JP2023023727A - Methods for producing transgenic plants - Google Patents

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 The technical field herein relates to methods for producing genetically modified plants.

Breeding has been carried out for the purpose of improving plant productivity or producing plants with new properties. Conventionally, breeding has been performed in anticipation of mutation, but in recent years, research and development have been conducted on breeding by genetic recombination technology or genome editing.

For example, in Patent Document 1, a process of cultivating a Nicotiana plant by hydroponics while circulating a nutrient solution, a process of cultivating the plant in a state where the circulation of the nutrient solution is stopped, A method for increasing the expression level of a recombinant protein, comprising the steps of infecting a bacterium, cultivating a plant infected with Agrobacterium while circulating a nutrient solution, and extracting a recombinant protein from leaves of the plant. (Paragraph [0005] of Patent Document 1). Agrobacterium carries polynucleotides encoding recombinant proteins.

JP 2015-57950 A

By the way, Agrobacterium is sometimes removed from Agrobacterium-infected plants. In that case, antibiotics may be used. However, when Agrobacterium is sterilized with antibiotics, sterilization may be insufficient. If the sterilization of Agrobacterium is insufficient, the Agrobacterium will proliferate in the medium, making it difficult to continue culturing the plant.

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 kill Agrobacterium that has infected plants during plant breeding. .

The method for producing a genetically modified plant in the first aspect comprises the steps of irradiating a first culture solution with atmospheric pressure plasma to produce a plasma-activated culture solution, and exposing at least part of the plant body to the second culture solution or a step of culturing in a solid medium to produce callus; a step of infecting the callus with Agrobacterium to produce an infectious agent; and mixing to sterilize the Agrobacterium.

In this genetically modified plant production method, a plasma-activated culture medium irradiated with atmospheric pressure plasma is supplied to an Agrobacterium-infected plant. Plasma-activated broth is sufficient to kill Agrobacterium.

The present specification provides a method for producing genetically modified plants capable of highly efficiently sterilizing Agrobacterium that has infected plants during plant breeding.

FIG. 3 is a conceptual diagram for explaining the configuration of a robot arm that scans the gas outlet of the plasma generator of the first embodiment; Figure 2. A is a sectional view showing the configuration of the first plasma generator, and FIG. B is a diagram showing the shape of the electrode. Figure 3. A is a cross-sectional view showing the configuration of the second plasma generator, and FIG. B is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma region. It is a figure which shows schematic structure of the 3rd plasma generator in 1st Embodiment. It is a schematic block diagram which shows the upper structure of the 3rd plasma generator in 1st Embodiment. FIG. 3 is a schematic configuration diagram showing a lower structure of a third plasma generator in the first embodiment; FIG. 4 is a diagram for explaining a case where a third plasma generator irradiates plasma in the first embodiment; Fig. 3 is a graph showing the relationship between solution treatment time and viable cell count; It is a graph which shows the relationship between the elapsed days after manufacturing a solution, and the number of viable bacteria. FIG. 10 is a diagram showing areas where solutions are added; It is a photograph showing the effect on the protocorm when a solution is added.

Specific embodiments will be described below by taking a method for producing a genetically modified plant as an example. However, the technology herein is not limited to these embodiments.

(First embodiment)
A first embodiment will be described. In the method for producing a genetically modified plant according to the first embodiment, the Agrobacterium used for genetic modification is sterilized by treatment with a plasma-irradiated culture solution. Therefore, first, a plasma generator for irradiating plasma will be described.

1. Plasma-activated culture fluid production device 1-1. Configuration of Plasma-Activated Culture Fluid Production Apparatus As shown in FIG. 1, the plasma-activated culture medium production apparatus PM of the first embodiment has a plasma irradiation device P1 and an arm robot M1. The plasma irradiation device P1 is for generating plasma and irradiating the solution with the plasma.

As shown in FIG. 1, the arm robot M1 can move the position of the plasma irradiation device P1 in each of the x-axis, y-axis, and z-axis directions. For convenience of explanation, the plasma irradiation direction is the -z-axis direction. Thereby, the distance between the liquid level of the solution and the plasma irradiation device P1 can be adjusted. In addition, this plasma-activated culture medium manufacturing apparatus PM can irradiate the plasma only for that time by setting the plasma irradiation time in advance.

As will be described later, the plasma irradiation device P1 has three types (first plasma generation device P10, second plasma generation device P20, and third plasma generation device P30). And either method may be used. The third plasma generator P30 does not have the robot arm M1 and the like shown in FIG.

1-2. First plasma generator Fig.2. A is a sectional view showing a schematic configuration of the plasma generator P10. Here, the plasma generator P10 is a first plasma generator that ejects plasma in dots. Figure 2. B is shown in FIG. FIG. 3B is a diagram showing details of the shape of electrodes 2a and 2b of the plasma generator P10 of A. FIG.

The plasma generator P10 has a housing portion 10, electrodes 2a and 2b, and a voltage application portion 3. As shown in FIG. The housing 10 is made of a sintered body made of alumina (Al2 O3). The shape of the housing portion 10 is cylindrical. The inner diameter of the housing part 10 is 2 mm or more and 3 mm or less. The thickness of the housing part 10 is 0.2 mm or more and 0.3 mm or less. The length of the housing part 10 is 10 cm or more and 30 cm or less. A gas introduction port 10i and a gas ejection port 10o are formed at both ends of the housing portion 10 . The gas inlet 10i is for introducing gas for generating plasma. The gas ejection port 10o is an irradiation section for irradiating the outside of the housing section 10 with plasma. The direction in which the gas moves is the direction of the arrow in the figure.

Electrodes 2a and 2b are a counter electrode pair arranged to face each other. The length of the electrodes 2a and 2b in the facing surface direction is smaller than the inner diameter of the casing 10. As shown in FIG. For example, it is about 1 mm. The electrodes 2a, 2b are shown in FIG. As shown in B, a large number of recesses (hollows) H are formed in each of the facing surfaces. Therefore, the facing surfaces of the electrodes 2a and 2b have fine irregularities. The depth of this recess H is about 0.5 mm.

The electrode 2a is arranged inside the casing 10 and near the gas introduction port 10i. The electrode 2b is arranged inside the casing 10 and in the vicinity of the gas ejection port 10o. Therefore, in the plasma generator P10, the gas is introduced from the opposite side of the facing surface of the electrode 2a and the gas is ejected to the opposite side of the facing surface of the electrode 2b. The distance between the electrodes 2a and 2b is, for example, 24 cm. The distance between the electrodes 2a, 2b may be smaller.

The voltage applying section 3 is for applying an AC voltage between the electrodes 2a and 2b. The voltage application unit 3 boosts the voltage to 9 kV using a commercial AC voltage of 60 Hz and 100 V, and applies a voltage between the electrodes 2a and 2b.

When argon is introduced from the gas introduction port 10 i and a voltage is applied between the electrodes 2 a and 2 b by the voltage applying section 3 , plasma is generated inside the housing section 10 . Figure 2. A plasma generation region P is defined as a region in which plasma is generated, as indicated by diagonal lines A. FIG. The plasma generation region P is covered with the housing portion 10 .

1-3. Second plasma generator Fig.3. A is a sectional view showing a schematic configuration of the plasma generator P20. Here, the plasma generator P20 is a second plasma generator that linearly ejects plasma. Figure 3. B is shown in FIG. 4 is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma generation region P of the plasma generator P20 of A. FIG.

The plasma generator P<b>20 has a housing portion 11 , electrodes 2 a and 2 b and a voltage application portion 3 . The housing portion 11 is made of a sintered body made of alumina (Al2 O3). A gas introduction port 11i and a large number of gas ejection ports 11o are formed at both ends of the housing portion 11 . The gas inlet 11i is shown in FIG. It has a slit shape with the left-right direction of A as the longitudinal direction. The width of the slit from the gas introduction port 11i to just above the plasma generation region P (the width in the left-right direction in FIG. 3.B) is, for example, 1 mm.

The gas ejection port 11o is an irradiation section for irradiating the outside of the housing section 11 with plasma. The gas ejection port 11o is cylindrical or slit-shaped. The gas ejection port 11o in the case of a cylindrical shape is formed in a straight line along the longitudinal direction of the plasma region. The inner diameter of the gas ejection port 11o is within the range of 1 mm or more and 2 mm or less. Moreover, in the case of a slit shape, it is preferable that the slit width of the gas ejection port 11o is 1 mm or less. A stable plasma is thereby formed. The gas introduction port 11i introduces gas in a direction intersecting a line connecting the electrodes 2a and 2b.

The electrodes 2a and 2b and the voltage applying section 3 are the same as those of the plasma generator P10 shown in FIG. Similarly, a commercial AC voltage is used to apply a voltage between the electrodes 2a and 2b. Thereby, the plasma can be ejected in a straight line.

The plasma generator P20 for ejecting plasma in a straight line is shown in FIG. By arranging them in a row in the left-right direction of B, plasma can be ejected in a plane over a certain rectangular area.

1-4. Third Plasma Generator FIG. 4 is a conceptual diagram showing a schematic configuration of the third plasma generator P30. The plasma generator P30 is for irradiating the contained solution with plasma.

As shown in FIG. 4, the plasma generator P30 includes a first electrode 110, a second electrode 210, a first potential application section 120, a second potential application section 220, and a first lead wire 130. , a second lead wire 230, a gas supply portion 140, a gas pipe coupling connector 150, a gas pipe 160, a first electrode protection member 170, a second electrode protection member 240, and a first electrode support member 180. , a sealing member 191 , a coupling member 192 , a container 250 , a sealing member 260 and a pedestal 270 .

1-4-1. Schematic Configuration of Electrode The first electrode 110 has a cylindrical portion 110a. Plasma gas can be supplied to the inside of the cylindrical portion 110a. That is, the inside of the first electrode 110 communicates with the gas supply section 140 . The first electrode 110 blows off gas toward the second electrode 210 from the cylindrical portion 110a. A tip portion of the first electrode 110 is shaped like an injection needle. That is, the tip portion of the first electrode 110 has an inclined surface that is inclined with respect to the direction perpendicular to the axial direction of the first electrode 110 . A micro hollow is formed at the tip of the first electrode 110 .

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 has a cylindrical shape. Alternatively, it may have a polygonal prism shape. Alternatively, it may be needle-shaped with a sharp tip. Here, the second electrode 210 has a tip portion 211 . A tip portion 211 of the second electrode 210 is made of an iridium alloy containing iridium. For example, 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, an iridium alloy is suitable for the tip portion 211 of the second electrode 210 . Also, platinum may be used instead of iridium. Alternatively, it may be palladium. Alternatively, it is preferably a metal or alloy containing at least one of iridium, platinum and palladium. Also, the tip portion 211 of the second electrode 210 may be gold. Also, during discharge, the second electrode 210 is immersed in the solution contained in the container 250 .

The first potential applying section 120 is for applying a periodically changing potential to the first electrode 110 . The second potential applying section 220 is for applying a periodically changing potential to the second electrode 210 . Here, either one of the first potential application section 120 and the second potential application section 220 may be grounded. The first lead wire 130 is for electrically connecting the first electrode 110 and the first potential application section 120 . The first lead wire 130 is preferably made of nickel alloy or stainless steel. The second lead wire 230 is for electrically connecting the second electrode 210 and the second potential application section 220 . The second lead wire 230 may be nickel alloy or stainless steel. Thereby, a high frequency voltage is applied between the first electrode 110 and the second electrode 210 . That is, the first potential applying section 120 and the second potential applying section 220 are voltage applying sections for applying a voltage between the first electrode 110 and the second electrode 210 .

1-4-2. Gas Supply Path The plasma generator P30 has the gas supply section 140, the gas pipe coupling connector 150, and the gas pipe 160, as described above. Therefore, the gas supply unit 140 supplies the plasma gas to the inside of the cylindrical portion of the first electrode 110 through the gas pipe 160 and the gas pipe coupling connector 150 . Here, the gas supply unit 160 supplies Ar gas, for example. Alternatively, other rare gas may be supplied. Alternatively, it may contain a small amount of other gas such as oxygen gas. Therefore, plasma gas is sprayed from the first electrode 110 toward the solution contained in the solution 250 .

1-4-3. Configuration of Upper Structure 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 . The distal end portion 111 is arranged at a position facing the second electrode 210, as shown in FIG. A tip portion 111 of the first electrode 110 has an inclined surface 111a. The inclined surface 111 a is a surface that is inclined with respect to a surface perpendicular to the axial direction of the first electrode 110 . Further, the tip portion 111 is formed with a micro hollow 111b. The micro hollow 111b is a minute recess having a length of 0.5 mm or more and 1 mm or less and a width of 0.3 mm or more and 0.5 mm or less.

Moreover, as described above, the plasma generator 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 is for sealing the inside of the container 250 . The connecting member 192 is a member for connecting the first electrode 110 and the gas pipe connecting connector 150 via the sealing member 191 or the like.

1-4-4. Configuration of Lower Structure FIG. 6 is a diagram showing the lower structure of the plasma generator P30. As described above, the plasma generator P30 has the container 250, the sealing member 260, and the pedestal 270. As shown in FIG. The container 250 can contain a solution inside. Here, the solution includes culture medium and organic solvent. Further, the container 250 accommodates the first electrode 110 and the second electrode 210 inside. Also, the container 250 may have a scale. This is for measuring the amount of solution contained inside the container 250 .

The sealing member 260 is for closing 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 member other than this may be applied as long as it ensures the tightness of the container 250 and prevents the solution from leaking to the bottom of the container 250 . The base 270 is for supporting the container 250 and other members.

2. Plasma generated by 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. Here, atmospheric pressure plasma refers to plasma having a pressure within the range of 0.5 to 2.0 atmospheres.

In this embodiment, Ar gas is mainly used as the plasma generating gas. Electrons and Ar ions are, of course, generated inside the plasma generated by the plasma generators P10 and P20. The Ar ions then generate ultraviolet rays. Moreover, since this plasma is emitted into the atmosphere, it generates oxygen radicals, nitrogen radicals, and the like.

The plasma density of this plasma is in the range of 1×10 ¹⁴ cm ⁻³ to 1×10 ¹⁷ cm ⁻³ . The plasma density of plasma generated by dielectric barrier discharge is about 1×10 ¹¹ cm ⁻³ or more and 1×10 ¹³ cm ^{−3 or} less. Therefore, the plasma density of the plasma generated by the plasma generators P10 and P20 is about three orders of magnitude higher than the plasma density of the plasma generated by the dielectric barrier discharge. Therefore, more Ar ions are generated inside this plasma. Therefore, a large amount of radicals and ultraviolet rays are generated. This plasma density is almost equal to the electron density inside the plasma.

The plasma temperature at the time of plasma generation is in the range of about 1000K or more and 2500K or less. Also, the electron temperature in this plasma is higher than the gas temperature. Moreover, although the electron density is in the range of 1×10 ¹⁴ cm ⁻³ to 1×10 ¹⁷ cm ⁻³ , the gas temperature is in the range of 1000 K to 2500 K. The temperature of this plasma is the temperature in the plasma generation region P where plasma is generated. Therefore, the plasma temperature at the position of the liquid surface can be made about room temperature by changing the plasma conditions and the distance from the gas outlet to the water surface.

2-2. Third Plasma Generator FIG. 7 is a diagram schematically showing how the plasma generator P30 generates plasma. The plasma generated by the plasma generator P30 is non-equilibrium atmospheric pressure plasma.

As shown in FIG. 7, the 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. FIG. The plasma generation region PG1 in FIG. 7 is conceptually depicted.

When the first potential applying section 120 and the second potential applying section 220 apply a voltage between the first electrode 110 and the second electrode 210, the second electrode 210 is placed inside the liquid. ing. Thus, between the first electrode 110 and the second electrode 210 is the liquid contained in the container 250 and the atmosphere. A line connecting the first electrode and the second electrode intersects the liquid surface LL1.

Therefore, plasma is generated between the liquid surface LL<b>1 and the first electrode 110 . At this time, the liquid surface LL1 of the liquid receives the wind pressure of the plasma gas emitted from the first electrode 110 in the direction of the arrow K1, and is depressed toward the liquid side. Then, inside the liquid, the solution is partially electrolyzed and vaporized. Plasma is also generated inside the vaporized gas. Also, the plasma generation region PG1 is in contact with the liquid surface LL1.

As described above, radicals derived from air or water are generated. Then, the solution is irradiated with radicals. The radicals thereby react with water molecules or solutes in the solution.

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

3-2. Plasma Irradiation Step Next, the first culture solution is irradiated with atmospheric pressure plasma generated in the plasma generation region by the plasma-activated culture solution manufacturing apparatus PM. The distance between the liquid surface and the plasma ejection port during plasma irradiation is, for example, 3 mm. Also, this distance may be changed within a range of, for example, 0.1 cm or more and 3 cm or less. The plasma density in the plasma generation region is in the range of 1×10 ¹⁴ cm ⁻³ to 1×10 ¹⁷ cm ⁻³ . The plasma temperature of this plasma is in the range of about 1000K or more and 2500K or less. However, this plasma temperature can be lowered to about room temperature (about 300 K) at the liquid surface. These plasma conditions are shown in Table 1. These conditions are only examples.

[Table 1]
Conditions Numerical range Liquid surface-spout distance 0.1 cm or more and 3 cm or less Plasma density 1×10 ¹⁴ cm ^-3 or more and 1×10 ¹⁷ cm ^-3 or less Plasma temperature 1000 K or more and 2500 K or less

Thus, a plasma-activated culture solution is produced by irradiating the first culture solution with atmospheric pressure plasma. It is considered that the components of the first culture solution react with radicals and the like derived from the plasma due to the irradiation of the atmospheric pressure plasma. In addition, nitrite ions and nitrate ions increase in the culture solution. It is thought that the components of the first culture solution also react with these ions and the like.

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

In this case, the plasma density time product per unit volume in the plasma-activated culture medium is 6×10 ¹⁴ sec·cm ⁻³ ·ml ⁻¹ or more and 1.2×10 ¹⁶ sec·cm ⁻³ ·ml ^{−1 or} more. It is below. Here, the plasma density time product per unit volume is (plasma density)×(irradiation time)/(volume of the first culture solution). That is, the plasma density time product per unit volume is the amount of plasma products irradiated to the first culture solution per unit volume.

4. Effect of Plasma-Activated Culture Solution The plasma-activated culture solution of the present embodiment is obtained by irradiating the first culture solution with plasma. The plasma-activated culture medium kills Agrobacterium, which will be described later.

5. Method for producing genetically modified plants using plasma-activated culture medium 5-1. Plasma-activated culture solution manufacturing process As described above, the plasma-activated culture solution is manufactured by irradiating the first culture solution with atmospheric pressure plasma.

5-2. Callus Generation Step On the other hand, at least part of the plant to be genetically modified is cultured in a second culture medium. A plant body to be genetically modified is, for example, a seed. This plant is, for example, a monocotyledonous plant. Examples of monocotyledonous plants include orchids. In addition, other plants may be used as long as they are commercially traded plants. Seeds are sown in a second broth. Here, the components of the second culture medium may be the same as or different from those of the first culture medium. The second culture medium contains amino acids and inorganic salts. This results in callus. In the case of orchids, orchid seeds are sown in the second culture medium. Several weeks after germination, cell clusters called protocorms are formed. The protocorm becomes callus.

5-3. Infectious body production step A culture medium containing Agrobacterium is added to the second culture medium in which the callus has been formed. This infects the callus with Agrobacterium. Infected organisms infected with Agrobacterium are thus produced. Agrobacterium DNA enters into a portion of the DNA of the infective. At this stage, the second culture contains the infectives.

5-4. Sterilization Step The plasma-activated medium is mixed with the second medium containing the infectious agent. Plasma-activated media kills Agrobacterium in infected organisms. The plasma-activated culture solution does not adversely affect plants such as orchids.

6. Effects of the First Embodiment In this genetically modified plant production method, a plasma-activated culture medium irradiated with atmospheric pressure plasma is supplied to an Agrobacterium-infected plant. Plasma-activated broth is sufficient to kill Agrobacterium.

6. Modification 6-1. Callus Generation Step Instead of culturing in the second culture solution, at least part of the plant body may be cultured in another medium. For example, solid media may be used. Alternatively, it may be a solid medium using the components of the second culture solution.

6-2. Dilution of Plasma-Activated Medium The plasma-activated medium may be diluted. In this case, the production method comprises a dilution step of diluting the plasma-activated broth. The dilution ratio is, for example, 1-fold or more and 10-fold or less. Of course, it may be other than this.

6-3. Third Plasma Generator A plasma generator P30 may be used to produce the plasma-activated culture solution. For this purpose, the first culture solution is irradiated with atmospheric pressure plasma generated in the plasma generation region by the plasma generator P30. A first electrode 110 is placed outside the first medium and a second electrode 210 is placed in the first medium. Then, the 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 that state.

6-4. First Electrode of 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, it is not limited to a cylindrical shape. As long as it has a tubular shape, it may have a polygonal shape.

6-5. Miniaturized Plasma Generator The plasma generators P10, P20, etc. may be further miniaturized. By sufficiently reducing the size, a pen-type plasma generator can be manufactured. Even in that case, plasma densities equivalent to those of the plasma generators P10 and P20 can be obtained.

6-6. Combinations Modifications of the first embodiment may be combined as appropriate.

(experiment)
A. Experiment 1
1. Experimental method 1-1. Solution manufacturing process Various solutions were manufactured using the plasma generator P20. The plasma density of the plasma generator P20 was 2×10 ¹⁶ cm ⁻³ . The plasma irradiation time was 5 minutes. The irradiation distance was 5 mm.

Table 2 shows the types of solutions prepared for the experiments. Solution 1 is obtained by irradiating an NP medium diluted to 1/2 with atmospheric pressure plasma. Solution 2 is obtained by irradiating atmospheric pressure plasma to NP medium diluted to 1/2 and then adding MES-KOH to adjust the pH. Solution 3 is NP medium diluted 1/2. Solution 4 is sterile deionized water exposed to atmospheric pressure plasma. Solution 5 is sterile deionized water itself. Solution 6 is a mixture of glycine and HCl aqueous solution.

[Table 2]
Solution type pH
Solution 1 Plasma activated culture solution 2.9-3.3
Solution 2 Plasma activated culture solution 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

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

[Table 3]
Ammonium nitrate Ammonium sulfate Boric acid Calcium nitrate Cobalt chloride hexahydrate Copper sulfate pentahydrate Disodium ethylenediaminetetraacetate dihydrate Iron sulfate heptahydrate Magnesium nitrate Manganese sulfate monohydrate Molybdate dihydrate Potassium iodide Potassium nitrate Potassium dihydrogen phosphate Zinc sulfate heptahydrate Gellan gum Glycine myo-inositol Niacin Pyridoxine hydrochloride Sucrose Thiamine hydrochloride

1-2. Callus Generation Process Orchid seeds were sown on NP medium and allowed to germinate. Cultivation was then continued to form a protocomb. Afterwards, protocorm callus was formed.

1-3. Infectious organism production process EHA105 strain Agrobacterium cultured overnight was diluted to OD ₆₀₀ =0.4 and suspended in NP medium. The suspension was left at room temperature for a period of time.

1-4. Sterilization Step A solution such as a plasma-activated culture medium was added to the Agrobacterium suspension. After that, the viable cell count was measured.

2. Experimental Results FIG. 8 is a graph showing the relationship between solution treatment time and viable cell count. The horizontal axis of FIG. 8 is the treatment time of the solution. The vertical axis in FIG. 8 is the number of viable bacteria.

As shown in FIG. 8, the number of viable bacteria decreased to about 10 ⁻³ by treating solution 1 for 10 minutes. By treating solution 1 for 30 minutes, the number of viable bacteria decreased to about 10 ⁻⁴ . By treating solution 1 for 60 minutes, the number of viable bacteria decreased to about 10 ⁻⁶ .

By treating the solution 2 for 10 minutes, the viable cell count decreased to about 10 ^-1 . By treating the solution 2 for 60 minutes, the viable cell count decreased to about 10 ^-2 to 10 ^-1 . By treating the solution 6 for 10 minutes, the viable cell count decreased to about 10 ^-1 . By treating the solution 6 for 60 minutes, the viable cell count decreased to about 10 ^-2 to 10 ^-1 .

As for other solutions 3 to 5, almost no bactericidal effect was observed.

FIG. 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 FIG. 9 represents the number of days elapsed since the solution was produced. The vertical axis in FIG. 9 is the number of viable bacteria. The processing time is 30 minutes.

As shown in FIG. 9, the bactericidal effect hardly changed by treating Solution 1 for 30 minutes even after 7 days from the production of Solution 1.

3. Summary of Experiments The plasma-activated culture medium (solution 1) exhibits a high bactericidal effect under acidic conditions. The plasma-activated culture medium (solution 2), pH-adjusted with MES-KOH, shows some bactericidal effect. Solution 3 also exhibits some bactericidal effect.

Thus, the plasma-activated culture medium exhibits a high bactericidal effect under acidic conditions. The following two factors are conceivable as the cause of the decrease in the bactericidal effect of Solution 2. The first cause is that the pH has changed. The second reason is that the bactericidal component was reduced by reacting with MES-KOH molecules. The bactericidal effect of solution 4 irradiated with plasma was sufficiently lower than the bactericidal effect of solution 1. Therefore, the sterilizing component is not considered to be radicals derived from water such as hydroxyl radicals.

B. Experiment 2
1. Experimental method 1-1. Solution manufacturing process Plasma-activated culture solution (solution 1) and NP medium (solution 3) were manufactured.

1-2. Callus Generation Process Orchid seeds were sown on NP medium and allowed to germinate. Cultivation was then continued to form a protocomb. Afterwards, protocorm callus was formed.

1-3. Solution addition step Plasma-activated culture medium (solution 1) and NP medium (solution 3) were added to the NP medium on which callus was formed. Processing time was 30 minutes.

2. Experimental Results FIG. 10 is a diagram showing areas to which solutions are added. As shown in FIG. 10, the plasma-activated culture medium (solution 1) was added to the left region of the petri dish and the protocorms treated for 30 minutes were placed, and the NP medium (solution 3) was added to the right region of the petri dish. The protocorms treated for 30 minutes were placed on the plate.

FIG. 11 is a photograph showing the effect on the protocorm when the solution was added. Protocorms treated with plasma-activated culture medium (solution 1) are placed in the left region of the petri dish, and protocorms treated with NP medium (solution 3) are placed in the right region of the petri dish.

3. Summary of Experiments The plasma-activated medium (solution 1) does not have a significant adverse effect on the protocorm, regardless of the plasma irradiation time. Therefore, even if the plasma-activated culture medium (solution 1) is used for sterilization of Agrobacterium, it is considered that plants that are the target of breeding will grow without problems.

(Appendix)
The method for producing a genetically modified plant in the first aspect comprises the steps of irradiating a first culture solution with atmospheric pressure plasma to produce a plasma-activated culture solution, and exposing at least part of the plant body to the second culture solution or a step of culturing in a solid medium to produce callus; a step of infecting the callus with Agrobacterium to produce an infectious agent; and mixing to sterilize the Agrobacterium.

In the method for producing a genetically modified plant according to the second aspect, the atmospheric pressure plasma has a plasma density of 1×10 ¹⁴ cm ⁻³ or more and 1×10 ¹⁷ cm ⁻³ or less.

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

In the method for producing a genetically modified plant in the fourth aspect, the plant body is a seed.

In the method for producing a genetically modified plant in the fifth aspect, the plant is a monocotyledonous plant.

P1... Plasma irradiation device M1... Robot arm PM... Plasma-activated culture medium production apparatus P10, P20, P30... Plasma generators 10, 11... Housing parts 10i, 11i... Gas introduction ports 10o, 11o... Gas ejection ports 2a, 2b... Electrode P... Plasma region H... Concave portion (hollow)
DESCRIPTION OF SYMBOLS 110... 1st electrode 120... 1st electric potential application part 130... 1st lead wire 140... Gas supply part 150... Gas pipe connection connector 160... Gas pipe 170... First electrode protection member 210... Second electrode 220... 2 potential application part 230... second lead wire 240... second electrode protection member 250... container 260... sealing member 270... pedestal

Claims (5)

irradiating the first culture solution with atmospheric pressure plasma to produce a plasma-activated culture solution;
Culturing at least part of the plant body in a second culture medium or solid medium to produce callus;
a step of infecting the callus with Agrobacterium to produce an infected body;
a step of mixing the plasma-activated culture solution with the second culture solution or the solid medium containing the infectious agent to sterilize the Agrobacterium;
A method for producing a genetically modified plant comprising: In the method for producing a genetically modified plant according to claim 1,
The plasma density of the atmospheric pressure plasma is
A method for producing a genetically modified plant, wherein the concentration is 1×10 ¹⁴ cm ⁻³ or more and 1×10 ¹⁷ cm ⁻³ or less. In the method for producing a genetically modified plant according to claim 1 or 2,
In the step of producing the plasma-activated culture solution,
A method for producing a genetically modified plant, comprising applying the atmospheric pressure plasma for 30 seconds or longer. In the method for producing a genetically modified plant according to any one of claims 1 to 3,
The plant body
A method of producing a genetically modified plant comprising being a seed. In the method for producing a genetically modified plant according to any one of claims 1 to 4,
The plant body
A method for producing a transgenic plant comprising being a monocotyledonous plant.

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