Methods for producing genetically modified plants

Abstract

To provide a method for producing a transgenic plant comprising efficiently killing Agrobacterium used for infecting the plant for breeding.SOLUTION: A method of plant production comprises the steps of: preparing a plasma-activated culture fluid by radiating plasma to a first culture fluid; culturing a plant seed in a second culture fluid or solid medium to form a callus; infecting the callus with an Agrobacterium to produce an infected callus; and mixing the plasma-activated culture fluid and the second culture fluid or solid medium containing the infected callus to kill the Agrobacterium.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The technical field of this specification relates to methods for producing genetically modified plants. [Background technology]

[0002] Plant breeding has been carried out with the aim of improving plant productivity or producing plants with new properties. Traditionally, breeding was carried out in the hope of generating mutations, but in recent years, research and development has been conducted on breeding using genetic engineering technology or genome editing.

[0003] For example, Patent Document 1 discloses a method for increasing the expression level of a recombinant protein, which comprises the steps of cultivating Nicotiana plants hydroponically while circulating a nutrient solution, cultivating the plants while stopping the circulation of the nutrient solution, infecting the resulting plants with Agrobacterium, cultivating the Agrobacterium-infected plants while circulating the nutrient solution, and extracting the recombinant protein from plant leaves (paragraph

[0005] of Patent Document 1). The Agrobacterium contains a polynucleotide that encodes the recombinant protein. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-57950 A Summary of the Invention [Problem to be solved by the invention]

[0005] Agrobacterium may be removed from plants infected with it. Antibiotics may be used to remove the Agrobacterium. However, when the Agrobacterium is sterilized with antibiotics, the sterilization may not be sufficient. If the Agrobacterium is not sterilized sufficiently, it will grow in the medium, making it difficult to continue culturing the plant.

[0006] The problem to be solved by the technology of the present specification is to provide a method for producing genetically modified plants that can highly efficiently sterilize Agrobacterium that has been infected into plants during plant breeding. [Means for solving the problem]

[0007] The method for producing a genetically modified plant in the first aspect includes the steps of: irradiating a first culture solution with atmospheric pressure plasma to produce a plasma-activated culture solution; culturing at least a part of the plant body in a second culture solution or solid medium to produce a callus; infecting the callus with Agrobacterium to produce an infective body; and mixing the plasma-activated culture solution with the second culture solution or solid medium containing the infective body and sterilizing the Agrobacterium. and the pH of the plasma activated culture medium is 3.3 or less.

[0008] This method for producing genetically modified plants involves supplying Agrobacterium-infected plants with a plasma-activated culture medium irradiated with atmospheric pressure plasma, which sufficiently sterilizes the Agrobacterium. [Effects of the Invention]

[0009] The present specification provides a method for producing genetically modified plants that can efficiently sterilize Agrobacterium that has been infected into plants during plant breeding. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 2 is a conceptual diagram for explaining the configuration of a robot arm that scans the gas nozzles of the plasma generation device of the first embodiment. [Figure 2] FIG. 2A is a cross-sectional view showing the configuration of the first plasma generating device, and FIG. 2B is a diagram showing the shape of the electrodes. [Figure 3] FIG. 3.A is a cross-sectional view showing the configuration of the second plasma generating device, and FIG. 3.B is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma region. [Figure 4] FIG. 2 is a diagram showing a schematic configuration of a third plasma generation device in the first embodiment. [Figure 5] FIG. 10 is a schematic configuration diagram showing an upper structure of a third plasma generation device in the first embodiment. [Figure 6] FIG. 4 is a schematic configuration diagram showing a lower structure of a third plasma generation device in the first embodiment. [Figure 7] FIG. 10 is a diagram for explaining a case where the third plasma generator is irradiating plasma in the first embodiment. [Figure 8] 1 is a graph showing the relationship between solution treatment time and viable cell count. [Figure 9] 1 is a graph showing the relationship between the number of days elapsed since the production of a solution and the number of viable bacteria. [Figure 10] FIG. 10 is a diagram showing an area where a solution is added. [Figure 11] Photographs showing the effect of adding a solution on protocorms. DETAILED DESCRIPTION OF THE INVENTION

[0011] Specific embodiments will be described below using a method for producing a genetically modified plant as an example, but the technology of the present specification is not limited to these embodiments.

[0012] (First embodiment) A first embodiment will be described. In the method for producing a genetically modified plant of the first embodiment, the Agrobacterium used for genetic modification is sterilized by treating the culture solution irradiated with plasma. Therefore, first, the plasma generator for irradiating the plasma will be described.

[0013] 1.Plasma activated culture medium manufacturing equipment 1-1.Configuration of plasma activated culture medium manufacturing equipment The plasma activated culture solution manufacturing apparatus PM of the first embodiment includes a plasma irradiation device P1 and an arm robot M1, as shown in Figure 1. The plasma irradiation device P1 is used to generate plasma and irradiate the plasma toward a solution.

[0014] As shown in FIG. 1, the arm robot M1 is capable of moving the position of the plasma irradiation device P1 in the x-axis, y-axis, and z-axis directions. For ease of explanation, the direction of plasma irradiation is assumed to be the -z-axis direction. This allows adjustment of the distance between the solution surface and the plasma irradiation device P1. Furthermore, this plasma activated culture solution manufacturing device PM can irradiate plasma for a predetermined plasma irradiation time.

[0015] As will be described later, there are three types of plasma irradiation device P1 (first plasma generator P10, second plasma generator P20, and third plasma generator P30). Any of these types may be used. Note that the third plasma generator P30 does not have the robot arm M1 shown in FIG. 1.

[0016] 1-2. First plasma generator Fig. 2.A is a cross-sectional view showing the schematic configuration of a plasma generator P10. Here, the plasma generator P10 is a first plasma generator that ejects plasma in a point-like manner. Fig. 2.B is a diagram showing the details of the shapes of electrodes 2a and 2b of the plasma generator P10 in Fig. 2.A.

[0017] The plasma generator P10 has a housing 10, electrodes 2a and 2b, and a voltage application unit 3. The housing 10 is made of a sintered body made of alumina (Al2O3). The housing 10 is cylindrical. The inner diameter of the housing 10 is 2 mm or more and 3 mm or less. The thickness of the housing 10 is 0.2 mm or more and 0.3 mm or less. The length of the housing 10 is 10 cm or more and 30 cm or less. A gas inlet 10i and a gas outlet 10o are formed on both ends of the housing 10. The gas inlet 10i is for introducing gas to generate plasma. The gas outlet 10o is an irradiation unit for irradiating the outside of the housing 10 with plasma. The direction of gas movement is indicated by the arrow in the figure.

[0018] The electrodes 2a and 2b are a pair of opposing electrodes arranged opposite each other. The length of the opposing surfaces of the electrodes 2a and 2b is smaller than the inner diameter of the housing 10, for example, about 1 mm. As shown in FIG. 2B, the opposing surfaces of the electrodes 2a and 2b each have a number of recesses (hollows) H formed therein. Therefore, the opposing surfaces of the electrodes 2a and 2b have a finely uneven shape. The depth of these recesses H is about 0.5 mm.

[0019] The electrode 2a is disposed inside the housing 10 near the gas inlet 10i. The electrode 2b is disposed inside the housing 10 near the gas outlet 10o. Therefore, in the plasma generator P10, gas is introduced from the opposite side of the opposing surface of the electrode 2a, and gas is ejected from the opposite side of the opposing surface of the electrode 2b. The distance between the electrodes 2a and 2b is, for example, 24 cm. The distance between the electrodes 2a and 2b may be shorter.

[0020] The voltage application unit 3 applies an AC voltage between the electrodes 2 a and 2 b. The voltage application unit 3 uses a commercial AC voltage of 60 Hz, 100 V, boosts it to 9 kV, and applies the voltage between the electrodes 2 a and 2 b.

[0021] When argon is introduced through the gas inlet 10i and a voltage is applied between the electrodes 2a and 2b by the voltage application unit 3, plasma is generated inside the housing 10. As shown by the diagonal lines in Figure 2A, the area where plasma is generated is referred to as the plasma generation area P. The plasma generation area P is covered by the housing 10.

[0022] 1-3. Second plasma generator Figure 3.A is a cross-sectional view showing the schematic configuration of plasma generator P20. Here, plasma generator P20 is a second plasma generator that linearly ejects plasma. Figure 3.B is a partial cross-sectional view of the plasma generation region P of plasma generator P20 in Figure 3.A, taken along a line perpendicular to the longitudinal direction.

[0023] The plasma generator P20 has a housing 11, electrodes 2a and 2b, and a voltage application unit 3. The housing 11 is made of a sintered body made of alumina (Al2O3). A gas inlet 11i and multiple gas outlets 11o are formed at both ends of the housing 11. The gas inlet 11i has a slit shape with its longitudinal direction extending left-right in FIG. 3A. The slit width from the gas inlet 11i to directly above the plasma generation region P (the width in the left-right direction in FIG. 3B) is, for example, 1 mm.

[0024] Gas outlet 11o is an irradiation part for irradiating plasma to the outside of housing 11. Gas outlet 11o is cylindrical or slit-shaped. In the case of a cylindrical shape, gas outlet 11o is formed in a straight line along the longitudinal direction of the plasma region. The inner diameter of gas outlet 11o is in the range of 1 mm to 2 mm. In addition, in the case of a slit-shaped gas outlet 11o, it is preferable that the slit width of gas outlet 11o is 1 mm or less. This allows stable plasma to be formed. In addition, gas inlet 11i is configured to introduce gas in a direction that intersects with the line connecting electrode 2a and electrode 2b.

[0025] The electrodes 2a, 2b and the voltage application unit 3 are the same as those in the plasma generator P10 shown in Fig. 1. Similarly, a commercial AC voltage is used to apply a voltage between the electrodes 2a, 2b. This allows the plasma to be ejected in a straight line.

[0026] Furthermore, if the plasma generators P20 that eject plasma in a straight line are arranged in a row in the left-right direction of FIG. 3B, plasma can be ejected in a plane over a certain rectangular area.

[0027] 1-4.Third plasma generator 4 is a conceptual diagram showing the general configuration of the third plasma generator P30. The plasma generator P30 is used to irradiate the contained solution with plasma.

[0028] As shown in FIG. 4, the plasma generation device P30 has a first electrode 110, a second electrode 210, a first potential application unit 120, a second potential application unit 220, a first lead wire 130, a second lead wire 230, a gas supply unit 140, a gas pipe coupling connector 150, a gas pipe 160, a first electrode protection member 170, a second electrode protection member 240, a first electrode support member 180, a sealing member 191, a coupling member 192, a container 250, a sealing member 260, and a stand 270.

[0029] 1-4-1. Schematic structure of the electrode The first electrode 110 has a cylindrical portion 110a. A plasma gas can be supplied to the inside of the cylindrical portion 110a. That is, the inside of the first electrode 110 is in communication with the gas supply unit 140. The first electrode 110 is configured to blow out gas from the cylindrical portion 110a toward the second electrode 210. The tip of the first electrode 110 has a syringe needle shape. That is, the tip of the first electrode 110 has an inclined surface that is inclined with respect to a direction perpendicular to the axial direction of the first electrode 110. A microhollow is formed at the tip of the first electrode 110.

[0030] The second electrode 210 is an electrode facing the first electrode 110. The second electrode 210 is a rod-shaped electrode. The second electrode 210 may be cylindrical, or may be polygonal, or may be needle-shaped with a pointed tip. The second electrode 210 has a tip 211. The tip 211 of the second electrode 210 is made of an iridium alloy containing iridium. For example, it is an alloy of iridium and platinum. Alternatively, it is an alloy of iridium, platinum, and osmium. Iridium alloys have high hardness and excellent heat resistance. Therefore, iridium alloys are suitable for the tip 211 of the second electrode 210. Alternatively, platinum may be used instead of iridium. Alternatively, palladium may be used. Alternatively, a metal or alloy containing at least one of iridium, platinum, and palladium may be used. Alternatively, the tip 211 of the second electrode 210 may be gold. During discharge, the second electrode 210 is immersed in the solution contained in the container 250.

[0031] The first potential application unit 120 is for applying a periodically changing potential to the first electrode 110. The second potential application unit 220 is for applying a periodically changing potential to the second electrode 210. Here, either the first potential application unit 120 or the second potential application unit 220 may be grounded. The first lead wire 130 is for electrically connecting the first electrode 110 and the first potential application unit 120. The first lead wire 130 is preferably made of a nickel alloy or stainless steel. The second lead wire 230 is for electrically connecting the second electrode 210 and the second potential application unit 220. The second lead wire 230 is preferably made of a nickel alloy or stainless steel. As a result, a high-frequency voltage is applied between the first electrode 110 and the second electrode 210. In other words, the first potential application unit 120 and the second potential application unit 220 are voltage application units for applying a voltage between the first electrode 110 and the second electrode 210 .

[0032] 1-4-2.Gas supply route As described above, the plasma generator P30 includes the gas supply unit 140, the gas pipe connector 150, and the gas pipe 160. Therefore, the gas supply unit 140 supplies plasma gas to the inside of the cylindrical portion of the first electrode 110 via the gas pipe 160 and the gas pipe connector 150. Here, the gas supply unit 160 supplies, for example, Ar gas. Alternatively, it may supply other rare gases. Alternatively, the gas may contain trace amounts of other gases, such as oxygen gas. Therefore, the plasma gas is sprayed from the first electrode 110 toward the solution contained in the solution 250.

[0033] 1-4-3. Superstructure configuration FIG. 5 is a diagram showing the upper structure of the plasma generator P30. As shown in FIG. 5, the first electrode 110 has a tip portion 111. As shown in FIG. 4, the tip portion 111 is disposed in a position facing the second electrode 210. The tip portion 111 of the first electrode 110 has an inclined surface 111a. The inclined surface 111a is a surface that is inclined with respect to a plane perpendicular to the axial direction of the first electrode 110. In addition, a microhollow 111b is formed in the tip portion 111. The microhollow 111b is a minute recess having a length of 0.5 mm to 1 mm and a width of 0.3 mm to 0.5 mm.

[0034] As described above, the plasma generation device P30 has the sealing member 191 and the coupling member 192. The sealing member 191 is attached to the container 250 shown in Fig. 4 and serves to seal the inside of the container 250. The coupling member 192 is a member that connects the first electrode 110 and the gas pipe coupling connector 150 via the sealing member 191 and the like.

[0035] 1-4-4. Substructure configuration FIG. 6 is a diagram showing the lower structure of the plasma generator P30. As described above, the plasma generator P30 has a container 250, a sealing member 260, and a stand 270. The container 250 is configured to be able to contain a solution therein. Here, the solution includes a culture solution and an organic solvent. The container 250 also contains a first electrode 110 and a second electrode 210 therein. The container 250 may also have a scale to measure the amount of solution contained in the container 250.

[0036] The sealing member 260 is for sealing the gap between the second electrode protection member 240 and the container 250. An example of the sealing member 260 is an O-ring. Any other member may be used as long as it ensures the airtightness of the container 250 and prevents the solution from leaking out to the bottom of the container 250. The stand 270 is for supporting the container 250 and other members.

[0037] 2. Plasma generated by a plasma generator 2-1. First plasma generator and second plasma generator The plasma generated by the plasma generators P10 and P20 is non-equilibrium atmospheric pressure plasma, where atmospheric pressure plasma refers to plasma at a pressure in the range of 0.5 atmospheres to 2.0 atmospheres.

[0038] In this embodiment, Ar gas is mainly used as the plasma generating gas. Electrons and Ar ions are naturally generated inside the plasma generated by the plasma generators P10 and P20. The Ar ions then generate ultraviolet rays. Furthermore, since this plasma is released into the atmosphere, oxygen radicals, nitrogen radicals, and the like are generated.

[0039] The plasma density of this plasma is 1×10 14 cm -3 More than 1×10 17 cm -3 The plasma density in the plasma generated by the dielectric barrier discharge is within the range of 1×1011 cm -3 More than 1×10 13 cm -3 The plasma density of the plasma generated by the plasma generators P10 and P20 is approximately three orders of magnitude greater than that of the plasma generated by dielectric barrier discharge. Consequently, more Ar ions are generated inside this plasma. As a result, the amount of radicals and ultraviolet rays generated is also large. The plasma density is approximately equal to the electron density inside the plasma.

[0040] The plasma temperature during plasma generation is in the range of approximately 1000K to 2500K. The electron temperature in this plasma is higher than the gas temperature. Moreover, the electron density is 1×10 14 cm -3 More than 1×10 17 cm -3 Although the temperature is within the range below, the gas temperature is within the range of approximately 1000K to 2500K. This plasma temperature is the temperature in the plasma generation region P where the plasma is being generated. Therefore, by changing the plasma conditions and the distance from the gas nozzle to the water surface, the plasma temperature at the liquid surface can be made to be approximately room temperature.

[0041] 2-2.Third plasma generator 7 is a diagram showing a schematic diagram of the plasma generator P30 generating plasma. The plasma generated by the plasma generator P30 is non-equilibrium atmospheric pressure plasma.

[0042] 7, plasma gas supplied from the gas supply unit 140 is emitted from the first electrode 110 in the direction of arrow K1. When a high-frequency voltage is applied between the first electrode 110 and the second electrode 210, a plasma generation region PG1 is formed between the first electrode 110 and the second electrode 210. The plasma generation region PG1 in FIG. 7 is depicted conceptually.

[0043] When the first potential application unit 120 and the second potential application unit 220 apply a voltage between the first electrode 110 and the second electrode 210, the second electrode 210 is disposed inside the liquid. In this manner, the liquid contained in the container 250 and the atmosphere are present between the first electrode 110 and the second electrode 210. The line connecting the first electrode and the second electrode intersects with the liquid level LL1 of the liquid.

[0044] Therefore, plasma is generated between the liquid surface LL1 and the first electrode 110. At this time, the liquid surface LL1 is depressed toward the liquid side by the wind pressure of the plasma gas emitted from the first electrode 110 in the direction of arrow K1. Then, the solution inside the liquid is partially electrolyzed and vaporized. Plasma is also generated inside the vaporized gas. In addition, the plasma generation region PG1 is in contact with the liquid surface LL1.

[0045] As a result, radicals derived from the air or water are generated, and the solution is irradiated with the radicals, which then react with water molecules or solutes in the solution.

[0046] 3. Method for producing plasma-activated culture medium 3-1.Culture solution preparation process First, a first culture solution is prepared. The first culture solution refers to the culture solution before plasma irradiation. The first culture solution contains amino acids. The first culture solution contains, for example, glycine, thiamine, sucrose, and inorganic salts.

[0047] 3-2.Plasma irradiation process Next, the first culture solution is irradiated with atmospheric pressure plasma generated in the plasma generation region by the plasma activated culture solution manufacturing device PM. The distance between the liquid surface and the plasma nozzle when irradiating the plasma is, for example, 3 mm. This distance may also be changed, for example, within a range of 0.1 cm to 3 cm. The plasma density in the plasma generation region is 1×10 14 cm -3 More than 1×10 17 cm -3The plasma temperature is within the range of approximately 1000K to 2500K. However, the plasma temperature at the liquid surface can be lowered to room temperature (approximately 300K). These plasma conditions are shown in Table 1. These conditions are merely examples.

[0048] [Table 1] Condition Numerical range Liquid level-spout distance 0.1cm or more 3cm or less Plasma density 1×10 14 cm -3 Over 1×10 17 cm -3 below Plasma temperature: 1000K or higher, 2500K or lower

[0049] In this way, a plasma-activated culture solution is produced by irradiating the first culture solution with atmospheric pressure plasma. It is believed that the irradiation of atmospheric pressure plasma causes the components of the first culture solution to react with radicals derived from the plasma. It is also believed that nitrite ions and nitrate ions increase in the culture solution. It is believed that the components of the first culture solution also react with these ions.

[0050] The plasma density of atmospheric pressure plasma is, for example, 2 × 10 16 cm -3 The atmospheric pressure plasma irradiation time is, for example, 30 seconds or more and 600 seconds or less. The volume of the first culture solution when irradiated with atmospheric pressure plasma is, for example, 10 ml or more and 1000 ml or less.

[0051] In this case, the plasma density-time product per unit volume in the plasma-activated culture medium is 6 x 10 14 sec cm -3 ml -1 Over 1.2 x 10 16 sec cm -3 ml -1Here, the plasma density-time product per unit volume is (plasma density) × (irradiation time) / (volume of first culture solution). In other words, the plasma density-time product per unit volume is the amount of plasma products irradiated to the first culture solution per unit volume.

[0052] 4. Effect of plasma-activated culture medium The plasma activated culture medium of this embodiment is the first culture medium irradiated with plasma. The plasma activated culture medium sterilizes Agrobacterium, which will be described later.

[0053] 5. Method for producing genetically modified plants using plasma-activated culture medium 5-1. Plasma activated culture medium manufacturing process As described above, the first culture solution is irradiated with atmospheric pressure plasma to produce a plasma activated culture solution.

[0054] 5-2. Callus generation process Meanwhile, at least a portion of the plant to be genetically modified is cultured in a second culture solution. The plant to be genetically modified is, for example, a seed. This plant is, for example, a monocotyledonous plant. An example of a monocotyledonous plant is an orchid. Other plants that are traded on the market may also be used. The seeds are sown in the second culture solution. Here, the components of the second culture solution may be the same as or different from the components of the first culture solution. The second culture solution contains amino acids and inorganic salts. This causes callus to form. In the case of orchids, orchid seeds are sown in the second culture solution. A cell mass called a protocorm is formed several weeks after germination. The protocorm then turns into callus.

[0055] 5-3. Infectious Agent Production Process A culture medium containing Agrobacterium is added to the second culture medium in which callus has formed. This infects the callus with Agrobacterium. In this way, an infective organism infected with Agrobacterium is produced. Agrobacterium DNA is introduced into part of the DNA of the infective organism. At this stage, the second culture medium contains the infective organism.

[0056] 5-4. Sterilization process The plasma-activated culture medium is mixed with the second culture medium containing the infective agent. The Agrobacterium in the infective agent is killed by the plasma-activated culture medium. The plasma-activated culture medium does not adversely affect plants such as orchids.

[0057] 6. Effects of the First Embodiment This method for producing genetically modified plants involves supplying Agrobacterium-infected plants with a plasma-activated culture medium irradiated with atmospheric pressure plasma, which sufficiently sterilizes the Agrobacterium.

[0058] 6. Variations 6-1. Callus generation process Instead of culturing in the second culture solution, at least a part of the plant may be cultured in another medium, such as a solid medium, or a solid medium containing the components of the second culture solution.

[0059] 6-2. Dilution of plasma-activated culture medium The plasma-activated culture medium may be diluted. In this case, the production method includes a dilution step of diluting the plasma-activated culture medium. The dilution ratio is, for example, 1 to 10 times. Of course, other dilutions are also possible.

[0060] 6-3.Third plasma generator A plasma generator P30 may be used to produce a plasma-activated culture solution. To do this, atmospheric pressure plasma generated in a plasma generation region by the plasma generator P30 is irradiated onto the first culture solution. The first electrode 110 is placed outside the first culture solution, and the second electrode 210 is placed inside the first culture solution. Then, gas is irradiated from the cylindrical portion 110a of the first electrode 110 toward the first culture solution. Then, a voltage is applied between the first electrode 110 and the second electrode 210 in this state.

[0061] 6-4. First electrode of the third plasma generator In the plasma generator P30 of the first embodiment, the cylindrical portion 110a of the first electrode 110 has a cylindrical shape. However, the shape is not limited to a cylindrical shape. It may also be a polygonal shape as long as it is a cylindrical shape.

[0062] 6-5. Miniaturized plasma generator The plasma generators P10, P20, etc. may be further miniaturized. By miniaturizing them sufficiently, a pen-shaped plasma generator can be manufactured. Even in this case, the same plasma density as that of the plasma generators P10, P20 can be obtained.

[0063] 6-6. Combinations In some cases, the modified examples of the first embodiment may be combined as appropriate.

[0064] (experiment) A. Experiment 1 1. Experimental Method 1-1. Solution manufacturing process Various solutions were produced using a plasma generator P20. The plasma density of the plasma generator P20 was 2 × 10 16 cm -3 The plasma irradiation time was 5 minutes, and the irradiation distance was 5 mm.

[0065] Table 2 shows the types of solutions prepared for the experiment. Solution 1 is NP medium diluted 1 / 2 and irradiated with atmospheric pressure plasma. Solution 2 is NP medium diluted 1 / 2 and irradiated with atmospheric pressure plasma, after which MES-KOH is added to adjust the pH. Solution 3 is NP medium diluted 1 / 2. Solution 4 is sterilized deionized water irradiated with atmospheric pressure plasma. Solution 5 is sterilized deionized water itself. Solution 6 is a mixture of glycine and an aqueous HCl solution.

[0066] [Table 2] Solution type pH Solution 1 Plasma activated culture medium 2.9-3.3 Solution 2 Plasma activated culture medium 5.2-5.4 Solution 3 NP medium 4.7-4.8 Solution 4 Plasma water 2.9~3.3 Solution 5 Sterile deionized water 7 Solution 6 Glycine HCl 3.0

[0067] Here, NP medium is Ichihashi New Phalaenopsis. The components of NP medium are shown in Table 3.

[0068] [Table 3] Ammonium nitrate Ammonium sulfate Boric acid calcium nitrate Cobalt chloride hexahydrate Copper sulfate pentahydrate Ethylenediaminetetraacetic acid disodium salt dihydrate Iron sulfate heptahydrate Magnesium nitrate Manganese sulfate monohydrate Molybdic Acid Dihydrate potassium iodide potassium nitrate Potassium dihydrogen phosphate Zinc sulfate heptahydrate Gellan gum glycine myo-inositol Niacin Pyridoxine hydrochloride sucrose Thiamine hydrochloride

[0069] 1-2. Callus generation process Orchid seeds were sown on NP medium and allowed to germinate. Culture was continued until protocorms formed, followed by callus formation.

[0070] 1-3. Infectious agent production process The OD of overnight cultured Agrobacterium strain EHA105 was 600 The suspension was diluted to a concentration of 0.4 and suspended in NP medium. The suspension was allowed to stand at room temperature for a certain period of time.

[0071] 1-4. Sterilization process A solution such as plasma activated culture medium was added to the Agrobacterium suspension, after which the viable cell count was measured.

[0072] 2. Experimental Results Figure 8 is a graph showing the relationship between the treatment time of the solution and the viable cell count. The horizontal axis of Figure 8 is the treatment time of the solution. The vertical axis of Figure 8 is the viable cell count.

[0073] As shown in Figure 8, by treating with Solution 1 for 10 minutes, the number of viable bacteria increased to 10 -3 By treating with Solution 1 for 30 minutes, the number of viable bacteria decreased to about 10 -4 By treating with Solution 1 for 60 minutes, the number of viable bacteria decreased to about 10 -6 It decreased to some extent.

[0074] By treating with Solution 2 for 10 minutes, the number of viable bacteria was 10 -1 By treating with Solution 2 for 60 minutes, the number of viable bacteria decreased to about 10 -2 ~10 -1 By treating with Solution 6 for 10 minutes, the number of viable bacteria decreased to about 10 -1 By treating with Solution 6 for 60 minutes, the number of viable bacteria decreased to about 10 -2 ~10 -1 It decreased to some extent.

[0075] The other solutions 3 to 5 showed almost no bactericidal effect.

[0076] Figure 9 is a graph showing the relationship between the number of days elapsed since the solution was produced and the number of viable bacteria. The horizontal axis of Figure 9 represents the number of days elapsed since the solution was produced. The vertical axis of Figure 9 represents the number of viable bacteria. The treatment time was 30 minutes.

[0077] As shown in FIG. 9, even after 7 days had passed since the preparation of Solution 1, the bactericidal effect remained almost unchanged by treating with Solution 1 for 30 minutes.

[0078] 3. Summary of the experiment The plasma-activated culture solution (Solution 1) exhibits a high bactericidal effect under acidic conditions. The plasma-activated culture solution (Solution 2) with its pH adjusted using MES-KOH exhibits a certain bactericidal effect. Solution 3 also exhibits a certain bactericidal effect.

[0079] Thus, plasma-activated culture medium exhibits a high bactericidal effect under acidic conditions. There are two possible reasons for the decrease in the bactericidal effect of Solution 2. The first is a change in pH. The second is that the bactericidal component was reduced by reacting with the MES-KOH molecules. The bactericidal effect of Solution 4, which was irradiated with plasma, was significantly lower than that of Solution 1. Therefore, it is thought that the bactericidal component is not a radical derived from water, such as a hydroxyl radical.

[0080] B. Experiment 2 1. Experimental Method 1-1. Solution manufacturing process Plasma-activated culture medium (solution 1) and NP medium (solution 3) were prepared.

[0081] 1-2. Callus generation process Orchid seeds were sown on NP medium and allowed to germinate. Culture was continued until protocorms formed, followed by callus formation.

[0082] 1-3. Solution addition process The plasma-activated culture solution (Solution 1) and NP medium (Solution 3) were added to the NP medium on which the callus had formed. The treatment time was 30 minutes.

[0083] 2. Experimental Results Figure 10 shows the regions where the solutions were added. As shown in Figure 10, protocombs treated with plasma-activated culture solution (Solution 1) for 30 minutes were placed in the left region of the dish, and protocombs treated with NP medium (Solution 3) for 30 minutes were placed in the right region of the dish.

[0084] Figure 11 shows the effect of adding solutions on protocombs. Protocombs treated with plasma-activated culture medium (Solution 1) are placed in the left region of the dish, and protocombs treated with NP medium (Solution 3) are placed in the right region of the dish.

[0085] 3. Summary of the experiment Regardless of the plasma exposure time, the plasma-activated culture solution (Solution 1) does not have a significant adverse effect on the protocorm. Therefore, even if the plasma-activated culture solution (Solution 1) is used to sterilize Agrobacterium, it is believed that the plants targeted for breeding will grow without any problems.

[0086] (Addendum) In a first aspect, the method for producing a genetically modified plant comprises the steps of irradiating a first culture solution with atmospheric pressure plasma to produce a plasma-activated culture solution, culturing at least a portion of the plant body in a second culture solution or solid medium to produce a callus, infecting the callus with Agrobacterium to produce an infective body, and mixing the plasma-activated culture solution with the second culture solution or solid medium containing the infective body to sterilize the Agrobacterium.

[0087] In the method for producing a genetically modified plant according to the second aspect, the plasma density of the atmospheric pressure plasma is 1×10 14 cm -3 More than 1×10 17 cm -3 The following is the result.

[0088] In the method for producing a genetically modified plant according to the third aspect, the atmospheric pressure plasma irradiation time is set to 30 seconds or more in the step of producing a plasma activated culture solution.

[0089] In the method for producing a genetically modified plant according to the fourth embodiment, the plant body is a seed.

[0090] In the method for producing a genetically modified plant according to the fifth aspect, the plant body is a monocotyledonous plant. [Explanation of symbols]

[0091] P1: Plasma irradiation device M1...Robot arm PM: Plasma activated culture medium manufacturing equipment P10, P20, P30...Plasma generator 10, 11...Housing 10i, 11i...Gas inlet 10o, 11o...Gas outlet 2a, 2b...electrodes P: Plasma region H...Hollow 110...1st electrode 120...first electric potential applying unit 130...First lead wire 140...Gas supply unit 150...Gas pipe connector 160...Gas pipe 170...First electrode protection member 210…Second electrode 220...second potential applying unit 230...Second lead wire 240...Second electrode protection member 250…container 260...Sealing member 270…mounting stand

Claims

1. a step of irradiating the first culture solution with atmospheric pressure plasma to produce a plasma activated culture solution; Cultivating at least a portion of the plant body in a second liquid or solid medium to produce callus; Infecting the callus with Agrobacterium to produce an infectivity; mixing the plasma activated culture solution with the second culture solution or the solid medium containing the infectious agent to sterilize the Agrobacterium; Including, The method for producing a genetically modified plant, wherein the pH of the plasma activated culture solution is 3.3 or less.

2. 2. The method for producing a genetically modified plant according to claim 1, The plasma density of the atmospheric pressure plasma is 1 x 10 １４ cm －３ 1x10 or more １７ cm －３ Is less than or equal to A method for producing a genetically modified plant, comprising:

3. 3. The method for producing a genetically modified plant according to claim 1 or 2, In the step of producing the plasma activated culture solution, The atmospheric pressure plasma irradiation time is set to 30 seconds or more. A method for producing a genetically modified plant, comprising:

4. The method for producing a genetically modified plant according to any one of claims 1 to 3, The plant body is Being a seed A method for producing a genetically modified plant, comprising:

5. The method for producing a genetically modified plant according to any one of claims 1 to 4, The plant body is Being a monocotyledonous plant A method for producing a genetically modified plant, comprising:

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