WO2020203806A1

WO2020203806A1 - Method for suppressing aggregation of isolated plant cells

Info

Publication number: WO2020203806A1
Application number: PCT/JP2020/014111
Authority: WO
Inventors: 松井　南; 龍史岡本; 加藤　紀夫; 雅子市川
Original assignee: 日本たばこ産業株式会社; 国立研究開発法人理化学研究所
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2020-10-08
Also published as: JP2022078383A

Abstract

This invention relates to a method for suppressing aggregation of isolated plant cells, and a method for introducing a substance to a plant. The method according to the present invention comprises bringing isolated plant cells into contact with a composition containing at least one type of gelling agent.

Description

How to suppress the aggregation of isolated plant cells

The present invention relates to a method of suppressing aggregation of isolated plant cells and a method of introducing a substance into a plant.

The use of gene recombination technology for plants, especially monocotyledonous plants, has rapidly become widespread since the method using Agrobacterium was developed in rice and corn in the 1990s. To date, various transformation methods have been developed. However, it is known that the efficiency of transformation differs greatly between species and varieties because many of them need to go through dedifferentiation and redifferentiation of plant tissues. Depending on the species and variety, the efficiency of transformation is low, and it is not possible to obtain a transformed plant with reproducibility. For example, B73, which is a very important line for breeding in maize, has been recognized as a typical difficult-to-culture variety, and it has been difficult to create a reproducible transformation method. Recently, a method for obtaining regenerated individuals even in difficult-to-culture varieties has been developed by introducing several genes, but it is essential to use gene transfer technology, and there remains concern about approval at the practical stage. Also, in other species, varieties and strains called difficult-to-culture varieties, which are considered to be difficult to regenerate by tissue culture, are universally present, which is an obstacle to breeding.

In recent years, it has become possible to efficiently edit the genome. However, the fact that the ease of tissue culture differs depending on the crop species and varieties has a great influence on the efficiency of genome editing, so that it can be put into practical use. It is a hindrance.

On the other hand, in the 1990s, artificial insemination (in vitro fertilization) was attempted in which sperm cells and egg cells were isolated from the plant and artificially fused with each other, and the plant was successfully produced. Non-Patent Document 1 describes a method of electrically fusing corn egg cells and sperm cells to produce fertilized egg cells (in vitro fertilized eggs) and culturing them into a plant. Non-Patent Document 1 uses a high-concentration mixture of plant tissue-degrading enzymes for the separation of egg cells. Further, Non-Patent Document 2 describes a method of producing a fertilized egg by electric fusion of male and female gametes of rice and culturing it into a plant body. Artificial insemination of plants (in vitro fertilization system) involves isolation of gamete cells (egg cells and sperm cells) from pre-pollination flowers, fusion of isolated cells (fertilization), and culture of fused cells (fertilized eggs). It consists of three steps. Typical successful examples are corn (Non-Patent Document 1) and rice (Non-Patent Document 2), and there are several reports of wheat and tobacco. Generally, egg cells are obtained by cutting the ovule before pollination or by degrading the ovule or ovule treated with a plant tissue degrading enzyme such as cellulase or pectinase under a microscope, and sperm cells are suitable. Obtained by bursting pollen in a flexible osmotic solution. The male and female gametes are then transferred to the fusion drop with a glass capillary. Three types of fusion methods for gamete cells have been reported: electrical fusion (Non-Patent Documents 1 and 2), calcium ion fusion (Non-Patent Document 3), and polyethylene glycol fusion (Non-Patent Documents 4 and 5). Has been done. However, it has been reported that a fertilized egg grows into an embryo and regenerates into a plant only for a fertilized egg produced by electrical fusion (Non-Patent Documents 1 and 2). These prior literatures indicate that it is possible to induce plants from fertilized egg cells that are artificially fused with male and female gametes. However, the points such as gene transfer and transformation into fertilized eggs are not described at all, and it is completely unknown whether transformation can be performed using in vitro fertilized eggs.

Fertilized offspring in species such as corn (Non-Patent Document 6), rice (Non-Patent Document 7), wheat (Non-Patent Document 8), barley (Non-Patent Documents 9 and 11), tobacco (Non-Patent Document 10). It is also known that fertilized eggs are taken out from bunches and ovules and cultured to produce plants. Non-Patent Documents 6 and 11 relating to maize and barley describe that DNA was introduced into fertilized egg cells by a microinjection method. However, the fact that a plant transformation method by a microinjection method for fertilized eggs has been put into practical use has not been reported. In addition, there is no knowledge about gene transfer by other methods.

The microinjection method can also introduce a gene into a cell having a cell wall, and it is not particularly necessary to remove the cell wall of the plant cell to be introduced by plant tissue degrading enzyme treatment or the like. However, it has the disadvantage that only one cell can be handled by one transfer operation, and is not suitable for medium to large-scale gene transfer experiments using a large number of plant cells. In addition, it is difficult to put it into practical use because it requires complicated operations under a microscope and requires skillful techniques. Other methods for introducing a gene into cells include a polyethylene glycol method (polyethylene glycol: PEG method), a peptide method (Non-Patent Document 12), an electroporation method, an Agrobacterium method, and the like, in addition to the microinjection method. The electroporation method, the peptide method and the PEG method, particularly the PEG method, are simpler than the microinjection method and have the advantage of being able to handle a large number of cells at one time. However, since plant cells have a cell wall, the tissues and cells are treated with plant tissue-degrading enzymes such as cellulase, pectinase, protease, and hemicellulase, especially when performing the PEG method or electroporation method. It is common to lyse the cell wall and protoplast the cells. For this reason, the above methods have so far targeted materials such as leaves, cultured cells, and callus that can obtain a large amount of protoplasts. (Non-Patent Documents 13 and 14)
On the other hand, unlike leaves, cultured cells, callus, etc., fertilized eggs take time to produce and isolate, and a large amount of protoplast cannot be obtained. In addition, egg cells and sperm cells immediately before fertilization do not start differentiation unless they are subjected to fusion treatment such as electrical treatment or addition of calcium solution. Further, it is considered that the cells of the fertilized egg obtained by such an in vitro fertilization system may be damaged by an artificial fertilization operation such as the above-mentioned fusion treatment. Furthermore, regarding fertilized egg cells, a method for removing the cell wall from the fertilized egg while maintaining the cell activity capable of continuing cell division and growing into a plant after removing the cell wall was unknown. Under these circumstances, it is speculated that egg cells, sperm cells, and fertilized egg cells are not suitable as targets for gene transfer, and it has been reported that gene transfer was performed by a method such as the PEG method, leading to cell division. I wasn't.

However, in recent years, an enzyme treatment method for removing a cell wall while maintaining cell activity has been found, and a method for introducing a substance into fertilized egg cells using PEG and further leading to individual regeneration has been established (Patent Document 1 WO2017). / 171092). Further, it has been found that a substance can be introduced into fertilized egg cells having an undeveloped cell wall immediately after in vitro fertilization with PEG without enzyme treatment (Patent Document 2 WO2018 / 143480). With these techniques, it has become possible to introduce substances into fertilized eggs and use the plants regenerated from the fertilized eggs for breeding and the like.

However, regardless of the elapsed time after fertilization, the fertilized egg may adhere to laboratory equipment such as slide glasses, cover glasses, and patterned needles during isolation / culture and substance introduction. In addition, fertilized eggs may aggregate with each other, and in such a case, the fertilized eggs obtained with great care may not be collected, and it may be difficult to use them for subsequent culture. Further, even if the cells can be recovered, the problem has been raised that the culture is not started due to the scratches made at the time of recovery, the culture efficiency is significantly lowered, and the efficiency of gene transfer is lowered.

In particular, when the purpose is to introduce a gene or the like into a plant cell, the PEG method has a major feature that a substance can be introduced into a large number of cells at the same time. However, when such aggregation occurs in fertilized egg cells, the advantages of the PEG method are lost because a large number of cells cannot be handled at one time and only a small amount of cells can be handled. In addition, the phenomenon of adhesion and aggregation observed in fertilized egg cells was also observed in plant culture using protoplasts, which are isolated plant cells, and in the case of substance introduction, as in the case of fertilized egg cells.

WO2017 / 171092 WO2018 / 143480

An object of the present invention is to provide a method for suppressing aggregation of isolated plant cells and a method for introducing a substance into a plant.

As a result of diligent research to solve the above problems, the present inventors suppress the aggregation of plant cells by contacting the isolated plant cells with a composition containing at least one gelling agent. We found that we could do it, and came up with the present invention. The present invention includes, but is not limited to, the following aspects.

[Aspect 1]
A method for suppressing the aggregation of isolated plant cells, which comprises contacting the isolated plant cells with a composition containing at least one gelling agent.
[Aspect 2]
The method according to aspect 1, wherein a part of the step of isolating and culturing the plant cells comprises isolating and culturing the isolated plant cells using a composition containing at least one gelling agent. ..
[Aspect 3]
The supplementary method according to aspect 1 or 2, comprising contacting the isolated plant cell with a composition containing at least one gelling agent prior to the step of culturing the plant cell.
[Aspect 4]
The method according to any one of aspects 1-3, wherein the isolated plant cell is a fertilized egg cell or an egg cell.
[Aspect 5]
The method according to any one of aspects 1-3, wherein the isolated plant cell is a fertilized egg cell that has not been treated with a plant tissue degrading enzyme.
[Aspect 6]
The method according to any one of aspects 1-3, wherein the isolated plant cell is a protoplastized plant cell.
[Aspect 7]
The isolated plant cells have the following steps:
(1-i) A fertilized egg cell is isolated from a tissue containing a fertilized egg cell of a plant, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme.
(1-ii) A tissue containing fertilized egg cells of a plant is treated with an enzyme solution containing a plant tissue degrading enzyme, and then the enzyme-treated fertilized egg cells are isolated.
(1-iii) The tissue containing the fertilized egg cells of the plant is treated with an enzyme solution containing a plant tissue degrading enzyme under low titer conditions, and at the same time, the enzyme-treated fertilized egg cells are isolated.
(1-iv) An egg cell and a sperm cell are isolated from a plant body and fused to produce a fertilized egg, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme, or
(1-v) Tissues containing plant egg cells are treated with an enzyme solution containing a plant tissue degrading enzyme, then the enzyme-treated egg cells are isolated and further fused with the isolated sperm cells.
It is a fertilized egg cell obtained by any of the steps of
The method according to any one of aspects 1-4 and 6.
[Aspect 8]
The method according to any one of aspects 1-7, comprising adding plant cells to a composition containing at least one gelling agent, and then transferring to a culture medium for culturing.
[Aspect 9]
The method according to any one of aspects 1-8, comprising introducing into a plant cell a substance selected from the group consisting of nucleic acids, proteins and peptides.
[Aspect 10]
Aspect 1-9, comprising contacting the plant cells with a composition comprising at least one gelling agent prior to the introduction of the substance, at the same time as the introduction of the substance and / or before culturing after the introduction of the substance. The method described in any one item.
[Aspect 11]
The method according to any one of aspects 1-10, wherein the culture efficiency of plant cells is improved.
[Aspect 12]
The method according to aspect 9 or 10, wherein the introduction efficiency of the substance is improved.
[Aspect 13]
A method of introducing substances into plants
(I) Isolate plant cells;
(Ii) Introduce a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells;
(Iii) Culturing plant cells into which supplies have been introduced,
The method comprising contacting an isolated plant cell with a composition comprising at least one gelling agent in any step, or between steps.
[Aspect 14]
Includes contacting plant cells with a composition comprising at least one gelling agent prior to introduction of the substance, at the same time as the introduction of the substance and / or before culturing after introduction of the substance.
[Aspect 15]
(Iii') Aspect 13 or 14, wherein the cells into which the substance has been introduced are called or embryoid-like by culturing plant cells, and the callus-like or embryoid-like tissue is redifferentiated in a redifferentiation medium. The method described.
[Aspect 16]
The method according to any one of aspects 13-15, wherein the substance introduction is performed using a PEG method or an electroporation method.
[Aspect 17]
The method according to any one of aspects 1-16, wherein the plant is a monocotyledonous plant.
[Aspect 18]
The method according to any one of aspects 1-17, wherein the plant is selected from the group consisting of corn, wheat, barley, rice, sorghum and rye.
[Aspect 19]
The method according to any one of aspects 1-18, wherein the composition containing at least one gelling agent is in a liquid or semi-solid state.

According to the present invention, when handling isolated plant cells such as fertilized eggs and protoplasts, the plant cells are contacted with a composition containing a gelling agent such as agarose in order to avoid aggregation of the plant cells. The culture efficiency can be improved. Furthermore, when a substance is introduced into a plant cell, the efficiency of introducing the substance can be improved.

1. 1. Method of Suppressing Aggregation of Isolated Plant Cells The present invention relates to a method of suppressing aggregation of isolated plant cells. The method of the present invention comprises contacting an isolated plant cell with a composition comprising at least one gelling agent.

(1) Plants The types of plants are not particularly limited. It may be either a dicotyledonous plant or a monocotyledonous plant, preferably a monocotyledonous plant, more preferably corn, wheat, barley, rice, sorghum, limewood, etc., and most preferably corn, wheat, rice.

Although not limited, the method of suppressing the aggregation of isolated plant cells can be used particularly for plants or varieties that are considered to be "difficult to culture". "Difficult-to-culture" means that it is difficult to culture, specifically, for example, it is difficult to culture cells isolated from a plant, formation of callus by treatment such as dedifferentiation, or re-culture from callus to a plant. It means that it is difficult to differentiate.

In general, monocotyledonous plants are more difficult to culture than dicotyledonous plants. "Difficult to culture" plants include, for example, soybeans, common beans, capsicum and the like. "Difficult-to-cultivate varieties" means varieties that are more difficult to cultivate than general research varieties of the same species (such as A188 for corn), for example, corn elite varieties derived from corn B73 and B73. , Elite varieties of wheat (for example, AC Barrie, TAM, etc.), varieties other than GoldenPromise and Igri of corn, 296B, C401, SA281, P88812, Pioneer 8505, Tx430 of sorghum, and the like.

In one embodiment, the plant is selected from the group consisting of corn, wheat, barley, rice, sorghum and rye.

(2) Isolated plant cells By contacting the isolated plant cells with a composition containing at least one gelling agent, such aggregation of plant cells is suppressed.

An "isolated plant cell" is not a cell in which almost all cells other than plasmodesma, such as plant cells existing in a plant tissue, are surrounded by a cell wall, but one cell or a very small number. Is a cell that exists in a state of being separated and isolated from other cells. For example, it includes cells in which some or all of the cells are not surrounded by a cell wall (plant cells in which some or all are protoplastized) isolated by enzyme treatment of plant tissue or the like. Alternatively, the isolated plant germ cells also include gametes for sexual reproduction, including egg cells, sperm cells.

As used herein, the term "egg cell" means a female gamete formed by meiosis of embryonic sac mother cells in a female sac. The method for isolating the egg cell is not limited, but for example, the ovary can be cut in a solution having an appropriate osmotic pressure, and the egg cell emerged from the cut surface can be isolated under a microscope using a glass capillary. As used herein, the term "sperm cell" means a male gamete formed by meiosis of pollen mother cells in anthers of a stamen. As used herein, the term "fertilized egg cell" means a cell in which a sperm cell and an egg cell are fused, but is not limited to the term, and includes an egg cell that starts development without fusion without undergoing meiosis. In an embodiment, the isolated plant germ cell is an isolated fertilized egg cell or egg cell. In particular, a fertilized egg cell formed by fertilization, in which cell wall formation has not started, or cell wall formation has started but has not yet been completed, and the fertilized egg cell or cell wall formation is incomplete. Contains fertilized egg cells that have been completed.

The cell wall formation of plant germ cells can be confirmed by known methods such as cellulose staining with calcoflor and aniline blue staining. Calcoflor is a non-specific fluorescent dye that binds to cellulose and chitin contained in the cell walls of plant cells, fungi and the like. The excitation is 300 to 440 nm (maximum 355 nm), and the maximum fluorescence of cellulose in 0.1 M phosphate buffer pH 7.0 is 433 nm. Non-limitingly, for example, the fluorescence brightness can be measured using a confocal laser scanning microscope, and the integrated brightness can be obtained by performing image analysis of the fluorescence intensity.

The "cell wall formation rate" is, for example, a ratio of brightness when compared with the brightness of a cell in which the cell wall formation of a plant cell is completely completed and the entire cell is covered with the cell wall. Can be expressed. “Isolated” means that the cell wall formation rate is preferably 80% or less, more preferably 70% or less, still more preferably 65% or less, still more preferably 63% or less, and particularly preferably. It means that it is 60% or less, particularly more preferably 50% or less, and most preferably 30% or less.

(3) Fertilized egg cell In one aspect of the method of suppressing the aggregation of isolated plant cells, a fertilized egg cell can be used as a plant germ cell. The method for obtaining fertilized egg cells is not particularly limited.

For example, a fertilized egg cell may be produced in a plant by a natural fertilization method, and the created fertilized egg cell may be obtained from the plant. The method for obtaining fertilized egg cells using the natural fertilization method is, for example, a method in which the stigma is exposed, pollen is attached and pollinated, and then the fertilized egg cells are isolated from the tissue containing the embryo sac. To isolate fertilized egg cells from a plant, the ovary immediately after fertilization is removed from the pollinated plant, the ovary is cut in a solution of appropriate osmotic pressure, and the fertilized egg cell that emerges from the cut surface is removed. It can be isolated under a microscope using a glass capillary or the like. Alternatively, after treating the ovule or ovule with an enzyme solution for a certain period of time, tissues such as the pearl core can be dissected, excised, and isolated under a microscope using, for example, a glass needle or the like. In the present specification, the natural fertilization method may be an artificial mating in which pollen is artificially attached to the stigma, or a natural mating.

Alternatively, after isolating the egg cell of the plant from the plant body in advance, the egg cell of the plant and the sperm cell may be fused in vitro to produce a fertilized egg cell. That is, an egg cell and a sperm cell may be first isolated from a plant body, and a fertilized egg cell may be produced in vitro by a known method such as an electrofusion method (also referred to as gamete fusion). In a non-limiting method for suppressing the aggregation of isolated plant cells, it is preferable to produce fertilized egg cells in vitro.

The electrical fusion method is a method in which two or more types of cells are fused in vitro by electrical stimulation. The ovary before pollination was cut in a solution of appropriate osmotic pressure, and the egg cells emerged from the cut surface were isolated under a microscope using a glass capillary or the like, and into a solution of appropriate osmotic pressure. Cell fusion can be triggered by submerging pollination and applying a pulse to the sperm cells released from the pollination under a microscope using a glass capillary or the like.

When cell fusion is performed by electrical fusion, conditions such as voltage and distance between electrodes can be appropriately determined by those skilled in the art according to the type of plant or the size of cells. When one fused cell (fertilized egg cell) is produced by electrical fusion of a sperm cell and an egg cell, the DC voltage is not limited, and the lower limit is preferably 10 kV or more, and the upper limit is 17 kV or less. Is preferable. The upper limit and the lower limit can be appropriately selected by those skilled in the art.

Further, the distance between the electrodes is not limited, and the lower limit is preferably 1.5 times or more the sum of the diameters of the egg cells and sperm cells to be fused, and the upper limit is preferably 6 times or less. As a method of measuring the cell diameter, there are a method of measuring the diameter using a microscopic eyepiece attached to a microscope, and a method of capturing an image taken by the microscope into a computer and measuring it with image analysis software. Alternatively, for the distance between the electrodes, for example, the lower limit is preferably 80 μm or more, and the upper limit is preferably 240 μm or less. The upper limit and the lower limit of the distance between the electrodes can be appropriately selected by those skilled in the art.

The osmotic pressure of the solution used for electrically fusing sperm cells and egg cells to prepare one fused cell can be appropriately selected according to the type of plant used. For example, it is preferable that the lower limit 380mosmol / kg _{H 2} O or more rice, more preferably, to 390mosmol / kg _{H 2} O or more, and is preferably not more than 470mosmol / kg _{H 2} O limit. For corn, the lower limit is preferably 500 mosmol / kg H ₂ O or more, and the upper limit is preferably 700 mosmol / kg H ₂ O or less. The upper and lower limits of the osmotic pressure of the solution can be appropriately selected by those skilled in the art.

Alternatively, other known cell fusion methods such as calcium fusion method and PEG fusion method may be used for cell fusion of egg cells and sperm cells. The "calcium fusion method" utilizes the property of the cell membrane that the fusion of the cell membrane is likely to occur depending on the calcium concentration. The "PEG fusion method" utilizes the fact that cells are treated with polyethylene glycol (polyethylene glycol, PEG) to bind cell membranes, and when PEG is removed, cells are fused.

In one embodiment, the isolated plant cells are subjected to the following steps:
(1-i) A fertilized egg cell is isolated from a tissue containing a fertilized egg cell of a plant, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme.
(1-ii) A tissue containing fertilized egg cells of a plant is treated with an enzyme solution containing a plant tissue degrading enzyme, and then the enzyme-treated fertilized egg cells are isolated.
(1-iii) The tissue containing the fertilized egg cells of the plant is treated with an enzyme solution containing a plant tissue degrading enzyme under low titer conditions, and at the same time, the enzyme-treated fertilized egg cells are isolated.
(1-iv) An egg cell and a sperm cell are isolated from a plant body and fused to produce a fertilized egg, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme, or
(1-v) Tissues containing plant egg cells are treated with an enzyme solution containing a plant tissue degrading enzyme, then the enzyme-treated egg cells are isolated and further fused with the isolated sperm cells.
It may be obtained by any of the steps of.

In one embodiment, a tissue containing an embryo sac (eg, an ovule) is cut in a solution of appropriate osmotic pressure, and (fertilized) egg cells emerged from the cut surface are isolated under a microscope using a glass capillary or the like. be able to. In this case, the isolated (fertilized) egg cell is treated with an enzyme solution for a certain period of time to obtain an enzyme-treated fertilized egg. Alternatively, after treating the tissue containing the ovule such as the ovule with an enzyme solution for a certain period of time, the tissue such as the ovule can be dissected under a microscope using, for example, a glass needle or the like, and mechanically removed and isolated. .. In this case, an enzyme-treated (fertilized) egg can be obtained without subsequent enzyme treatment. The enzyme treatment for obtaining a fertilized egg by fusing the isolated egg cell and the sperm cell may be performed at any stage before, at the same time, or after the fusion with the sperm cell.

The method of the present invention is characterized in that (fertilized) egg cells are treated with a low titer condition with an enzyme solution containing a plant tissue degrading enzyme from a tissue containing (fertilized) egg cells of a plant. The enzyme treatment may be performed at any time before the (fertilized) egg cell is isolated from the tissue, at the same time as the isolation, or after the isolation, but preferably at the same time as the isolation or after the isolation. Is.

The cell wall of a plant has a basic skeleton made of cellulose embedded in a substrate (matrix, substrate gel) made of other polysaccharides and proteins. The polysaccharides that make up the substrate are traditionally divided into pectin, which is extracted with hot water or an acidic buffer, and hemicellulose, which is an alkali-soluble component. Recently, matrix polysaccharides (matrix polysaccharides) It is often summarized as a matrix (polysaccharide). The term "plant tissue-degrading enzyme" as used herein is a general term for enzymes that directly or indirectly act on and decompose pectin, cellulose, hemicellulose, other matrix polysaccharides, phospholipids, proteins, etc. around plant tissues and cells. is there.

As "plant tissue degrading enzyme", for example, non-limitingly, protoplast preparation enzyme, phospholipase that decomposes cell membrane, tannase that is considered to be useful for tissue decomposition, rice and other components contained in the type II cell wall are decomposed. Ferrulic acid esterase, protease and the like are included. In particular, various protoplast preparation enzymes that are used to lyse the cell walls of plant cells to prepare protoplasts can be used. For example, pectinase, cellulase, protease, hemicellulase (hemicellulase is a general term for enzymes that hydrolyze hemicellulose), glucuronidase, zymolidase, chitinase, glucanase, xylanase, galactanase, arabinase and ligninase. , Or a mixture thereof (a mixture of two or more of these enzyme groups) is included. Pectinases include, for example, polygalacturonase (galacturonase), pectin lyase and pectin methyl esterase.

Alternatively, in one embodiment, the isolated plant cell may be a fertilized egg cell that has not been treated with a plant tissue degrading enzyme as described above. As the "fertilized egg cell not treated with the plant tissue degrading enzyme", for example, the isolated plant germ cell described in Patent Document 2 (WO2018 / 143480) can be used. Alternatively, the isolated plant cell is a plant tissue-degrading enzyme that comprises a fertilized egg cell, a tissue containing a plant fertilized egg cell, or a tissue containing a plant egg cell, as described in Patent Document 1 (WO2017 / 171092). It may be treated under low titer conditions with an enzyme solution containing.

(4) Protoplast In one embodiment, the isolated plant cell may be a protoplastized plant cell. A "protoplast" is a cell from which a cell wall has been removed from a plant cell, which is generally spherical and weak, and has the property of being destroyed by a slight impact. Utilizing the properties of protoplasts, it is possible to introduce substances into cells and fuse them by treatment with PEG or electrical stimulation. Callus can be obtained by culturing and growing protoplasts. However, for example, when the purpose is to introduce a substance, problems such as protoplasts tending to aggregate with each other due to PEG treatment and adhesion to a test instrument may occur. In such a case, undesired aggregation can be suppressed by contacting the protoplast with a composition containing at least one gelling agent.

(5) Gelling agent The gelling agent used in the method for suppressing the aggregation of plant cells may be a known gelling agent, and a gelling agent known in the art can be used. Non-limiting examples of such gelling agents include, for example, agarose, agar and gellan gum, gellite, alginic acid, gelatin, fighter gel and the like.

The concentration of the gelling agent is appropriately selected depending on the type of the gelling agent. Preferably, the composition containing at least one gelling agent is selected to have a concentration that is liquid or semi-solid. For example, in the case of agarose, 0.05-2.0% is preferable, 0.1-1.0% is more preferable, 0.1-0.5% is further preferable, and 0.15-0.3% is preferable. Most preferred.

In the present specification, "liquid state" indicates that the composition containing the gelling agent (for example, a solution) is liquid, and indicates a state in which a fluid having no fixed shape is taken. "Semi-solid" refers to a state that has both liquid and solid attributes, is defined as a semi-fluid that is closer to solid than liquid, and is characterized by being viscous and freely deformable, so-called gel-like. Show things.

On the other hand, a solid aspect such as agarose beads that can maintain a constant shape for a certain period of time is not included in the present invention.

(6) Composition Containing At least One Gelling Agent The composition containing at least one gelling agent is not particularly limited as long as it is a composition containing a gelling agent. In one embodiment of the method of suppressing plant cell aggregation, in a part of the step of isolating and culturing plant cells, the isolated plant cells are isolated using a composition containing at least one gelling agent. , Including culturing. Non-limitingly comprising contacting the isolated plant cells with a composition comprising at least one gelling agent prior to the step of culturing the plant cells. Plant cells may be added to a composition containing at least one gelling agent and then transferred to a culture medium for culturing. The gelling agent contained in the composition may be one type or two or more types (for example, two types, three types, four types, or more).

In one embodiment, the composition comprising at least one gelling agent is a solution that transfers isolated isolated plant cells prior to transplantation into a medium for culture. Non-limiting examples thereof include solutions (also referred to as MMG solutions) containing osmoregulators such as mannitol and sucrose, pH regulators such as MES and / or magnesium salts as main components.

In addition, in this specification, a "medium" is a medium for culturing a plant cell, and contains a carbon source, a nitrogen source, a salt, etc. necessary for culturing the plant cell (limited). However, for example, it refers to M6 medium, B5 medium, N6 medium, ZMS medium, etc.). The "medium" for culturing is distinguished from the above-mentioned solutions for treating isolated plant cells prior to the start of culturing, such as MMG solutions.

For example, when the isolated plant cells are protoplasts, the plant tissues are enzymatically treated to form protoplasts, and then added to a solution containing mannitol or sucrose for isolation or washing with protoplasts. The composition containing at least one gelling agent may be a solution containing the mannitol or sucrose. The composition containing at least one gelling agent may contain a component that induces dedifferentiation (callus formation) or redifferentiation.

For example, when the plant cell is a fertilized egg cell, the fertilized egg enzymatically or mechanically isolated from the plant tissue or the fertilized egg obtained by electrically fusing the egg cell and the sperm cell is isolated or obtained. , A composition containing at least one gelling agent, which is added to a solution containing mannitol or shoe cloth (such as, but not limited to, MMG solution) once for washing or incubation before transfer to the medium. May be the solution.

In one embodiment, the composition containing at least one gelling agent is in liquid or semi-solid form.

The method of suppressing the aggregation of plant cells may include introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells. For example, when a substance is introduced into a plant cell using the PEG method, the composition containing at least one gelling agent is an isolated plant cell, a PEG solution containing the substance to be introduced, and / or the said. It may be a solution to be moved before the PEG solution. For example, a fertilized egg enzymatically or mechanically isolated from a plant tissue, or a fertilized egg obtained by electrically fusing an egg cell and a sperm cell, may be subjected to either or both of the following (i) and (ii). A gelling agent may be included. Preferably, both (i) and (ii) may contain a gelling agent.

(I) A solution containing mannitol or sucrose (such as, but not limited to, MMG solution) for once washing and incubation after isolation or acquisition of the fertilized egg and before transfer to the medium; / Or
(Ii) A solution used for introducing a substance to be introduced and a substance by the PEG method containing PEG.

(7) Suppression of aggregation The aggregation of isolated plant cells is suppressed by the method of the present invention. "Aggregation of plant cells" means, for example, aggregation of plant cells, for example, aggregation of fertilized eggs or protoplasts, adhesion of plant cells to a test instrument (for example, cover glass), cell aggregation, adhesion, etc. It means that it includes close contact widely.

"Suppressing plant cell aggregation" means that the isolated plant cells are brought into contact with a composition containing at least one gelling agent, as compared to the case where the plant cells are not aggregated. Means that is suppressed. As a result, preferably, for example, the recovery rate of a single fertilized egg cell is improved, the culture efficiency of plant cells is improved, and the introduction efficiency of a substance when a substance is introduced is improved. ..

(8) Introduction of substance The method for suppressing aggregation of isolated plant cells may include a step of introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells.

The step of "introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells" is described, for example, in Patent Document 1 (WO2017 / 171092) and Patent Document 2 (WO2018 / 143480). It can be carried out.

A substance introduced into a plant is a substance of a size and properties that can be supplied into a target cell. It may be naturally occurring or artificially manufactured. Examples include various biomolecules and compounds. Examples of biomolecules include nucleic acids, proteins, peptides, polysaccharides, lipids, organelles and the like. In one aspect, the substance is selected from the group consisting of nucleic acids, proteins and peptides. Nucleases such as Cas9 nuclease for genome editing and proteins such as modifying enzymes and antibodies can also be introduced.
Two or more kinds of nucleic acids, proteins, peptides, polysaccharides, lipids and the like, and substances such as metal ions and compounds may be introduced in combination. For example, the nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different species of substances such as nucleic acids and proteins may be introduced simultaneously or as a complex.

The method for introducing a substance into a plant is not particularly limited as long as it is a known method capable of introducing a desired substance into a plant, and can be appropriately selected depending on the type of plant. For example, a physicochemical method (direct DNA introduction method) such as the PEG method, electroporation method, particle gun method, microinjection method, whisker method, or a biological method such as the Agrobacterium method (indirect introduction method of DNA). ) Can be preferably used. A direct introduction method is preferable, and a PEG method or an electroporation method is more preferable. The PEG method is more preferable. The PEG method can be carried out with reference to a known method such as Non-Patent Document 13 (Yoo et al. (2007)).

The time when the plant cells are brought into contact with the composition containing at least one gelling agent is not particularly limited. In one embodiment, the method of suppressing the aggregation of isolated plant cells is to gel at least one type of plant cell prior to the introduction of the substance, at the same time as the introduction of the substance, and / or before culturing after the introduction of the substance. Includes contact with a composition containing an agent.

2. Method of Introducing a Substance into a Plant The present invention relates to a method of introducing a substance into a plant. The method of the present invention
(I) Isolate plant cells;
(Ii) Introduce a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells;
(Iii) Culturing plant cells into which supplies have been introduced,
Including the process. In the present invention, the isolated plant cells are brought into contact with a composition containing at least one gelling agent in any of the steps (i)-(iii) above, or between steps. It is characterized by including.

The definitions of "plant cells", "substances", and "introduction of substances" are as described in the section "1. Method of suppressing aggregation of isolated plant cells".

The time when the plant cells are brought into contact with the composition containing at least one gelling agent is not particularly limited. The gelling agent may be contained in the solution used in any of the steps (i)-(iii) above. Alternatively, a step of contacting the isolated plant cell with the composition containing at least one gelling agent may be included between the steps (i)-(iii) above. In one embodiment, the method of suppressing the aggregation of isolated plant cells is to gel at least one type of plant cell prior to the introduction of the substance, at the same time as the introduction of the substance, and / or before culturing after the introduction of the substance. Includes contact with a composition containing an agent.

In one embodiment, the substance is introduced using the PEG method or the electroporation method.

In one embodiment, the method of introducing a substance into a plant is to callus or embryoidize the cells into which the substance has been introduced by culturing (iii') plant cells, and then callus or embryoidize the tissue. It may include a step of redifferentiating with a redifferentiation medium.

The callus formation or embryoid body formation step and the redifferentiation step are not particularly limited, and a known method for regenerating a plant body from a plant cell can be used. For example, the method described in Patent Document 2 (WO2018 / 143480) may be used. In one embodiment, the step of inducing division and proliferating the substance-introduced fertilized egg cell to form callus or embryo-like body is not particularly limited because the optimum conditions differ depending on the plant, but a nurse culture method in which Feeder cells are added is used. be able to. For example, the material introduced fertilized egg cell, mannitol droplets (e.g., 450mosmol / kg _H 2 O) were washed and put into the process before performing the sterilization, as well as transferring material introduced fertilized egg cell in the liquid medium, standing After that, the culture is shaken, and the culture in a liquid medium is included. It is preferable to add feeder cells to the liquid medium and perform co-culture (nurse culture method). As the medium, for example, a liquid MS medium, B5 medium, N6 medium or the like to which auxin such as 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid is added can be used. By the culturing step, spherical embryoid bodies having a diameter of about 50 to 200 μm are formed 4 to 14 days after the start of culturing the fertilized egg stick.

The redifferentiation step can also be carried out according to a known redifferentiation step. For example, the method described in Patent Document 2 (WO2018 / 143480) may be used. As one embodiment, the spherical embryoid body is transferred to the medium to which the feeder cells are not added, further cultured for about 10 to 14 days, and then placed in an arbitrary medium to which auxin is not added, for example, MS medium, and the plant is cultured. To form. Examples of the medium include MS medium, B5 medium, N6 medium, and solid medium using agarose, agar, gellan gum, gellite, and the like.

3. 3. Improvement of plant cell culture efficiency and improvement of substance introduction efficiency By using the method of suppressing the aggregation of isolated plant cells of the present invention or the method of introducing a substance into a plant, the plant cell culture efficiency is improved. And / or, the effect of improving the introduction efficiency of the substance can be obtained.

"Improved culture efficiency of plant cells" means that the isolated plant cells are brought into contact with a composition containing at least one gelling agent, as compared to the case where the plant cells are not contacted. Means that is cultivated more efficiently. For example, culturing fertilized eggs increases the proportion of fertilized eggs that normally initiate division (division initiation rate), increases the rate of formation of globular or callus-like embryos, and increases the probability of survival in subsequent culturing steps. It can be confirmed by events such as. For example, the split initiation rate is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30 as compared with the case where the composition containing at least one gelling agent is not added. It rises by more than%.

"Improved substance introduction efficiency" means that the isolated plant cells are brought into contact with the composition containing at least one gelling agent, as compared to the case where the substance is not brought into contact with the plant. It means that the probability of being introduced will increase. For example, the substance introduction efficiency is 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25, as compared with the case where the composition containing at least one gelling agent is not added. % Or more, 30% or more, 35% or more, increase.

4. Substance-introduced plants The present invention also includes substance-introduced plants obtained by methods of introducing substances into plants. Prior to the present invention, it was difficult or impossible to obtain a substance-introduced plant, especially for plants and varieties that are considered to be “difficult to culture”. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently obtain a substance-introduced plant for such plants and varieties by a simple method.

Further, the substance-introduced plant obtained by the method of the present invention is not limited to nucleic acids such as plasmids and gene sequence fragments, proteins for genome editing, and plants in which peptides are introduced and retained in plants. In particular, it includes transformed plants obtained by introducing a gene, plants whose genome has been edited by introducing a genome editing-related substance such as Cas9 or guide RNA, and their progeny and clones.

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples. Those skilled in the art can easily modify or modify the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 Isolation of fertilized maize egg cells In this example, fertilized maize egg cells were isolated.
The ears of corn grown in a greenhouse at the appropriate mating time were collected, the foreskin of the ears was removed, the ovary was exposed, and the length of silk (corn whiskers, pistils) was trimmed to about 12 cm. Then, the ears were divided into two in the vertical direction, and the tip of the silk was pollinated with pollen collected from the ears of corn. The mating time was around 9 am. The ears after mating were placed on a sugar-free MS agar medium and placed in an environment of about 25 ° C.

Twelve hours after mating, the ears were moved to an environment of about 5 degrees and kept under that condition until isolation work was performed.

During the isolation operation, the ears were moved from 5 ° C to room temperature. From ovules ears were excised and nucellus sections containing the embryo sac, was placed in a 10% mannitol solution 1mL in 3.5cm plastic petri dish _{(650mosmol / kg H 2 O)} . 0.5 mL of the enzyme mixture was placed in a 3.5 cm plastic petri dish to prepare a 1.5 mL enzyme solution, which was left at room temperature for 5 to 45 minutes.

The enzyme solution is a 3-fold dilution of a 650 mosmol / kg mannitol aqueous solution containing 1% cellulase (manufactured by Worthington), 0.3% macerozyme (manufactured by Yakult Honsha), and 0.05% pectriase (manufactured by Shengshin Pharmaceutical Co., Ltd.). I used the one that I did.

After leaving it in the enzyme solution for 20 to 30 minutes, the enzyme solution was removed with a pipette and washed twice with a 10% mannitol solution. The enzyme-treated and washed pearl core sections were placed in 1.5 mL of a mannitol solution having the same concentration and used for the separation of fertilized eggs.

The fertilized egg was isolated using two glass needles. Fertilized egg cells were isolated by fixing the bead core section with one glass needle to immobilize it and scraping the tissue in the area where the fertilized egg cell is presumed to be present with the other glass needle. As for the estimation of the region, when fertilization was performed, the one invaded by the pollen tube among the two existing helper cells was denatured and turned dark brown, so that was used as a marker. The isolated fertilized egg cells were transferred to droplets on a cover glass or a glass-bottomed charley after washing the micropipette with mannitol solution.

The droplets on the cover glass were created by the following method.

1) Immerse the periphery of the cover glass in a 1,1,1-trichloroethane solution containing 5% dichloromethylsilane and dry it;
2) Place 0.2-0.3 mL of mineral oil (Embryo Culture-tested Grade, Sigma-Aldrich, 1001279270) on the center of the cover glass;
3) Insert 1 ~ 2 [mu] L of 10% mannitol solution into the mineral oil (650mosmol / kg _H 2 O) with a micropipette.

Droplets on the glass bottom charley were created by the following method.

1) Place 0.4-0.5 mL of mineral oil (Embryo Culture-tested Grade, Sigma-Aldrich, 1001279270) on the center of the cover glass;
2) Insert 1 to 2 μL of 10% mannitol solution (650 mosmol / kg H2O) into the mineral oil with a micropipette.

Example 2 Nucleic acid introduction into fertilized egg In this example, nucleic acid was introduced into the fertilized egg obtained in Example 1. In the process of introducing nucleic acid, the fertilized egg was brought into contact with the composition containing the gelling agent.

Specifically, the fertilized egg cells isolated in Example 1 were transferred to droplets (about 2 μL) of MMG solution (15 mM MgCl ₂ , 4 mM MES (pH 5.7), 650 mosmol / kg H ₂ O mannitol), and then transferred. Nucleotide sequence to be introduced into MMG, 35S promoter :: signal sequence :: GFP :: endoplasmic reticulum residual signal (HDEL) :: transferred to a droplet to which a plasmid containing a nosterminator (Non-Patent Document 11) was added. Then the liquid droplet and the PEG solution containing fertilized egg cells (12.5 mL mannitol solution _{(650mosmol / kg H 2 O)} , so as to 25mg with distilled water added 1M calcium chloride PEG4000,2.5mL of 7.5g Droplets (about 2 μL) of (adjusted) were mixed and stirred 30-50 times with a glass capillary.

However, it was left in the droplet for 5 to 30 minutes in the process before and after the PEG treatment. For the droplets under the standing condition, a group in which agarose was added and a group in which agarose was not added were provided so that the final concentration was 0.3%, and a comparative experiment for confirming the effect of adding agarose was carried out in Example 3 below. ..

Example 3 Culturing of fertilized egg cells subjected to nucleic acid introduction treatment The fertilized egg cells subjected to nucleic acid introduction treatment in Example 2 were cultured in this example.

Specifically, the fertilized egg cells subjected to the nucleic acid introduction treatment were transferred to 2 μl of the prepared medium for fertilized cells and statically cultured in a dark place. The medium for fertilized cells is ZMS medium (Kranz, 1993). MS medium and of changes, 165mg _{/ L} NH 4 NO3, as organic, 1.0 mg / L nicotinic acid, 10.0 mg / L thiamine _· H 2 O, 1mg / L pyridoxine · HCl, 750mg / L glutamine, 150 mg / L Proline, 100 mg / L asparagine, and 100 mg / L myo-inositol were added. The 2mg / L 2,4-D was added as a plant hormone, was further adjusted adding osmotic glucose to 600mosmol / kg _{H 2} O. The pH was 5.7.

The prepared medium for fertilized cells was placed in a Millicell CM insert (manufactured by Millipore) having a diameter of 12 mm, and placed in a 3.5 cm plastic petri dish containing 2 mL of the medium. Further, 40 to 60 μL of a floating rice cell culture (Line Oct, manufactured by RIKEN BioResource Center) was added to the charley as a feeder cell.

Using the cleaning and sterilization by micro capillary, the isolated embryo cells were put into fresh 9% mannitol droplets _{(600mosmol / kg H 2 O)} , then on a membrane in the CM insert containing the fertilized cell medium Moved to.

The fertilized egg cells were allowed to stand in a dark place at 26 ° C. for 1 day, and then shake culture was started according to WO2018 / 143480. The number of fertilized eggs that normally started to divide as a result of shaking culture is shown below.

As shown in Table 1, in the group in which agarose was added to the MMG solution (used group), there were clearly more fertilized eggs that started to divide normally than in the group not added (non-used group), and the culture efficiency was increased. Was shown to be improving.

Example 4 Isolation of fertilized egg cells of wheat In this example, fertilized egg cells of wheat were isolated.

From wheat (variety Fielder) grown in a greenhouse, Kumlehn et al. Fertilized egg cells were isolated according to (1997), Plant Journal, 12 (6): 1473-1479 (Non-Patent Document 15). Males were removed 1 to 3 days before flowering, and 4 to 6 days later, the stigmas of the removed florets were artificially crossed by contacting anthers of the same variety. Three to four hours after mating, the ears were sterilized by immersing them in 1-2% sodium hypochlorite for 6 minutes and then washing them three times with sterile distilled water. The florets were aseptically disassembled and the ovary was collected on a 35 mm diameter plastic petri dish containing 4 mL of 0.55 M mannitol solution. While submerging the ovary, a scalpel was used at the bottom of the plastic petri dish to cut the bottom of the ovary. The ovule section was removed from the ovule, the ovule section was fixed on one of the two glass needles, and the fertilized egg cell was removed by gently pressing the tissue in the area where the fertilized egg cell was presumed to be present on the other side. When fertilization was performed, the region was estimated because the pollen tube invaded the two existing helper cells denatured and turned dark brown, which was used as a marker.

Example 5 Washing and culturing of fertilized wheat egg cells The fertilized wheat egg cells isolated in Example 4 were washed and cultured in this example. In the washing step, the fertilized egg was brought into contact with the composition containing the gelling agent.

Specifically, 0.55 M mannitol solution (control group) or 0.15% agarose (Type) as a solution for washing fertilized eggs on the cover glass part of a 35 mm glass-based dish φ12 mm (3971-035 manufactured by IWAKI). 200 μL of 0.55 M mannitol solution (test group) containing IX Agarose, A2576 manufactured by Sigma Co., Ltd.) was filled. The fertilized wheat cells isolated in Example 4 were sequentially transferred to the cover glass using a glass capillary and allowed to stand (washing step).

After 3 hours, fertilized egg cells were collected in a glass capillary, and Kumlehn et al. Culturing was started with reference to (2016), Methods in Molecular Biology, 1359: 503-514 (Non-Patent Document 16). Specifically, a 12 mm diameter Millicell-CM insert (manufactured by Millipore) was placed in a 35 mm diameter plastic petri dish, and the 2,4-D concentration was changed to 0.02 mg / L inside the Millicell-CM insert. 0.2 mL of the modified N6Z liquid medium (Non-Patent Document 16) was added, and 2 mL of the above-mentioned modified N6Z medium was added to the outside.

The collected fertilized egg cells were transferred to the inside of the Millicell-CM insert. The number of fertilized egg cells that could be cultured was counted by collecting them in a glass capillary as a single fertilized egg cell without adhesion between multiple fertilized egg cells. The results are shown in Table 2.

As shown in Table 2, in the group in which the fertilized egg cells were washed with the mannitol solution (control group), fertilized egg cells adhered to each other and it was difficult to recover, but the group in which agarose was added to mannitol was observed. In (test plot), all fertilized egg cells could be collected individually and the culture could be started. Therefore, the effect of suppressing the aggregation of plant cells by including the gelling agent in the solution for handling fertilized egg cells was also observed in the fertilized egg cells of wheat.

According to the present invention, it has become possible to suppress the aggregation of isolated plant cells, and it has become possible to more efficiently culture and introduce substances of plant cells. As a result, even plants that could not be imparted with useful traits because transformation was difficult due to difficulties in culturing, etc., were easily, stably, and reproducibly transformed or genome-edited individuals. Can be obtained.

Claims

The method for suppressing the aggregation of isolated plant cells, which comprises contacting the isolated plant cells with a composition containing at least one gelling agent.
The first aspect of claim 1, wherein a part of the step of isolating and culturing the plant cells includes isolating and culturing the isolated plant cells using a composition containing at least one gelling agent. Method.
The supplementary method according to claim 1 or 2, which comprises a step of contacting the isolated plant cell with a composition containing at least one gelling agent before the step of culturing the plant cell.
The method according to any one of claims 1-3, wherein the isolated plant cell is a fertilized egg cell or an egg cell.
The method according to any one of claims 1-3, wherein the isolated plant cell is a fertilized egg cell that has not been treated with a plant tissue degrading enzyme.
The method according to any one of claims 1-3, wherein the isolated plant cell is a protoplastized plant cell.
The isolated plant cells have the following steps:
(1-i) A fertilized egg cell is isolated from a tissue containing a fertilized egg cell of a plant, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme.
(1-ii) A tissue containing fertilized egg cells of a plant is treated with an enzyme solution containing a plant tissue degrading enzyme, and then the enzyme-treated fertilized egg cells are isolated.
(1-iii) The tissue containing the fertilized egg cells of the plant is treated with an enzyme solution containing a plant tissue degrading enzyme under low titer conditions, and at the same time, the enzyme-treated fertilized egg cells are isolated.
(1-iv) An egg cell and a sperm cell are isolated from a plant body and fused to produce a fertilized egg, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme, or
(1-v) Tissues containing plant egg cells are treated with an enzyme solution containing a plant tissue degrading enzyme, then the enzyme-treated egg cells are isolated and further fused with the isolated sperm cells.
It is a fertilized egg cell obtained by any of the steps of
The method according to any one of claims 1-4 and 6.
The method according to any one of claims 1-7, which comprises adding plant cells to a composition containing at least one gelling agent, and then transferring the plant cells to a culture medium for culturing.
The method according to any one of claims 1-8, which comprises introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells.
Claim 1-9, which comprises contacting the plant cells with a composition containing at least one gelling agent prior to the introduction of the substance, at the same time as the introduction of the substance and / or before culturing after the introduction of the substance. The method according to any one of the above.
The method according to any one of claims 1-10, which improves the culturing efficiency of plant cells.
The method according to claim 9 or 10, wherein the introduction efficiency of the substance is improved.
A method of introducing substances into plants
(I) Isolate plant cells;
(Ii) Introduce a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells;
(Iii) Culturing plant cells into which supplies have been introduced,
The method comprising contacting an isolated plant cell with a composition comprising at least one gelling agent in any step, or between steps.
Includes contacting plant cells with a composition containing at least one gelling agent prior to the introduction of the substance, at the same time as the introduction of the substance and / or before culturing after the introduction of the substance.
(Iii') Callus or embryoid body of the substance-introduced cell by culturing plant cells, and redifferentiating the cast or embryoid body tissue in a redifferentiation medium, claim 13 or 14. The method described in.
The method according to any one of claims 13-15, wherein the substance is introduced using the PEG method or the electroporation method.
The method according to any one of claims 1-16, wherein the plant is a monocotyledonous plant.
The method according to any one of claims 1-17, wherein the plant is selected from the group consisting of corn, wheat, barley, rice, sorghum and rye.
The method according to any one of claims 1-18, wherein the composition containing at least one gelling agent is in a liquid or semi-solid state.