNAi library. A shows transformed line # 1 (T1 generation). The target traits were stick-shaped leaves (arrowheads) and trumpet-shaped leaves (arrows). B shows transformed line # 3 (T1 generation). A trait that can be distinguished from the host individual could not be confirmed. The figure which shows each example of the determination method and determination result of T-DNA introduce | transduced into each individual | organism | solid of HDT-RNAi Arabidopsis thaliana library. The figure which shows the gene expression analysis in a transformation system | strain # 1.

Claims (12)

A method for analyzing gene functions of plants ,
Any of the following combinations:
(A) two or more constructs each holding a polynucleotide region that suppresses the expression of two or more different endogenous genes so as to be able to be integrated into the host plant chromosome.
(B) two or more constructs each holding a polynucleotide region for expressing two or more different exogenous genes so that they can be integrated into the host plant chromosome,
Co-infecting a plant with two or more types of Agrobacterium each holding
Regenerating a plant individual from the plant body that has undergone the infection step;
When the construct (a) is used, a library comprising plant individuals or their progeny in which the expression of one or more endogenous genes selected from the two or more endogenous genes is suppressed is prepared. When the construct (b) is used, a step of preparing a library comprising plant individuals or their progeny whose expression of one or more foreign genes selected from the two or more foreign genes is promoted A method comprising:   The method according to claim 1, wherein the two or more constructs of (a) are three or more constructs, and the two or more constructs of (b) are three or more constructs. The method according to claim 1 or 2, wherein the construct (a) is used . The method according to claim 1 or 2, wherein the construct (b) is used. The method in any one of Claims 1-4 provided with the process of selecting or classifying a transformed plant body by using as a parameter | index the phenotype of the said plant individual | organism | solid or its progeny. Further, when the construct (a) is used, the endogenous gene whose expression is suppressed in the regenerated plant individual is determined, and when the construct (b) is used, the endogenous gene is expressed in the regenerated plant individual. The method according to any one of claims 1 to 5, further comprising the step of determining the foreign gene whose expression has been promoted . A plant gene screening method comprising :
Any of the following combinations:
(A) two or more constructs each holding a polynucleotide region that suppresses the expression of two or more different endogenous genes so as to be able to be integrated into the host plant chromosome.
(B) two or more constructs each holding a polynucleotide region for expressing two or more different exogenous genes so that they can be integrated into the host plant chromosome,
Co-infecting a plant with two or more types of Agrobacterium each holding
Regenerating a plant individual from the plant body that has undergone the infection step;
When the construct (a) is used, a library comprising plant individuals or their progeny in which the expression of one or more endogenous genes selected from the two or more endogenous genes is suppressed is prepared. When the construct (b) is used, a step of preparing a library comprising plant individuals or their progeny whose expression of one or more foreign genes selected from the two or more foreign genes is promoted When,
Selecting or classifying transformed plants using as an index the phenotype of the regenerated plant individual or its progeny;
When the construct (a) is used, the endogenous gene whose expression is suppressed in the regenerated plant individual is determined, and when the construct (b) is used, the expression is promoted in the regenerated plant individual. Determining the exogenous gene, and
A method comprising:   The method according to claim 7, wherein the two or more constructs of (a) are three or more constructs, and the two or more constructs of (b) are three or more constructs. A method for producing a library of plant individuals,
Any of the following combinations:
(A) two or more constructs each holding a polynucleotide region that suppresses the expression of two or more different endogenous genes so as to be able to be integrated into the host plant chromosome.
(B) two or more constructs each holding a polynucleotide region for expressing two or more different exogenous genes so that they can be integrated into the host plant chromosome,
Co-infecting a plant with two or more types of Agrobacterium each holding
Further, when the construct (a) is used, a library comprising plant individuals or their progeny in which expression of one or more endogenous genes selected from the two or more endogenous genes is suppressed is obtained. When the construct (b) is used, a library composed of plant individuals or their progeny whose expression of one or more foreign genes selected from the two or more foreign genes is promoted is prepared. And a process of
A method comprising:   The method according to claim 9, wherein the two or more constructs of (a) are three or more constructs, and the two or more constructs of (b) are three or more constructs.   The method according to claim 9 or 10, wherein the construct (a) is used.   The method according to claim 9 or 10, wherein the construct (b) is used.