Method for introducing a target molecule into target plant cells or target tissue.

Abstract

This provides a novel method to improve the efficiency of gene and molecule introduction into plant cells and tissues. [Solution] A method for introducing a target molecule into target plant cells or target tissue by bringing target plant cells or target tissue into contact with an introduction solution containing a target molecule, and irradiating the target plant cells or target tissue with plasma by selecting a first frequency range of 5 kHz or less, or a second frequency range higher than the first frequency range, depending on the impedance of the target plant cells or target tissue, wherein when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, the plasma of the first frequency range is irradiated, the plasma of the second frequency range is irradiated and thereafter the plasma of the second frequency range is irradiated at least once, or the plasma of the second frequency range is irradiated and thereafter the plasma of the first frequency range is irradiated, and when irradiating fish eggs with plasma, the plasma of the second frequency range is irradiated.

Description

[Technical Field]

[0001] The present invention relates to a method for introducing a target molecule into target plant cells or target tissue. [Background technology]

[0002] In recent years, problems in the agricultural sector have included increased food demand due to population growth, a decline in agricultural workers, and supply shortages and instability due to extreme weather events. Solutions to these problems include mass production of plants, increased yields, and reduced production costs. Therefore, gene and molecular introduction technologies, such as genetic modification and genome editing, are crucial in plant breeding and seedling production to achieve these goals.

[0003] One of the gene transfer technologies used to date is the Agrobacterium method, which involves introducing useful genes into target cells by infecting them with the soil bacterium Agrobacterium. While the Agrobacterium method is simple for introducing genes into dicotyledonous plants, it has limitations in monocotyledonous plants, such as a limited number of host plants, and is difficult to use in grains. Another gene transfer technology known as the particle gun method is that it can be used to introduce genes into plant species that are difficult to introduce using the Agrobacterium method. However, the particle gun method has disadvantages, such as being damaging because it directly injects gold particles, and having low introduction efficiency.

[0004] In contrast to these methods, the microplasma method, which involves treating cells with plasma to introduce external genes and molecules into the cells, has been attempted for some time (see, for example, Japanese Patent Publication No. 3585124 (Patent Document 1)). The microplasma method is highly safe because it leaves no genes other than the one to be introduced, and the damage to cells after plasma irradiation is low. Furthermore, it is easy to operate because the process can be completed in a short time (several ms to tens of ms), and because it does not use chemicals, it requires fewer steps. It is a method that can safely introduce genes into cells that are difficult to introduce using conventional methods.

[0005] However, when attempting to apply the microplasma method to plant cells with cell walls, cell tissues composed of multiple cells, or even fish eggs with egg membranes thicker than those of animal cells, there was a problem in that the introduction efficiency was insufficient compared to that of animal cells. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 3585124 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention was made to solve the above problems, and its objective is to provide a novel method for improving the efficiency of gene and molecule introduction into plant cells and tissues (including fish eggs) using plasma, such as the microplasma method and surface discharge method. [Means for solving the problem]

[0008] The present invention relates to a method for introducing the target molecule into target plant cells or target tissue by bringing target plant cells or target tissue into contact with an introduction solution containing the target molecule, and irradiating the target plant cells or target tissue with plasma by selecting a first frequency range of 5 kHz or less, or a second frequency range higher than the first frequency range, depending on the impedance of the target plant cells or target tissue. (1) When irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) Irradiating with plasma in the first frequency range, (B) Irradiate with plasma in the second frequency range, and then irradiate with plasma in the second frequency range at least once, or (C) Irradiating with plasma in the second frequency range, and then irradiating with plasma in the first frequency range. (2) When irradiating fish eggs with plasma, the plasma is characterized by being irradiated in the second frequency range.

[0009] In the method of the present invention, it is preferable that the first frequency range is in the range of 100 Hz to 5 kHz.

[0010] In the method of the present invention, it is preferable that the second frequency range is in the range of 10 to 200 kHz.

[0011] In the method of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) it is preferable to irradiate with plasma in the second frequency range at least once after irradiation with plasma in the first frequency range, and it is more preferable that the amount of the target molecule introduced increases with increasing the number of irradiations of plasma in the second frequency range.

[0012] In the method of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) it is preferable to irradiate with plasma in the first frequency range at least once after irradiation with plasma in the first frequency range, and it is more preferable that the amount of the target molecule introduced increases with increasing the number of irradiations of plasma in the first frequency range.

[0013] In the method of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (B) when irradiating with plasma in the second frequency range, and then irradiating with plasma in the second frequency range at least once, it is preferable that the amount of the target molecule introduced increases with increasing the number of times the plasma in the second frequency range is irradiated.

[0014] In the method of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (C) when irradiating with plasma in the second frequency range, and then irradiating with plasma in the first frequency range, it is preferable to irradiate with plasma in the first frequency range at least once after the irradiation with plasma in the first frequency range, and it is more preferable that the amount of target molecule introduced increases with increasing the number of irradiations of plasma in the first frequency range.

[0015] In the method of the present invention, (1) when the target of irradiation is a target plant cell or target tissue (excluding fish eggs) and plasma is irradiated, (C) when plasma of the second frequency range is irradiated, and then plasma of the first frequency range is irradiated, it is preferable to irradiate with plasma of the second frequency range at least once after irradiation with plasma of the first frequency range, and it is more preferable that the amount of the target molecule introduced increases with increasing the number of irradiations of plasma of the second frequency range.

[0016] In the method of the present invention, (2) when irradiating fish eggs with plasma in the second frequency range, it is preferable to irradiate with plasma in the second frequency range at least once after the irradiation with plasma in the second frequency range, and it is more preferable that the amount of the target molecule introduced increases with an increase in the number of irradiations with plasma in the second frequency range.

[0017] In the method of the present invention, (2) when irradiating fish eggs with plasma in the second frequency range, it is preferable to include at least one of the following steps before irradiating with plasma of the second frequency: a step to induce hatching of the fish eggs, and a step to increase the conductivity of the introduced solution. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide a method for introducing target molecules into target plant cells or target tissues, which has been conventionally inefficient in introducing genes and molecules into plant cells and tissues (including fish eggs), and which has been significantly improved. [Brief explanation of the drawing]

[0019] [Figure 1] SEM photograph (magnification: 200 times) showing the results in Experimental Example 3. Fig. 1(a) shows the case of plasma irradiation at a frequency of 500 Hz, Fig. 1(b) shows the case of plasma irradiation at a frequency of 20 kHz, and Fig. 1(c) shows the control case (without plasma irradiation). [Figure 2] Fig. 2(a) is a diagram showing the impedance locus of cell tissue, and Fig. 2(b) is a diagram showing the electric circuit representing the cell tissue. [Figure 3] Fig. 3(a) shows the case where low-frequency plasma (plasma in the first frequency range) on the arc Z2 is selected in the impedance locus of cell tissue, and Fig. 3(b) is a diagram schematically showing the flow of low-frequency current in the electric circuit of Fig. 2(b) when the low-frequency plasma selected in Fig. 3(a) is irradiated. [Figure 4] It is a diagram schematically showing the flow of low-frequency current and high-frequency current in cell tissue. [Figure 5] Fig. 5(a) is a diagram showing a simple equivalent circuit model of plant cells, and Fig. 5(b) is a diagram schematically showing the flow of current in plant cells according to the frequency range. [Figure 6] In Experimental Example 1, it is a graph showing the plotted frequencies examined in the measurement results and the frequencies at the maximum and minimum parts. [Figure 7] It is a schematic diagram of the experimental apparatus used in Experimental Example 2. [Figure 8] It is a graph plotting the results of Experimental Example 2. [Figure 9] SEM photograph (magnification: 30000 times) showing the results in Experimental Example 3. Fig. 9(a) shows the case of plasma irradiation at a frequency of 500 Hz, Fig. 9(b) shows the case of plasma irradiation at a frequency of 20 kHz, and Fig. 9(c) shows the control case (without plasma irradiation). [Figure 10] It is a schematic diagram showing that, as predicted from the results of Experimental Example 3, gaps are generated on the cell surface by plasma treatment, allowing molecules to reach the cell membrane. [Figure 11]This diagram shows the procedure for Experiment Example 4. [Figure 12] This graph plots the results of Experiment Example 4. [Figure 13] This is a schematic diagram showing the introduction pathway in the microplasma method, as predicted from the results of Experimental Example 4. [Figure 14] This graph plots the results of Experiment Example 5. [Figure 15] This schematic diagram shows the expected result from Experimental Example 5, illustrating how low-frequency plasma creates gaps on the cell surface, allowing molecules to reach the cell membrane and be introduced via endocytosis. [Figure 16] This graph plots the results of Experiment Example 6. [Figure 17] This graph plots the results of Experiment Example 7. [Figure 18] This is a schematic diagram showing the structure of fish eggs. [Figure 19] Figure 19(a) shows a simplified equivalent circuit model of fish eggs, and Figure 19(b) schematically shows how current flows through fish eggs in different frequency ranges. [Figure 20] Figure 20(a) shows a photograph of normal fish eggs, and Figure 20(b) shows a photograph of fish eggs after using hatching enzymes. [Figure 21] This graph shows the impedance for each incubation enzyme usage time in Experiment Example 8. [Figure 22] This diagram schematically shows the circuit of the experimental system used in Experimental Example 9. [Figure 23] These are photographs showing the results of Experimental Example 9. Figure 23(a) shows hatched larvae using a FITC-Dextran solution with a conductivity of 0.03 S / m, and Figure 23(b) shows hatched larvae using a FITC-Dextran solution with a conductivity of 1.85 S / m. [Figure 24] This figure shows a circuit model of the structure of fish eggs. [Figure 25] Figure 25(a) is a magnified view of the area around the egg membrane of the circuit model shown in Figure 24, and Figure 25(b) is a schematic diagram showing the current flow around the egg membrane during plasma irradiation. [Figure 26]Figure 26(a) is a magnified view of the area around the embryo in the circuit model shown in Figure 24, and Figure 26(b) is a schematic diagram showing the current flow around the embryo during plasma irradiation. [Modes for carrying out the invention]

[0020] The present invention provides a method for introducing a target molecule into plant cells or tissue by bringing target plant cells or tissue into contact with an introduction solution containing a target molecule, and irradiating the target plant cells or tissue with plasma by selecting a first frequency range of 5 kHz or less, or a second frequency range higher than the first frequency range, depending on the impedance of the target plant cells or tissue. The present invention provides a method for introducing a target molecule into target plant cells or tissue that selectively realizes the formation of a molecular introduction pathway and the induction of membrane transport such as endocytosis by applying a stimulus to the cell tissue that is suitable for the electrical characteristics of the cell tissue, thereby significantly improving the introduction efficiency of genes and molecules into plant cells and tissues, which was previously insufficient.

[0021] In the method for introducing the target molecule of the present invention, "target plant cells" refer to plant cells that are targeted for introduction of the target molecule. There are no particular restrictions on the type of plant cell; unlike the conventional Agrobacterium method, introduction into monocots is not difficult, and the plant cells may be derived from monocots or dicots. In the present invention, the target molecule can be introduced into multiple target plant cells simultaneously. These target plant cells may be of a single type or a mixture of two or more types.

[0022] Furthermore, the term "target tissue" used in the method for introducing the target molecule of the present invention refers to the tissue to which the selected molecule is introduced, and is not limited to any particular type of tissue. Specific examples of such target tissues include pre-differentiation plant tissues constructed by callus culture, and fish eggs. These target tissues may be of a single type or a mixture of two or more types.

[0023] The target molecule used in the method for introducing the target molecule of the present invention refers to a molecule selected for introduction into target plant cells or target tissues, and is not limited to a specific type of molecule. Specific examples of such target molecules include DNA, RNA, other nucleic acid molecules or their derivatives, and macromolecular compounds such as proteins and peptides and their derivatives, including signaling proteins and transcription factors. DNA or RNA may be single-stranded or double-stranded, and may be linear or cyclic. Derivatives of nucleic acid molecules include vectors, antisense polynucleotides, decoy polynucleotides, ribozymes, and siRNA. The molecular weight of polynucleotides is not particularly limited. Protein molecules and protein molecule derivatives include signaling factors, transcription factors, various enzymes, various receptors, antibodies or the Fab portion of antibodies, genome editing proteins, and protein pharmaceuticals that cannot be administered orally.

[0024] Other target molecules include low-molecular-weight physiologically active substances and drug candidates. Among these, physiologically active low-molecular-weight compounds such as pharmaceuticals that are difficult to introduce into tissues or plant cells by other introduction methods are preferred. Low-molecular-weight compounds that are difficult to introduce into tissues or plant cells by other introduction methods include low molecules with a molecular weight of 1,000 Da or more, molecules with low membrane permeability, etc.

[0025] Furthermore, the various target molecules mentioned above may be used individually or as a mixture of two or more types.

[0026] The introductory solution used in this invention is preferably suspended in water or a suitable medium such as an aqueous solution. Examples of solvents or dispersion media for the aqueous solution or suspension include physiological saline and pH buffer solutions.

[0027] When introducing a solution containing the target molecule into target plant cells, methods such as dropping the liquid containing the target molecule onto the target plant cells or mixing the liquid containing the target molecule with the target plant cells can be used. Alternatively, the liquid containing the target molecule can be added to a dispersion or suspension containing the target plant cells.

[0028] In the method for introducing the target molecule of the present invention, the first frequency range is not particularly limited as long as it is 5 kHz or less, but it is preferably in the range of 100 Hz to 5 kHz, and more preferably in the range of 500 Hz to 1 kHz.

[0029] In the method for introducing the target molecule of the present invention, the second frequency range is not particularly limited as long as it is higher than the first frequency range, but it is preferably in the range of 10 to 200 kHz, and more preferably in the range of 20 to 50 kHz.

[0030] (1) When irradiating target plant cells or target tissue (excluding fish eggs) with plasma in this case, (A) Irradiating with plasma in the first frequency range, (B) Irradiate with plasma in the second frequency range, and then irradiate with plasma in the second frequency range at least once, or (C) Irradiating with plasma in the second frequency range, and then irradiating with plasma in the first frequency range. Do one of the following:

[0031] Here, Figure 1 shows SEM images (magnification: 200x) of the results from Experimental Example 3, which will be described later. Figure 1(a) shows the case when plasma was irradiated at a frequency of 500 Hz, Figure 1(b) shows the case when plasma was irradiated at a frequency of 20 kHz, and Figure 1(c) shows the control case (no plasma irradiation). In the control case (Figure 1(c)) and the 20 kHz case (Figure 1(b)), a membrane covers the cell surface, whereas at 500 Hz, the membrane covering the cell surface is peeled off (Figure 1(a)). Thus, in this invention, we have experimentally demonstrated that the efficiency of gene and molecule introduction into plant cells and tissues can be improved by selectively supplying frequency-controlled electrical stimulation to sites with different impedances. This technology of applying variable frequencies to cells makes it possible to introduce genes and molecules that can lead to plant breeding and seedling cultivation.

[0032] Plant cells have a cell membrane surrounded by a cell wall, and cell tissue has interstitial tissue (intercellular matrix) between cells. Here, Figure 2(a) shows the impedance trajectory of cell tissue, and Figure 2(b) shows the electrical circuit representing cell tissue. In Figure 2(a), the vertical axis is reactance (X) (unit: Ω), and the horizontal axis is resistance (R) (unit: Ω). As revealed in Experimental Example 1 described later, the change in impedance on the cell surface resulted in two arcs Z1 and Z2, as shown in Figure 2(a). We believe that this is because the cell (arc Z1) and the intercellular matrix (arc Z2) are separated, resulting in two arcs. It is thought that the two arcs appear because the current to the intercellular matrix and the cell changes as the frequency changes. When cell tissue is represented as an electrical circuit, as shown in Figure 2(b), it is composed of resistors and capacitors. On the interstitial side, as the frequency gradually changes from low to high, the current to the capacitor increases, causing an arc resembling a dummy cell to appear. During this time, the current to the cell basically flows only through the resistive component, so the first arc Z2 appears. Subsequently, as the frequency increases, the current in the interstitial side basically flows only through the capacitor, while on the cell side, current gradually flows through the capacitor as well, and it is thought that the second arc Z1 appears.

[0033] The impedance trajectory of the cell tissue shown in Figure 2(a) indicates that low-frequency current affects the interstitial tissue. Here, Figure 3(a) shows the case where a low-frequency plasma (plasma in the first frequency range) on the arc Z2 is selected in the impedance trajectory of the cell tissue, and Figure 3(b) is a schematic diagram showing how the low-frequency current flows in the electrical circuit of Figure 2(b) when the low-frequency plasma selected in Figure 3(a) is irradiated. As described above, it is thought that the low-frequency current flows in the electrical circuit shown in Figure 2(b) as shown in Figure 3(b).

[0034] Figure 4 schematically illustrates the flow of low-frequency and high-frequency currents in cell tissue. As shown in Figure 4, high-frequency currents flow in a straight line without distinction between cells and interstitial tissue, while low-frequency currents are thought to flow through cells. It is believed that this flow of low-frequency currents resulted in the peeling of the membrane covering the cell surface, as shown in Figure 1(a).

[0035] Figure 5(a) shows a simplified equivalent circuit model (Hayden model) of a plant cell, and Figure 5(b) schematically shows how current flows in a plant cell depending on the frequency range. If I1 is the current flowing inside the cell and I2 is the current flowing outside the cell, then for high-frequency currents, I1 > I2, and for low-frequency currents, I2 > I1, meaning that the amount of current flowing inside and outside the cell can be controlled by frequency. By irradiating with plasma in the frequency range to which low-frequency currents belong (i.e., the first frequency range), even a single irradiation can stimulate the intercellular matrix and cell surface (cell wall), forming an introduction pathway in the cell wall while stimulating the inside of the cell to induce endocytosis. On the other hand, if plasma in the frequency range to which high-frequency currents belong (i.e., the second frequency range) is irradiated only once, an introduction pathway is not formed in the cell wall, and therefore it is thought that molecules do not reach the cell membrane.

[0036] The method for introducing the target molecule of the present invention is based on the experimental findings described above. (1) When irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) by irradiating with plasma in the first frequency range, the membrane covering the cell surface is peeled off, as shown in Figure 1(a), and the target molecule can be introduced into the target plant cells or target tissue (excluding fish eggs).

[0037] The conditions for plasma irradiation in the first frequency range are not particularly limited; they can be carried out under the same conditions as conventional microplasma methods, except that the frequency is within the first frequency range. From the viewpoint of increasing cell viability, the irradiation time is preferably very short, between 0.1 ms and 100 ms. The number of times plasma irradiation in the first frequency range is performed can be once or multiple times (preferably 2 to 5 times), and is not particularly limited.

[0038] In the method for introducing the target molecule of the present invention, the first frequency range is determined according to the impedance of the target plant cell or target tissue (excluding fish eggs). As described above, the impedance trajectory will result in the appearance of two arcs Z1 and Z2, but the specific centers and radii of the arcs will differ for each target plant cell or target tissue. In experimental example 3 described later, a frequency f2 of 500 Hz on arc Z2 in Figure 2(a) is used as the plasma (low-frequency plasma) in the first frequency range.

[0039] In the method for introducing the target molecule of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) when irradiating with plasma in the first frequency range, it is preferable to irradiate with plasma in the second frequency range at least once after irradiating with plasma in the first frequency range. By irradiating with plasma in the second frequency range after irradiating with plasma in the first frequency range as described above, the plasma in the first frequency range penetrates the intercellular matrix and cell wall, and the plasma in the second frequency range promotes the introduction of more target molecules, as experimentally demonstrated in Experimental Example 5 described later (see Figure 14).

[0040] The conditions for plasma irradiation in the second frequency range are not particularly limited and can be the same as those for conventional microplasma methods. From the viewpoint of increasing cell viability, the irradiation time is preferably very short, between 0.1 ms and 100 ms. The number of times plasma irradiation in the second frequency range is performed can be once or multiple times (preferably 2 to 5 times) and is not particularly limited.

[0041] In the method for introducing the target molecule of the present invention, the second frequency range is determined according to the impedance of the target plant cell or target tissue, similar to the first frequency range. As mentioned above, the impedance trajectory will result in the appearance of two arcs, Z1 and Z2, but the specific centers and radii of the arcs will differ for each target plant cell or target tissue. In Experimental Example 5, described later, a frequency f1 of 20 kHz on arc Z1 in Figure 2(a) is used as the plasma (high-frequency plasma) in the second frequency range.

[0042] Furthermore, in the method for introducing the target molecule of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) when irradiating with plasma in the first frequency range, the irradiation with plasma in the first frequency range may be performed at least once after the irradiation with plasma in the first frequency range. As mentioned above, the number of times the plasma in the first frequency range is irradiated may be once or multiple times (preferably 2 to 5 times), but it has been found that the amount of target molecule introduced increases with increasing number of irradiations when the plasma in the first frequency range is irradiated multiple times. Here, Figure 16 is a graph showing the results of Experimental Example 6, where the vertical axis is the average fluorescence and the horizontal axis is the number of irradiations, and experimental results were obtained showing that the amount of target molecule introduced by irradiation with plasma in the first frequency range (500 Hz) increases from the second time to the first time, and from the third time to the second time.

[0043] In the method for introducing the target molecule of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (B) irradiating with plasma in the second frequency range, and then irradiating with plasma in the second frequency range at least once, is also possible. As described above, the molecule does not reach the cell membrane with only one irradiation of plasma in the second frequency range, but experimental results were obtained showing that the introduction of the target molecule was confirmed by irradiating with plasma in the second frequency range two or more times. Here, Figure 16 is a graph showing the results of Experimental Example 6, where the vertical axis is the average brightness and the horizontal axis is the processing order. In the processing order, "H" means high-frequency plasma irradiation (only the first time), "HH" means high-frequency plasma irradiation → high-frequency plasma irradiation, and "HHH" means high-frequency plasma irradiation → high-frequency plasma irradiation → high-frequency plasma irradiation, and the high-frequency plasma is specifically 20 kHz. In this case as well, it is preferable that the amount of the target molecule introduced increases by increasing the number of times the plasma in the second frequency range is irradiated onto the target plant cells or target tissue (excluding fish eggs).

[0044] Furthermore, in the method for introducing the target molecule of the present invention, (1) when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (C) irradiating with plasma in the second frequency range and then irradiating with plasma in the first frequency range may be performed. Here, Figure 17 is a graph showing the results of Experimental Example 7, where the vertical axis represents the average brightness and the horizontal axis represents the processing order. In the processing order, "LH" means low-frequency plasma irradiation → high-frequency plasma irradiation, "HL" means high-frequency plasma irradiation → low-frequency plasma irradiation, and "L" means low-frequency plasma irradiation (only the first time), where the low-frequency plasma is specifically 500 Hz and the high-frequency plasma is specifically 20 kHz. As shown in Figure 17, even when plasma irradiation in the second frequency range is performed first and then plasma irradiation in the first frequency range, a higher amount of the target molecule was introduced compared to when only the first plasma irradiation in the first frequency range was performed, and this case is also included in the present invention. The conditions for plasma irradiation in this case are the same as those described above for the case where plasma irradiation in the first frequency range is performed first, and are not particularly limited. In this case, plasma irradiation in the first frequency range may be performed at least once after the plasma irradiation in the first frequency range, or plasma irradiation in the second frequency range may be performed at least once after the step of irradiating with plasma in the first frequency range. In this case, it is preferable that the amount of target molecule introduced is increased by increasing the number of times the plasma in the first frequency range or the second frequency range is irradiated onto the target plant cells or target tissue.

[0045] (2) When irradiating fish eggs with plasma Here, Figure 18 is a schematic diagram showing the structure of fish eggs, Figure 19(a) is a diagram showing a simplified equivalent circuit model of fish eggs, and Figure 19(b) is a diagram schematically showing how current flows in fish eggs depending on the frequency range. As shown in Figure 18, fish eggs have a structure consisting of an embryo, an egg membrane covering it, and a periovarian space between them. When fish eggs are viewed as an electrical circuit, the capacitor part is represented by the egg membrane and the solution part by the resistor. In surface discharge, a fluorescent molecular solution surrounds the fish eggs, so a simplified circuit can be considered a parallel circuit of the fish egg external solution (processing solution) and the fish eggs. If the current flowing inside the fish egg is I1 and the current flowing on the surface of the egg membrane is I2, then in the case of high-frequency current, I1 > I2, and in the case of low-frequency current, I2 > I1, so the amount of current flowing inside and outside the fish egg can be controlled by frequency. As will be described later in Experimental Example 8, in the case of fish eggs, which have an egg membrane that is significantly thicker than the cell membrane, endocytosis can be induced by irradiating them with plasma in the frequency range to which high-frequency currents belong (i.e., the second frequency range) and stimulating the egg membrane, periovular coelence, and embryo in that order. On the other hand, when plasma in the frequency range to which low-frequency currents belong (i.e., the first frequency range) is irradiated, it is thought that endocytosis will not be induced because the electrical stimulation does not reach the embryo.

[0046] As for the fish eggs, for example, in Experiment Example 8 described later, medaka fish eggs were used, but there are no particular restrictions on the type of fish eggs used, whether they are from freshwater fish or saltwater fish.

[0047] (2) When irradiating fish eggs with plasma in the second frequency range, it is preferable to irradiate with plasma in the second frequency range at least once after the initial irradiation. In this case, it is more preferable that the amount of target molecule introduced increases with increasing the number of irradiations of plasma in the second frequency range.

[0048] Furthermore, (2) when irradiating fish eggs with plasma in the second frequency range, it is preferable to include at least one of the following steps before irradiation with plasma of the second frequency: a step to induce hatching of the fish eggs and a step to increase the conductivity of the introduction solution, and it is more preferable to include both the step to induce hatching of the fish eggs and the step to increase the conductivity of the introduction solution. Including at least one of these steps (and even both) can further improve the introduction efficiency into the fish eggs. The means used in the step to induce hatching of the fish eggs are not particularly limited as long as they can induce hatching of the fish eggs, but for example, a known appropriate hatching enzyme can be used on the fish eggs after egg collection. The method for increasing the conductivity of the introduction solution can be to change the composition of the introduction solution as done in Experimental Example 9 described later, but is not limited to this.

[0049] Furthermore, in the method for introducing the target molecule of the present invention, it is preferable that the target molecule be introduced into target cells or target tissues via endocytosis. This is because when the target molecule is introduced into target plant cells, etc., via endocytosis, early endosomes containing the target molecule are formed, and the target molecule can be stabilized by inhibiting the lysosomal degradation of these early endosomes via late endosomes.

[0050] The present invention will be further explained with experimental examples below, but the present invention is not limited to these.

[0051] <Experimental Example 1: Frequency Investigation using Impedance Analysis> It is known that plant cells exhibit changes in impedance when the frequency is changed. For example, studies such as "Research on the Use of Electrical Impedance Information of Plant Cell Tissues - Analysis of Impedance Characteristics by an Equivalent Circuit Model" by Kiyohiko Toyoda et al., and "Effects of Hot Water Immersion Treatment on the Thermophysical and Electrical Properties of Potatoes and Radishes" by Teppei Imaizumi et al. have reported obtaining Cole-Cole plots by measuring the impedance of plant cells. Therefore, we investigated the optimal frequency to introduce by analyzing the impedance of plant cells. Plant cells can be considered using the simplified equivalent circuit model (Hayden model) shown in Figure 5.

[0052] The actual measurement was performed by gripping a callus (purchased from RIKEN) with tweezers. Figure 6 shows the measurement results plotted against the frequencies examined and the frequencies at the maximum and minimum regions. Similar results were obtained for most calluses.

[0053] The change in impedance at the cell surface resulted in two arcs, Z1 and Z2, as shown in Figure 2(a). It was hypothesized that this is due to the separation of the cell (arc Z1) and the intercellular matrix (arc Z2). The change in frequency is thought to alter the current flowing to the intercellular matrix and the cell, leading to the appearance of these two arcs. Specifically, in the circuit shown in Figure 2(b), the intercellular matrix gradually changes from low frequency, increasing the current to the capacitor, resulting in the appearance of a dummy cell-like arc. During this time, the current to the cell flows primarily through the resistive component, thus creating the first arc, Z2. Subsequently, as the frequency increases, the intercellular matrix primarily flows only through the capacitor, while the cell gradually begins to receive current through the capacitor, leading to the appearance of the second arc, Z1. These results suggest that low-frequency currents may affect the intercellular matrix.

[0054] <Experimental Example 2: Frequency Dependence Evaluation> As a starting point for exploring molecular introduction conditions, we evaluated the frequency dependence of molecular introduction using fluorescent molecules. The experimental apparatus shown in the schematic diagram in Figure 7 was used, and the procedure was as follows: First, *Calliphora japonica* was collected from the culture medium, and the sample was left to stand for 30 minutes in a pretreatment solution (a mixture of physiological saline and surfactant with adjusted pH and salinity). Then, the sample was placed in the center of a 3.5 cm petri dish, and the following conditions were met. • Applied voltage: 11kVpp • Application time: 12ms • Frequency: 0.5~50kHz • Opposing electrode: Copper plate ·Introduced substance: FITC-dextran (250kDa) • Cells used: *Cells of *Minato-kamojigusa* • Conductivity of pre-treatment solution: 0.3 S / m Plasma irradiation was performed.

[0055] After irradiation, FITC-dextran solution was added to the sample. After 30 minutes, the sample was washed with tap water and observed.

[0056] The graph plotting the results is shown in Figure 8. In Figure 8, the vertical axis represents brightness and the horizontal axis represents frequency (kHz). Below 5 kHz (circled in the graph), the brightness is higher than that of the untreated control, suggesting a high introduction rate at low frequencies and indicating that the current flowing through the intercellular matrix is ​​involved in the introduction. In other words, it was considered that the low-frequency plasma had some effect on the cell surface and intercellular matrix. The current flow in the circled area of ​​Figure 8 is as shown in Figure 3(b), and as shown in Figure 4, it was considered that the low-frequency current flows through the cells in a weaving manner.

[0057] <Experimental Example 3: Surface Observation using SEM> Next, surface observation using a scanning electron microscope (SEM) was performed to investigate the effects of plasma treatment on the cell surface. The experimental setup shown in the schematic diagram in Figure 7 was used, and the procedure was as follows: First, in the same manner as in Experimental Example 2, under the following conditions • Applied voltage: 11kVpp • Application time: 12ms • Frequency: 500Hz, 20kHz • Opposing electrode: Copper plate • Cells used: *Cells of *Minato-kamojigusa* Plasma irradiation was performed.

[0058] After irradiation, SEM samples were prepared using the t-butanol freeze-drying method and observed. Figure 1 shows SEM images (magnification: 200x) of the results from Experiment Example 3. Figure 1(a) shows the case when plasma was irradiated at a frequency of 500 Hz, Figure 1(b) shows the case when plasma was irradiated at a frequency of 20 kHz, and Figure 1(c) shows the control case (no plasma irradiation). In the control case (Figure 1(c)) and the 20 kHz case (Figure 1(b)), a membrane covered the cell surface, whereas at 500 Hz, the membrane covering the cell surface was peeled off (Figure 1(a)).

[0059] Figure 9 shows SEM images (magnification: 30,000x) of the results from Experiment Example 3. Figure 9(a) shows the case when plasma was irradiated at a frequency of 500 Hz, Figure 9(b) shows the case when plasma was irradiated at a frequency of 20 kHz, and Figure 9(c) shows the control case (no plasma irradiation). In the control case, the cells were densely packed without gaps, as shown by the black fill (Figure 9(c)). However, at 500 Hz, countless gaps were observed on the cell surface (Figure 9(a)). Furthermore, at 20 kHz, the surface state was observed to be intermediate between the control and 500 Hz (Figure 9(b)). From these results, it was thought that at 500 Hz, gaps were created on the cell surface, allowing molecules to penetrate the intercellular matrix and cell wall. Based on these results from Experiment Example 3, it was predicted that, as shown in the schematic diagram in Figure 10, plasma treatment creates gaps on the cell surface, allowing molecules to reach the cell membrane.

[0060] <Experimental Example 4: Evaluation of the Contribution of Endocytosis> We investigated the molecular introduction pathway by evaluating the contribution of endocytosis to *Epipactis thunbergii* callus in microplasma using an endocytosis inhibitor. FITC-Dextran was used as the introduced substance, and ES9-17 (a clathrin-dependent endocytosis inhibitor) was used as the endocytosis inhibitor. Using the experimental apparatus shown schematicly in Figure 7, and following the procedure shown in Figure 11, *Epipactis thunbergii* callus was collected from the culture medium and divided into three groups of samples. The first group's sample (F+P+ES) was inoculated with the endocytosis inhibitor and allowed to stand for 30 minutes, while the second group's sample (F+P) was used as is. For F+P+ES and F+P, the samples were placed in the center of a 3.5 cm petri dish, and the following conditions were met. Voltage: 11kVpp • Application time: 12ms • Frequency: 1kHz • Opposing electrode: Copper plate ·Introduced substance: FITC-Dextran • Inhibitor: ES9-17 (30 μM) • Cells used: *Cells of *Minato-kamojigusa* • Conductivity of pre-treatment solution: 0.3 S / m Plasma irradiation was performed.

[0061] For F+P+ES and F+P, after plasma irradiation, samples from the three groups (F (control)) were taken from the culture medium, and FITC-Dextran solution was added dropwise to the sample. After standing for 30 minutes, the sample was washed with water.

[0062] The results are plotted in a graph shown in Figure 12. In Figure 12, the vertical axis represents brightness. As can be seen from Figure 12, when an endocytosis inhibitor was used, the results were similar to the control, while P+F had a 66.4% higher brightness than P+F+E. This suggests that the majority of the introduction pathway into *Cymbidium goeringii* callus in the microplasma method is clathrin-dependent endocytosis.

[0063] Based on the results of Experimental Example 4, the introduction pathway in the microplasma method was predicted to be as shown in the schematic diagram in Figure 13. That is, it was thought that the molecules were introduced by plasma treatment, which allowed them to reach the cell membrane, and then endocytosis was induced.

[0064] <Experimental Example 5: Introduction experiment using two irradiations (low frequency → high frequency)> In conjunction with impedance analysis, and with an aim to improve introduction by focusing on low and high frequencies in plants, the experiment was conducted using the experimental apparatus shown in the schematic diagram in Figure 7, following the procedure below. First, the callus of *Echinococcus mutabilis* was collected from the culture medium, and the sample was placed in the center of a 3.5 cm petri dish, and then the following conditions were met. • Applied voltage: 11kVpp • First application time: 12ms • Second application time: 4ms • First frequency: 500Hz • Second frequency test: 20kHz • Opposing electrode: Copper plate ·Introduced substance: FITC-Dextran (250kDa) • Cells used: *Cells of *Minato-kamojigusa* Plasma irradiation was performed twice. After the second plasma irradiation, FITC-dextran solution was added to the sample, and after 30 minutes, the sample was washed with tap water and then observed.

[0065] The graph plotting the results is shown in Figure 14. In Figure 14, the vertical axis represents brightness. As can be seen from Figure 14, the brightness after one irradiation was approximately 12,000, while the brightness after two irradiations was approximately 20,000. From these results, it was thought that low-frequency plasma penetrated the intercellular matrix and cell wall, and that high-frequency plasma, which has also been confirmed to be used for introduction in animal cells, promoted a greater degree of introduction. The introduction mechanism revealed from these results is shown in the schematic diagram in Figure 15, which indicates that low-frequency plasma created gaps on the cell surface, and after reaching the cell membrane, molecular introduction occurred by endocytosis. Furthermore, it was thought that the introduction rate improved in the introduction experiment with two irradiations due to other factors such as ion aggregation and dispersion caused by low-frequency and high-frequency electrical stimulation.

[0066] <Experimental Example 6: Introduction experiment using multiple irradiations at the same frequency> The experiment was conducted using the experimental apparatus shown in the schematic diagram in Figure 7, following the procedure below. First, the callus of *Minato-kamojigusa* was collected from the culture medium, and the sample was placed in the center of a 3.5 cm petri dish, and then the following conditions were met. • Applied voltage: 11kVpp • First application time: 12ms • Second application time: 12ms • Application time (3rd time): 12ms • Opposing electrode: Copper plate ·Introduced substance: FITC-Dextran (250kDa) • Cells used: *Cells of *Minato-kamojigusa* Plasma irradiation was performed three times. The frequency of low-frequency plasma irradiation was set to 500 Hz, and the frequency of high-frequency plasma irradiation was set to 20 kHz. FITC-dextran solution was added to the sample after the first plasma irradiation (for the first irradiation only), after the second plasma irradiation (for the second irradiation), and after the third plasma irradiation (for the third irradiation). After 30 minutes, the sample was washed with tap water and then observed.

[0067] Figure 16 is a graph showing the results of Experimental Example 6, where the vertical axis represents average brightness and the horizontal axis represents the processing order. In the processing order, "L" means low-frequency plasma irradiation (first time only), "LL" means low-frequency plasma irradiation → low-frequency plasma irradiation, "LLL" means low-frequency plasma irradiation → low-frequency plasma irradiation → low-frequency plasma irradiation, "H" means high-frequency plasma irradiation (first time only), "HH" means high-frequency plasma irradiation → high-frequency plasma irradiation, and "HHH" means high-frequency plasma irradiation → high-frequency plasma irradiation → high-frequency plasma irradiation. In the case of 500 Hz irradiation, the experimental results showed that the amount of introduced substance increased from the second time to the first time, and from the third time to the second time. On the other hand, when irradiation was performed three times at 20 kHz, the introduction of the introduced substance was hardly observed.

[0068] <Experimental Example 7: Introduction experiment using two irradiations> The experiment was conducted using the experimental apparatus shown in the schematic diagram in Figure 7, following the procedure below. First, the callus of *Minato-kamojigusa* was collected from the culture medium, and the sample was placed in the center of a 3.5 cm petri dish, and then the following conditions were met. • Applied voltage: 11kVpp • First application time: 12ms • Second application time: 12ms • Opposing electrode: Copper plate ·Introduced substance: FITC-Dextran (250kDa) • Cells used: *Cells of *Minato-kamojigusa* Plasma irradiation was performed twice. The frequency of the low-frequency plasma irradiation was set to 500 Hz, and the frequency of the high-frequency plasma irradiation was set to 20 kHz. For comparison, the same experiment was also performed when only the first low-frequency plasma irradiation was performed. In the case of only one irradiation, FITC-dextran solution was added to the sample after the first plasma irradiation, and in the case of two irradiations, after the second plasma irradiation. After 30 minutes, the sample was washed with tap water and observed.

[0069] Here, Figure 17 is a graph showing the results of Experimental Example 7, where the vertical axis represents average brightness and the horizontal axis represents the processing order. In the processing order, "LL" means low-frequency plasma irradiation → low-frequency plasma irradiation, "LH" means low-frequency plasma irradiation → high-frequency plasma irradiation, "HL" means high-frequency plasma irradiation → low-frequency plasma irradiation, "HH" means high-frequency plasma irradiation → high-frequency plasma irradiation, and "L" means low-frequency plasma irradiation (only the first time). As shown in Figure 17, when plasma irradiation in the second frequency range was performed first, followed by plasma irradiation in the second frequency range, when plasma irradiation in the second frequency range was performed first, followed by plasma irradiation in the first frequency range, and when plasma irradiation in the first frequency range was performed first, compared to the first time plasma irradiation in the first frequency range, a higher amount of target molecule was introduced.

[0070] <Experimental Example 8> As shown in Figure 18, medaka fish eggs have a structure consisting of an embryo, an egg membrane surrounding it, and a periovular space between them. When the fish egg and solution are viewed as a single electrical circuit, the egg membrane acts as a capacitor and the solution acts as a resistor. By changing the frequency during discharge, it should be possible to determine which current path, I1 or I2, in the circuit shown in Figure 19(a) is more likely to be taken. Therefore, impedance analysis of the fish egg was performed to investigate frequency conditions that increase the current I1 flowing through the fish egg.

[0071] After collecting the medaka eggs, hatching was induced by using a hatching enzyme (purchased from NBRP Medaka) for 10, 20, and 30 minutes. Figure 20(a) shows normal fish eggs before using the hatching enzyme, and Figure 20(b) shows fish eggs after using the hatching enzyme for 1 hour. It can be seen that inducing hatching thins the egg membrane and improves the efficiency of introduction. As a control, skipjack tuna eggs were used without the use of the hatching enzyme. Figure 21 shows a graph of impedance for each duration of hatching enzyme use, obtained from 801 measurement points between 40 Hz and 200 kHz. In the graph in Figure 21, the vertical axis is the reactance (kΩ) of the fish eggs, and the horizontal axis is the resistance (kΩ). As shown in Figure 21, it was found that when the hatching enzyme was used, the impedance decreased with increasing time, i.e., as the egg membrane thinned. The impedance decreases at higher frequencies, and the points in the diagram represent impedances around 10kHz, but it was observed that they gradually approach the impedance of bonito eggs.

[0072] <Experimental Example 9> Figure 22 schematically shows the circuit of the experimental system used in Experimental Example 9. Because there was a possibility of a problem with the amount of current flowing through the fish eggs during plasma irradiation, the absolute amount of current was increased by using a fluorescent molecule solution with increased conductivity (reduced solution resistance). First, medaka eggs were collected, adhesive threads were removed, and the same hatching enzyme as in Experimental Example 8 was used for 30 minutes. After standing in a dish until just before hatching, the eggs were placed in the center of a 3.5 cm dish, FITC-Dextran solution was added dropwise, and plasma irradiation was performed twice. After standing for 1 hour, the eggs were washed and the hatched juveniles were observed. The experimental conditions were as follows:

[0073] • Resistance: 100kΩ • Number of irradiations: 2 ·Introduced molecule: FITC-Dextran (10kDa) • Concentration of introduced molecule: 10 μg / μl • Frequency: 10kHz ·FITC solution volume: 800μl • Conductivity of FITC-Dextran solution: 0.03 S / m, 1.85 S / m • Fish eggs used: Late-stage embryos • Hatching enzyme: 30 minutes The FITC-Dextran solution (introduction solution) has the following composition: • Composition of FITC-Dextran solution with conductivity of 0.03 S / m: Freshwater • Composition of FITC-Dextran solution with conductivity of 1.85 S / m: Artificial seawater: Distilled water = 1:1 The conductivity was changed accordingly.

[0074] Figure 23 is a photograph showing the results of Experimental Example 9. Figure 23(a) shows hatched larvae when using a FITC-Dextran solution with conductivity of 0.03 S / m, and Figure 23(b) shows hatched larvae when using a FITC-Dextran solution with conductivity of 1.85 S / m. In the case of conductivity of 0.03 S / m, the discharge current was 593 mA and the introduction rate was 0 / 20, whereas in the case of conductivity of 1.85 S / m, the discharge current was 3287 mA, a difference of nearly six times, and the introduction rate was 6 / 53.

[0075] Based on the results of Experimental Examples 8 and 9, an equivalent circuit analysis of the structure of fish eggs suggests a circuit model for the impedance of fish eggs, as shown in Figure 24. This model consists of three parallel circuits of resistors and pseudocapacitors connected in series, with a resistor connected in series to each. In this equivalent circuit, the current path is thought to change depending on the discharge frequency. Since the inside of the fish egg cells is thought to correspond to one of the pseudocapacitor regions, introduction into the fish egg is likely to occur under conditions where current flows through the pseudocapacitor region. In Figure 24, from left to right, the circuit resistance of the cable, the electrode interface (around 100 Hz), the area around the egg membrane (around 1000 Hz), and the area around the embryo (around 150 kHz) are shown, respectively.

[0076] Figure 25(a) is a magnified view of the area around the egg membrane of the circuit model shown in Figure 24, and Figure 25(b) is a schematic diagram showing the current flow around the egg membrane during plasma irradiation. The current flowing on the R2 side, which is the surface of the egg membrane, in the circuit model around the egg membrane is I R2 The current flowing through the CPE2 side, which is the inside of the egg membrane, is I CPE2Then, when the total current flowing through is set to 100, the shunt ratio at a frequency of 100 Hz is I R2 :I CPE2 = 93:7, and at a frequency of 10,000 Hz, I R2 :I CPE2 = 12:88. It can be seen that by irradiating the plasma in the frequency range to which the high-frequency current belongs (i.e., the second frequency range), a large amount of current flows inside the egg membrane.

[0077] FIG. 26(a) is a diagram showing an enlarged view of the periphery of the embryo of the circuit model shown in FIG. 24, and FIG. 26(b) is a diagram schematically showing the flow of current during plasma irradiation of the periphery of the embryo. Let the current flowing through the R3 side, which is the embryo surface, among the circuit models around the embryo be I R3 and the current flowing through the CPE3 side, which is inside the embryo, be I CPE3 Then, when the total current flowing through is set to 100, the shunt ratio at a frequency of 100 Hz is I R3 :I CPE3 = 30:70, and at a frequency of 10,000 Hz, I R3 :I CPE3 = 1:99. It can be seen that by irradiating the plasma in the frequency range to which the high-frequency current belongs (i.e., the second frequency range), a large amount of current flows inside the embryo. Also, it was found that even for low-frequency currents around the embryo, the current flowing inward is large, and once the current flows into the egg membrane, there is little current that escapes into the perivitelline cavity without passing through the embryo, even for low frequencies.

[0078] All the embodiments disclosed this time should be considered as illustrative in all respects and not restrictive. The scope of the present invention is shown not by the above description but by the claims, and it is intended that all modifications within the meaning and scope equivalent to the claims are included.

Claims

1. A method for introducing the target molecule into target plant cells or target tissue by bringing target plant cells or target tissue into contact with an introduction solution containing the target molecule, and irradiating the target plant cells or target tissue with plasma by selecting a first frequency range of 5 kHz or less, or a second frequency range higher than the first frequency range, depending on the impedance of the target plant cells or target tissue, (1) When irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) Irradiating with plasma in the first frequency range, (B) Irradiating with plasma in the second frequency range, and then irradiating with plasma in the second frequency range at least once, (C) Irradiating with plasma in the second frequency range, and then irradiating with plasma in the first frequency range. (2) A method of irradiating fish eggs with plasma, wherein the plasma is in the second frequency range.

2. The method according to claim 1, wherein the first frequency range is in the range of 100 Hz to 5 kHz.

3. The method according to claim 1, wherein the second frequency range is in the range of 10 to 200 kHz.

4. (1) The method according to claim 1, wherein when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) after irradiation with plasma in the first frequency range, the irradiation with plasma in the second frequency range is performed at least once.

5. The method according to claim 4, wherein the amount of the target molecule introduced is increased by increasing the number of irradiations with the plasma in the second frequency range.

6. (1) The method according to claim 1, wherein when irradiating target plant cells or target tissue (excluding fish eggs) with plasma, (A) after irradiation with plasma in the first frequency range, the irradiation with plasma in the first frequency range is performed at least once.

7. The method according to claim 6, wherein the amount of the target molecule introduced is increased by increasing the number of irradiations with the plasma in the first frequency range.

8. (1) The method according to claim 1, in which a plasma is irradiated onto a target plant cell or target tissue (excluding fish eggs), and (B) the plasma in the second frequency range is irradiated, and thereafter the plasma in the second frequency range is irradiated at least once, wherein the amount of the target molecule introduced is increased by increasing the number of times the plasma in the second frequency range is irradiated.

9. (1) The method according to claim 1, in which a plasma is irradiated onto a target plant cell or target tissue (excluding fish eggs), and (C) the step of irradiating with a plasma in the second frequency range, and then irradiating with a plasma in the first frequency range, wherein the irradiation with the plasma in the first frequency range is performed at least once after the irradiation with the plasma in the first frequency range.

10. The method according to claim 9, wherein the amount of the target molecule introduced is increased by increasing the number of irradiations with the plasma in the first frequency range.

11. (1) The method according to claim 1, in which the target of irradiation is a target plant cell or target tissue (excluding fish eggs) and plasma is irradiated, and (C) plasma of the second frequency range is irradiated, and then plasma of the first frequency range is irradiated, wherein the irradiation of plasma of the second frequency range is performed at least once after the irradiation of plasma of the first frequency range.

12. The method according to claim 11, wherein the amount of the target molecule introduced is increased by increasing the number of irradiations with the plasma in the second frequency range.

13. (2) The method according to claim 1, wherein when irradiating fish eggs with plasma in the second frequency range, the irradiation with plasma in the second frequency range is performed at least once after the irradiation with plasma in the second frequency range.

14. The method according to claim 13, wherein the amount of the target molecule introduced is increased by increasing the number of irradiations with plasma in the second frequency range.

15. (2) The method according to claim 1, 13, or 14, wherein, when irradiating fish eggs with plasma in the second frequency range, the method further comprises at least one of the steps of inducing the hatching of fish eggs and increasing the conductivity of the introduced solution before irradiating with plasma of the second frequency range.

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