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

To provide a plant cell injection system that is automatically controlled under conditions specialized for plants.SOLUTION: A method for introducing a substance into a plant cell comprises: a) a step of positioning a nano-pipette at a cell-corresponding position; b) a step of 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 of moving the nano-pipette toward the cell for a configured puncture distance and puncturing the cell; d) a step of applying a voltage to discharge the substance into the cell; and e) a step of extracting the nano-pipette.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[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. [Background technology]

[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, a method for introducing a complex of gold particles and genome editing tools into individual plants using a particle gun method described in Patent Documents 3 and 4. The iPB method introduces the genome editing tool randomly into random cells and may cause damage to the introduced cells or introduced substances, raising concerns about the stability of gene modification efficiency and invasiveness to individual plants. 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. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2014 / 160036 [Patent Document 2] International Publication No. 2013 / 012452 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-205103 [Patent Document 4] Japanese Patent Application Laid-Open No. 2017-205104 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0005] [1] A method for introducing a substance into a plant cell, comprising: a) positioning a nanopipette filled with a substance at a plant cell-corresponding location in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 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 substance for genome editing. [4] the substance is a positively charged substance or a negatively charged substance, If the substance is a positively charged substance, step d) is carried out by applying a voltage so that the interior of the nanopipette is at a positive potential and the electrolyte is at a negative potential; When the substance is a negatively charged substance, step d) is carried out by applying a voltage so that the inside of the nanopipette is at a negative potential and the electrolyte is at a positive potential; The method described in [2] above. [5] Step d) is carried out by applying a voltage at a set voltage of -11 V or more and +11 V or less for a set application time of 0.1 seconds or more and 10.0 seconds or less. The method described in [4] above. [6] A method for producing a genetically modified plant or plant cell, comprising: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 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. [7] 1. A genetically modified plant or plant cell produced by a method comprising: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 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. [8] A control program for an apparatus for introducing a substance into a plant cell, the control program including instructions for: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; b) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 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. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0007] [Figure 1] 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. [Figure 2] FIG. 1 is a conceptual diagram of a pipette puncturing step in the method of the present disclosure. [Figure 3] FIG. 1 is a conceptual diagram of the pipetting material ejection step in the method of the present disclosure. [Figure 4] FIG. 1 illustrates an example control mechanism for a system for use in the methods of the present disclosure. [Figure 5] Photographs of rice callus genome-edited using the method of the present disclosure are shown. The top and bottom images show two separate samples. The right image shows the whitened areas framed. [Figure 6] Photographs of barley plants genome-edited using the method of the present disclosure are shown. The bottom image shows an enlarged version of the top image. The right image shows the whitened area framed. [Figure 7] 1 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 various conditions of injection voltage. DETAILED DESCRIPTION OF THE INVENTION

[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 substances into plant cells] 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 (a method for introducing a substance into a plant cell and a method for producing genetically modified plant cells) will be described. The system of the present disclosure includes, for example, the following components: · Nanopipettes; A three-dimensional (xyz) moving pipette holder for moving the nanopipette in three dimensions (sometimes referred to as "xyz" for convenience); An electrode that can come into contact with the liquid filled inside the nanopipette (hereinafter referred to as the "pipette electrode"); An electrode that can come into contact with the electrolyte in the cell holder (hereinafter referred to as the "reference electrode"); · Current measurement circuit for measuring the current between the pipette electrode and the reference electrode; 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) The term "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 approximately 10 nm to approximately 500 nm, preferably approximately 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. Preferably, the nanopipette has a shape or scale that allows insertion of an electrode into the nanopipette that contacts the solution inside the nanopipette.

[0013] Nanopipettes have a single flow path (sometimes called a "barrel" or "bore") or multiple parallel flow paths within a tube. A nanopipette with a single flow path within a tube is sometimes called a "single-barreled nanopipette." A nanopipette with multiple parallel flow paths within a tube is sometimes called a "multi-barreled nanopipette." A nanopipette with two parallel flow paths within a tube is sometimes called a "double-barreled nanopipette." The nanopipette used in the method of the present disclosure is preferably a single-barreled nanopipette from the viewpoints 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 in, for example, Patent Document 1 (WO 2014 / 160036) and Patent Document 2 (WO 2013 / 012452).

[0016] (Three-dimensional (xyz) moving pipette holder) A "three-dimensional (xyz) moving 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 moving pipette holder so that two of the three dimensions (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 moving 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 can be roughly positioned, such as a three-dimensional actuator 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 electromagnetically driven actuators such as motor-based actuators (rotary motors, linear motors).

[0018] A "fine actuator" is a three-dimensional actuator capable of fine positioning, such as a three-dimensional actuator with a stroke of approximately 100–500 μm and a setting resolution of approximately 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 is sufficient for the target cell size, the fine actuator may be limited to one dimension, the Z axis, for 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 in contact with the electrolyte in a cell holder. In addition, 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 used to measure 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 preferably has a current measurement range of approximately 100 pA to 100 nA and is capable of measuring current changes (reductions) of approximately 2% to 50% for cell surface detection. For example, if the current (steady-state current) at a sufficient distance from the cell surface is 10 nA, and cell surface detection is set as a 20% decrease in current, the point at which the current drops to 8 nA is measured. A low-noise amplifier circuit or other suitable circuit is also used to accurately detect a very small steady-state reference current and its changes. Low noise can 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 tip 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 can be automatically controlled 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"), allowing the nanopipette tip to automatically pause immediately adjacent to the cell.

[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) Although the cell holder for holding plant cells is not particularly limited, a transparent holder with an openable top is usually used. Examples of such holders include cell culture vessels with an openable lid such as cell culture dishes and multi-well plates, and flat plates such as glass slides.

[0026] (cell immersion electrolyte) Examples of electrolytes that can be used to immerse cells in the cell holder include liquid media (e.g., Murashige and Skoog (MS) medium, Gamborg B5 medium, Chu (N6) medium), and buffered saline solutions (e.g., phosphate-buffered saline (PBS), HEPES-buffered saline (HBS), Hank's balanced salt solution (HBSS), etc.).

[0027] (Substances introduced into plant cells) The substance to be introduced into plant cells is not particularly limited, but examples thereof include proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, and pigments. The substance to be introduced into plant cells is not particularly limited, but a substance that dissolves or suspends in an electrolyte solution is preferred. Furthermore, a charged substance is also preferred. The charged substance may be a single charged substance, a mixture or complex of multiple substances that is charged overall, or a substance (a single substance or a mixture or complex of multiple substances) that charges the entire solution when dissolved or suspended in an electrolyte solution. Charged substances include positively charged substances and negatively charged substances. The substance to be introduced into plant cells (e.g., proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, pigments, etc.) is preferably either a positively charged substance or a negatively charged substance. For example, proteins, mixtures or complexes of proteins, mixtures or complexes containing nucleic acids and proteins, etc. may be either a positively charged substance or a negatively charged substance, for example, a positively charged substance. 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 or negatively charged substances, for example, negatively charged substances. Furthermore, the substance introduced into plant cells 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 may be an aqueous electrolyte or a non-aqueous electrolyte, with an aqueous electrolyte being preferred. Examples of the aqueous electrolyte include those exemplified above as the electrolyte into which the cell is immersed.

[0029] [Method for introducing substances into plant cells] The method for introducing a substance into a plant cell according to the present disclosure comprises the following steps. a) a step of positioning a nanopipette filled with a substance at a position corresponding to a plant cell in an electrolyte solution (pipette positioning step); b) a step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 50% (pipette approach step); c) a step of 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) A step of 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) A step of 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 then centrifuging the centrifuge holder with the nanopipette attached in a centrifuge. The suction method may be performed by aspirating 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 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 XY axis using the microscope stage.

[0031] (Step b): Pipette approach step) In step b), the current between the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the rate of decrease from the steady-state current becomes the set current decrease rate (the "pipette pause position (P2)" in Figure 1). The "direction toward the plant cell" refers to the direction toward the plant cell on the "z-axis." The current (ionic current) between the nanopipette and the electrolyte can be measured by a current measurement circuit as the current between the pipette electrode and the reference electrode. Furthermore, in step b), a low voltage (set approach voltage) is preferably applied between the nanopipette and the electrolyte (i.e., between the pipette electrode and the reference electrode) to prevent the filling solution from flowing out due to electroosmotic flow. The change in the current between the nanopipette and the electrolyte is explained with reference to Figure 1. When the nanopipette tip is positioned outside the electrolyte, the nanopipette is disconnected from the electrolyte, and the current (I) becomes zero (I0). When the nanopipette tip reaches the electrolyte surface position (P0) and enters the electrolyte, the nanopipette is connected to the electrolyte, and the current (I) increases. For a while after this, the current (I) remains steady without significant changes even when the nanopipette is advanced. The current (I) at this point is called the steady current (I1). When the nanopipette tip reaches a position very close to the cell surface position (P3) (the "current drop starting point position (P1)"), the current begins to drop rapidly according to the distance between the cell and the nanopipette tip. The nanopipette is then advanced to a position (the "pipette pause position (P2)") where the current (I) drops from the steady current (I1) to a current (I2) that is the set current drop rate (R). The nanopipette may be temporarily stopped at the pipette pause position (P2). The nanopipette movement in step b) can be performed using either a rough actuator, a fine actuator, or a combination of these. At least, movement beyond the current drop starting point (P1) is preferably performed by a fine actuator. The nanopipette movement in step b) is preferably performed automatically by a program. By presetting the current drop rate according to the plant species, it is possible to minimize damage to cells and achieve highly efficient substance introduction into cells.

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

[0033] <Approach Voltage> The set approach voltage is a value optimized for plant cells. 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 nanopipette movement in the pipette puncture step is usually performed by a fine actuator under automatic program control. Furthermore, the nanopipette movement in the pipette puncture step is preferably performed at a faster 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] <Setting puncture 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 so that it can reach the L2 layer of the shoot apical meristem.

[0036] (Process d): Substance discharge process) Step d) is a step of applying a voltage between the inside of the nanopipette and the electrolyte solution to discharge the substance into the plant cell. 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 a set injection time (set application time: T1).

[0037] <Injection Voltage> The injection voltage setting is optimized for plant cells. For positively charged substances, the injection voltage setting is preferably −11 to +11 V, more preferably −10 to +10 V, and most preferably 4 V to 10 V. Furthermore, the injection voltage setting can be optimized depending on the plant species, as described in the “Examples of Setting Parameters” below.

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

[0039] (Step e): Pipette removal step) Step e) is the step of removing the nanopipette. "Away from the plant cell" refers to the direction away from the plant cell (inside 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] <Retraction Distance> The set extraction distance is optimized for plant cells. The set extraction 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 as a method for producing genetically modified plants or plant cells. The method of the present disclosure for producing genetically modified plants or plant cells includes the following steps. a) a step of positioning a nanopipette filled with a gene modification substance at a position corresponding to a plant cell in an electrolyte solution (pipette positioning step); b) a step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 50% (pipette approach step); c) a step of 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) A step of 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) A step of 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 device for introducing substances into plant cells] The present disclosure also provides a control program for an apparatus for introducing a substance into a plant cell. The program of the present disclosure includes instructions for performing the following steps: a) a step of positioning a nanopipette filled with a gene modification substance at a position corresponding to a plant cell in an electrolyte solution (pipette positioning step); b) a step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the current drop rate from the steady state current is between 2% and 50% (pipette approach step); c) a step of 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) A process of applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the plant cell (substance discharge process); e) A step of 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 (applied 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] [Example of setting parameters] [Example 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 V 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 Extraction 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 V 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 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 Extraction distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0049] <<Raphanus>> Example: Radish (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 reduction 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 Extraction 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 reduction 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 to 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 Extraction 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 reduction 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 Extraction distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0052] <<Genus 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 reduction 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 to 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 Extraction 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 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 3.5 seconds, more preferably 0.8 to 3.2 seconds, most preferably 1 to 3 seconds Extraction 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 reduction 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 Extraction 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 Extraction distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0056] [Example 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 reduction 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 Extraction distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm

[0057] <Brassicaceae> Example: Raphanus>> 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 reduction 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 Extraction 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 reduction 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 Extraction 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 reduction 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 Extraction 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 reduction 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 Extraction 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 reduction 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 Extraction 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 reduction 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 Extraction distance: preferably 40 to 60 μm, more preferably 45 to 55 μm, most preferably 50 μm [Example]

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

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

[0066] (Injection of reagent solution into plant sample cells) The plant sample was placed under the microscope. The nanopipette filled with the reagent solution was inserted into the plant cell using a device for introducing substances into the plant cells (YOKOGAWA Single Cellome). TM The nanopipette was attached to the SU10 System Unit head 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 detection 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 the location corresponding to the target cell in the sample. The SU10 software was switched from measurement mode to delivery mode. The parameters for the plant sample listed in Table 1 were set. Pressing Start in the SU10 software allowed delivery of the reagent solution to the target cell. Next, the joystick was used to position the nanopipette at the location corresponding to the next target cell, and delivery to each cell was performed. After delivery was complete, the sample was removed with tweezers and transferred to culture medium (agar medium, simple medium containing water, soil, etc.). Plant culture and growth were then initiated.

[0067] [Table 1]

[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"), which consists 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. 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 times or more in a clean bench and then 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 proliferate secondary callus. Small, highly viable callus was picked from the proliferated secondary callus and embedded in agar medium for use.

[0070] [Table 2]

[0071] (Preparation of genome editing tools (RNP)) In this study, genome editing was performed targeting the rice Phytoene desaturase (OsPDS) gene. The base sequence of the gRNA used for genome editing (SEQ ID NO: 1) is shown in Table 3. The 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 the genome editing tool (RNP) used in the study. Complexation was performed by incubating in 1x PBS buffer at room temperature for at least 10 minutes.

[0072] [Table 3]

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

[0074] Delivery was performed using the SU10 system on 2 to 5 calli for each 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 complete, the calli 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 (protocol) are as follows: ●Protein Protocol App V=1V, Inj V=4~5V, CDR=14%, Pen D=20μm, Inj T=3~5sec, Ret D=50μm Plasmid Protocol App V=-1V, Inj V=-4V, CDR=14%, Pen D=20μm, Inj T=3sec, Ret D=50μm

[0076] [Table 4]

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

[0078] Furthermore, 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 (sequence number 3) appeared at a rate of approximately 2% compared to the wild type (sequence number 2). Furthermore, the sequence change due to genome editing was confirmed at a site slightly shifted from the theoretical mutation site (cut site). Reanalysis also confirmed the same mutation, confirming that it was an off-target but definite mutation. [ka]

[0079] (Test of introducing genome editing tools into barley growing points) Using the SU10 system, we introduced a genome editing tool (Cas9 / gRNA complex: RNP) into barley (Hordeum vulgare) seed meristems. The target gene was the barley phytoene desaturase gene (HvPDS). Disruption of this gene by genome editing results in the appearance of white patches.

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

[0081] [Table 5]

[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 the HvPDS (LOC123449634). Candidate gRNAs were located on exons and eight sequences containing restriction enzyme sites near the Cas9 cleavage active site (three bases from the PAM sequence) were selected. Furthermore, gRNA candidates were further narrowed down by sequence analysis of the actual barley seeds to be used. These were synthesized using the Guide-it CRISPR / Cas9 Systems (Takatakarabiola Bio), combined with the Cas9 protein to form an RNP complex, and in vitro cleavage activity was measured. As a result, the candidate gRNA sequence to be used was determined to be SEQ ID NO: 4 (Table 5).

[0084] 2. Preparation of Transduction RNP - SU10-mediated Transduction The determined gRNA was mixed with Cas9 protein to synthesize RNP. The RNP was prepared at a concentration of 1 μM. The mixture was prepared using 1x PBS buffer and incubated at room temperature for at least 10 minutes to form a complex. The synthesized RNP was then introduced into 30 cells of the meristem of barley seeds that had been watered overnight.

[0085] 3. Cultivation of Introduced Barley Seeds RNP-transfected seeds were grown on MS medium. Roots began to grow 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 a cell tray filled with vermiculite and seedling soil in a 1:1 ratio and grown there.

[0086] (Results of a test introducing genome editing tools into barley growing points) After introducing the genome editing tool into the barley meristem (shoot apical meristem), we confirmed that some barley plants developed successfully and developed into individual plants, but the center of the stem became white (Figure 6). This whiteness was a phenotype seen in PDS gene mutations induced by genome editing.

[0087] (Search for optimal parameters for direct injection of reagent solutions into individual plants by injecting them into the growing point of barley) In order to establish parameters for highly efficient genome editing, we explored the optimal parameters for direct injection into individual plants. In exploring the optimal parameters, we used the index of optimization as the ability to deliver the reagent solution to plants with high efficiency and ensure the survival rate of the plants after injection.

[0088] Target sample: Barley (Hordeum vulgare) Delivery substance: GFP-protein (Abcam reagent) Number of deliveries = 10 times per individual N number = 2 (however, when the injection voltage is 5V and the time is 3 to 5 seconds, N number = 4) Microscope: Stereo microscope (Evident SZX10) Nanopipette: NP02 (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 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. This suggests 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] [Table 6] (-: Data not available)

[0092] Target sample: Barley (Hordeum vulgare) Delivery substance: FITC dextran Number of deliveries = 10 times per individual N=3 Microscope: Stereo microscope (Evident SZX10) Nanopipette: NP02 (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 out of a total of 30 times (10 times) in which a fluorescent reagent (FITC dextran) was delivered to the shoot apical meristem of barley individuals (N=3 each). The success rate for substance delivery was determined as follows: ◎: 20% or more △: 10% or more but less than 20% ×: Less than 10%

[0095] [Table 7] [Industrial Applicability]

[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. [Explanation of symbols]

[0097] 101 Nanopipette 102 Pipette Electrode 103 Three-dimensional (xyz) moving pipette holder 111 Substances introduced into plant cells 201 Cell holder 202 Reference electrode 211 Plant Cell 212 Electrolyte 301 Current measurement circuit 302 Voltage application circuit 401 Optical microscope observation section 402 Optical microscope stage D Setting puncture distance I ion current I0 base current I1 steady state current I2 Current decreased at the set current decrease rate P0 Electrolyte surface position P1 Current drop starting point position P2 Pipette pause position P3 cell surface position P4 Material discharge position R Set current reduction rate T time T1 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 a substance at a location corresponding to a plant cell in an electrolyte solution; 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 decrease rate from the steady state current is between 2% and 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 of 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 of claim 2, wherein the substance is a genome editing substance.

4. the substance is a positively charged substance or a negatively charged substance, If the substance is a positively charged substance, step d) is carried out by applying a voltage such that the interior of the nanopipette is at a positive potential and the electrolyte is at a negative potential; When the substance is a negatively charged substance, step d) is carried out by applying a voltage so that the interior of the nanopipette is at a negative potential and the electrolyte is at a positive potential; The method of claim 2.

5. Step d) is carried out by applying a voltage at a set voltage of −11 V or more and +11 V or less for a set application time of 0.1 seconds or more and 10.0 seconds or less. The method of claim 4.

6. A method for producing a genetically modified plant or plant cell, comprising: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; 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 decrease rate from the steady state current is between 2% and 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.

7. 1. A genetically modified plant or plant cell produced by a method comprising: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; 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 decrease rate from the steady state current is between 2% and 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.

8. A control program for an apparatus for introducing a substance into a plant cell, the control program including instructions for: a) positioning a nanopipette filled with a gene modification substance at a location corresponding to a plant cell in an electrolyte solution; 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 decrease rate from the steady state current is between 2% and 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.

Images