JP2012110280A - Method for forming gene transferred plant body - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently redifferentiate a transformed plant body.SOLUTION: The method for forming a gene transferred plant body includes: transferring an agrobacterium into a tissue of a leaf blade by returning the leaf blade of a leaf having a leafstalk such as Jatropha curcas in a state having the leafstalk, and immersing with a fungus liquid of the agrobacterium from the state of more decompressed state than atmospheric pressure to pressure of the atmospheric pressure; then contacting an antibiotic and the agrobacterium in the tissue of the leaf blade by immersing the leaf blade into an aqueous solution of the antibiotic; and then contacting the leaf blade with a bactericide other than the antibiotic such as a hypochlorous acid. After that, callus induction is performed on a culture medium including the antibiotic using a part of the leaf blade into which the agrobacterium is transferred as a tissue fragment.

Description

  The present invention relates to a method for forming a transgenic plant.

  In recent years, many attempts have been made to produce biodiesel fuel from plants such as corn and sugarcane in preparation for the depletion of fossil fuel resources such as oil. As this plant body, plants of the genus Jatropha have attracted attention. Jatropha genus plants are non-edible plants that are highly resistant to pests and pathogens and can be cultivated on thin land, so there is no competition with food and farmland, and Jatropha seeds are about 30-40% This is because there are various advantages that oil and fat can be recovered with a high yield.

  However, Jatropha plants can only be cultivated in subtropical to tropical regions, the number of fruiting is once a year, and the size of the fruit is small.

  Therefore, attempts have been made to stably produce plant cultured cells (callus) and to produce biodiesel fuel from callus. For example, Patent Document 1 (Japanese Unexamined Patent Application Publication No. 2009-148192) discloses a method for extracting jatropha oil from callus cells derived from a plant of the genus Jatropha.

  However, this method has a small amount of fats and oils contained in callus and is not suitable for producing a large amount of fats and oils. In order to increase the production amount from the cultivated Jatropha plant, it is desired to improve, for example, stress resistance such as drought resistance and cold resistance and to improve oil and fat in seeds. For these improvements, artificial mating and mutation can be used, but its feasibility is low. Therefore, a method using cell engineering is introduced that introduces a target gene into a plant cell and imparts a desired property.

  A method using Agrobacterium is widely used as a method for introducing a target gene into a plant body, not limited to Jatropha plants. For example, in Non-Patent Document 1, after infecting Agrobacterium by coagulating a cotyledon of a Jatropha plant in a liquid containing Agrobacterium at room temperature and normal pressure, it is rooted through callus induction and shoot formation. It shows how to regenerate plants. Further, in Patent Document 2 (Japanese Patent Application Laid-Open No. 2004-357574), after infecting Agrobacterium by immersing a tissue piece of a crab plant seedling under reduced pressure in a liquid containing Agrobacterium, A method for regenerating plant bodies by performing callus induction and redifferentiating the callus is shown. Patent Document 3 (Japanese Patent Laid-Open No. 2003-174830) discloses a method of regenerating a plant body through somatic embryogenesis after infecting callus of a cyclamen plant with Agrobacterium.

  As described above, a method for introducing (redifferentiating) a plant body by introducing a target gene into the plant body using Agrobacterium and inducing callus and shoot formation has been already known.

JP 2009-148192 A JP 2004-357574 A JP 2003-174830 A

Li et al., Establishment of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas, Plant Cell Tiss. Organ. Cult., 2008, Vol.92, No2, pp.173-181)

  By the way, in order to increase the regeneration efficiency of plants into which genes have been introduced, increasing the efficiency of gene introduction by Agrobacterium infection is also an important factor. Efficient selection is also an important factor.

  In order to confirm that a tissue piece has been infected, in addition to the target gene, the kanamycin resistance gene, hygromycin in addition to the target gene, including the method described in Patent Document 2, A marker gene such as a resistance gene and a reporter gene such as a GUS gene were introduced together, and introduction of the target gene was confirmed by selection using the marker gene and expression of the reporter gene.

  However, the method using a marker gene or a reporter gene cannot confirm the presence or absence of infection only by contacting with Agrobacterium. For this purpose, callus induction was performed using a callus induction medium containing an antibiotic for selection by arbitrarily selecting a piece of tissue contacted with Agrobacterium. Therefore, if infection by Agrobacterium can be confirmed before callus induction, a tissue piece with high certainty of infection can be selected efficiently, and the plant body can be efficiently regenerated.

  In addition, in the gene transfer method by Agrobacterium infection, after Agrobacterium infection, Agrobacterium sterilization is performed using a callus induction medium containing antibiotics that exhibit antibacterial or bactericidal properties. Had gone. In addition, even after callus induction, antibiotics were added to the medium for shoot formation to ensure sterilization.

  However, sterilization with a callus induction medium containing antibiotics causes many remaining Agrobacterium, and the low rate of transformed callus formation is thought to be due to the large amount of remaining Agrobacterium. In addition, it was thought that the long-term contact of antibiotics with tissue fragments to sufficiently kill Agrobacterium had an effect on the low rate of callus formation. Therefore, it is desired that the sterilization operation be completed as completely and in a short period as possible. It was also desirable to contact antibiotics at low concentrations if possible.

  The present invention has been made on the basis of the above problems, and the method for forming a transgenic plant of the present invention is a method in which at least part of the leaves of the plant is immersed in an Agrobacterium solution. Agrobacterium is introduced into the leaf blade tissue by returning from atmospheric pressure to atmospheric pressure, and at least a part of the leaf blade into which the Agrobacterium has been introduced is brought into contact with a disinfectant other than antibiotics. This is a method of performing callus induction after the treatment.

  According to the formation method of the present invention, not only the efficiency of introduction into leaf blades used for callus induction is enhanced, but also sterilization after infection with infected Agrobacterium can be reliably performed in a short period of time. Also, sterilization is achieved by contact with low concentrations of antibiotics. As a result, when the plant tissue is regenerated into the plant body, not only the work efficiency is improved, but also the regeneration efficiency is increased.

FIG. 1 is an image showing leaves before and after immersion under reduced pressure of Agrobacterium, (a) is an image of leaves before immersion, and (b) is an image of leaves after immersion. FIG. 2 is an image showing that the GUS gene is expressed in leaves after growth.

  The method for forming a transgenic plant of the present invention comprises a step of immersing at least a part of a plant leaf in an Agrobacterium solution and introducing Agrobacterium into the leaf blade; A step of bringing at least a part of the introduced leaf blade into contact with a disinfectant other than an antibiotic, and a step of inducing callus of the leaf blade in contact with the disinfectant. That is, the method for forming a transgenic plant according to the present invention introduces a gene with high efficiency by infiltrating Agrobacterium into the leaf tissue of the plant and contacts Agrobacterium with a fungicide before callus induction. In this way, the certainty of sterilization is improved.

  The method for forming a transgenic plant of the present invention is a method for regenerating a plant from the leaves of a plant, and is applied to a method for performing transformation using Agrobacterium.

  In the present invention, plant leaves are used as a target for gene introduction. The leaves of the plant body are composed of the leaf body that is the body of the leaf that performs photosynthesis, the handle-like petiole that connects the leaf body and the stem, and the bamboo leaf that is an accessory part at the base of the leaf. In the calligraphy, leaf means one with leaf blades and petiole, regardless of the presence or absence of cocoon leaves. The plant body that is the subject of the present invention may be either a monocotyledonous plant or a dicotyledonous plant, and any plant that has a petiole (stalked leaf) is preferably used. This is to ensure the introduction of Agrobacterium using the petiole as described later. Examples of such plants include Jatropha curcas plants such as Jatropha curcas, soybean plants such as soybeans, and asteraceae plants. In view of utility value as an alternative energy source, Jatropha plants are preferably used. It is done.

  As a target to be transformed, a tissue piece obtained by cutting a leaf into an appropriate size can be used. In the present invention, a leaf having a petiole without cutting into a tissue piece is preferably used. This is because Agrobacterium can be introduced efficiently, and the introduced site can be easily identified.

(Introduction of Agrobacterium)
In the present invention, Agrobacterium is first introduced into the leaves. This introduction is performed by a so-called reduced pressure dipping method. The reduced pressure infiltration method is a known method, and is performed by immersing a leaf to be introduced into an Agrobacterium solution in an environment (under reduced pressure) reduced from atmospheric pressure. The introduction is performed by immersing at least a part of the leaf blade in the bacterial solution or by inserting a petiole and immersing it in a leaf state. When immersing the leaf blades, the whole including the petiole may be immersed in the bacterial solution, or only the leaf blades may be immersed in the bacterial solution. Moreover, when immersing in the cutting leaf state, the petiole is immersed in the bacterial solution. In the specification of the present application, the cutting leaf state refers to a state in which the entire leaf is not immersed in various liquids such as a bacterial solution, but the leaf blade is inserted in the liquid without being immersed in the liquid, and is in a place close to the petiole. It does not matter if some of the leaf blades are immersed in the liquid. The leaf may be either a young leaf or an old leaf, but a young leaf is preferable from the viewpoint of easy introduction of Agrobacterium, and specifically, a young leaf of 5th to 4th or more is suitable for a Jatropha plant.

  The Agrobacterium used is not limited as long as it is used for gene introduction, and various types of Agrobacterium are used. For example, Agrobacterium tumefaciens C58C1Rif strain and GV3101KR strain are exemplified. Agrobacterium includes a vector into which a gene to be introduced is integrated. As the vector, commonly used vectors such as pBI121, pGV2260, and pMP90 are used. The vector may have various reporter genes and marker genes in addition to the target gene. Methods for preparing vectors and introduction of genes into Agrobacterium can be performed by known methods.

The method for preparing the bacterial solution for immersing the leaves is not different from the method for preparing the bacterial solution used in the known reduced pressure infiltration method, and the medium liquid is particularly limited as long as it is a liquid in an environment where Agrobacterium can survive. It is not a thing. For example, distilled water, isotonic solution, buffer solution, LB medium for tissue culture, YBS medium and the like are exemplified. The OD ₆₀₀ is 0.05 to 0.5, preferably 0.1 to 0.3, or 1.0 × 10 ^{2 to} 1.0 × 10 ⁸ cfu in the bacterial solution in which the leaves are immersed. , Preferably 1.0 × 10 ^{4 to} 1.0 × 10 ⁶ cfu / ml.

  Immersion under reduced pressure is performed in a chamber whose pressure is reduced from atmospheric pressure by a vacuum pump or the like. For example, in the case of immersing the whole leaf or leaf blades at 0.01 to 50 KPa (about 0.5 atm), preferably 0.1 to 10 KPa at a pressure, the petiole is inserted in a leaf state for about 1 to 10 minutes. When soaking, soak for about 5 minutes to 2 hours. And the pressure in a chamber is returned to atmospheric pressure in the immersed state. But these conditions are suitably changed according to the kind of plant and the state of a leaf.

  A surfactant is preferably added to the Agrobacterium solution in order to promote penetration into the leaves. As the surfactant, a nonionic surfactant is preferable. For example, Silwet-L77 (trade name) which is one kind of polyether-modified silicone, Tween 20 (trade name) which is one kind of polyoxyethylene sorbitan fatty acid ester, Examples include Tween 80 (trade name).

(Leaf growth)
After this, the leaves are grown in a solution that does not contain fungicides and antibiotics and Agrobacterium to ensure the introduction of Agrobacterium. The term “growth” as used herein means maintaining the leaves alive so that the leaves do not die, and it is not always necessary to grow them. The growing liquid does not need to contain bactericides and antibiotics, and examples thereof include a culture liquid containing water, nutrients required for plant growth such as nitrogen, phosphorus, and potassium. The growth may be performed under the condition that leaves do not die. For example, the pH of the solution is 5 to 8, and the environment is room temperature, preferably about 15 to 30 ° C., for example, about 1 day to 10 weeks, preferably 3 to 7. Leave for days. At this time, it may be grown in the dark, but it is preferable to provide a light time of about 8 to 16 hours. The light conditions are, for example, 200 to 2,000 lux, preferably 500 to 1,000 lux.

  By this growth process, it is considered that Agrobacterium that has entered the leaf blade from the stoma and / or petiole of the leaf diffuses throughout the leaf blade. By this diffusion, the color in the part where Agrobacterium is introduced is discolored to such an extent that it can be distinguished from the surrounding color, and it becomes possible to visually determine whether or not it is infected with Agrobacterium. . Moreover, since it can be recognized as a block (section) surrounded by the veins, it is easy to produce a tissue piece for callus induction. However, the growth process is an arbitrary process, and if the discoloration can be visually discerned by immersion in a bacterial solution containing Agrobacterium, the process can proceed to the next sterilization process without passing through the process.

(Disinfection with antibiotics)
In the present invention, it is desirable to immerse the leaves in an aqueous solution containing antibiotics before sterilization with a bactericide. This is to promote sterilization of Agrobacterium from inside the blade. Although this sterilization step is also an optional step, it is a step that is desired to be implemented from the viewpoint of reducing the concentration of antibiotics added to the callus induction medium. This step may be performed after sterilization with a bactericide, or may be performed before and after sterilization with a bactericide. Here, the sterilization is an operation for reducing the number of bacteria remaining after the sterilization operation, and the killing of Agrobacterium is not guaranteed. The same meaning is used in the sterilization with the following fungicides.

  The antibiotic is not particularly limited as long as it is an antibiotic having a sterilizing effect on Agrobacterium. For example, cefotaxime, moxalactam, merovenem, carbenicillin and the like are exemplified. One or a combination of two or more of these is used for sterilization. Nutrients required for plant growth such as nitrogen, phosphorus and potassium may be added to the aqueous solution containing antibiotics.

  The concentration of antibiotic varies depending on the type of antibiotic, but is higher than the concentration in the medium used for callus induction according to the present invention, preferably the concentration in the medium used for the previous callus induction and a comparable level. For example, 400 to 500 μg / ml for cefotaxime and 40 to 600 μg / ml for carbenicillin 100 μg / ml meropenem.

  For sterilization, a petiole is inserted and immersed in an aqueous solution containing antibiotics in a leaf state. The method of sterilization in the state of cutting leaves is easy to handle, and according to this method, the antibiotic penetrates into the inside of the leaf blade through the petiole vascular bundle, and the antibiotic works effectively.

  Immersion is performed at room temperature, and the immersion time varies depending on the type and concentration of the antibiotic, but is generally 12 to 24 hours, preferably 16 to 20 hours. The light condition is preferably weak light to normal indoor brightness.

(Bacteria disinfection with bactericides)
The leaf into which Agrobacterium has been introduced by the above steps is brought into contact with a fungicide. As the disinfectant, a disinfectant excluding antibiotics for sterilization is used. As the bactericidal agent, a drug showing a bactericidal effect against Agrobacterium in a relatively short time is selected, for example, hypochlorite such as hypochlorous acid, sodium hypochlorite, potassium hypochlorite, Examples include hydrogen peroxide, benzalkonium chloride, and benzethonium chloride. Of these, hypochlorous acid, hypochlorite, and hydrogen peroxide are preferably used for the reason that a bactericidal effect can be obtained in a short time and there is little fear of remaining.

  By immersing the leaves in an aqueous solution of these germicides, contact with the germicide is performed. The entire leaf including the petiole may be immersed in an aqueous solution of the bactericide, or only the leaf blades may be immersed in the aqueous solution of the bactericide. This removes all Agrobacterium remaining on the surface of the leaf blades. This step may be performed before or before sterilization with antibiotics.

  The concentration of the disinfectant varies depending on the type of disinfectant, but hypochlorous acid or hypochlorite is, for example, 0.1 to 1 w / v%, preferably 0.6 w / v% as hypochlorous acid. is there. The immersion time is about 2 to 20 minutes, and the immersion time is adjusted by the concentration of the bactericide.

(Callus induction)
Next, callus induction is performed from the leaf blade obtained above. Callus induction is performed by a known method. The medium to be used may be any medium containing a solidifying agent such as a carbon source, a nitrogen source, inorganic salts, and agar. For example, MS (Murashige and Skoog) medium and SH123 medium are exemplified. In addition, 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid), TDZ, BA (Benzyl adenine), IA (Indole acetic acid), IBA (Indole butyric acid) In addition, one or more plant growth regulators such as BAP (6-Benzyl amino purine) are added as necessary. Furthermore, antibiotics for sterilization and antibiotics for markers are added to the medium. At this time, the concentrations of basic nutrients such as carbon source and nitrogen source, plant growth regulators, and antibiotics for markers are almost the same as the concentrations in the known callus induction medium. The concentration of the antibiotic is ½ to 1/100 of the concentration in a conventional callus induction medium, for example, less than 100 μg / ml for cefotaxime and less than 5 μg / ml for meropenem. In some cases, a medium containing no antibiotics can be used. This is because the viable count of Agrobacterium decreases due to contact with bactericides and antibiotics.

  For callus induction, a leaf into which Agrobacterium has been introduced by the above-described process, preferably a slice of leaf blade is used. At this time, the discolored portion is used. This is because Agrobacterium is considered to have been introduced reliably. Usually, the whole blade is discolored, so the whole can be used.

  Callus induction is performed in an environment of room temperature, preferably about 15 to 30 ° C., for example, for about 2 to 4 weeks, preferably about 2 weeks. In addition, for example, it is preferable to culture under a light condition of 1,000 to 5,000 lux with a light time of about 8 to 16 hours per day and a dark time of 16 to 8 hours.

(Redifferentiation)
The induced callus is subsequently redifferentiated through shoot formation, shoot elongation, and route formation. Known processes are used as they are for the shoot formation, shoot elongation, and route formation, and are appropriately selected according to the type of plant. The medium to be used may be any medium containing a solidifying agent such as a carbon source, a nitrogen source, inorganic salts, and agar. For example, MS culture medium, SH123 culture medium, etc. are illustrated. In addition, one or more plant growth regulators such as 2,4-D, NAA, TDZ, BA, IBA, and BAP are added to the medium as necessary. At this time, the concentrations of basic nutrients such as a carbon source and a nitrogen source and plant growth regulators are the same as the concentrations in a known shoot-forming medium. Moreover, since Agrobacterium is considered to be almost killed by callus induction, shoots can be formed using a medium that does not contain antibiotics. However, in order to make death more reliable, a medium supplemented with antibiotics for sterilization can also be used. In the case of adding an antibiotic for sterilization, the concentration is 1/2 to 1/100 of the concentration in a known shoot-forming medium, for example, less than 100 μg / ml for cefotaxime, and less than 5 μg / ml for merobenem It is.

  As described above, in the present invention, since the step of efficiently introducing Agrobacterium into the leaf blade tissue by the reduced pressure infiltration method and then contacting with the bactericidal agent is provided, not only the gene introduction probability is increased, but also the leaf A very large amount of Agrobacterium present in the body is effectively sterilized. As a result, not only the influence of Agrobacterium residue is eliminated, but also the influence of antibiotics for sterilization can be eliminated because the amount of antibiotics used and the number of times of use can be reduced. Therefore, the regeneration rate from plant tissue to plant body is increased.

  In particular, in the present invention, since a leaf having a petiole is used, the petiole can be immersed in a solution containing Agrobacterium or antibiotics or a growth solution in an inserted leaf state. In the state of cutting leaves, it is easier to handle than when handling tissue pieces, not only can a large amount of leaves be processed at once, but also each liquid absorbed from the petiole spreads through the veins to every corner of the leaf blade, spreading Agrobacterium and Helps spread antibiotics and promotes Agrobacterium infection and sterilization. In addition, the part where Agrobacterium is introduced is observed as several sections surrounded by the veins, and the introduction of Agrobacterium can be easily discriminated visually to improve the work efficiency. Furthermore, since there is less possibility of contamination by other bacteria compared to the case of introducing a gene into a tissue piece from which a leaf has been cut, the disinfection operation on the leaf, which is performed as a pretreatment for introducing Agrobacterium, can be omitted. .

  EXAMPLES Next, the present invention will be further described based on examples. However, the present invention is not limited to the following examples, and all modifications that are included in the scope of the claims and equivalents thereof are included in the present invention. Is intended.

(Introduction of Agrobacterium)
As a plant leaf, the 4th leaf of Jatropha curcas grown in a greenhouse was used. As the Agrobacterium, LBA4404 strain (MW Bevan et al. Nucleic Acids Res. 12: 8711-8721 (1984)) obtained by detoxifying the Ti plasmid was used. In order to confirm the transformation, a commercially available β-glucuronidase (GUS) gene was incorporated into a binary vector and a plasmid pBI121 was purchased and used to transform Agrobacterium (RA Jefferson et al. EMBO J. 6: 3901-3907 (1987)). Agrobacterium LBA4404 / pBI121 carrying the β-glucuronidase (GUS) gene was cultured overnight at 30 ° C. in an L-liquid medium containing kanamycin as a marker at a concentration of 50 μg / ml. This culture solution was diluted with the suspension solution so that OD ₆₀₀ was 0.1. The bacterial concentration of this bacterial solution was 1.2 × 10 ⁸ cfu / ml. The composition of the suspension solution is 10 mM MES pH 5.6, 10 mM MgCO ₂ , Acetosyringone 20 mg / L, 0.005% Silwet-L77.

    The whole leaf blade of the leaf having the above-mentioned petiole is immersed in a bacterial solution placed in a beaker, depressurized at 25 ° C. and 0.1 KPa for 5 minutes, and then the pressure is rapidly returned to the atmospheric pressure, whereby an Agrobacterium solution is obtained. Infiltrated into the leaf tissue. The photograph after infiltration is shown in FIG. As shown in FIG. 1, discolored portions of Jatropha curcas leaf leaves that had been infiltrated under reduced pressure were observed compared to the leaf blades before infiltration.

(Leaf growth)
Next, the petiole of leaves introduced with Agrobacterium was immersed in tap water in a container and immersed in a leaf state, and left for 5 days at 30 ° C., 16 hours light period, 8 hours dark period, and about 750 lux illumination. . Thereafter, a tissue piece was collected from the treated leaf from a portion that was faded compared to the surrounding portion, and the expressed glucuronidase activity was examined to confirm gene transfer. The result is shown in FIG. As shown in FIG. 2, color development indicating the expression of the GUS gene was observed in the tissue piece.

    At this time, Agrobacterium was sterilized using a bactericide on the grown leaves. A commercially available hypochlorous acid bleach (trade name Kitchen Hiter, containing 6 w / v% sodium hypochlorite) was diluted to 10% with distilled water. The whole leaf blade of the grown leaf was immersed in this solution at room temperature for 20 minutes. When the number of Agrobacterium cells remaining in the leaf blades after immersion was measured, it was about 1700 per 2 mm diameter leaf disk.

(Disinfection with antibiotics)
Next, the grown leaves were sterilized with antibiotics. The petioles of the grown leaves were inserted into an aqueous solution containing 500 μg / ml of cefotaxime and immersed in a leaf state overnight for about 8 hours. Thereafter, sterilization treatment was performed under the same conditions as described above. When the number of cells after this sterilization treatment was measured, it was about 190 per 2 mm diameter leaf disc, and the number of cells was reduced to about 1/10 compared to the case where sterilization was not performed by immersion in an antibiotic solution. did. Thus, before the callus induction, by sterilizing with antibiotics absorbed from the petiole, the Agrobacterium count could be reduced to nearly 10%.

(Callus induction)
Subsequently, the leaf blades from which Agrobacterium was sterilized as described above were sterilized using a bactericidal agent, and a leaf disk having a diameter of about 6 mm was prepared, and 3 mg / L BA and 0.1 mg / L were added to the MS medium. The cells were cultured for 2 weeks on a solid medium (containing 0.7% agar) containing L IBA, 3% sucrose, 20 μg / mL cefotaxin, and the marker kanamycin at 20 μg / ml. Callus was formed after 2 weeks of culture. When the number of cells of this callus was measured, the number of cells was 0. Moreover, callus was formed from all the leaf discs, and GUS activity was observed in those callus.

  In this way, Agrobacterium that has penetrated into the leaf tissue is killed by immersion in the antibiotic solution, so callus can be induced in a medium containing a low concentration of antibiotic, and contact with the antibiotic can be prevented. It can be finished in a short time. Furthermore, since Agrobacterium that has penetrated into the leaf tissue for bleaching treatment is almost completely killed, the adverse effect of Agrobacterium is eliminated and callus can be induced efficiently. In addition, since a tissue piece in which introduction of Agrobacterium can be visually confirmed can be obtained, callus induction can be performed with high probability.

  According to the present invention, various transformed plants such as Jatropha plants imparted with cold resistance and drought resistance by gene transfer can be efficiently produced by tissue culture.

Claims (13)

A step of introducing Agrobacterium into the tissue of the leaf blade by returning at least a part of the leaves of the plant body to the atmospheric pressure from the pressure reduced from the atmospheric pressure in a state of being immersed in the Agrobacterium bacterial solution,
Contacting at least part of the leaf blade introduced with the Agrobacterium with a bactericide other than antibiotics;
A method for forming a transgenic plant comprising a step of inducing callus of the leaf blade in contact with the fungicide.   The method for forming a transgenic plant according to claim 1, wherein the bacterial solution of Agrobacterium contains a surfactant.   The method for forming a transgenic plant according to claim 2, wherein the surfactant is a nonionic surfactant.   The method for forming a transgenic plant according to any one of claims 1 to 3, wherein the fungicide is at least one of hypochlorous acid, a salt thereof, and hydrogen peroxide.   The method for forming a transgenic plant according to any one of claims 1 to 4, wherein in the step of introducing Agrobacterium, a leaf blade having a petiole is immersed in an Agrobacterium fungus.   The method for forming a transgenic plant according to any one of claims 1 to 4, wherein, in the step of introducing Agrobacterium, a petiole is immersed in an Agrobacterium solution in a leaf state.   The method for forming a transgenic plant according to claim 5 or 6, wherein the leaf into which the Agrobacterium has been introduced is contacted with a fungicide and / or after that, the petiole is immersed in a solution containing an antibiotic in a leaf state. .   6. The method according to claim 5, further comprising the step of immersing and growing the petiole in a liquid containing no fungicide and antibiotics in a defoliation state after the introduction of Agrobacterium and before and / or after contacting the leaf blade with the fungicide. 7. A method for forming a transgenic plant according to item 6.   9. The transgenic plant according to claim 8, wherein the leaf is dipped in a solution containing antibiotics in a defoliation state before the leaves grown by soaking in the liquid not containing the fungicide and antibiotics are brought into contact with the fungicide. Forming method   The method for forming a transgenic plant according to any one of claims 1 to 9, wherein the callus induction step is a step performed in a medium containing a reduced concentration of antibiotic.   The method for forming a transgenic plant according to any one of claims 1 to 10, further comprising a step of redifferentiating the induced callus in a medium not containing an antibiotic.   The method for forming a transgenic plant according to any one of claims 1 to 11, wherein the plant is a Jatropha plant.   A transgenic plant produced by the method for forming a transgenic plant according to any one of claims 1 to 12.