Method for introducing substance into plant cell, genetically modified plant or plant cell and methods for producing same, and program for controlling device for introducing substance into plant cell

Abstract

The present invention provides a plant cell injection system that is automatically controlled under conditions specialized for plants. This method for introducing a substance into a plant cell comprises: a) a step for positioning a nano-pipette at a cell-corresponding position; b) a step for moving the nano-pipette to a predetermined position toward a cell on the basis of an electric current between the inside of the nano-pipette and an electrolytic solution; c) a step for moving, for a configured puncture distance, the nano-pipette toward the cell and puncturing the cell; d) a step for applying a voltage to discharge the substance into the cell; and e) a step for extracting the nano-pipette.

Description

Method for introducing a substance into a plant cell, genetically modified plants or plant cells, methods for producing the same, and control program for an apparatus for introducing a substance into a plant cell

[0001] The present disclosure relates to a method for introducing a substance into a plant cell, a genetically modified plant or plant cell and a method for producing the same, and a control program for an apparatus for introducing a substance into a plant cell.

[0002] Genome editing tools such as the CRISPR-Cas9 system are expected to be easily used for genetic modification of individual organisms. In particular, methods for introducing genome editing tools into individual plants include, for example, the nanopipette injection method described in Patent Documents 1 and 2, and the in planta particle bombardment (iPB) method, which involves introducing a complex of gold particles and a genome editing tool into an individual plant using a particle gun method described in Patent Documents 3 and 4. With the iPB method, the genome editing tool is introduced into random cells, and damage to the introduced cells and introduced substances may occur, raising concerns about the stability of gene modification efficiency and invasiveness to the individual plant. While nanopipette injection methods are expected to improve gene modification efficiency, stability, and minimal invasiveness, injection conditions specific to plants have not yet been established.

[0003] International Publication No. 2014 / 160036 International Publication No. 2013 / 012452 JP 2017-205103 A JP 2017-205104 A

[0004] The present disclosure aims to establish injection conditions specific to plants in a method for injecting into plant cells using a nanopipette, and to provide an automatically controlled injection system for plant cells.

[0005] [1] A method for introducing a substance into a plant cell, comprising: a) positioning a nanopipette filled with a substance at a position corresponding to the plant cell in an electrolyte; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady current is a set current drop rate of 2% to 50%; c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the plant cell; and e) removing the nanopipette. [2] The method according to [1] above, wherein the substance is a protein, a mixture or complex containing a protein, a mixture or complex containing a protein and a nucleic acid, a nucleic acid, or a dye. [3] The method according to [2] above, wherein the substance is a genome editing substance. [4] The method according to [2] above, wherein the substance is a positively charged substance or a negatively charged substance, and when the substance is a positively charged substance, step d) is carried out by applying a voltage so that the inside of the nanopipette has a positive potential and the electrolyte has a negative potential, and when the substance is a negatively charged substance, step d) is carried out by applying a voltage so that the inside of the nanopipette has a negative potential and the electrolyte has a positive potential. [5] The method according to [4] above, wherein step d) is carried out by applying a voltage at a set applied voltage of not less than -11 V and not more than +11 V for a set application time of not less than 0.1 seconds and not more than 10.0 seconds. [6] A method for producing genetically modified plants or plant cells, comprising: a) positioning a nanopipette filled with a genetic modification substance at a position corresponding to a plant cell in an electrolyte; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady current is a set current drop rate of 2% to 50%; c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte to expel the substance into the plant cell; and e) removing the nanopipette.[7] A genetically modified plant or plant cell produced by a method comprising the following steps: a) positioning a nanopipette filled with a genetic modification substance at a position corresponding to the plant cell in an electrolyte; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady current is a set current drop rate of 2% to 50%; c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte to expel the substance into the plant cell; and e) removing the nanopipette. [8] A control program for an apparatus for introducing a substance into a plant cell, the control program including instructions for performing the following: a) positioning a nanopipette filled with a gene modification substance at a position in an electrolyte corresponding to the plant cell; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady-state current is a set current drop rate of 2% to 50%; c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte to expel the substance into the plant cell; and e) removing the nanopipette.

[0006] According to the present disclosure, in a method for injecting into plant cells using a nanopipette, injection conditions specific to plants can be established, and an automatically controlled injection system for plant cells can be provided.

[0007] 1 is a conceptual diagram of a system for use in the method of the present disclosure and a pipette approach step in the method of the present disclosure. FIG. 2 is a conceptual diagram of a pipette puncture step in the method of the present disclosure. FIG. 3 is a conceptual diagram of a pipette material ejection step in the method of the present disclosure. FIG. 4 is a diagram showing an example of a control mechanism for a system for use in the method of the present disclosure. FIG. 5 is a photograph of rice callus genome-edited using the method of the present disclosure. The upper and lower figures each show two different samples. The whitened area is boxed in the right figure. FIG. 6 is a photograph of an individual barley plant genome-edited using the method of the present disclosure. The lower figure is an enlarged photograph of the upper figure. The whitened area is boxed in the right figure. FIG. 7 is a graph showing the survival rate of barley into which a reagent solution was introduced using the method of the present disclosure, depending on each injection voltage condition.

[0008] The present invention will be described in detail below, with reference to the drawings as necessary. However, the drawings are merely examples for explaining the present invention, and the technical scope of the present invention is not limited by the examples shown in the drawings.

[0009] [System for introducing a substance into a plant cell] A system (a system for introducing a substance into a plant cell, hereinafter referred to as the "system of the present disclosure") for use in the methods of the present disclosure (methods for introducing a substance into a plant cell and methods for producing genetically modified plant cells) will be described. The system of the present disclosure includes, for example, the following components: a nanopipette; a three-dimensional (xyz) movement pipette holder for moving the nanopipette in three dimensions (sometimes referred to as "xyz" for convenience); an electrode (hereinafter referred to as the "pipette electrode") that can come into contact with the liquid filled inside the nanopipette; an electrode (hereinafter referred to as the "reference electrode") that can come into contact with the electrolyte in the cell holder; a current measurement circuit for measuring the current between the pipette electrode and the reference electrode; and a voltage application circuit for applying a voltage between the pipette electrode and the reference electrode.

[0010] The system of the present disclosure may further include a position input device for manually moving the three-dimensional movement module. The system of the present disclosure is used by being attached to an optical microscope. The system of the present disclosure is used by placing a cell holder that holds plant cells and an electrolyte solution in which the plant cells are immersed on the optical microscope, and by holding a solution containing a substance to be introduced into the plant cells inside the nanopipette.

[0011] Each component will be described below.

[0012] (Nanopipette) A "nanopipette" refers to a tubular structure having a nanoscale tip opening. A nanoscale tip opening is, for example, a conical tip opening (i.e., a nanopore) of about 10 nm to about 500 nm, preferably about 50 nm (±20%). Nanopipettes are made of inert, non-biological materials such as glass or quartz. The inner wall of the nanopipette may be surface-treated to suppress adsorption of substances (e.g., nucleic acids, proteins) filled inside the nanopipette. Furthermore, the nanopipette preferably has a shape or scale that allows the insertion of an electrode into the nanopipette that contacts the solution inside the nanopipette.

[0013] Nanopipettes have a single flow path (sometimes referred to as a "barrel" or "bore") within their tube, or multiple parallel flow paths. A nanopipette with a single flow path within its tube is sometimes referred to as a "single-barreled nanopipette." A nanopipette with multiple parallel flow paths within its tube is sometimes referred to as a "multi-barreled nanopipette." A nanopipette with two parallel flow paths within its tube is sometimes referred to as a "double-barreled nanopipette." The nanopipette used in the method of the present disclosure is preferably a single-barreled nanopipette, from the standpoints of ease and reliability of the operation of filling the nanopipette with a substance, and accuracy of current measurement and voltage application.

[0014] Nanopipettes are commercially available (for example, Yokogawa, product number SU10ACC-NP01, etc.) Nanopipettes can also be fabricated by, for example, pulling a capillary tube made of glass or quartz with a laser.

[0015] Details of the nanopipette are described, for example, in Patent Document 1 (WO 2014 / 160036) and Patent Document 2 (WO 2013 / 012452).

[0016] (Three-dimensional (xyz) movement pipette holder) A "three-dimensional (xyz) movement pipette holder" is a pipette holder to which a nanopipette is attached and which moves the attached nanopipette in three dimensions by driving a rough actuator and a fine actuator. The nanopipette is attached to the three-dimensional movement pipette holder so that two of the three directions (sometimes referred to as the "x-axis direction" and the "y-axis direction" for convenience) are perpendicular or nearly perpendicular to the long axis of the nanopipette, and the remaining direction (sometimes referred to as the "z-axis direction" for convenience) is parallel or nearly parallel to the long axis of the nanopipette. The three-dimensional movement pipette holder may be composed of, for example, a holder stage driven by a rough actuator and a holder head driven by a fine actuator, mounted on the holder stage, and to which the nanopipette is attached.

[0017] A "rough actuator" is a three-dimensional actuator that allows for rough positioning, and examples of such actuators include those with a stroke on the order of 10 to 100 mm and a setting resolution on the order of 0.1 to 1 μm. Examples of rough actuators include electromagnetic force-driven actuators such as motor-based (rotary motor, linear motor) actuators.

[0018] A "fine actuator" is a three-dimensional actuator capable of fine positioning, such as a three-dimensional actuator with a stroke on the order of 100 to 500 μm and a setting resolution on the order of 1 nm. Examples of fine actuators include piezoelectric effect-driven actuators, such as piezoelectric element-based actuators. Note that if the XY axis setting resolution of the rough actuator has sufficient performance for the target cell size, the fine actuator may be limited to one dimension, the Z axis, to specialize in precise approach and puncture of cells.

[0019] (Position Input Device) The position input device is a device for manually inputting and indicating the position of the nanopipette. Examples of the position input device include a pointing device such as a joystick, a key input device such as a keyboard, and a combination of these.

[0020] (Pipette Electrode, Reference Electrode) Examples of the pipette electrode and reference electrode include a gold electrode, a silver electrode (e.g., a silver tetrakis(4-chlorophenyl)borate (AgTBACI) electrode, an Ag / AgCl electrode, etc.), and a platinum electrode. The reference electrode may be used so as to come into contact with the electrolyte in a cell holder. Furthermore, when the nanopipette is a multi-barrel (e.g., double-barrel) nanopipette, the pipette electrode may be placed in a flow path that holds a substance to be introduced into a plant cell, and the reference electrode may be placed in a separate flow path.

[0021] (Current measurement circuit, voltage application circuit) The current measurement circuit is a circuit for measuring the ionic current between the pipette electrode and the reference electrode (i.e., the current between the inside of the nanopipette and the electrolyte). The current measurement circuit has a current measurement range of approximately 100 pA to 100 nA, and is preferably capable of measuring current changes (reductions) of approximately 2% to 50% for cell surface detection. For example, in a case where the current (steady-state current) is 10 nA at a sufficient distance from the cell surface, if cell surface detection is set as a 20% decrease in current, the point at which the current reaches 8 nA is measured. Also, a low-noise amplifier circuit or the like is used to accurately detect a very small steady-state reference current and its changes. Low noise may also be achieved using software-based digital filtering techniques.

[0022] The current measured by the current measurement circuit is used as an indicator of the distance between the cell and the nanopipette tip based on the principles of scanning ion conductance microscopy (SICM). When the nanopipette tip comes into contact with the electrolyte in the cell holder, an ionic current begins to flow between the nanopipette and the reference electrode. Moving the nanopipette tip closer to the cell does not significantly change the current value if the nanopipette tip is sufficiently far from the cell surface. This current value is called the "steady-state current." As the nanopipette tip approaches the cell very close, the current rapidly decreases in proportion to the distance between the cell and the nanopipette tip. This is due to the highly insulating nature of the cell membrane. Therefore, the nanopipette tip can be automatically paused in close proximity to the cell by automatically controlling it to pause when the rate of decrease from the steady-state current reaches a preset value (hereinafter referred to as the "set current decrease rate").

[0023] (Voltage application circuit) The voltage application circuit is a circuit for applying a voltage between the pipette electrode and the reference electrode (between the inside of the nanopipette and the electrolyte). The voltage application circuit is preferably a circuit capable of applying a voltage on the order of -11 to +11 V with time control on the order of 0.01 seconds.

[0024] (Plant Cells) The method of the present disclosure is applicable to plant cells. The form of the plant cells is not particularly limited, and may be cultured cells or cells present in callus, seeds, sprouts, plant tissue, plant tissue fragments, etc. Examples of plant tissues include the shoot apical meristem of sprouts (e.g., L2 layer, etc.).

[0025] (Cell Holder) The cell holder for holding plant cells is not particularly limited, but typically a transparent holder with an openable top is used. Examples of such holders include cell culture vessels with an openable top, such as cell culture dishes and multi-well plates, and flat plates such as glass slides.

[0026] (Electrolyte for Cell Immersion) Examples of the electrolyte for immersing cells in the cell holder include liquid media (e.g., Murashige and Skoog (MS medium), Gamborg B5 medium, Chu (N6) medium), buffered saline (e.g., phosphate buffered saline (PBS), HEPES buffered saline (HBS), Hank's balanced salt solution (HBSS)), etc.).

[0027] (Substances to be introduced into plant cells) The substances to be introduced into plant cells are not particularly limited, but examples include proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, and pigments. The substances to be introduced into plant cells are not particularly limited, but a substance that dissolves or suspends in an electrolyte solution is preferred. Furthermore, a charged substance is preferred. The charged substance may be a single charged substance, a mixture or complex of multiple substances that is charged as a whole, or a substance that charges the entire solution when dissolved or suspended in an electrolyte solution (a single substance or a mixture or complex of multiple substances). Charged substances include positively charged substances and negatively charged substances. The substances to be introduced into plant cells (e.g., proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, and pigments) are preferably either positively or negatively charged substances. For example, proteins, mixtures or complexes of proteins, mixtures or complexes containing nucleic acids and proteins, etc. may be either positively or negatively charged substances, for example, positively charged substances. Examples of mixtures or complexes containing nucleic acids and proteins include genome editing substances. Genome editing substances include CRISPR-Cas9, CRISPR-Cas3, ZFN, TALEN, and PPR systems. Furthermore, for example, nucleic acids (e.g., DNA, RNA, etc.) may be either positively charged or negatively charged substances, for example, they may be negatively charged substances. Furthermore, the substance introduced into the plant cell may be a marker substance such as a dye, or may contain a marker substance.

[0028] The substance introduced into the plant cell is usually in the form of a liquid (e.g., a solution). When the substance introduced into the plant cell is a solution, the solvent can be an aqueous electrolyte or a non-aqueous electrolyte, but an aqueous electrolyte is preferred. Examples of the aqueous electrolyte include those exemplified above as the electrolyte into which the cell is immersed.

[0029] [Method for Introducing a Substance into a Plant Cell] The method for introducing a substance into a plant cell according to the present disclosure comprises the following steps: a) positioning a nanopipette filled with a substance at a position in an electrolyte corresponding to the plant cell (pipette positioning step); b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the current drop rate from the steady state current is a set current drop rate of 2% to 50% (pipette approach step); c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell (pipette puncturing step); d) applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the plant cell (substance discharging step); and e) removing the nanopipette (pipette removal step).

[0030] (Step a): Pipette Positioning Step) Step a) is a step of positioning a nanopipette filled with a substance at a plant cell-corresponding position in the electrolyte. Methods for filling the nanopipette with a substance include, for example, centrifugation and suction. The centrifugation method may be performed by attaching the nanopipette to a centrifuge holder, filling the nanopipette with a substance through the base opening, and centrifuging the centrifuge holder with the nanopipette attached in a centrifuge. The suction method may be performed by suctioning the substance through the tip opening. For positioning, the nanopipette filled with a substance may be attached to, for example, the three-dimensional (xyz) moving pipette holder described above. The "plant cell-corresponding position" refers to a position on the "z-axis" from the location of the target plant cell. Positioning may be performed using a rough actuator, a fine actuator, or a combination of these. The rough actuator and the fine actuator may be driven manually via a position input device or automatically by a program. Furthermore, there is also a method of positioning the X and Y axes using the stage of the microscope.

[0031] (Step b): Pipette Approach Step) Step b) is a step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the rate of decrease from the steady current becomes a set current decrease rate ("pipette pause position (P2)" in Figure 1). The "direction of approaching the plant cell" refers to the direction toward the plant cell on the "z-axis." The current (ionic current) between the inside of the nanopipette and the electrolyte can be measured by a current measurement circuit as the current between the pipette electrode and the reference electrode. In addition, in step b), in order to measure the current, it is preferable to apply a low voltage (set approach voltage) between the inside of the nanopipette and the electrolyte (i.e., between the pipette electrode and the reference electrode) that is set to a level that prevents the filling solution from flowing out due to electroosmotic flow. The change in the current between the inside of the nanopipette and the electrolyte will be explained with reference to Figure 1. When the tip of the nanopipette is located outside the electrolyte, the inside of the nanopipette and the electrolyte are disconnected, and the current (I) is zero (I ０ ) When the tip of the nanopipette reaches the electrolyte surface position (P0) and enters the electrolyte, the inside of the nanopipette and the electrolyte are connected, so the current (I) increases. For a while after that, even if the nanopipette is advanced, the current (I) does not change significantly and becomes steady. The current (I) at this time is called the steady current (I １ When the tip of the nanopipette reaches a position ("current drop starting point position (P1)") that is extremely close to the cell surface position (P3), the current begins to drop rapidly according to the distance between the cell and the tip of the nanopipette. After that, the current (I) becomes a steady current (I １ ) by the set current reduction rate (R) ２) ("pipette pause position (P2)"). The nanopipette may be paused at the pipette pause position (P2). The nanopipette movement in step b) may be performed using a rough actuator, a fine actuator, or a combination of these. At least the movement from the current drop start point position (P1) onwards is preferably performed using a fine actuator. The nanopipette movement in step b) is preferably performed automatically by a program. By presetting the set current drop rate according to the plant species, it is possible to suppress damage to cells and achieve highly efficient substance introduction into cells.

[0032] <Current Drop Rate> The current drop rate used is a value optimized for plant cells. The current drop rate is preferably 2 to 50%, more preferably 3 to 40%, and most preferably 5 to 20%. Furthermore, the current drop rate can be a value optimized for the plant species, as described in the "Examples of Setting Parameters" below.

[0033] <Set Approach Voltage> A set approach voltage optimized for plant cells is used. The set approach voltage is preferably −2 to +2 V, more preferably −1.0 to +1.0 V. Furthermore, the set approach voltage can be a value optimized depending on the plant species, as described in the “Examples of Set Parameters” below.

[0034] (Step c): Pipette Puncture Step) Step c) is a step of moving the nanopipette from the position (i.e., the pipette pause position (P2)) toward the cell by a set puncture distance to puncture the plant cell. "Toward the cell" refers to the direction toward the plant cell (the interior of the plant cell) on the "z-axis." The movement of the nanopipette in the pipette puncture step is usually performed by a fine actuator under automatic program control. Furthermore, it is preferable that the movement of the nanopipette in the pipette puncture step be performed at a faster movement speed than in the pipette approach step. By presetting the puncture distance according to the plant species, it is possible to suppress damage to the cell and achieve highly efficient substance introduction into the cell.

[0035] <Set Penetration Distance> The set puncture distance is a value optimized for plant cells. The set puncture distance is preferably 1 to 50 μm, more preferably 3 to 40 μm. Furthermore, the set puncture distance can be a value optimized for the plant species, as described in the "Examples of Set Parameters" below. When a genome editing substance is introduced to obtain a genetically modified plant, it is preferable to set the puncture distance to a value that can reach the L2 layer of the shoot apical meristem.

[0036] (Step d): Substance Discharge Step) Step d) is a step of discharging a substance into a plant cell by applying a voltage between the inside of the nanopipette and the electrolyte solution. Depending on the type of substance to be introduced into the plant cell (e.g., protein, a mixture or complex containing protein, a mixture or complex containing protein and nucleic acid, nucleic acid, and dye, etc.), the voltage is applied so that the inside of the nanopipette is at a positive potential and the electrolyte solution is at a negative potential, or so that the inside of the nanopipette is at a negative potential and the electrolyte solution is at a positive potential. When the substance to be introduced into the plant cell is a positively charged substance, the voltage is applied so that the inside of the nanopipette is at a positive potential and the electrolyte solution is at a negative potential. When the substance to be introduced into the plant cell is a negatively charged substance, the voltage is applied so that the inside of the nanopipette is at a negative potential and the electrolyte solution is at a positive potential. The voltage is applied at a set injection voltage (set applied voltage: V Ｉ) and the set injection time (set application time: T １ ) is preferably performed.

[0037] <Set Injection Voltage> The set injection voltage is a value optimized for plant cells. In the case of a positively charged substance, the set injection voltage is preferably −11 to +11 V, more preferably −10 to +10 V, and most preferably 4 V to 10 V. Furthermore, the set injection voltage can be a value optimized depending on the plant species, as described in the "Examples of Set Parameters" below.

[0038] <Set Injection Time> A set injection time optimized for plant cells is used. The set injection time is preferably 0.1 to 10 seconds, more preferably 0.5 to 5.0 seconds. Furthermore, the set injection time can be optimized depending on the plant species, as described in "Examples of Set Parameters" below.

[0039] (Step e): Pipette Removal Step) Step e) is a step of removing the nanopipette. The "direction away from the plant cell" refers to the direction away from the plant cell (interior of the plant cell) on the "z-axis." The nanopipette may be moved at a set removal distance. The nanopipette movement in the pipette removal step is usually performed by a fine actuator under automatic program control.

[0040] <Set Retraction Distance> The set retraction distance is a value optimized for plant cells. The set retraction distance is preferably within a range of 40 μm or more. The set puncture distance can be set to the above value for all plant species.

[0041] [Method for producing genetically modified plants or plant cells] The method of the present disclosure can also be used to produce genetically modified plants or plant cells. The method for producing genetically modified plants or plant cells of the present disclosure includes the following steps: a) positioning a nanopipette filled with a gene modification substance at a position corresponding to a plant cell in an electrolyte (pipette positioning step); b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady current is a set current drop rate of 2% to 50% (pipette approach step); c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell (pipette puncture step); d) applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the plant cell (substance discharge step); and e) removing the nanopipette (pipette removal step).

[0042] Examples of gene modification substances include positively charged gene modification substances and negatively charged gene modification substances. Positively charged gene modification substances include gene modification substances based on mixtures or complexes containing proteins and nucleic acids, such as genome editing substances (e.g., Cas9-sgRNA-RNP complexes used in CRISPR-Cas9 systems, as well as substances used in genome editing systems such as CRISPR-Cas3, ZFN, TALEN, and PPR). Negatively charged gene modification substances include nucleic acids (DNA, RNA). Examples of nucleic acids include DNA such as plasmid vectors, and RNA such as antisense RNA and siRNA.

[0043] The conditions for the method for producing genetically modified plants or plant cells can be the same as those described for the method for introducing a substance into plant cells of the present disclosure.

[0044] [Control Program for Apparatus for Introducing a Substance into a Plant Cell] The present disclosure also provides a control program for an apparatus for introducing a substance into a plant cell. The program includes instructions for performing the following steps: a) positioning a nanopipette filled with a gene modification substance at a position in the electrolyte corresponding to the plant cell (pipette positioning step); b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the drop rate from the steady current is a set current drop rate of 2% to 50% (pipette approach step); c) moving the nanopipette toward the cell at a set puncture distance of 1 μm to 50 μm to puncture the plant cell (pipette puncturing step); d) applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the plant cell (substance discharge step); and e) removing the nanopipette (pipette removal step).

[0045] The program of the present disclosure may further include instructions for controlling the approach voltage, the injection voltage (applied voltage), the injection time (application time), and the withdrawal distance.

[0046] The conditions controlled by the program of the present disclosure can be the conditions described in the method of introducing a substance into a plant cell of the present disclosure.

[0047] [Examples of Setting Parameters] [Examples of Setting Parameters for Introducing Positively Charged Substances] <Plants in general> Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 2 to 11 V, more preferably 3 to 10.5 V, most preferably 4 to 10 V Current reduction rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 8 to 32 μm, more preferably 9 to 31 μm, most preferably 10 to 30 μm Injection time: preferably 0.5 to 6 seconds, more preferably 0.8 to 6 seconds, most preferably 1 to 5 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0048] <Brassicaceae> Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 11 V, more preferably 4.5 to 10.5 V, most preferably 5 to 10 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 5.5 seconds, more preferably 0.8 to 5.2 seconds, most preferably 1 to 5 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0049] <<Raphanus genus (Raphanus)>> Example: radish (sprouts) (Raphanus sativus var. hortensis) Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 6 V, more preferably 4.5 to 5.5 V, most preferably 5 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0050] <Poaceae> Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 11 V, more preferably 4.55 to 10.5 V, most preferably 5 to 10 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 8 to 42 μm, more preferably 9 to 41 μm, most preferably 10-40 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0051] <<Hordeum>> Example: barley (Hordeum vulgare) Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 2 to 8 V, more preferably 3 to 7 V, most preferably 4 to 5 V Current decrease rate: preferably 13 to 15%, more preferably 13.5 to 14.5%, most preferably 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 6 seconds, more preferably 1 to 6 seconds, most preferably 3 to 5 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0052] <<Zea>> Example: corn (Zea mays) Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 6 to 11 V, more preferably 6.5 to 10.5 V, most preferably 7 to 10 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 18 to 42 μm, more preferably 19 to 41 μm, most preferably 20-40 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0053] <Solanaceae> Example: Solanum Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 8 V, more preferably 4.5 to 8.5 V, most preferably 5 to 7 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 8 to 32 μm, more preferably 9 to 31 μm, most preferably 10 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0054] Example: Tomato (Solanum lycopersicum) Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 8 V, more preferably 4.5 to 8.5 V, most preferably 5 to 7 V Current drop rate: preferably 13 to 15%, more preferably 13.5 to 14.5%, most preferably 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 2.5 to 3.5 seconds, more preferably 2.8 to 3.2 seconds, most preferably 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0055] Example: potato (Solanum tuberosum) Approach voltage: preferably 0.5 to 1.5 V, more preferably 0.8 to 1.2 V, most preferably 1 V Injection voltage: preferably 4 to 8 V, more preferably 4.5 to 8.5 V, most preferably 5 to 7 V Current reduction rate: preferably 19 to 21%, more preferably 19.5 to 20.5%, most preferably 20% Puncture distance: preferably 8 to 32 μm, more preferably 9 to 31 μm, most preferably 10 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0056] [Examples of setting parameters for introducing negatively charged substances] <Plants in general> Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -2 to -11 V, more preferably -2.5 to -10.5 V, most preferably -3 to -10 V Current decrease rate: preferably 9 to 21%, more preferably 9.5 to 20.5%, most preferably 10 to 20% Puncture distance: preferably 8 to 32 μm, more preferably 9 to 31 μm, most preferably 10 to 30 μm Injection time: preferably 0.3 to 3.5 seconds, more preferably 0.4 to 3.2 seconds, most preferably 0.5 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0057] <Brassicaceae> Example: Raphanus genus>> Example: Radish (Raphanus sativus var. hortensis) Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -2 to -8 V, more preferably -2.5 to -7.5 V, most preferably -5 to -7 V Current decrease rate: preferably 13 to 21%, more preferably 13.5 to 20.5%, most preferably 14 to 20% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Pull-out distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0058] <Poaceae> Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -3 to -8 V, more preferably -3.5 to -7.5 V, most preferably -4 to -7 V Current decrease rate: preferably 13 to 15%, more preferably 13.5 to 14.5%, most preferably 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0059] <<Hordeum>> Example: barley (Hordeum vulgare) Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -3 to -8 V, more preferably -3.5 to -7.5 V, most preferably -4 to -7 V Current decrease rate: preferably 13 to 15%, more preferably 13.5 to 14.5%, most preferably 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0060] <Solanaceae> Example: Solanum Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -6 to -11 V, more preferably -6.5 to -10.5 V, most preferably -7 to -10 V Current decrease rate: preferably 9 to 15%, more preferably 9.5 to 14.5%, most preferably 10 to 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0061] Example: Tomato (Solanum lycopersicum) Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -6 to -11 V, more preferably -6.5 to -10.5 V, most preferably -7 to -10 V Current decrease rate: preferably 13 to 15%, more preferably 13.5 to 14.5%, most preferably 14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0062] Example: potato (Solanum tuberosum) Approach voltage: preferably -0.3 to -0.7 V, more preferably -0.4 to -0.6 V, most preferably -0.5 V Injection voltage: preferably -6 to -11 V, more preferably -6.5 to -10.5 V, most preferably -7 to -10 V Current drop rate: preferably 9 to 15%, more preferably 9.5 to 14.5%, most preferably 10-14% Puncture distance: preferably 18 to 32 μm, more preferably 19 to 31 μm, most preferably 20 to 30 μm Injection time: preferably 0.5 to 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Withdrawal distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0063] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0064] (Preparation of nanopipette filled with reagent solution) A nanopipette was prepared. A genome editing tool reagent solution was prepared by dissolving a genome editing tool (Cas9-RNP) (10 ng / μL to 1 μg / μL) and a fluorescent reagent (FITC-labeled dextran) (10 μg / μL) in PBS. 3 μL or more of the reagent solution was taken using a microloader attached to a centrifuge holder, filled into the top of the nanopipette, and centrifuged for 30 to 60 seconds in a tabletop centrifuge. The silver wire attached to the nanopipette was inserted through the hole at the top filled with the reagent solution and fixed with a dedicated jig. At this time, it was confirmed that the silver wire was immersed in the reagent solution.

[0065] (Preparation of plant samples) The plant species listed in Table 1 were used. The shoot apical meristems of the plants were exposed and arranged on a dish or the like. The plants were fixed using tape or the like to prevent movement. An electrolyte solution was added so that the entire arranged plants (especially the shoot apical meristems) were submerged.

[0066] (Injection of reagent solution into cells of plant sample) The plant sample was set on a microscope. The nanopipette filled with the reagent solution was inserted into a plant cell material introduction device (Yokogawa Single Cellome). ＴＭThe nanopipette was attached to the head of the SU10 System Unit (SU10) and the rotary angle was adjusted. The reference electrode attached to the SU10 head was placed in the sample medium, and the SU10 software (measurement mode) was started. Using either manual or liquid detect mode, the nanopipette was immersed in the medium, and the software confirmed that the current value had increased from 0 nA. The SU10 joystick was used to position the nanopipette at a location corresponding to the target cell in the sample. The SU10 software was switched from measurement mode to delivery mode. The parameters corresponding to the plant sample listed in Table 1 were set. Start was pressed in the SU10 software, and the reagent solution was delivered to the target cell. The joystick was then used to position the nanopipette at a location corresponding to the next target cell, and delivery to each cell was performed one after another. After delivery, the sample was removed with tweezers and transferred to a culture medium (agar medium, simple medium containing water, soil, etc.), after which plant cultivation and growth began.

[0067]

[0068] (Genome editing tool introduction test into rice callus) A genome editing tool (Cas9 / gRNA complex: RNP) was introduced into rice callus using the same material introduction system (hereinafter referred to as the "SU10 system") consisting of the same material introduction device, nanopipette, microscope, etc. as described above. The target gene was the rice Phytoene desaturase gene (OsPDS). When this target gene is disrupted by genome editing, it exhibits a phenotype of white callus appearance. A plasmid carrying an expression cassette for a drug (hygromycin) resistance gene was co-introduced, and selective culture was performed to screen the introduced callus.

[0069] (Preparation of test rice callus) Callus induction was performed from rice (Oryza sativa, cultivar: Nipponbare) seeds. The husks were removed from the seeds and sterilized using 70% ethanol and 10% Haiter (registered trademark) (Kao) solution. The sterilized seeds were rinsed with distilled water five or more times in a clean bench and transplanted onto callus induction medium (Table 2). Callus induction was performed on the sterile sown seeds in a dark environment at 25°C. Callus cultured for three weeks or more was transplanted onto new callus induction medium to grow secondary callus. Small, highly active callus was picked from the grown secondary callus and embedded in agar medium for use.

[0070]

[0071] (Preparation of genome editing tool (RNP)) In this test, genome editing was performed targeting the rice Phytoene desaturase (OsPDS) gene. The base sequence (SEQ ID NO: 1) of the gRNA used for genome editing is shown in Table 3. gRNA was synthesized using Guide-it CRISPR / Cas9 Systems (Takara Bio). The synthesized gRNA was complexed with Guide-it Recombinant Cas9 (Takara Bio) to prepare a genome editing tool (RNP) and used in the test. Complexation was performed by incubating in 1x PBS buffer at room temperature for 10 minutes or more.

[0072]

[0073] (Introduction and analysis of genome editing tool (RNP) using the SU10 system) A genome editing tool (RNP) injection test into fixed rice callus was conducted using the SU10 system. The complexed RNP was filled into a nanopipette at a concentration of 0.1 to 1 μM and placed in the SU10 system. A section was also set up in which a plasmid containing a selection marker was simultaneously introduced at 50 to 100 mg / L. Callus fixed on solid medium was treated so that there were 30 shots per callus. MS medium was used as the buffer applied to the medium surface, and an antibacterial agent (Plant Preservative Mixture (PPM) (Plant Cell Technology)) was added before the operation.

[0074] Delivery was performed with the SU10 system to 2 to 5 calli per RNP concentration group under the conditions listed in Table 4. The treated calli were incubated overnight at 35°C to promote genome editing. They were then cultured under standard callus induction conditions. After recovery culture was completed, they were transferred to a drug (hygromycin) selection medium and sampled. DNA was extracted from the sampled calli, and gene editing was analyzed by next-generation sequencing (NGS).

[0075] In Table 4, the injection parameter conditions (protocols) are as follows: Protein protocol: App V = 1 V, Inj V = 4 to 5 V, CDR = 14%, Pen D = 20 μm, Inj T = 3 to 5 sec, Ret D = 50 μm Plasmid protocol: App V = -1 V, Inj V = -4 V, CDR = 14%, Pen D = 20 μm, Inj T = 3 sec, Ret D = 50 μm

[0076]

[0077] (Test results for introducing genome editing tools into rice calli) Drug selection culture was performed on a drug (hygromycin) selection medium, and drug-resistant calli were obtained as calli in which plasmid delivery to plant cells was successful ("Number of selected calli" in Table 4). It was confirmed that the introduction of the DNA-free genome editing tool (RNP) resulted in the appearance of calli exhibiting a partially white phenotype due to the target gene PDS mutation. Photographs of calli (two samples) exhibiting a partially white phenotype from test plot #17 are shown in Figure 5.

[0078] In addition, genetic analysis (NGS) confirmed that the introduction of a DNA-free genome editing tool (RNP) resulted in a change in the base sequence of the target gene PDS mutation. A mutation (SEQ ID NO: 3) appeared at a rate of approximately 2% compared to the wild type (SEQ ID NO: 2). Furthermore, sequence changes due to genome editing were confirmed at a site slightly shifted from the theoretical mutation site (Cut site). Reanalysis also confirmed the same mutation, confirming a definite mutation, albeit off-target.

[0079] (Test for introducing a genome editing tool into the barley meristem) Using the SU10 system, a genome editing tool (Cas9 / gRNA complex: RNP) was introduced into the barley (Hordeum vulgare) seed meristem. The target gene was the barley Phytoene desaturase gene (HvPDS). When this target gene is disrupted by genome editing, it exhibits a phenotype of white areas.

[0080] To perform CAPS analysis of the edited individuals, the gRNA sequence on the HvPDS was searched for. A gRNA sequence (SEQ ID NO: 4, Table 5) was designed on the exon of HvPDS, and this gRNA was complexed with the Cas9 protein to form a complex (RNP), which was then introduced into the barley meristem using the SU10 system. The introduced individuals were cultured for about 2 days after recovery and then grown in cell trays.

[0081]

[0082] Specifically, each step was carried out as follows.

[0083] 1. Selection and Sequencing of gRNA Candidates Based on information from the barley database, gRNAs were searched for in HvPDS (LOC123449634). Candidate gRNAs were located on exons, and eight sequences containing restriction enzyme sites around the Cas9 cleavage active site (the third base from the PAM sequence) were selected as candidates. Furthermore, gRNA candidates were further narrowed down by sequence analysis of the barley seeds actually used, and synthesized using Guide-it CRISPR / Cas9 Systems (Takata Kara Bio La Bio), complexed with the Cas9 protein (RNP), and in vitro cleavage activity measurements were performed. As a result, the candidate gRNA sequence to be used was determined to be SEQ ID NO: 4 (Table 5).

[0084] 2. Preparation of RNP for Induction - Introduction with SU10 The determined gRNA and Cas9 protein were mixed to synthesize RNP. RNP was prepared at a concentration of 1 μM. The mixture was prepared using 1x PBS buffer, and the complex was formed by incubating at room temperature for 10 minutes or more. The synthesized RNP was introduced into 30 cells of the meristem of barley seeds that had been watered overnight.

[0085] 3. Cultivation of transfected barley seeds The RNP-transfected seeds were grown on MS medium. Roots grew within 1-2 days of cultivation, and the seeds were then transplanted onto a Kimtowel (Nippon Paper Crecia) containing sterilized water to prevent bacterial and mold growth. The next day, the seeds were transplanted into cell trays filled with a 1:1 ratio of vermiculite to nursery soil, and then cultivated.

[0086] (Test results for introducing a genome editing tool into the meristem of barley) After introducing a genome editing tool into the meristem (shoot apical meristem) of barley, we confirmed that individuals that had successfully grown and matured into plants developed a white center of the stem (Figure 6). This whiteness was a phenotype seen in PDS gene mutations caused by genome editing.

[0087] (Search for optimal parameters for direct injection into individual plants by injecting a reagent solution into the growing point of barley) Aiming to establish parameters for highly efficient genome editing, we searched for optimal parameters for direct injection into individual plants. In searching for optimal parameters, we used the ability to deliver a reagent solution to plants with high efficiency and ensure the survival rate of plants after introduction as indicators of optimization.

[0088] Target sample: Barley (Hordeum vulgare) Delivery substance: GFP-protein (Abcam reagent) Number of deliveries = 10 times per individual N number = 2 (however, for injection voltage 5V and time 3-5 seconds, N number = 4) Microscope: Stereo microscope (Evident SZX10) Nanopipette: NP02 (manufactured by Yokogawa Electric Corporation)

[0089] As parameters, the combination of injection voltage and injection time, which are thought to affect delivery efficiency, was narrowed down to the optimal parameters.

[0090] A good success rate for substance delivery was confirmed under conditions of an injection voltage of 3V to 7V and an injection time of 3 to 5 seconds. The delivery success rate per individual was confirmed to be particularly high, reaching a maximum of 50% when the injection voltage was 5V and the application time was 3 seconds, and a maximum of 70% when the injection voltage was 5V and the injection time was 5 seconds. Meanwhile, a tendency for the delivery success rate to decrease was confirmed when the injection voltage was 2V or less and 8V or more. It was suggested that the amount of substance delivered per cell may depend on the injection voltage and injection time. The results of the delivery success rate are shown in Table 6. The results of the survival rate of plants after delivery are shown in Figure 7.

[0091] (-: Data not acquired)

[0092] Target sample: barley (Hordeum vulgare) Delivery substance: FITC dextran Number of deliveries = 10 times per individual N number = 3 Microscope: stereo microscope (Evident SZX10) Nanopipette: NP02 (manufactured by Yokogawa Electric Corporation)

[0093] As a parameter, the set current reduction rate, which is thought to affect delivery efficiency, was narrowed down to the optimal parameter.

[0094] A good success rate for substance delivery was confirmed when the set current drop rate (CDR) was between 3% and 40%, and an even better success rate for substance delivery was confirmed when the set current drop rate was between 5% and 20%. The results of the delivery success rate are shown in Table 7. The success rate for substance delivery is the percentage of cases where fluorescence was observed immediately after delivery of a fluorescent reagent (FITC dextran) out of a total of 30 times (10 times) when the fluorescent reagent was delivered to the shoot apical meristem of barley individuals (N=3 each). The success rate for substance delivery was judged as follows: ◎: 20% or more △: 10% or more but less than 20% ×: Less than 10%

[0095]

[0096] According to the present disclosure, in a method for injecting into plant cells using a nanopipette, injection conditions specific to plants can be established, and an automatically controlled injection system for plant cells can be provided.

[0097] 101 Nanopipette 102 Pipette electrode 103 Three-dimensional (xyz) moving pipette holder 111 Substance to be introduced into plant cell 201 Cell holder 202 Reference electrode 211 Plant cell 212 Electrolyte 301 Current measurement circuit 302 Voltage application circuit 401 Observation part of optical microscope 402 Stage of optical microscope D Set puncture distance I Ion current I ０ Base current I １ Steady-state current I ２ Current reduced at the set current reduction rate P0 Electrolyte surface position P1 Current reduction start point position P2 Pipette temporary stop position P3 Cell surface position P4 Substance discharge position R Set current reduction rate T Time T １ Injection time (application time) V Voltage V Ａ Approach voltage V Ｉ Injection voltage (applied voltage)

Claims

1. A method for introducing a substance into a plant cell, comprising: a) positioning a nanopipette filled with the substance at a position corresponding to the plant cell in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte solution and moving the nanopipette in the cell direction to a position with a set current reduction rate of 2% or more and 50% or less from the steady current; c) moving the nanopipette in the cell direction at a set penetration distance of 1 μm or more and 50 μm or less to penetrate the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte solution to discharge the substance into the plant cell; and e) removing the nanopipette.

2. The method according to claim 1, wherein the substance is a protein, a mixture or complex containing a protein, a mixture or complex containing a protein and a nucleic acid, a nucleic acid, or a dye.

3. The method according to claim 2, wherein the substance is a substance for genome editing.

4. The substance is a positively charged substance or a negatively charged substance. When the substance is a positively charged substance, step d) is performed by applying a voltage such that the inside of the nanopipette becomes a positive potential and the electrolyte solution becomes a negative potential. When the substance is a negatively charged substance, step d) is performed by applying a voltage such that the inside of the nanopipette becomes a negative potential and the electrolyte solution becomes a positive potential. The method according to claim 2.

5. The method according to claim 4, wherein step d) is performed by applying a voltage at a set applied voltage of -11 V or more and +11 V or less and a set applied time of 0.1 second or more and 10.0 seconds or less.

6. A method for producing a genetically modified plant or plant cell, comprising: a) positioning a nanopipette filled with a substance for genetic modification at a position corresponding to the plant cell in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte solution and moving the nanopipette in the cell direction to a position with a set current reduction rate of 2% or more and 50% or less from the steady current; c) moving the nanopipette in the cell direction at a set penetration distance of 1 μm or more and 50 μm or less to penetrate the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolyte solution to discharge the substance into the plant cell; and e) removing the nanopipette.

7. A genetically modified plant or plant cell produced by a method comprising: a) positioning a nanopipette filled with a substance for genetic modification at a position corresponding to a plant cell in an electrolytic solution; b) measuring a current between the inside of the nanopipette and the electrolytic solution and moving the nanopipette in the cell direction to a position where the reduction rate from the steady current is within a set current reduction rate of 2% or more and 50% or less; c) moving the nanopipette in the cell direction at a set piercing distance of 1 μm or more and 50 μm or less to pierce the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolytic solution to discharge the substance into the plant cell; and e) removing the nanopipette.

8. A control program for a substance introduction device into a plant cell, comprising instructions for performing: a) positioning a nanopipette filled with a substance for genetic modification at a position corresponding to a plant cell in an electrolytic solution; b) measuring a current between the inside of the nanopipette and the electrolytic solution and moving the nanopipette in the cell direction to a position where the reduction rate from the steady current is within a set current reduction rate of 2% or more and 50% or less; c) moving the nanopipette in the cell direction at a set piercing distance of 1 μm or more and 50 μm or less to pierce the plant cell; d) applying a voltage between the inside of the nanopipette and the electrolytic solution to discharge the substance into the plant cell; and e) removing the nanopipette.

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