HK1154333B - Method of producing transformed plant by using agrobacterium strain - Google Patents

Description

Method for preparing transformed plant by using agrobacterium

Technical Field

The present application claims priority from Japanese patent application 2008-094049, filed on 31/3/2008.

The present invention relates to a novel method for producing transformed plants using Agrobacterium.

Background

As a method for transforming monocotyledons such as corn and rice, which are major cereals, electroporation method, particle gun method and the like have been conventionally known. However, these physical gene transfer methods have the following problems: multiple copies of the gene are introduced, the insertion of the gene is not in an intact form, and transformed plants are frequently malformed, sterile and the like.

The gene transfer method using Agrobacterium is widely used as a method for transforming dicotyledonous plants. The host of Agrobacterium bacteria is limited to dicotyledonous plants, which are not thought to be able to colonize monocotyledonous plants (De Cleene and De Ley 1976), but attempts are being made to transform monocotyledonous plants with Agrobacterium.

Grimsley et al report: the infection with maize streak virus was confirmed by inserting the DNA of maize streak virus (maize) into the T-DNA of Agrobacterium and inoculating the DNA to the growing point of maize, whereas the infection symptoms could not be confirmed by inoculating only the DNA of maize streak virus. This can be interpreted as: the above phenomena indicate that Agrobacterium is capable of introducing DNA into maize (Grimsley et al, 1987). However, the virus still has the possibility of propagation without integration into the nuclear genome, and this result does not indicate the integration of T-DNA into the nucleus. Grimsley et al further disclose: the infection efficiency was highest when inoculated to the apical growing point of maize (Grimsley et al, 1988), infection requiring the Vir C gene of the plasmid of Agrobacterium (Grimsley et al, 1989).

Gould et al, after damaging the growing point of corn with a needle, inoculated a strongly pathogenic Agrobacterium EHA1 carrying a kanamycin resistance gene and a GUS gene, and selected the growing point after the treatment with kanamycin, and as a result, obtained a plant showing resistance. Southern analysis was performed to confirm that the progeny seeds had the introduced gene, and as a result, the introduced gene was confirmed for some seeds (Gould et al, 1991). This suggests that: in a plant obtained by selecting a growth point treated with Agrobacterium with kanamycin, transformed cells and non-transformed cells were present in a mixed manner (chimerism).

Mooney et al attempted to introduce a kanamycin resistance gene into wheat embryos using Agrobacterium. First, the embryo is damaged in its cell wall by treating it with an enzyme, and then inoculated with Agrobacterium. The treated callus proliferated very few calli considered to have kanamycin resistance, but these calli could not be regenerated into plants. In addition, structural mutations of the introduced gene were observed in all resistant calli when the presence of the kanamycin resistance gene was confirmed by Southern analysis (Mooney et al, 1991).

After damaging the rice blastoderm, Raineri et al treated 8 rice cultivars with highly pathogenic Agrobacterium A281(pTiBo542), and observed the proliferation of tumor-like tissues in 2 cultivars, Nipponbare and Tabanum 5. Further, the rice embryo was inoculated with Agrobacterium harboring a plasmid obtained by inserting a kanamycin resistance gene and a GUS gene into a Ti plasmid obtained by removing a hormone synthesis gene from T-DNA, and the proliferation of kanamycin-resistant callus was observed. In the resistant callus, the expression of GUS gene was confirmed, but transformed plants could not be obtained. These can be interpreted as: T-DNA of Agrobacterium is introduced into cells of rice (Raineri et al, 1990).

As described above, there are research reports that: in rice crops such as rice, corn and wheat, gene transfer can be performed using Agrobacterium. However, they all have a problem in reproducibility, and furthermore, confirmation of the introduced gene is incomplete and no convincing result is given (Potrykus 1990).

Chan et al report: after 2 days of rice immature embryos cultured in the coexistence of 2, 4-D were damaged, Agrobacterium carrying nptII gene and GUS gene was inoculated in the medium containing potato suspension culture cells. The treated immature embryos were cultured on a medium containing G418, and as a result, redifferentiated plant bodies were obtained from the callus produced by induction. When the presence of GUS gene was confirmed in the redifferentiated plant and the progeny thereof by Southern analysis, the presence of the transgene was confirmed in both the redifferentiated and the progeny (Chan et al, 1993). This result supports the transformation of rice with Agrobacterium, but the transformation efficiency was very low, only 1.6%, and only 1 individual of the regenerated plants showed normal growth relative to the 250 immature embryos tested. Since a large amount of labor is required to extract rice immature embryos, it is hard to say that such low transformation efficiency reaches a practical level.

In recent years, there have been reports of: by using a super binary vector having a part of pathogenic genes of highly pathogenic Agrobacterium, stable and efficient transformation can be performed even in monocotyledons such as rice and maize (Hiei et al, 1994, Ishida et al, 1996). These reports suggest that transformation with Agrobacterium has the following advantages in addition to stable and efficient transformation: the obtained transformed plant has few mutations, and the copy number of the introduced gene is small and is often in intact form. Following the success in rice and maize, Agrobacterium transformation has also been reported in the major cereals wheat (Cheng et al, 1997), barley (Tingay et al, 1997) and milo (Zhao et al, 2000).

As an attempt to improve the efficiency of transforming corn with agrobacterium, in addition to the above, there are: selection of transformed cells in N6 minimal Medium (Zhao et al, 2001), addition of AgNO to the Medium₃And carbenicillin (ZHao et al, 2001, Ishida et al, 2003), addition of cysteine to the coculture medium (Frame et al, 2002), and the like. Thus, by changing the composition of the medium and selecting the marker gene, the efficiency of transforming rice or corn with Agrobacterium is improved.

In the case of the methods reported so far for transforming rice and maize with Agrobacterium, transformed plants are basically obtained by the following steps: transformation of callus derived from an embryonic disc inoculated with Agrobacterium or callus induced from an immature embryo inoculated with Agrobacterium is carried out by selective proliferation using a medium containing a herbicide component and an antibiotic, and the resulting transformed cell mass is implanted in a redifferentiation medium to redifferentiate (Deji et al, 2000; Negrotto et al, 2000; Nomura et al, 2000 a; Nomura et al, 2000 b; Taniguchi et al, 2000; Frame et al, 2002; Zhang et al, 2003; Frame et al, 2006).

The selection of transformed cells is essential for the production of transformed plants in terms of the method of transformation of plants, without which successful transformation of plants is not possible (Potrykus et al, 1998; Erikson et al, 2005; Joersbo et al, 2001). In most cases, selection of transformed cells is performed using the following method: a gene showing resistance to a drug which inhibits the proliferation of a non-transformed cell is introduced into a plant material, and the plant material is cultured in a medium containing the drug, thereby selectively proliferating only the transformed cell in which the drug-resistant gene is recombined into the genome of the plant cell and expressed.

Among the genes used for selection of transformed cells (selectable marker genes), the most commonly used gene is a gene that confers resistance to herbicides or antibiotics (Kuiper et al, 2001). As genes conferring resistance to herbicides, the bar gene, the EPSP gene (De Block et al, 1987; Comai et al, 1985) are frequently used as selectable marker genes for plant transformation, and as genes conferring resistance to antibiotics, the NPTII gene, the HPT gene (Bevan et al, 1983; Waldron et al, 1985) are frequently used as selectable marker genes for plant transformation. In addition, it has been reported in recent years that PMI gene, XylA gene (Joersbo et al, 1998; Haldrup et al, 1998), etc., which utilize specific ability to metabolize sugar, are also effective as selectable marker genes. As a selectable marker gene utilizing a mechanism for selectively proliferating a transformed cell, many genes have been reported in addition to the above-mentioned genes. Furthermore, it is considered that a selection step for selectively growing transformed cells is essential in a transformation method using these genes.

In the Inplanta (インプランタ) transformation method in which floral bud tissue is transformed by reduced pressure infiltration, transformed seeds (Bent, 2000) can be obtained without a selection step. However, in order to obtain a transformed seed as a target from among seeds mixed with many non-transformed seeds, a selection step using an antibiotic resistance gene or the like is necessary.

The following methods have been reported: transformed plants were obtained by introducing the GFP gene and visually selecting the site of transformation using cells emitting fluorescence under ultraviolet irradiation as an index (Elliott et al, 1998; Zhu et al, 2004). In this method, although it is not necessary to select transformed cells from mixed non-transformed cells based on the difference in proliferation, a selection step of distinguishing transformed cells from non-transformed cells based on the presence or absence of expression of a GFP gene and further separating them is necessary.

As techniques for removing a selection marker gene from a transformed plant, co-transformation systems (Komari et al, 1996), MAT vector systems (Ebinuma et al, 1997) and the CreLox system (Gleave et al, 1999; Zhang et al, 2003) have been reported. By using these systems, transformed plants free of a selectable marker gene can be obtained. However, in the process of producing a transformed plant without a selection marker, a step of discriminating and selecting transformed cells from non-transformed cells using a drug resistance gene, a plant hormone synthesis gene, or the like, which has been conventionally employed, is necessary.

As described above, in the methods hitherto used for plant transformation, a selection step for selecting transformed cells and non-transformed cells is indispensable. In this selection step, as described above, a selection marker gene for selection must be present in addition to the target gene (GOI gene). By utilizing the reaction caused by the function of the protein or enzyme produced by the expression of the selection marker gene, for example, herbicide resistance, antibiotic resistance or fluorescence, a few transformed cells among a large number of non-transformed cells can be distinguished and then propagated to obtain transformed plants. However, the selectable marker gene is not required for the transformed plant to be produced, and if the transformed plant is left to contain the selectable marker gene, there is no risk that the herbicide resistance gene or the antibiotic resistance gene will be transmitted to a general non-recombinant plant through a transformant, and thus there are many general consumers who are anxious to apply the selectable marker gene to the transformed plant. In addition, although many types of selectable marker genes have been reported, the selectable marker genes are limited by the types of plants to which they are applied, and there is a problem in introducing the genes separately. Further, although there have been reports on techniques for removing a selectable marker gene from a transformant, these methods require considerable labor, for example, a long culture period as compared with conventional transformation methods, and selection of individuals without a selectable marker from progeny plants is required.

As described above, although there have been many reports on the production of transformed plants, there has been no report on a method for obtaining a transformant without a selection step of selecting transformed cells, tissues, organs or individuals from untransformed cells, tissues, organs or individuals. That is, it can be said that it has not been possible to obtain a transformed plant by introducing only a GOI (Gene of interest) gene.

Patent document 1: WO98/54961

Patent document 2: WO02/12520

Patent document 3: WO02/12521

Patent document 4: WO2005/017169

Patent document 5: WO2005/017152

Patent document 6: WO2007/069643

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Disclosure of Invention

Problems to be solved by the invention

In the conventional gene transfer method into monocotyledonous plants using Agrobacterium, a selection step of a transformed cell, tissue, organ or individual using a selection marker gene transfer is indispensable. It is an object of the present invention to develop and provide a method for obtaining transformed plants which is not subjected to such a selection step.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that: by treating monocotyledons for improving transformation efficiency, transformed plants can be obtained with practical efficiency without introducing a selection marker gene.

Specifically, the present inventors have noted that, in general, in the transformation step of a plant, the number of plant cells into which a foreign gene is recombined is very small, and have considered that: if the transformation efficiency can be greatly improved, the proportion of transformed cells in non-transformed cells can be increased, and the transformed plants can be maintained until the plant body is redifferentiated, transformed plants can be obtained without a selection step using a selectable marker gene. As a result of intensive studies, it was found that: by appropriately carrying out the transformation-enhancing treatment, transformed plants can be obtained with sufficient practical efficiency, and the present invention has been completed.

The present invention is not intended to be limited thereto, but includes the following embodiments as preferred embodiments.

[ embodiment 1]

A method for producing a transformed plant using Agrobacterium, comprising:

(i) a co-culture step of culturing the agrobacterium-inoculated plant material in a co-culture medium; and

(ii) (ii) culturing the tissue obtained in (i) without callus proliferation culture or after callus proliferation culture, with a redifferentiation medium, and redifferentiating the cultured tissue into a plant;

wherein, in the method for producing the transformed plant,

1) carrying out conversion improvement treatment; and

2) selection of transformed cells using a selection drug is not performed in any step from coexistence until redifferentiation.

[ embodiment 2]

The method for producing a transformed plant according to embodiment 1, wherein,

3) the step of culturing the co-cultured tissue with a callus growth medium is not included between the co-culturing step and the redifferentiation step.

[ embodiment 3]

The method of producing a transformed plant according to embodiment 1 or 2, wherein the nucleic acid introduced by agrobacterium does not include a resistance gene to a selective drug.

[ embodiment 4]

The method for producing a transformed plant according to any one of embodiments 1 to 3, wherein 1) the transformation-improving treatment is: a treatment for improving the efficiency of introducing a target gene into a plant cell, a treatment for improving the induction rate of a callus derived from an immature embryo or the like, or a treatment for improving the redifferentiation efficiency of a transformed callus.

[ embodiment 5]

The method for producing a transformed plant according to any one of embodiments 1 to 4, wherein 1) the transformation-enhancing treatment is selected from the group consisting of:

heat treatment;

centrifuging;

heat and centrifugal treatment;

carrying out pressurization treatment;

adding silver nitrate and/or copper sulfate to the coculture medium;

treating with Agrobacterium in the presence of the powder;

adding carbenicillin to the culture medium in the callus proliferation and/or redifferentiation step after the coexistence step;

adding N6 inorganic salt into callus proliferation culture medium; and

cysteine was added to the coculture medium.

[ embodiment 6]

The method according to embodiment 1 to 5, wherein the co-existence step comprises adding a compound belonging to a benzoic acid herbicide.

[ embodiment 7]

The method according to embodiment 6, wherein the compound that is a benzoic acid herbicide is of a benzoic acid type, a salicylic acid type, or a picolinic acid type.

[ embodiment 8]

The method for producing a transformed plant according to embodiment 6 or 7, wherein the compound that is a benzoic acid herbicide is 3, 6-Dichloro-2-methoxybenzoic acid (3, 6- ジク Kokou-o- ア Di シン acid, 3, 6-dichoro-o-anisic acid) or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

[ embodiment 9]

The method of producing a transformed plant according to embodiment 1 to 8, wherein the plant is a monocotyledon.

[ embodiment 10]

The method of producing a transformed plant according to embodiment 1 to 9, wherein the plant is maize or rice.

[ preferred embodiments for practicing the invention ]

The present invention provides a method for producing a transformed plant using Agrobacterium. The present invention is based on the following findings: in the method of gene transfer into a plant using Agrobacterium, the transfer efficiency is improved, and thus, the transfer of a selectable marker gene is not required. The method of the present invention is a method for producing a transformed plant using agrobacterium, the method comprising:

(i) a co-existence step of culturing the Agrobacterium-inoculated plant material with a co-existence medium, and

(ii) (ii) culturing the tissue obtained in (i) without callus proliferation culture or after callus proliferation culture, in a redifferentiation medium to redifferentiate the tissue into a plant;

the method of the invention is characterized in that:

1) performing a conversion-enhancing treatment, and

2) selection of transformed cells using a selection drug is not performed in all steps from coexistence until redifferentiation.

Conversion enhancing treatment

One of the features of the present invention is to perform a conversion-enhancing treatment. In the method of the present invention, the "transformation-enhancing treatment" means: by performing this treatment, the proportion of the resulting transformed plants is increased. Without being limited thereto, specific examples thereof include: a treatment for improving the efficiency of introducing a target gene into a plant cell, a treatment for improving the callus induction rate of an immature embryo or the like, a treatment for improving the redifferentiation efficiency of a transformed callus, and the like. The conversion-improving treatment is not limited, and includes, for example, the following treatments or a combination thereof.

Heat treatment (see: WO98/54961),

Centrifugal treatment (see: WO02/12520),

Thermal and centrifugal treatment (see: WO02/12521),

Pressure treatment (see: WO2005/017169),

Silver nitrate and/or copper sulfate (see AgNO) were added to the coculture medium₃(ZHao et al, 2001, Ishida et al, 2003; CuSO₄(WO2005/017152)、

Treatment of Agrobacterium in the presence of a powder (cf. WO2007/069643),

After the coculture step, carbenicillin (see: Zhao et al, 2001, Ishida et al, 2003) and/or carbenicillin (see: Zato et al, 2001, Ishida et al, 2003) are added to the medium during the callus growth and/or redifferentiation step,

Addition of N6 inorganic salt to callus growth Medium (Chu 1978) (Zhao et al, 2001), and

treatment in which cysteine was added to the coculture medium (Frame et al, 2002).

Among them, the heat treatment, the centrifugal treatment, the heat and centrifugal treatment, the pressure treatment, and the addition of the powder are all treatments for improving the gene transfer efficiency, and the addition of silver nitrate, copper sulfate, and carbenicillin has an effect of improving the callus induction rate. In addition, the redifferentiation efficiency can be improved by adding copper sulfate to the redifferentiation medium.

Without being limiting, the heat treatment can be carried out, for example, by the method described in WO 98/54961. For example, before contacting the plant material with Agrobacterium, the treatment is carried out at 33 to 60 ℃ and preferably 37 to 52 ℃ for 5 seconds to 24 hours, preferably 1 minute to 24 hours.

Centrifugation may be carried out, for example, as described in WO 02/12520. For example, before contacting the plant material with Agrobacterium, the plant material is treated at a centrifugal acceleration of 100G to 25 ten thousand G, preferably 500G to 20 ten thousand G, more preferably 1000G to 15 ten thousand G for 1 second to 4 hours, more preferably 1 second to 2 hours.

The heat and centrifugation treatment may be carried out using, for example, the method described in WO 02/12521. The conditions for the heat treatment and the centrifugation treatment may be, for example, the above-mentioned conditions.

The pressure treatment may be carried out by the method described in, for example, WO 2005/017169. The pressurization treatment is preferably carried out in a range of 1.7 atm to 10 atm, more preferably in a range of 2.4 atm to 8 atm, without being limited thereto.

Addition of silver nitrate and/or copper sulfate to the coculture medium is described in, for example, Zhao et al, 2001, Ishida et al, 2003, WO 2005/017152. Silver nitrate and/or copper sulfate may be added to the coculture medium at a concentration of, for example, 1. mu.M to 50. mu.M, preferably 1. mu.M to 10. mu.M.

Treatment of the Agrobacterium in the presence of the powder may be carried out, for example, as described in WO 2007/069643. Specifically, the following method can be employed, for example: mixing the suspension with the powder to inoculate plant material, or mixing the plant with the powder and inoculating it with Agrobacterium. The powder is not limited, and may be porous powder, glass wool, or activated carbon, preferably porous ceramic, glass wool, or activated carbon, and more preferably hydroxyapatite, silica gel, or glass wool.

The treatment of adding N6 inorganic salt to the callus growth medium (Zhao et al, 2001) can be carried out by adding N6 inorganic salt to the callus growth medium (Chu 1978).

The cysteine may be added to the coculture medium at a concentration of 10mg/L to 1g/L, preferably 50mg/L to 750mg/L, and more preferably 100mg/L to 500 mg/L.

The addition of carbenicillin to the medium after the coculture step, during the callus propagation and/or redifferentiation step, can be carried out by the method described by Zhao et al, 2001 or Ishida et al, 2003. Carbenicillin may be added at a concentration of, for example, 50 to 500mg/L, preferably 100 to 300mg/L, in the callus growth medium and/or in the redifferentiation step. Moreover, carbenicillin, although an antibiotic, is substantially non-toxic to plants and can be used to prevent the proliferation of microorganisms in the culture medium.

Those skilled in the art can perform these treatments at appropriate timing/conditions. Further, from the viewpoint of improving the conversion efficiency, it is more preferable to appropriately combine them. Further, for example, in the case of corn, it is preferable to combine heat and centrifugation, powder treatment, addition of AgNO to the coculture medium₃And/or CuSO₄And (3) 2 or more treatments of adding carbenicillin as an antibiotic to the callus growth medium and/or the redifferentiation medium. In the case of rice, it is preferable to include heat treatment and/or centrifugation treatment, and it is most preferable to combine the treatment of adding carbenicillin as an antibiotic to the callus growth medium and/or the redifferentiation medium.

Therefore, the preferable transformation-enhancing treatment is heat treatment, centrifugation, heat and centrifugation, pressure treatment, addition of AgNO to the coculture medium₃And/or CuSO₄Or a treatment with agrobacterium in the presence of a powder, a treatment with carbenicillin as antibiotic in the callus proliferation medium and/or the redifferentiation medium, or a combination of said treatments.

The present inventors succeeded in sufficiently increasing the number of transformed individuals in the newly differentiated individuals finally obtained by performing these treatments, and found that sufficient transformed individuals can be obtained without performing a selection treatment of transformed cells, and established a non-selection transformation method which meets practical requirements.

The transformed individual can be easily obtained by confirming the presence or absence of the introduced gene by PCR or the like, or by confirming the phenotype of the introduced gene in the case of a progeny individual.

Selection of transformed cells

The invention is characterized in that: selection of transformed cells using the characteristics of the nucleic acid introduced by Agrobacterium is not performed in any step for plant transformation from coexistence to redifferentiation.

Examples of selection of transformed cells using the characteristics of nucleic acids introduced by Agrobacterium include selection of transformed cells using a selective drug resistance gene and a selective drug.

"selection of transformed cells using the selection drug resistance gene and the selection drug" means: in any step for plant transformation from coexistence to redifferentiation, culture is performed with a medium to which a drug for selecting transformed plants is added, and transformed cells are selected by the presence or absence of resistance to the selection drug. The present invention does not include such a step at all.

Examples of drugs of choice used in the conventional art are antibiotics and/or herbicides. As the antibiotic, an antibiotic toxic to plants, such as Hygromycin (ハ イ グ ロ マ イ シン, Hygromycin), kanamycin (カ ナ マ イ シン), blasticidin S (ブラストサイジン S, blasticidin S), or the like; as the herbicide, for example, glufosinate (フ オ ス フ イ ノ ス ラ イ シン, phosphinothricin), bialaphos (ビアラフオス, bialaphos), glyphosate (グリホセ - ト, glyphosate), or the like is used.

In order to carry out this step, the DNA inserted into the T-DNA in Agrobacterium must contain not only the gene desired to be expressed in the plant but also a selective drug resistance gene. Such selective drug resistance genes are well known in the art. For example, in the case of a redifferentiation step in a redifferentiation medium containing hygromycin as a selective drug, the hygromycin resistance gene must be introduced into the plant by Agrobacterium.

In the present invention, since a selection step using a drug is not performed, it is not necessary to have a nucleic acid into which a selection drug resistance gene, i.e., a selection marker gene, is introduced by agrobacterium. Thus, without limitation, in a preferred embodiment, the invention does not include such nucleic acids.

Alternatively, selection of transformed plants can be performed based on "auxotrophic (requirement) selection" of plant cells, for example, "sugar auxotrophy (sugar requirement)" as a conventional method.

Regarding sugar auxotrophy, sucrose (シユ - クロ - ス), glucose, and the like are known as sugars that can be used by plant cells, but mannose cannot be used. Therefore, when plant tissues are cultured in a medium containing only mannose as a carbon source, the plant tissues die because there is no available sugar. Selection based on sugar auxotrophy takes advantage of this principle. In selecting a transformed plant based on sugar auxotrophy, a gene that can utilize a sugar that is not normally utilized by plant cells is introduced into a plant tissue by Agrobacterium. Such genes are well known in the art, and for example, PMI gene, xylose isomerase gene, and the like can be used. In the present invention, the introduced nucleic acid does not necessarily contain such a gene.

Alternatively, as a conventional method, a gene which can be easily detected may be introduced as an index for screening, and selection may be made by the presence or absence of expression of the gene. Examples of the gene as an index for such screening include the GFP gene. In the present invention, the introduced nucleic acid does not necessarily contain such a gene.

In the method of the present invention, the selectable marker gene refers to: genes other than the desired gene to be transformed (GOI gene) are introduced into plants, and the purpose of introducing a selection marker gene is to select transformed cells among a large number of non-transformed cells. The selection marker gene is not limited, and examples of the selection marker gene include herbicide resistance genes, antibiotic resistance genes, fluorescent genes, and the like.

Method for producing transformed plant

The method for producing a transformed plant using Agrobacterium generally comprises all or a part of the following steps I to V.

I. Preparation of plant Material

Preparation and inoculation procedure of Agrobacterium

Coexistence step

Callus proliferation step

V. redifferentiation step

I. Preparation of plant Material

The plant to be treated in the present invention is a plant to which a method for transformation and introduction by Agrobacterium can be applied. Preferably monocotyledonous plants. The monocotyledonous plant used in the method of the present invention is preferably a plant of the family oryza, including, but not limited to, rice, corn, barley, wheat, milo, and the like. The most preferred plants for use in the methods of the invention are rice or maize.

In the method of the present invention, the "plant material" includes all forms of plants such as cells, leaves, roots, stems, buds, flowers (including stamens, pistils, etc.), fruits, seeds, germinated seeds, or plant tissues of any other parts, growing points, explants, immature embryos, callus or adventitious embryo-like tissue (hereinafter, referred to as callus or the like or simply callus in the present specification), or complete plant bodies of the plants to be tested for transformation of the plants by the agrobacterium method.

As the form of the plant material used in the method of the present invention, immature embryos or callus are preferable, and immature embryos are most preferable. In the present specification, expressions such as a plant cell, a plant tissue, and a complete plant body have meanings generally adopted in the art. In this specification, immature embryos refer to: embryos and blastoderms of immature seeds in the process of maturation after pollination. In addition, the period of the immature embryo used in the method of the present invention (mature period) is not particularly limited, and may be collected at any period after powdering. Most preferably, the immature embryos are after 2 days after pollination. Preferably, an immature embryonic blastoderm is used which can be proliferated by the method described later after transformation described later and can induce a callus having the ability to regenerate a normal individual. Furthermore, the immature embryos are preferably inbred (bred), F1 between inbreds, F1 between inbreds and natural receptacle varieties, or commercial F1. In the present specification, callus means: a mass of cells in an undifferentiated state that proliferate disorderly. In order to obtain callus, differentiated cells of plant tissues may be obtained by culturing them in a medium containing a plant growth regulator such as auxin (e.g., 2, 4-D) or cytokinin (referred to as dedifferentiation medium). This treatment for obtaining callus is called dedifferentiation treatment, and further, this process is called dedifferentiation process.

In this step, plant tissues, immature embryos and the like are taken out of plant bodies, seeds and the like as necessary, and plant materials suitable for transformation are prepared. The preparation of the plant material can be carried out according to known methods. If necessary, the plant material may be cultured before it is infected with Agrobacterium.

Preparation and inoculation procedure of Agrobacterium

The plant material used in the present invention is inoculated with agrobacterium. As used herein, "inoculation" means: various methods of inoculating Agrobacterium are known in the art for contacting Agrobacterium with plant material. Examples of the method include: a method of adding a plant material to a suspension obtained by suspending agrobacterium in a liquid medium, a method of directly dropping an agrobacterium suspension onto a plant material on a coculture medium, a method of injecting an agrobacterium suspension into a plant material, a method of immersing a plant material in an agrobacterium suspension and reducing pressure, and the like. However, the Agrobacterium-inoculated plant material used in the present invention is not limited to Agrobacterium-inoculated plant materials using these methods.

In the inoculation step of agrobacterium, various additives such as acetosyringone (acetosyringone), a surfactant, and porous ceramics may be included in the suspension of agrobacterium in order to improve the transformation efficiency by agrobacterium.

The Agrobacterium which can be used in the present invention may be any known Agrobacterium bacterium, but is preferably Agrobacterium tumefaciens (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (Agrobacterium rhizogenes). In a preferred embodiment of the present invention, the agrobacterium is, for example, LBA4404, EHA101 and AGL1, C58C1, and the like, but is not limited thereto.

It has long been known that the Agrobacterium tumefaciens (Agrobacterium tumefaciens) is capable of causing crown gall disease (crown gall disease) in a variety of dicotyledonous plants, and in the 70's of the twentieth century, Ti plasmids were found to be associated with pathogenicity, and T-DNA, which is part of Ti plasmids, was recombined into the plant genome. It was thereafter understood that genes related to the synthesis of hormones (cytokinins and auxins) necessary for tumor induction exist on the T-DNA, which are expressed in plants in spite of bacterial genes. In the excision of T-DNA and transmission to plants, a gene group present in the Virulence (Virulence) region (vir region) on the Ti plasmid is necessary, and in addition, in order to make T-DNA excisable, border sequences (ボ - ダ side-by-side) present on both ends of T-DNA are necessary. Agrobacterium rhizogenes, which is another Agrobacterium bacterium, also has the same system based on the Ri plasmid (e.g., FIGS. 3 and 4 of Japanese patent laid-open No. 2000-342256).

Since T-DNA can be recombined into a plant genome due to infection with Agrobacterium, it is expected that a desired gene will be recombined into a plant genome when inserted into the T-DNA. However, since Ti plasmids are very large, at least 190kb, it is difficult to insert genes into T-DNA on the plasmid using standard genetic engineering methods. Therefore, methods for inserting foreign genes into T-DNA have been developed.

First, LBA4404 (see Hoekema, A., et al, (1983), Nature, Vol.303, p.179-180), C58C1(pGV3850), GV3Ti11SE, etc. were prepared as disarmed strains in which hormone synthesis genes were removed from T-DNA of a tumor Ti plasmid. By using these, 2 methods of introducing a desired gene into Ti plasmid T-DNA of Agrobacterium or introducing T-DNA having a desired gene into Agrobacterium have been developed. One of them is a method in which a desired gene can be inserted by easy genetic manipulation, by introducing an intermediate vector replicable in E.coli into the T-DNA region of the relieved type Ti plasmid of Agrobacterium by homologous recombination by the triparental hybridization method (referred to as the intermediate vector method).

Yet another method is called binary vector (binary vector) method, which is based on the following results: in the case of T-DNA recombination into plants, the vir region is required, but it is not necessary for the function to be present on the same plasmid. The vir region includes virA, virB, virC, virD, virE and virG (plant バイオテクノロジ A (エンタプライズ Kabushiki Kaisha (1989))), and the vir region includes all of virA, virB, virC, virD, virE and virG. Therefore, the binary vector is obtained by recombining T-DNA into a small plasmid that can replicate in both Agrobacterium and Escherichia, and can be used by introducing it into Agrobacterium having a relieved type Ti plasmid.

The introduction of the binary vector into Agrobacterium can be carried out by a known method such as electroporation or triple crossing. Among the binary vectors, there are pBIN19, pBI121, pGA482, etc., and based on these, a large number of novel binary vectors have been constructed for transformation. In addition, in the Ri plasmid system, the same vector was constructed for transformation.

The agrobacterium A281 is a strong-pathogenic (super-viral) strain, has a wide host range and has higher transformation efficiency than other strains. This property is due to the Ti plasmid pTiBo542 possessed by A281. To date, 2 new systems have been developed using pTiBo 542. Strains EHA101 and EHA105 using the pTiBo542 release type Ti plasmid are suitable for the above binary vector system, and are used as systems having high transformation efficiency for transformation of various plants.

The other uses a super binary vector system. Super-binary vectors ('super-binary' vectors) are described, for example, in the following documents cited in the present specification.

Hiei, Y., et al, (1994), The Plant Journal, Vol.6, p.271-282;

ishida, Y., et al, (1996), Nature Biotechnology, Vol.4, p.745-750;

komari, T.and Kubo T., (1999), Methods of Genetic Transformation: agrobacterium tumefaciens. in Vasil, I.K. (ed.) Molecular improvement of cellular crops, Kluwer Academic Publishers, Dordrecht, p.43-82; and

a pamphlet of international publication No. 95/06722,

a super binary vector system is described in, for example, FIG. 4 of Japanese patent laid-open No. 2000-342256.

This system is composed of a disarmed Ti plasmid having vir regions (virA, virB, virC, virD, virE and virG (hereinafter also referred to as "vir fragment regions", respectively)) and a plasmid having T-DNA, and is therefore one of binary vector systems. However, this is different from the case of using a super binary vector, which is a plasmid having a T-DNA side, that is, a binary vector, in which a fragment of the vir region (preferably a fragment containing at least virB or virG, more preferably a fragment containing both virB and virG) obtained by substantially removing at least one of the vir fragment regions is recombined. Furthermore, when a T-DNA region in which a desired gene is recombined is introduced into Agrobacterium having a super binary vector, homologous recombination by the three-line hybridization method can be used as an easy method.

In the method of the present invention, there is no particular limitation on the Agrobacterium as a host, and Agrobacterium tumefaciens (for example, the above-mentioned Agrobacterium tumefaciens LBA4404 (see Hoekema, A., et al, (1983), Nature, Vol.303, p.179-180) and EHA101) can be preferably used.

The method of the present invention is not particularly limited as long as it is a gene transfer system based on the expression of a gene group of pathogenic (vir) regions in the bacterium belonging to the genus Agrobacterium, and the effects of the present invention can be obtained.

For example, any vector system among the aforementioned intermediate vectors, binary vectors, strongly pathogenic binary vectors, super binary vectors and the like can be used, and the effects of the present invention can be obtained. However, from the viewpoint of further improving transformation efficiency, a strongly pathogenic binary vector or a super binary vector is preferable (particularly, when the plant to be introduced is maize, a super binary vector is preferably used). The same applies to different vector systems obtained by modifying these vectors (for example, a portion or all of the vir region of Agrobacterium is excised, then episomally incorporated into a plasmid, and a portion or all of the vir region is excised, and introduced into Agrobacterium as a new plasmid).

The gene desired to be introduced into a plant can be recombined into the restriction enzyme site in the T-DNA region of the above plasmid by a conventional method. In the case of a large-sized DNA having a plurality of restriction sites, it is sometimes not easy to introduce the desired DNA into the T-DNA region by a conventional subcloning method. In such a case, the target DNA can be introduced by homologous recombination in the cells of Agrobacterium by the three-line hybridization method. Although not limited thereto, the size of the introduced gene is preferably about 100bp to 200 kbp.

The introduction of the plasmid into Agrobacterium such as Agrobacterium tumefaciens can be carried out by a conventional method, and examples thereof include the above-mentioned three-line hybridization method, electroporation method, electroinjection method and chemical treatment method by PEG or the like.

As in the prior art, the gene to be introduced into a plant is basically located between the right and left border sequences of T-DNA. However, since the plasmid is circular, the number of border sequences may be 1, and when a plurality of genes are to be arranged at different positions, the number of border sequences may be 3 or more. In the case of Agrobacterium, the DNA may be placed on a Ti or Ri plasmid, or may be placed on another plasmid. Even more, it can be configured on a plurality of plasmids.

Inoculation of the plant material with agrobacterium bacteria can be carried out, for example, by simply contacting the plant material with agrobacterium bacteria. The inoculation may be carried out by a conventional inoculation, and further, may be carried out by a dropwise inoculation.

The conventional inoculation is a method of inoculating by the following manner: the plant material is mixed with an agrobacterium suspension (inoculum) and immersed in the suspension, and the immersed plant material is taken out and allowed to grow on a culture medium for coculture. This can be done, for example: preparation 10⁶～10¹¹A plant material is immersed in an Agrobacterium suspension having a cell concentration of about cfu/ml for about 3 to 10 minutes, and then cultured on a solid medium for several days. The drop inoculation is a method of performing inoculation by the following method: the suspension of Agrobacterium is dropped on the plant material seeded on the culture medium, and after the dropped suspension is dried, the plant material is seeded on other positions of the culture medium or other culture media for co-culture.

Coexistence step

In this step, the DNA is introduced into the plant cells from Agrobacterium reliably by culturing the Agrobacterium-inoculated plant cells in the presence of Agrobacterium in a medium containing auxins. Preferably, the plant material is co-cultured with the agrobacterium at the same time as or after infection of the plant material with the agrobacterium and before removal of the agrobacterium.

The medium used in this step is referred to as "coculture medium" in the present specification. In the coculture, a known medium can be used. For example, LS-AS medium, nN6-AS medium, N6S3-AS medium, 2N6-AS medium (see Hiei, Y., et al, (1994), the plant Journal, Vol.6, p.271-282) and the like are known.

In the present invention, it is preferable to add an auxin to the coculture medium. Auxins generally have an effect of dedifferentiating plant materials, and in this step, substantially all or part of the plant materials become dedifferentiated tissues (callus). Examples of auxin compounds include 3, 6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3, 5, 6-trichloropyridinecarboxylic acid (picloram), 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) and/or triiodobenzoic acid (TIBA). In a preferred embodiment of the present invention, the coculture medium does not contain dicamba, picloram, 2, 4-D, and auxins other than 2, 4, 5-T.

Without limitation, the total amount of auxins such as dicamba, picloram, 2, 4-D, and 2, 4, 5-T in the coculture medium is preferably 0.1 to 5.0mg/L, more preferably 0.5 to 3.0mg/L, still more preferably 1.0 to 2.0mg/L, and most preferably 1.5 mg/L.

The inventors of the present invention found that: when the plant material is corn, the transformation efficiency is further improved by adding a substance exhibiting the activity of an auxin, particularly an auxin belonging to a benzoic acid herbicide, among the auxins to the coculture medium, and a transformed plant can be obtained without introducing a selection marker gene.

Benzoic acid herbicides can be classified into (i) benzoic acid type, (ii) salicylic acid type, (iii) picolinic acid type, (iv) terephthalic acid type (Takematsu, 1982) according to their basic structure. However, the (iv) terephthalic acid type does not show auxin activity, and thus is preferably a herbicide belonging to any one of the (i) benzoic acid type, (ii) salicylic acid type, (iii) picolinic acid type, more preferably any one of the (ii) salicylic acid type, (iii) picolinic acid type. More preferably Dicamba (3, 6-dichoro-o-anisic acid) or Picloram (Picloram) (4-amino-3, 5, 6-trichlorocolitic acid). Therefore, it is most preferable to add a substance exhibiting the activity of an auxin belonging to a benzoic acid herbicide to the coculture medium for corn.

Alternatively, in the case where the plant material is rice, 2, 4-dichlorophenoxyacetic acid (2, 4-D) is preferably added.

The term "culturing" in this step means: to implant plant tissues (Bed) on the solidified coexisting culture medium or in the liquid coexisting culture medium, and growing and breeding in an appropriate temperature, light and dark conditions and time (period). The solidification of the coculture medium can be carried out by adding a solidifying agent known in the art, and for example, agarose or the like is known as such a solidifying agent. The temperature for the culture in this step may be suitably selected, and is preferably carried out at 20 ℃ to 35 ℃, more preferably at 25 ℃. Further, the culture in this step is preferably carried out in a dark place, but is not limited thereto. The culture time in this step may be suitably selected, and is preferably 1 to 10 days, more preferably 7 days.

Callus proliferation step

In the plant transformation method using Agrobacterium, a callus proliferation step is generally considered to be necessary.

By transferring to a callus growth medium and growing the callus, a "cell mass of a population containing transformed cells" can be obtained. The callus proliferation culture medium refers to: a medium containing a plant hormone and a nutrient suitable for dividing and proliferating cells in a dedifferentiated state is also used as a "selective medium" for selectively proliferating transformed cells by adding a drug (selective pressure) that inhibits the proliferation of untransformed cells in a general transformation test. Therefore, the callus growth step is usually a step of culturing the plant material having undergone the above-mentioned coexistence step in a medium containing auxins, and selecting a transformant by the presence or absence of gene transfer. The medium used in this step is referred to as "selective medium" in the present specification, and includes a selective drug or the like for selection by the presence or absence of gene transfer.

This step is carried out by changing the composition of the medium in the conventional method and repeating the steps several times. For example, in the selection step of a plurality of times, by increasing the concentration of the selection drug in each selection step, the thoroughness of drug selection can be increased, and the possibility of obtaining a transformed plant can be increased. This selection step is preferably carried out at least 2 times, more preferably 3 times. When the step is carried out for a plurality of times, the step needs about 10 days to 3 weeks for 1 time, and the step needs about 5 to 10 weeks for carrying out a plurality of selections. Therefore, in the transformation method of plants using Agrobacterium, this step is the most time-consuming step.

In the conventional plant transformation method using Agrobacterium, this step can be considered as an essential step. However, the present inventors have found for the first time that: the present inventors have also conceived the present invention that the transformation can be efficiently performed by "transformation-promoting treatment" in which the selection of transformed cells using a selection drug is not performed in any step from the coexistence to the redifferentiation including the callus proliferation step. Therefore, it is preferable in the present invention that this step can be omitted. That is, the step of culturing the tissue after the coculture in the callus growth medium is not included between the coculture step and the redifferentiation step. The following are found: this can further improve the working efficiency and allow the transformation step to be carried out in a shorter time, thereby obtaining the transformed plant more efficiently. Examples 1 to 4 and 6 to 7 in the present specification describe examples in which, in particular, in maize, callus growth steps are omitted and successful plant transformation is achieved.

Alternatively, the callus growth step may be performed without adding a selective drug to the callus growth medium. In this case, although callus proliferation occurs, the "selection" of transformants is not performed.

V. redifferentiation step

This step is a step of redifferentiating the obtained cell mass, growing and breeding redifferentiated individuals, and obtaining complete plant bodies as required. In order to regenerate a complete Plant body from The resulting transformed cells, it can be carried out according to a known method (e.g., Hiei, Y., et al, (1994), The Plant Journal, Vol.6, p.271-282; and Ishida, Y., et al, (1996), Nature Biotechnology, Vol.14, p.745-750).

This step is an essential step in both the conventional method and the present invention. Conventionally, it is considered that selection of transformants using a selection drug is necessary even in the redifferentiation step. The transformed plant can be selected by culturing the plant material subjected to the co-existence step with a redifferentiation medium containing a selection drug, and depending on the presence or absence of resistance to the selection drug. However, it is a feature of the present invention that in any step from the coexistence to the transformation of a redifferentiated plant, a selection of transformed cells is not performed using a selective drug. Therefore, in the present invention, even in the redifferentiation step, selection is not performed using a selective drug.

The medium used in this step is referred to as a "redifferentiation medium" in the present specification, and examples of the redifferentiation medium include a medium containing no auxin. Examples of the medium that can be used as the redifferentiation medium include a medium containing LS inorganic salts and N6 inorganic salts as essential components, and specifically, LSZ medium and the like can be used. However, the "redifferentiation medium" does not contain a selective drug.

The "redifferentiation" in the present invention means: the fully or partially dedifferentiated plant material regains the properties of the original plant material or plant body. When the dedifferentiation step is provided, the dedifferentiated tissue is redifferentiated, and a completely transformed plant can be obtained. Whether or not redifferentiation of the plant has occurred can be easily determined by observing the morphology of the plant. For example, it can be determined whether a specific differentiated plant organ such as a stem or a leaf appears from a dedifferentiated tissue.

In the present specification, "vigor (vigor)" means the degree of growth of the redifferentiated plant. The vigor of the plant can be determined by known methods known in the art. For example, the average value of all transformed plant tissues can be determined by scoring the maize, wherein the transformed plant tissues that are not redifferentiated after the redifferentiation step are 0 point, the transformed plant tissues that have the maximum length of redifferentiated shoots of less than 5mm are 1 point, the transformed plant tissues that have the maximum length of redifferentiated shoots of 5mm or more and less than 2cm are 2 points, and the transformed plant tissues that have the maximum length of redifferentiated shoots of 2cm or more are 3 points. However, the method of evaluating the vitality is not limited to this, and a known method may be appropriately modified depending on the evaluation target or the like.

The term "culturing" in this step means: plant tissues are planted on the solidified redifferentiation culture medium or the liquid redifferentiation culture medium and grown and bred in a suitable temperature, light and shade condition and time (period). Solidification of the redifferentiation medium can be carried out by using agar or the like, for example, as described above. The temperature for the culture in this step may be suitably selected, and is preferably carried out at 20 ℃ to 35 ℃, more preferably 25 ℃. Further, the cultivation in this step is preferably carried out under illumination for 16 to 24 hours/day, but not limited thereto. The culture time in this step may be suitably selected, and is preferably 7 to 21 days, more preferably 14 days.

After this step, a completely transformed plant can be easily obtained by a method known in the art. This is a step of confirming the presence or absence of the introduced gene in the redifferentiated individual obtained and specifying the transformed individual. Although not limited thereto, PCR, Southern analysis, or the like can be preferably used. Furthermore, selection can be easily performed by confirming the phenotype of the introduced gene.

ADVANTAGEOUS EFFECTS OF INVENTION

By the present invention, a desired plant can be stably and efficiently transformed in a method for transforming a monocotyledon without introducing a plant selection marker gene.

Drawings

FIG. 1 shows the results of Southern blot analysis of a snow-light redifferentiated plant body obtained by non-selective transformation.

Genomic DNA was extracted from the redifferentiated plant body transformed with Agrobacterium LBA4404(pSB134) and obtained without selection, and digested with restriction enzyme HindIII. The digested DNA was subjected to agarose gel electrophoresis and hybridized with a GUS probe. Seed-derived Yukihikari (control) (lane C), GUS-positive expression (uniform expression of GUS) redifferentiated plant bodies (lanes 1 to 11), and GUS spot expression redifferentiated plant bodies (lanes 12 to 17).

FIG. 2 shows the results of Southern blot analysis of A188 redifferentiated plant bodies obtained by non-selective transformation.

Genomic DNA was extracted from redifferentiated plant bodies (maize) transformed with Agrobacterium LBA4404(pSB124) and obtained without selection, and digested with the restriction enzyme BamHI. The digested DNA was subjected to agarose gel electrophoresis and hybridized with a GUS probe. Seed-derived A188 (control) (lane C), GUS positive expression (uniform expression of GUS), redifferentiated plant bodies (lanes 1-13).

FIG. 3 shows the results of Southern blot analysis of A188 redifferentiated current generation plant bodies and T1 progeny plant bodies obtained by non-selection transformation.

A redifferentiated current generation plant body (maize) transformed with Agrobacterium LBA4404(pSB124) and obtained without selection was mated with pollen of an untransformed A188 plant, and genomic DNA was extracted from the obtained T1 progeny plant body and digested with the restriction enzyme BamHI. The digested DNA was subjected to agarose gel electrophoresis and then hybridized with a GUS probe. Plant of the present generation (T0) line No. 195 (lane 1), plant of the progeny of the GUS positive expression (T1) line No. 195 (lanes 2-6), plant of the GUS negative progeny (T1) line No. 195 (lane 7). Plant of the current generation (T0) line No. 169 (lane 8), plant of the progeny of the GUS positive expression (T1) line No. 169 (lanes 9-13), and plant of the progeny of the GUS negative expression (T1) line No. 169 (lane 14).

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Gene expression in plants indiscriminately redifferentiated from maize immature embryos inoculated by conventional methods

1. Materials and methods

Immature embryos (1.0-1.5 mm in size) of maize (variety: A188) harvested 7-14 days after pollination were aseptically harvested and washed 1 time with LS-inf liquid medium (Ishida et al, 1996). As the transformation-enhancing treatment, pretreatment for enhancing the gene transfer efficiency (heat treatment at 46 ℃ for 3 minutes and centrifugation at 20,000Xg for 10 minutes) was carried out (Hiei et al, 2006). About 1.0X10 in LS-inf liquid medium containing 100. mu.M acetosyringone⁹cfu/ml Agrobacterium suspension strain LBA4404(pSB134) (Hiei and Komari, 2006) was used as the inoculum.

The heat/centrifugation treated immature embryos were added with an inoculum, stirred for 30 seconds and then allowed to stand at room temperature for 5 minutes. In addition to 2, 4-D (2, 4-dichlorphenoxy-acetic acid), 5 mu M AgNO₃And 5. mu.M CuSO₄Dicamba (3, 6-dichoro-o-anisic acid) was added to LS-AS medium (Ishida et al, 1996, 8g/L agarose AS a solidifying agent) at a concentration of 1.5mg/L to obtain a co-productThe immature embryos inoculated with Agrobacterium are seeded on the coculture medium and the blastoderm is allowed to stand.

Immature embryos obtained by culturing for 7 days at 25 ℃ in the dark are implanted in a medium containing 10. mu.M CuSO₄The LSZ medium of (Ishida et al, 1996), was cultured under illumination conditions at 25 ℃ for about 2 weeks. A part of the leaf of the plant body in which redifferentiation was observed was cut out, immersed in 0.1M phosphate buffer (pH6.8) containing 0.1% Triton X-100, and allowed to stand at 37 ℃ for 1 hour. After removing the phosphate buffer, a phosphate buffer containing 1.0mM 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ℃ for 24 hours, the expression of GUS gene was examined.

The conditions of example 1 are summarized below.

Materials: immature embryo of corn

The method comprises the following steps: inoculation of Agrobacterium by conventional methods

And (3) conversion improvement treatment: heat and centrifugal treatment, adding Ag to the culture medium⁺、Cu²⁺Treatment of ions auxin added to the coculture Medium: dicamba

After the coculture, the callus was directly subjected to the redifferentiation step without going through the callus proliferation step.

Carrier: super binary vector with pSB134GUS gene, hygromycin resistance gene (selection marker gene) and partial pathogenic gene of strong pathogenic strain

Selecting and processing: all the way is without

2. Results

Redifferentiation of the plant body was observed for all 16 immature embryos implanted in the LSZ medium. 4-13 leaves were collected from plants redifferentiated from each immature embryo, and the expression of GUS gene was examined.

All of the 2 immature embryo-derived leaves were GUS-negative. From among leaves collected from the remaining 14 immature embryos, at least 1 leaf was able to observe the expression of GUS gene. Leaves showing punctate expression were observed in 5 immature embryos. Streaky leaves expressing the GUS gene were observed in 6 immature embryos. 2 immature embryos were found to have both punctate and striated expressed leaves. Here, the streak-like expressed leaves are considered to be a chimera of transformed cells and non-transformed cells, while the dot-like expressed leaves are considered to be a portion of the transformed cells in which gene silencing has occurred. From 1 immature embryo, GUS-positive leaves with incisions uniformly stained blue were obtained.

The results of this example show that a maize plant obtained by transformation with the GUS gene under conditions where no selection step using antibiotics or the like is performed in any step from coexistence until redifferentiation is obtained.

Example 2 Gene expression in plants indiscriminately redifferentiated from maize immature embryos inoculated with the dropping method

1. Materials and methods

To 1ml of the inoculum source of Agrobacterium strain LBA4404(pSB134) prepared according to the method of example 1, about 80mg of hydroxyapatite (Bio-Rad) was added. As the transformation-enhancing treatment, pretreatment for enhancing the gene transfer efficiency (heat treatment at 46 ℃ for 3 minutes and centrifugation at 20,000Xg for 10 minutes) was performed.

The immature embryos (variety A188) after the above treatment were seeded on a coculture medium containing 5. mu.M AgNO in addition to 2, 4-D and allowed to set on the blastoderm₃And 5. mu.MCuSO₄In LS-AS medium (Ishida et al, 1996, 8g/L agarose AS a solidifying agent) of (1.5 mg/L).

Stirring was performed with a Vortex Mixer (Vortex Mixer) to uniformly disperse hydroxyapatite in the inoculation source, and then 5. mu.l of the inoculation source was dropped onto the immature embryo. After the dropwise added inoculum source has dried, the immature embryos are moved to other locations on the same medium. The incubator was sealed and then incubated at 25 ℃ in the dark for 7 days. Immature embryos after coculture were cultured in the same manner as in example 1 to obtain redifferentiated plants, and the leaves of the redifferentiated plants were examined for GUS gene expression.

The conditions of example 2 are summarized below.

Materials: immature embryo of corn

The method comprises the following steps: inoculation of Agrobacterium by the titration method

And (3) conversion improvement treatment: powder treatment, heat and centrifugation, and addition of Ag to the culture medium⁺、Cu²⁺Treatment of ions

Auxin added in the coexistence step: dicamba

After the coculture, the callus was directly subjected to the redifferentiation step without going through the callus proliferation step.

Carrier: super binary vector with pSB134GUS gene, hygromycin resistance gene (selection marker gene) and partial pathogenic gene of strong pathogenic strain

Selecting and processing: all the way is without

2. Results

Redifferentiation of the plant body was observed for all 12 immature embryos implanted in the LSZ medium. 3-17 leaves were collected from plants redifferentiated from each immature embryo, and the expression of GUS gene was examined. All leaves from 3 immature embryos were GUS negative. For leaves harvested from the remaining 9 immature embryos, the expression of GUS gene was observed in at least 1 leaf. Among 3 immature embryos, leaves showing punctate expression were observed. Among 4 immature embryos, it was observed to have both a spotted expression leaf and a striped expression leaf. From 2 immature embryos, GUS-positive leaves were obtained in which the incisions were uniformly stained blue.

The results of this example show that a maize plant obtained by transformation with the GUS gene under conditions where no selection step using antibiotics or the like is performed in any step from coexistence until redifferentiation is obtained.

Example 3 Effect of auxin in Co-Presence Medium on Gene expression in plants indiscriminately redifferentiated from maize immature embryos

1. Materials and methods

Immature embryos (1.0-1.5 mm in size) of maize (variety: A188) harvested 7-14 days after pollination were collected aseptically and washed 1 time with LS-inf liquid medium. As the transformation-enhancing treatment, pretreatment (heat treatment at 46 ℃ for 3 minutes) for enhancing the gene transfer efficiency was performed. The soil rod strain LBA4404(pSB124) (Komari et al, 1996) was suspended as an inoculum in LS-inf liquid medium containing 100. mu.M acetosyringone.

The heat-treated immature embryo was added with an inoculation source, stirred for 30 seconds, and then allowed to stand at room temperature for 5 minutes. Immature embryos inoculated with Agrobacterium are seeded on a co-culture medium containing 5. mu.M AgNO in addition to 2, 4-D (2, 4-dichlorphenoxy-acetic acid) and the blastoderm is oriented upwards to co-seed 24 immature embryos₃And 5. mu.M CuSO₄In LS-AS medium (Ishida et al, 1996, with a solidifier of 8g/L agarose) at a concentration of 6.8. mu.M, with dicamba (3, 6-dichoro-o-anisic acid) or Picloram (Picloram) (4-amino-3, 5, 6-trichlorocolitic acid).

On the other hand, the composition is also used in a composition containing 5. mu.M AgNO₃And 5. mu.M CuSO₄The LS-AS medium of (1.5 mg/L (6.8. mu.M)) of 2, 4-D was used AS the growth hormone medium for the experiments. 24 immature embryos were implanted.

Immature embryos obtained by culturing for 7 days at 25 ℃ in the dark are implanted in a medium containing 10. mu.M CuSO₄LSZ medium (Ishi)da et al, 1996), and culturing under illumination at 25 ℃ for about 2 weeks. A part of the leaf of the plant body in which redifferentiation was observed was cut out, immersed in 0.1M phosphate buffer (pH6.8) containing 0.1% Triton X-100, and allowed to stand at 37 ℃ for 1 hour. After removing the phosphate buffer, a phosphate buffer containing 1.0mM 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ℃ for 24 hours, the expression of GUS gene was examined.

The conditions of example 3 are summarized below.

Materials: immature embryo of corn

The method comprises the following steps: inoculating Agrobacterium by conventional method

And (3) conversion improvement treatment: heat treatment, adding Ag to the culture medium⁺、Cu²⁺Treatment of ions

Auxin added to the coculture Medium: dicamba, picloram or 2, 4-D

After the coculture, the callus was directly subjected to the redifferentiation step without going through the callus proliferation step.

Carrier: super binary vector having pSB124GUS gene, but no selection marker gene, and having a part of pathogenic gene of highly pathogenic strain

Selecting and processing: all the way is without

2. Results

Redifferentiation of the plant body was observed for 24 immature embryos implanted on the LSZ medium for immature embryos cultured with the coculture medium containing dicamba. Leaves were collected from plants redifferentiated from each immature embryo, and the expression of GUS gene was examined. All of the leaves from 13 immature embryos were GUS negative. From among leaves collected from the remaining 11 immature embryos, at least 1 leaf was able to observe the expression of GUS gene. Individuals with incomplete expression, such as punctate expression, striped expression, etc., were also counted.

In immature embryos cultured in the co-culture medium containing picloram, redifferentiation of the plant body was also observed on the LSZ medium in its entirety. Leaves from 15 immature embryos were all GUS negative. For leaves collected from the remaining 9 immature embryos, GUS gene expression was observed in at least 1 leaf.

Among 24 immature embryos co-cultured with a medium containing 2, 4-D, 3 immature embryos were not observed for redifferentiation of the plant on LSZ medium. Of the 21 immature embryos in which redifferentiation was observed, 4 immature embryos were able to observe the expression of GUS gene in at least 1 leaf.

Thus, in particular, in immature embryos co-cultured with a medium containing dicamba and picloram, efficient redifferentiation of plant bodies was observed, and the obtained redifferentiated plants showed a high proportion of the expression of the introduced gene. In contrast, the expression of the transferred gene was observed in immature embryos co-cultured in a medium containing 2, 4-D, but to a slightly lesser extent.

The results of this example show that a maize plant obtained by transformation with the GUS gene under conditions where no selection step using antibiotics or the like is performed in any step from coexistence until redifferentiation is obtained. 2, 4-D, dicamba and picloram are all substances that exhibit auxin activity among organic compounds with herbicidal activity. The difference is that 2, 4-D belongs to the phenoxy class of herbicides, whereas dicamba and picloram belong to the benzoic class of herbicides (Zhusong, 1982). The above results show that, in corn, in order to efficiently obtain transformed plants from immature embryos without selection, a substance belonging to a benzoic acid herbicide is more suitable than a substance belonging to a phenoxy herbicide in terms of auxin contained in a medium in which immature embryos inoculated with agrobacterium are co-cultured.

Example 4 Gene expression in plants redifferentiated without selection by Heat and centrifugation treatment of immature embryo of maizeAchieved effect

1. Materials and methods

Immature embryos (1.0-1.5 mm in size) of maize (variety: A188) harvested 7-14 days after pollination were collected aseptically and washed 1 time with LS-inf liquid medium. As the transformation-enhancing treatment, pretreatment for enhancing the gene transfer efficiency (heat treatment at 46 ℃ for 3 minutes and centrifugation at 20,000Xg for 10 minutes at 4 ℃) was performed. As a control, an immature embryo which had not been subjected to the above pretreatment was provided. The soil rod strain LBA4404(pSB124) was suspended as an inoculum in LS-inf liquid medium containing 100. mu.M acetosyringone.

The immature embryos subjected to heat and centrifugal pretreatment and the control immature embryos not subjected to pretreatment were added with an inoculation source, respectively, stirred for 30 seconds and then allowed to stand at room temperature for 5 minutes. The immature embryos inoculated with Agrobacterium are seeded on a co-cultivation medium containing 5. mu.M AgNO in addition to 2, 4-D (2, 4-dichloro-acetic acid) and the blastodisc is oriented upwards₃And 5. mu.M CuSO₄In LS-AS medium (Ishida et al, 1996, with a solidifier of 8g/L agarose) at a concentration of 6.8. mu.M, with dicamba (3, 6-dichoro-o-anisic acid). 75 immature embryos were provided, respectively. The test was performed 2 times.

Immature embryos obtained by culturing for 7 days at 25 ℃ in the dark are implanted in a medium containing 10. mu.M CuSO₄The LSZ medium of (Ishida et al, 1996), was cultured under illumination conditions at 25 ℃ for about 2 weeks. A part of the leaf of the plant body in which redifferentiation was observed was cut out, immersed in 0.1M phosphate buffer (pH6.8) containing 0.1% Triton X-100, and allowed to stand at 37 ℃ for 1 hour. After removing the phosphate buffer, a phosphate buffer containing 1.0mM 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ℃ for 24 hours, the expression of GUS gene was examined.

The conditions of example 4 are summarized below.

Materials: immature embryo of corn

The method comprises the following steps: inoculating Agrobacterium by conventional method

And (3) conversion improvement treatment:

(1) heat and centrifugal treatment, adding Ag to the culture medium⁺、Cu²⁺Treatment of ions

(2) No heat and centrifugation treatment was performed, but addition of Ag to the medium was performed⁺、Cu²⁺Treatment of ions

Auxin added to the coculture Medium: dicamba

After the coculture, the callus was directly subjected to the redifferentiation step without going through the callus proliferation step.

Carrier: super binary vector having pSB124GUS gene, but no selection marker gene, and having a part of pathogenic gene of highly pathogenic strain

Selecting and processing: all the way is without

2. Results

In trial 1, 296 shoots (shoots) were redifferentiated from 75 immature embryos that had been subjected to heat and centrifugation. Among these, 5 individuals showed GUS gene expression in the whole leaf tissue. On the other hand, 291 seedlings were redifferentiated from 75 immature embryos which were not subjected to pretreatment, and the number of individuals showing GUS gene expression in the whole leaf tissue was 0. In the 2nd experiment, 243 seedlings were redifferentiated from the immature embryo subjected to heat/centrifugation treatment, and among them, GUS-positive individuals in which the tissues of 4 leaves showed GUS gene expression in their entirety were obtained.

266 shoots were observed redifferentiated from the immature embryos which were not subjected to the heat/centrifugation treatment, and among them, 1 individual was a GUS-positive individual whose leaf tissue showed GUS gene expression as a whole. These results show that, particularly, by heat/centrifugation treatment of immature embryos before inoculation with Agrobacterium, it is possible to efficiently obtain individuals expressing an introduced gene in the whole plant tissue, which are re-differentiated without selection. However, the reason why the result of gene transfer was obtained for only 1 individual was considered to be the effect of adding silver nitrate and copper sulfate to the coculture medium.

Example 5 non-selection transformation of Rice

1. Materials and methods

Immature seeds of rice (variety: snowy light) 8 to 12 days after flowering are glume-removed, treated with 70% ethanol for several seconds, and then treated with 1% sodium hypochlorite containing 1 drop of Tween20 (registered trademark) for 5 minutes. The immature seeds were washed several times with sterilized water, and immature embryos 1.3-1.8mm long were collected. Next, as transformation enhancement treatment, in order to enhance the gene transfer efficiency, the immature embryo was centrifuged at 20,000Xg for 10 minutes (Hiei et al, 2006). About 1.0x10 in AA-inf liquid medium (Hieiand Komari, 2006) containing 100. mu.M acetosyringone⁹concentration of cfu/ml Agrobacterium LBA4404(pSB134) (Hiei and Komari, 2006) was used as an inoculum. The centrifuged immature embryos were seeded in blastoderm-side up on nN6-As medium (Hiei et al, 2006). Mu.l of an inoculum was added dropwise to each immature embryo, and the embryos were cultured in the dark at 25 ℃ for 7 days in the coexistence.

After the cocultivation, the immature embryos are divided into 4 to 5 parts by a scalpel. The dissected immature embryos were seeded on nN6 medium (Hiei et al, 2006) containing 250mg/L cefotaxime (cefotaxime) and 100mg/L carbenicillin in a blastoderm-side up manner and cultured at 30 ℃ for about 10 days under bright conditions. The sections that are enlarged mainly by the proliferation of blastodermal cells are divided into 4 to 5 parts. At this stage, 18-25 sections were obtained for each immature embryo. The sections were seeded in blastoderm-side up in nN6 medium (Hiei et al, 2006) containing 250mg/L cefotaxime and 100mg/L carbenicillin, and cultured at 30 ℃ for about 2 weeks under bright conditions.

After the coculture, the callus was proliferated by 2 cultures, and the cells of each blastoderm were proliferated about 140 times, and the sections were covered with the callus. The growth rate of cells can be estimated from the size of each section. From the calli formed on the sections, only 1 callus (0.5-1mm large) was taken per section, and seeded on N6R redifferentiation medium (Hiei et al, 2006) and cultured for 2 weeks under the bright conditions at 30 ℃. The reason why only 1 callus was seeded on the redifferentiation medium from each section was to obtain a plant derived from random and independent cells. The cluster shoots (shoot) redifferentiated from each callus were transplanted into N6F rooting medium (Hiei et al, 2006), and cultured under the bright conditions at 30 ℃ for about 10 days to obtain completely redifferentiated plants. Furthermore, the above medium does not contain any selective drugs such as hygromycin and bialaphos.

A part of the leaf of the obtained plant was cut, immersed in 0.1M phosphate buffer (pH6.8) containing 0.1% Triton X-100 (registered trademark), and left to stand at 37 ℃ for 1 hour. After removing the phosphate buffer, a phosphate buffer containing 1.0mM 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ℃ for 24 hours, the expression of GUS gene was examined. Furthermore, the examination of GUS gene expression was carried out in1 leaf from the largest 1 plant produced from 1 section. Transformation efficiency was evaluated by the ratio of the number of plants showing GUS positivity (uniform expression of GUS)/the number of plants examined for GUS gene expression.

Furthermore, in order to confirm whether or not the introduced gene has been recombined in the genome of the redifferentiated plant body, Southern blot analysis was performed by the following method. DNA was extracted from the leaves of redifferentiated plants by the method of Komari (1989), and 7. mu.g of DNA was digested with restriction enzyme HindIII for each plant. The digested DNA was subjected to 0.7% agarose gel electrophoresis (1.5V/cm, 15 hours). Southern hybridization was carried out according to the method of Sambrook et al (1989). Furthermore, the probe used was a SalI-SacI (1.9kb) fragment of pGL2-IG (Hiei et al, 1994) as a GUS gene fragment.

The conditions of example 5 are summarized below.

Materials: rice immature embryo

The method comprises the following steps: inoculating Agrobacterium by conventional method

And (3) conversion improvement treatment: centrifuging, and adding cefotaxime and carbenicillin into culture medium

Auxin added in coexistence step: 2, 4-D

After the coculture, the callus is proliferated, and then redifferentiation is performed.

Carrier: super binary vector having pSB134GUS gene, hygromycin resistance gene (selection marker gene) and part of pathogenic gene of highly pathogenic strain

Selecting and processing: all the way is without

2. Results

The test was performed 2 times (tests 1 and 2). The results of test 1 are shown in table 1. In test 1, 100 divided sections were obtained from 5 immature embryos, and from this 100 calli were implanted in redifferentiation medium, respectively, to obtain 92 plants. Among 73 plants, 1 leaf was taken out and the expression of GUS gene was examined, and 9 plants among them were (GUS-positive) transformants which expressed GUS uniformly in the entire leaf tissue. The transformation efficiency was 12.3% with respect to the redifferentiated plant body.

The results of test 2 are shown in table 2. In experiment 2, 107 divided sections were obtained from 5 immature embryos, and from these 107 calli were implanted in redifferentiation medium, 100 plants were obtained. Among these, 1 leaf from each of 95 plants was examined for the expression of GUS gene, and 16 plants were transformants which expressed GUS uniformly. The transformation efficiency was 16.8% with respect to the redifferentiated plant body. As described above, a transformant was obtained with a very high efficiency of 10% or more relative to a redifferentiated plant body without any selection step.

In both of test 1 and test 2, the GUS gene expression was spotted in leaves of several plants (tables 1 and 2). The abnormal expression may be considered to be caused by gene silencing. Moreover, the plant showing punctate expression is not considered to be a transformant.

The results of the Southern blot analysis are shown in FIG. 1. In redifferentiated plants showing positive GUS gene expression, the presence of the introduced GUS gene was confirmed in all of the 11 individuals examined (FIG. 1). In redifferentiated plants in which GUS gene expression was spotted, the presence of the GUS gene was confirmed in all of the 6 individuals examined, but the number of copies of the introduced gene tended to be large in all of the individuals examined (fig. 1). Furthermore, although Southern blot analysis was also performed on redifferentiated plants that were negative for GUS gene expression, no band that hybridized with the GUS probe was detected in any of the 7 plants tested.

In this example, blastodermal cells of immature embryos after gene introduction were proliferated, and redifferentiated plants were obtained from calli randomly selected therefrom. Among these, 10% or more of these are transformed plants. This fact indicates that more than 10% of blastodermal cells of immature embryos infected by Agrobacterium are transformed.

[ Table 1]

Use of Rice immature embryos with Agrobacterium LBA4404(pSB134)

Preparation of non-selection transformant (test 1)

[ Table 2]

Use of Rice immature embryos with Agrobacterium LBA4404(pSB134)

Preparation of non-selection transformant (test 2)

Example 6 Southern analysis in Current generations of transformed maize plants obtained without selection

1. Materials and methods

The transformed maize (variety: A188) obtained in examples 3 and 4 was cultivated in a greenhouse. DNA was extracted from the leaves of these plants by the method of Komari et al (Komari et al, (1989)), and 10. mu.g of the DNA was digested with the restriction enzyme BamHI for each plant. The digested DNA was subjected to 0.7% agarose gel electrophoresis (1.5V/cm, 15 hours). Southern hybridizations were performed according to the method of Sambrook et al (Sambrook, J., et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.). Furthermore, the probe used was a SalI-SacI (1.9kb) fragment of pGL2-IG (Hiei et al, 1994) as a GUS gene fragment.

2. Results

Any of the transformants showed a band hybridizing to the GUS probe. This pattern varied from transformant to transformant, indicating that the introduced gene was randomly inserted into the chromosome of the plant. Thus, it was confirmed by a molecular level method that the maize plant obtained by the non-selection method was a transformant (FIG. 2).

Example 7 introduction of Gene into transgenic maize obtained without selection to inheritance in progeny plants

1. Materials and methods

The transformed maize (variety: A188) obtained in examples 3 and 4 was cultivated in a greenhouse. Collecting pollen of untransformed A188 plant and villi of transformed plant () Mating to obtainGeneration seed (T1 generation).

The seeds were sown in a vinyl pot to which culture soil was added. Leaves of seedlings on the 11 th day after sowing were taken, immersed in 0.1M phosphate buffer (pH6.8) containing 0.1% Triton X-100, and allowed to stand at 37 ℃ for 1 hour. After removing the phosphate buffer, a phosphate buffer containing 1.0mM 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ℃ for 24 hours, the expression of GUS gene was examined.

DNA was extracted from the leaves of the current generation plants and the above-mentioned T1 plants by the method of Komari (1989), and 10. mu.g of the DNA was digested with BamHI, which is a restriction enzyme, for each plant. The digested DNA was subjected to 0.7% agarose gel electrophoresis (1.5V/cm, 15 hours). Southern hybridization was carried out according to the method of Sambrook et al (1989). Furthermore, the probe used was a SalI-SacI (1.9kb) fragment of pGL2-IG (Hiei et al, 1994) as a GUS gene fragment.

2. Results

Table 3 shows the results of examining the segregation of GUS gene in progeny plants of maize transformed plants obtained by non-selection transformation.

[ Table 3]

Of maize transformed plants obtained by non-selection transformation

Isolation of GUS Gene in progeny plants

As shown in Table 3, for any of the 4 lines of transformed plants examined, the expression of the GUS gene in the progeny plants showed positive and negative segregation. The number of plants showing positive expression of GUS gene and the number of plants showing negative expression were in a ratio of 1: 1 or 3: 1 in any of the lines, and it was confirmed that the GUS gene introduced by non-selection transformation was inherited to progeny plants according to the Mendelian method.

DNA extracted from the transformed contemporary plant of line number 195 showed 1 hybrid band. The DNA extracted from the plants of 5 individuals showing GUS positivity among the T1 progeny plants all showed 1 band of the same size as the T0 plants. On the other hand, no hybridization band was detected by using DNA extracted from a plant showing GUS-negativity.

DNA extracted from the transformed contemporary plant of line No. 169 showed 5 hybrid bands. The DNA extracted from the plants of 5 individuals showing GUS-positivity among the T1 progeny plants all showed bands of the same size as those of the T0 plant, but the number thereof differed by 1, 4 and 5 bands. GUS gene expression in T1 plants of line No. 169 showed two-factor segregation, and it was considered that 4 copies and 1 copy of the GUS gene were integrated into different chromosomes, respectively. Using DNA extracted from a plant showing GUS-negativity, no hybridization band was detected (FIG. 3).

From the above, it was confirmed that the gene introduced into the maize transformed plant obtained by the non-selection transformation was inherited to the progeny plant according to the Mendelian rule.

Claims (21)

1. A method for producing transformed corn or rice using agrobacterium, comprising:

(i) a step of culturing the Agrobacterium-inoculated corn or rice material in a co-culture medium; and

(ii) (ii) culturing the tissue obtained in (i) in a redifferentiation medium without subjecting the tissue to a callus growth culture to redifferentiation into corn or rice, or culturing the tissue obtained in (i) in a redifferentiation medium after subjecting the tissue to a callus growth culture to redifferentiation into corn or rice;

wherein, in the method for producing transformed corn or rice,

1) performing a conversion-enhancing treatment, and

2) in any step from coexistence to redifferentiation, the transformed cells are not selected using the characteristics of the nucleic acid introduced by Agrobacterium,

wherein 1) the conversion-enhancing treatment is selected from:

heat treatment;

centrifuging;

heat and centrifugal treatment;

carrying out pressurization treatment;

adding silver nitrate and/or copper sulfate to the coculture medium;

treating with Agrobacterium in the presence of the powder;

adding carbenicillin to the culture medium in the callus proliferation and/or redifferentiation step after the coexistence step;

adding N6 inorganic salt into callus proliferation culture medium; and

cysteine was added to the coculture medium.

2. The method of producing transformed corn or rice according to claim 1, wherein in the method of producing transformed corn or rice,

3) the step of culturing the co-cultured tissue with a callus growth medium is not included between the co-culturing step and the redifferentiation step.

3. The method of producing transformed maize or rice according to claim 1 or 2, wherein selection of transformed cells using the characteristics of the nucleic acid introduced by Agrobacterium is performed using a selection drug resistance gene and a selection drug.

4. The method of producing transformed maize or rice according to claim 1 or 2,

1) the transformation-enhancing treatment is a treatment for enhancing the efficiency of introducing a gene of interest into cells of maize or rice, a treatment for enhancing the callus induction rate of immature embryos, or a treatment for enhancing the redifferentiation efficiency of transformed calli.

5. The method of producing transformed maize or rice according to claim 3,

1) the transformation-enhancing treatment is a treatment for enhancing the efficiency of introducing a gene of interest into cells of maize or rice, a treatment for enhancing the callus induction rate of immature embryos, or a treatment for enhancing the redifferentiation efficiency of transformed calli.

6. The method for producing transformed corn or rice according to claim 1 or 2, wherein the co-existence step comprises adding a compound belonging to benzoic acid herbicides.

7. The method of producing transformed corn or rice according to claim 3, wherein the co-existence step comprises adding a compound belonging to benzoic acid herbicides.

8. The method of producing transformed corn or rice according to claim 4, wherein the co-existence step comprises adding a compound belonging to benzoic acid herbicides.

9. The method of producing transformed corn or rice according to claim 5, wherein the co-existence step comprises adding a compound belonging to benzoic acid herbicides.

10. The method for producing transformed maize or rice according to claim 6, wherein the compound belonging to a benzoic acid herbicide is of a benzoic acid type, a salicylic acid type or a picolinic acid type.

11. The method for producing transformed maize or rice according to claim 7, wherein the compound belonging to the benzoic acid herbicide is of the benzoic acid type, salicylic acid type or picolinic acid type.

12. The method for producing transformed maize or rice according to claim 8, wherein the compound belonging to the benzoic acid herbicide is of the benzoic acid type, salicylic acid type or picolinic acid type.

13. The method for producing transformed maize or rice according to claim 9, wherein the compound belonging to the benzoic acid herbicide is of the benzoic acid type, salicylic acid type or picolinic acid type.

14. The method for producing transformed maize or rice according to claim 6, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

15. The method for producing transformed maize or rice according to claim 7, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

16. The method for producing transformed maize or rice according to claim 8, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

17. The method for producing transformed maize or rice according to claim 9, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

18. The method for producing transformed maize or rice according to claim 10, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

19. The method for producing transformed maize or rice according to claim 11, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

20. The method for producing transformed maize or rice according to claim 12, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.

21. The method for producing transformed maize or rice according to claim 13, wherein the compound belonging to benzoic acid herbicides is 3, 6-dichloro-2-methoxybenzoic acid or 4-amino-3, 5, 6-trichloropyridinecarboxylic acid.