Method for introducing substance into plant cell, method for culturing plant cells, and method for producing plant transformant or genome-edited plant body

Abstract

The present invention provides a highly efficient and simple method for introducing a substance into a plant cell, a method for culturing plant cells, and a method for producing a plant transformant or a genome-edited plant body, all of which are applied to wide-ranging plant cells.　The present invention also provides: a method for introducing a substance into a plant cell, the method being characterized by bringing the plant cell into contact with a polyvinylpyrrolidone solution; a method for culturing plant cells; and a method for producing a plant transformant or a genome-edited plant body.

Description

Method for introducing substances into plant cells, method for culturing plant cells, and method for producing plant transformants or genome-edited plants.

[0001] The present invention relates to a method for introducing substances into plant cells, a method for culturing plant cells, and a method for producing plant transformants or genome-edited plants.

[0002] The world population is projected to increase from approximately 8 billion today to approximately 9.7 billion by 2050. Furthermore, in recent years, extreme weather events such as high temperatures, droughts, and heavy rainfall have become more frequent around the world, indicating significant changes in the global environment. Additionally, there is a need to adapt to the diversification of diets (including high-functional foods and plant-based meats). Therefore, it is expected that the sustainable and / or stable supply of food will become increasingly important in the future.

[0003] Regarding the sustainable and / or stable supply of food, attempts are being made, for example, to breed plants that can be produced efficiently by transforming plant cells. When transforming plant cells, it is sometimes necessary to culture the plant cells, and various methods have been developed to improve efficiency.

[0004] For example, it is known that phenolic compounds secreted from plant tissues during cultivation can inhibit callus formation in plant tissues or cause browning of callus, thereby reducing cultivation efficiency. To address this problem, a method has been used in various plant species, such as sorghum, tobacco, and kiwifruit, to suppress the adverse effects of phenolic compounds by adding polyvinylpyrrolidone (PVP) to a solid culture medium and adsorbing the phenolic compounds onto the PVP (Non-Patent Document 1, Patent Document 1, Patent Document 2).

[0005] However, these methods vary greatly depending on the plant species and variety, and all of them involve many steps and are complex. Therefore, there is a need for highly efficient and simple methods for introducing substances into plant cells, culturing plant cells, and producing transformed plants or genome-edited plants that can be used across a wide range of plant cells.

[0006] Chinese Patent Application Publication No. 107996402, Chinese Patent Application Publication No. 108522277

[0007] Gurel et al. 2009 Plant cell rep 28:429-444

[0008] The present invention aims to solve the aforementioned problems in the conventional approach and achieve the following objectives. Specifically, the present invention provides a highly efficient and simple method for introducing substances into plant cells, a method for culturing plant cells, and a method for producing transformed plants or genome-edited plants, targeting a wide range of plant cells.

[0009] As a result of diligent research to achieve the above objective, the present inventors have found that a method for introducing substances into plant cells, a method for culturing plant cells, and a method for producing transformed plants or genome-edited plants, all of which are applicable to a wide range of plant cells, can be provided by including a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells; a method for culturing plant cells, and a method for producing transformed plants or genome-edited plants, all of which are applicable to a wide range of plant cells.

[0010] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are as follows: <1> A method for introducing a substance into plant cells, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells. <2> A method for culturing plant cells, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution. <3> A method for producing a transformed plant or a genome-edited plant, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells. This specification encompasses the disclosures of Japanese Patent Application No. 2025-043540, which forms the basis of the priority of this application.

[0011] According to the present invention, the aforementioned problems in the conventional method can be solved, the aforementioned objectives can be achieved, and a highly efficient and simple method for introducing substances into plant cells, a method for culturing plant cells, and a method for producing transformed plants or genome-edited plants can be provided for a wide range of plant cells.

[0012] (Method for introducing substances into plant cells) The method for introducing substances into plant cells includes a contact step and an introduction step, and may further include other steps A described later. There are no particular restrictions on the order of the contact step and the introduction step, and they can be appropriately selected according to the purpose. The introduction step may be performed after the contact step, the contact step may be performed after the introduction step, or the contact step and the introduction step may be performed simultaneously. Among these, it is preferable to perform the contact step and the introduction step simultaneously in order to provide a highly efficient and simple method.

[0013] When the introduction step is performed after the contact step, there are no particular restrictions on the time between the contact step and the introduction step, and it can be appropriately selected depending on the purpose. However, from the standpoint of providing a highly efficient and simple method, it is preferable to have 24 hours or less, more preferably 12 hours or less, even more preferably 6 hours or less, even more preferably 1 hour or less, particularly preferably 30 minutes or less, and most preferably 10 minutes or less.

[0014] When the contact step is performed after the introduction step, there are no particular restrictions on the time between the introduction step and the contact step, and it can be appropriately selected according to the purpose. However, from the standpoint of providing a highly efficient and simple method, it is preferable to have 24 hours or less, more preferably 12 hours or less, even more preferably 6 hours or less, even more preferably 1 hour or less, particularly preferably 30 minutes or less, and most preferably 10 minutes or less.

[0015] - Contact process - The contact process is a process of bringing plant cells into contact with a polyvinylpyrrolidone solution.

[0016] The plant cells mentioned above are not particularly limited as long as they are derived from plants, and can be appropriately selected according to the purpose. The plants themselves are not particularly limited and can be appropriately selected according to the purpose, and examples include angiosperms such as monocots and dicots, or gymnosperms.

[0017] There are no particular restrictions on the monocotyledonous plants mentioned above, and they can be appropriately selected depending on the purpose. Examples include grasses, lilies, banana plants, pineapples, and orchids.

[0018] Examples of grasses include rice, wheat, barley, corn, oats, turfgrass, sorghum, rye, millet, and sugarcane. Examples of lilies include onions and asparagus. Examples of bananas include bananas. Examples of pineapples include pineapples. Examples of orchids include orchids.

[0019] There are no particular restrictions on the dicotyledonous plants mentioned above, and they can be appropriately selected according to the purpose. Examples include plants of the Brassicaceae family, Fabaceae family, Solanaceae family, Cucurbitaceae family, Convolvulaceae family, Rosaceae family, Moraceae family, Malvaceae family, Asteraceae family, Amaranthaceae family, and Polygonaceae family.

[0020] Examples of Brassicaceae plants include Arabidopsis thaliana, Chinese cabbage, rapeseed, cabbage, cauliflower, and radish. Examples of Fabaceae plants include soybeans, adzuki beans, kidney beans, peas, cowpeas, and alfalfa. Examples of Solanaceae plants include tomatoes, eggplants, potatoes, tobacco, and chili peppers. Examples of Cucurbitaceae plants include cantaloupe, cucumbers, melons, and watermelons. Examples of Convolvulaceae plants include morning glories, sweet potatoes, and bindweed. Examples of Rosaceae plants include roses, strawberries, and apples. Examples of Moraceae plants include mulberries, figs, and rubber trees. Examples of Malvaceae plants include cotton and kenaf. Examples of Asteraceae plants include sunflowers and lettuce. Examples of Amaranthaceae plants include sugar beets. Examples of Polygonaceae plants include buckwheat.

[0021] Examples of the aforementioned gymnosperms include pine, cedar, ginkgo, and cycad.

[0022] Among these, angiosperms are preferred because they provide a highly efficient method, and among monocots, grasses are more preferred, with rice, wheat, barley, sorghum, or maize being particularly preferred, and wheat or maize being most preferred. Among dicots, potatoes, soybeans, tobacco, cotton, or sunflowers are preferred, with potatoes being most preferred.

[0023] There are no particular restrictions on the type of plant cell, and it can be appropriately selected depending on the purpose. Examples include germ cells such as egg cells, sperm cells, and fertilized egg cells, and somatic cells. Among these, germ cells are preferred, and fertilized egg cells are more preferred, in order to provide a highly efficient method. The plant cells may be tissues such as embryos, leaves, roots, stems, fruits, shoot apices, and tubers, and may be tissues at any stage of maturation. The plant cells may be processed plant cells. They may also be callus derived from the plant tissue. Alternatively, they may be suspension culture cells or protoplasts derived from plant tissue or callus.

[0024] The polyvinylpyrrolidone solution is not particularly limited as long as it contains polyvinylpyrrolidone (PVP) and can be appropriately selected depending on the purpose.

[0025] There are no particular restrictions on the lower limit of the weight-average molecular weight of the polyvinylpyrrolidone (PVP), and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient method, it is preferably 1,000 or more, more preferably 5,000 or more, and even more preferably 10,000 or more. The lower limit of the weight-average molecular weight may also be 20,000 or more, 30,000 or more, or 40,000 or more. There are no particular restrictions on the upper limit of the weight-average molecular weight of the polyvinylpyrrolidone (PVP), and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient method, it is preferably 1,000,000 or less, more preferably 750,000 or less, even more preferably 500,000 or less, and particularly preferably 400,000 or less. Among these, from the standpoint of providing a highly efficient method, a value of 1,000 to 1,000,000 is preferred, 5,000 to 1,000,000 is more preferred, 10,000 to 750,000 is even more preferred, 10,000 to 500,000 is particularly preferred, and 10,000 to 400,000 is particularly preferred.

[0026] The polyvinylpyrrolidone (PVP) may be used alone or in combination of two or more types. Commercially available polyvinylpyrrolidone (PVP) can be used.

[0027] There are no particular restrictions on the lower limit of the concentration of polyvinylpyrrolidone (PVP) in the polyvinylpyrrolidone solution, and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient method, a value of 0.001% (w / v) or higher is preferred, 0.005% (w / v) or higher is more preferred, and 0.01% (w / v) or higher is even more preferred. There are no particular restrictions on the upper limit of the concentration of polyvinylpyrrolidone (PVP) in the polyvinylpyrrolidone solution, and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient method, a value of 20% (w / v) or lower is preferred, 10% (w / v) or lower is more preferred, 5% (w / v) or lower is even more preferred, and 1% (w / v) or lower is particularly preferred. Among these, from the viewpoint of providing a highly efficient method, a value of 0.001% (w / v) or higher and 5% (w / v) or lower is preferred, and 0.01% (w / v) or higher and 1% (w / v) or lower is even more preferred.

[0028] The solvent for the polyvinylpyrrolidone solution is not particularly limited as long as it is a liquid, and can be appropriately selected depending on the purpose. Examples include water, mannitol solution, buffer solution, and plant cell culture medium. The solvent for the polyvinylpyrrolidone solution may be used alone or in combination of two or more.

[0029] The polyvinylpyrrolidone solution may contain other solutes besides polyvinylpyrrolidone (PVP), such as acetosyringone and Agrobacterium strains, but it is preferable that it does not contain gelling agents, milrinone, phosphodiesterase (PDE) III inhibitors, citric acid, activated carbon, tidiazurone, or naphthaleneacetic acid.

[0030] The aforementioned contact is not particularly limited and can be appropriately selected depending on the purpose. Examples include adding the plant cells to the polyvinylpyrrolidone solution, adding the polyvinylpyrrolidone solution to the plant cells, or replacing the solution in which the plant cells are immersed with the polyvinylpyrrolidone solution. Among these, the method of adding the plant cells to the polyvinylpyrrolidone solution is preferred. The method of adding is not particularly limited and can be appropriately selected depending on the purpose. It may be added or replaced in stages, in one step, or continuously.

[0031] There are no particular restrictions on the lower limit of the contact time between the plant cells and the polyvinylpyrrolidone solution, and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient and simple method, 30 seconds or more is preferred, 1 minute or more is more preferred, 2 minutes or more is even more preferred, and 3 minutes or more is particularly preferred. There are no particular restrictions on the upper limit of the contact time between the plant cells and the polyvinylpyrrolidone solution, and it can be appropriately selected depending on the purpose. However, from the viewpoint of providing a highly efficient and simple method, 24 hours or less is preferred, 1 hour or less is more preferred, 30 minutes or less is even more preferred, 20 minutes or less is even more preferred, 15 minutes or less is particularly preferred, and 10 minutes or less is most preferred. Among these, from the viewpoint of providing a highly efficient and simple method, 30 seconds or more and 24 hours or less is preferred, 1 minute or more and 1 hour or less is more preferred, 2 minutes or more and 30 minutes or less is even more preferred, 3 minutes or more and 20 minutes or less is even more preferred, 3 minutes or more and 15 minutes or less is particularly preferred, and 3 minutes or more and 10 minutes or less is most preferred.

[0032] -Introduction Process- The introduction process is a process of introducing substances into plant cells.

[0033] There are no particular restrictions on the introduction method, and it can be appropriately selected depending on the purpose. However, from the standpoint of providing a highly efficient and simple method, the introduction method using microinjection, macroinjection, particle bombardment, electroporation, polyethylene glycol (PEG), whisker, lipofection, soniporation, magnetofection, laser microbeam, membrane-permeable peptide, nanoparticles, viral vector, or Agrobacterium is preferred, with the Agrobacterium method being more preferred.

[0034] The aforementioned microinjection method and macroinjection method are methods of injecting a substance into cells by microscopic manipulation through a microglass injection needle, and are also called microinjection methods. The aforementioned microinjection method and macroinjection method can be carried out according to known protocols.

[0035] The particle gun method is a method of introducing a substance into cells by using a projectile made of metal nanoparticles such as gold or tungsten coated with a substance, which is then ejected at high speed. It is also called the particle gun method, biolistic method, or microprojectile method. High-pressure gas such as helium can be used to eject the metal nanoparticles. The ejection intensity of the metal nanoparticles can be adjusted by controlling the gas pressure and the distance between the metal nanoparticles and the sample, making it possible to introduce the substance into various types of cells. The particle gun method can be carried out according to known protocols.

[0036] The electroporation method described above is a method of introducing substances by applying electrical pulses to a cell suspension to create tiny holes in the cell membrane, thereby delivering the introduced substance from the cell suspension into the cell. The electroporation method can be carried out according to known protocols.

[0037] The PEG method described above is a method of introducing a substance into cells by applying polyethylene glycol (PEG) to them. The PEG method can be carried out according to known protocols.

[0038] The whisker method is a method in which plant cells, whiskers and an introduced substance are vigorously stirred to allow the introduced substance to be taken into the plant cells damaged by the whiskers. Said whisker method can be carried out according to a known protocol.

[0039] The lipofection method is a method in which positively charged lipids and the like form a complex with the introduced substance through electrical interaction, and the complex is taken into cells. Said lipofection method can be carried out according to a known protocol.

[0040] The sonoporation method is a method for introducing a substance by forming micropores in a cell membrane using low-frequency (kilohertz) or high-frequency (megahertz) ultrasound. Said sonoporation method can be carried out according to a known protocol.

[0041] The magnetofection method is a method in which the introduced substance is attached to magnetic nanoparticles, and the substance is introduced into cells using a magnetic plate. Said magnetofection method can be carried out according to a known protocol.

[0042] The laser microbeam method is a method for introducing a substance by forming micropores through irradiating cells with laser light. Said laser microbeam method can be carried out according to a known protocol.

[0043] The method using said membrane-permeable peptide is a method in which a substance is bound to a membrane-permeable peptide containing many basic amino acids, an amphipathic membrane-permeable peptide containing basic amino acids and hydrophobic amino acids, or a membrane-permeable peptide containing many hydrophobic amino acids, and then introduced into cells. Said method using a permeable peptide can be carried out according to a known protocol.

[0044] The aforementioned nanoparticle method involves attaching a substance to particles with a diameter of 1 nm to 100 nm made of materials such as lipids, magnetic materials, metals, and carbon materials, and then introducing them into cells. The nanoparticle method can be used in combination with methods such as particle gun method, soniporation method, electroporation method, magnetofection method, PEG method, and vortex mixer method. The aforementioned nanoparticle method can be carried out according to known protocols.

[0045] The aforementioned viral vector method is a method for expressing foreign genes in a plant body by utilizing the infectivity and replication ability of viruses, and vectors derived from retroviruses, lentiviruses, adeno-associated viruses, etc., are used. The aforementioned viral vector method can be carried out according to known protocols.

[0046] The aforementioned Agrobacterium method is a method of introducing substances by infecting plant tissue with the fungus Agrobacterium.

[0047] There are no particular restrictions on the Agrobacterium bacteria, and they can be appropriately selected depending on the purpose. For example, strains of Agrobacterium tumefaciens such as LBA4404, EHA101, EHA105, AGL0, AGL1, and C58C1 can be used.

[0048] There are no particular restrictions on the plasmid used for Agrobacterium bacteria, and it can be appropriately selected depending on the purpose. Examples include pLC41, pSB131, U0009B, U0017S, pSB134, pNB131, and pIG121Hm. The Agrobacterium method can be carried out according to a known protocol.

[0049] In the present invention, the substance introduced into plant cells means a substance of a size and properties that can be supplied into the target cells. The substance is not particularly limited and can be appropriately selected depending on the purpose, and may be naturally occurring or artificially manufactured, and examples include biomolecules, metal ions, and compounds.

[0050] The biomolecules mentioned above are not particularly limited and can be appropriately selected depending on the purpose. Examples include nucleic acids, peptides, polysaccharides, lipids, and organelles. Among these, nucleic acids or peptides are preferred.

[0051] The nucleic acid or peptide is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be a nucleic acid or peptide that encodes a target peptide to be introduced into the plant cells.

[0052] The nucleic acid is not particularly limited and can be appropriately selected depending on the purpose. Examples include RNA, DNA, RNA-DNA conjugates, and mixtures of RNA and DNA. Among these, circular DNA, linear DNA, circular RNA, or linear RNA are preferred. The nucleic acid may also be a vector. The nucleic acid can be of any length depending on the transformation method used.

[0053] The aforementioned peptide is a general term for molecules in which various amino acids are linked together in a specific order by amide bonds (also called "peptide bonds"), and is a substance that includes proteins (polypeptides) and oligopeptides. There are no particular restrictions on the aforementioned protein, and it can be appropriately selected depending on the purpose. Examples include nucleases such as Cas9 nuclease for genome editing, modifying enzymes, and antibodies. There are no particular restrictions on the molecular weight of the aforementioned protein, and it can be appropriately selected depending on the purpose, but it is preferably 300 kDa or less, and more preferably 200 kDa or less.

[0054] There are no particular restrictions on the length of the peptide, and it can be appropriately selected depending on the purpose, but it may be 100 amino acids or less, or 50 amino acids or less.

[0055] The aforementioned polysaccharides are a general term for substances in which two or more monosaccharide molecules are polymerized by glycosidic bonds. For example, they exhibit properties different from those of the monosaccharides that make up starch.

[0056] The aforementioned polysaccharides are not particularly limited and can be appropriately selected depending on the purpose. Examples include starch (amylose, amylopeptin), glycogen, cellulose, chitin, agarose, carrageenan, heparin, hyaluronic acid, pectin, xyloglycan, and glucomannan.

[0057] Lipids, as mentioned above, are a general term for substances isolated from living organisms that are insoluble in water. They are defined by their solubility, rather than by specific chemical or structural properties. In biochemical terms, they are "molecules that exist in or originate from living organisms and have long-chain fatty acids or hydrocarbon chains."

[0058] There are no particular restrictions on the lipids mentioned above, and they can be appropriately selected depending on the purpose. Examples include (i) simple lipids formed by the esterification of only alcohols and fatty acids (acylglycerols, ceramides, etc.), (ii) complex lipids containing phosphate or sugar in the molecule, generally with sphingosine or glycerol as the backbone (phospholipids, glycolipids, lipoproteins, etc.), and (iii) hydrophobic derived lipids (fatty acids, tenpenoids, steroids, carotenoids, etc.) derived from simple lipids or complex lipids by hydrolysis.

[0059] The aforementioned compounds are not particularly limited and can be appropriately selected depending on the purpose. Examples include fluorescent dye compounds such as uranine and FITC (fluorescein isothiocyanate), and compounds that exhibit effects such as growth promotion, cell cycle regulation, and genome editing efficiency enhancement.

[0060] The aforementioned substance is not particularly limited and can be appropriately selected depending on the purpose, but biomolecules or compounds are preferred, nucleic acids, peptides, or compounds are more preferred, and nucleic acids or peptides are even more preferred.

[0061] The aforementioned substances may be used individually or in combination of two or more. For example, the nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different types of substances, such as nucleic acids and peptides, may be introduced simultaneously or as a complex.

[0062] -Other Steps A- The other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples include the isolation step of the plant cells before the contact step and the introduction step, the standing step after the introduction step, and the culture step after the introduction step. Furthermore, other steps A may further include known methods for improving the efficiency of gene introduction into plant cells (referred to herein as "gene introduction efficiency improvement treatment"). The gene introduction efficiency improvement treatment may be performed before the introduction step, simultaneously with the introduction step, or after the introduction step.

[0063] There are no particular restrictions on the standing step after the introduction step, and it can be appropriately selected depending on the purpose. For example, this could involve standing the plant cells in contact with the polyvinylpyrrolidone solution.

[0064] There are no particular restrictions on the lower limit of the standing time after the introduction process, and it can be appropriately selected depending on the purpose. However, from the standpoint of providing a highly efficient and simple method, it is preferable to have 10 seconds or more, more preferably 30 seconds or more, and even more preferably 1 minute or more. There are no particular restrictions on the upper limit of the standing time after the introduction process, and it can be appropriately selected depending on the purpose. However, from the standpoint of providing a highly efficient and simple method, it is preferable to have 10 days or less, more preferably 7 days or less, even more preferably 1 day or less, even more preferably 3 hours or less, especially preferably 1 hour or less, and most preferably 45 minutes or less. Among these, from the standpoint of providing a highly efficient and simple method, it is preferable to have 10 seconds or more and 10 days or less, more preferably 30 seconds or more and 1 day or less, and even more preferably 1 minute or more and 1 day or less.

[0065] There are no particular restrictions on the culture step after the introduction step, and it can be appropriately selected according to the purpose. For example, a step of culturing the plant cells after the introduction step in a culture medium can be used. The culture medium may be a medium that does not contain polyvinylpyrrolidone. A solid culture medium to which polyvinylpyrrolidone has been added may also be used.

[0066] If the aforementioned standing step is performed, the culture step after the introduction step can be performed after the standing step.

[0067] Examples of gene transfer efficiency improvement treatments include a) centrifugation, b) addition of silver nitrate and / or copper sulfate to the co-culture medium, c) heat treatment, d) heat and centrifugation, e) pressurization, f) inoculation with Agrobacterium in the presence of powder, g) addition of cysteine ​​to the co-culture medium, and h) physical / chemical damage to one or more selected parts from the radicle, bud, and hypocotyl simultaneously with and / or after the co-culture step. While not limited thereto, for example, a) centrifugation may be performed using the method described in WO2002 / 012520, b) addition of silver nitrate and / or copper sulfate to the co-culture medium may be performed using the method described in WO2005 / 017152, d) heat and centrifugation may be performed using the method described in WO2002 / 012521, and h) treatment to physically / chemically damage one or more selected sites from the radicle, pupae, and hypocotyl simultaneously with and / or after the co-culture step may be performed using the method described in WO2011 / 013764. These methods may be used individually or in combination of two or more. Therefore, the method for introducing substances of the present invention may include one or more of the above-mentioned gene transfer efficiency improving treatments.

[0068] (Method for culturing plant cells) The method for culturing plant cells includes a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution, and may include other steps B. The contact step is as described in the above-mentioned (Method for introducing substances into plant cells) - Contact step -.

[0069] -Plant Cells- There are no particular restrictions on the type of plant cells used in the plant cell culture method described above, and they can be appropriately selected according to the purpose. Plant cells described in (Method for introducing substances into plant cells) may be used, or transformed or genome-edited plant cells may be used. Here, the transformed or genome-edited plant cells may have acquired a phenotype due to transformation or genome editing when subjected to the plant cell culture method, or may acquire a phenotype based on transformation or genome editing by going through the culture method described above. There are no particular restrictions on the method of transformation or genome editing for the transformed or genome-edited plant cells used in the plant cell culture method described above, and they can be appropriately selected according to the purpose.

[0070] -Other Process B- There are no particular restrictions on the other processes, and they can be appropriately selected according to the purpose. Examples include the isolation process of the plant cells before the contact process, the culture medium exchange process after the contact process, the culture process before or after the contact process, the introduction process before or after the contact process, the standing process after the introduction process, and the culture process after the introduction process.

[0071] The isolation step of the plant cells prior to the contact step is as described in the above-mentioned (method for introducing substances into plant cells) - Other Steps A -.

[0072] The introduction step before or after the contact step is as described in the introduction step of the above-mentioned (method for introducing substances into plant cells).

[0073] The settling step after the introduction step and the culture step after the introduction step are as described in the above-mentioned (Method for introducing substances into plant cells) - Other Steps A -.

[0074] There are no particular limitations on the culture medium exchange step after the contact step, and it can be appropriately selected depending on the purpose. For example, this could include a step of transferring the plant cells to a culture medium that does not contain polyvinylpyrrolidone or a culture medium that contains polyvinylpyrrolidone, or a step of removing the polyvinylpyrrolidone solution and adding a culture medium that does not contain polyvinylpyrrolidone.

[0075] There are no particular restrictions on the culture step before or after the contact step, and it can be appropriately selected depending on the purpose. For example, one example is a step of culturing the plant cells in a culture medium before or after the contact step.

[0076] (Method for producing a plant transformant or a genome-edited plant) The method for producing the plant transformant or a genome-edited plant includes a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells, and may include other step B. Furthermore, the method for producing the plant transformant or a genome-edited plant may also include one or more of the gene transfer efficiency improvement treatments.

[0077] The plant cells, the contact step, the introduction step, other step B, and the gene transfer efficiency improvement treatment are as described above in (Method for introducing substances into plant cells) and (Method for culturing plant cells).

[0078] The above-mentioned method for producing a plant transformant or a genome-edited plant yields a plant transformant or a genome-edited plant.

[0079] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0080] <Production of Maize Transformants (1)> (Comparative Example 1: Maize Transformation) Transformation of immature embryos of maize variety A188 was carried out according to the Agrobacterium method described in Ishida et al., NATURE PROTOCOLS, 2007, Vol. 2, No. 7, pp. 1614-1621, with some modifications.

[0081] Specifically, immature embryos of corn variety A188, with a long side length of 0.8 mm–1.5 mm, were aseptically removed from parent plants grown in a greenhouse and collected in a 2 mL microcentrifuge tube containing 2 mL of liquid medium LS-inf (Ishida et al., 2007, NATURE PROTOCOLS, Vol. 2, No. 7, pp. 1614–1621). The centrifuge tube containing the immature embryos was transferred to a water bath and heat-treated at 46°C for 3 minutes. After cooling on ice, the immature embryos were washed once with the same liquid medium. Next, centrifugation at 20,000 × g was performed at 4°C for 10 minutes.

[0082] The T-DNA region of pLC41 (Accession No. LC215698.1) was modified to produce pLC41 GUS-Bar, which has a T-DNA containing a GUS gene (Pubi-Iubi-IGUS-Tnos) mediated by a castor catalase intron and a Bar gene (P35S-bar-T35S) driven by a cauliflower mosaic virus 35S promoter. pVGW9, described in International Publication No. 2014157541, was introduced into Agrobacterium strain LBA4404 via electroporation and used for maize transformation.

[0083] The Agrobacterium strain was spread onto YP medium (Yeast extract 5 g / L, Peptone 10 g / L, NaCl 5 g / L, Agar 15 g / L, pH 6.8: Ishida et al., 2007, NATURE PROTOCOLS, Vol. 2, No. 7, pp. 1614-1621), incubated at 28°C for 2 days, and then an Agrobacterium suspension was prepared in LS-inf medium containing 100 μM acetosyringone so that the absorbance at 600 nm was 1.5.

[0084] The immature embryos, which had undergone the aforementioned heat and centrifugation treatment, were immersed in the Agrobacterium suspension, lightly mixed with a vortex mixer, and then allowed to stand for 5 minutes. Next, the immature embryos were transferred to an empty sterile 6 cm petri dish along with the Agrobacterium suspension, and the immature embryos with the Agrobacterium suspension attached were placed in coexistence medium LS-As (Ishida et al., NATURE PROTOCOLS, 2007, Vol. 2, No. 7, pp. 1614-1621) with the blastocyst side facing upward. After culturing in the dark at 25°C for 5 days, eight of the immature embryos cultured in coexistence medium were subjected to GUS staining.

[0085] The GUS staining test involved washing immature embryos once with 0.1 M phosphate buffer (pH 6.8) containing 0.1% (v / v) Triton X-100, then immersing them in phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc), and allowing them to react at 28°C for 24 hours. The extent of the blastodisc tissue that exhibited blue staining was then examined.

[0086] On day 7 of culture in the co-existence medium, the remaining plant tissues were treated with AgNO ３ Modified LSD1.5A medium without (LS inorganic salt, 0.5 mg / L; Nicotinic acid, 0.5 mg / L; Pyridoxine hydrochloride, 1 mg / L; Thiamine hydrochloride, 100 mg / L; Myo-inositol, 1.5 mg / L; 2,4-D, 20 g / L; Sucrose, 0.7 g / L; Proline, 0.5 g / L; MES, 250 mg / L; Cefotaxime, 100 mg / L; Carbenicillin, 5 mg / L; Phosphinothricin, 8 g / L; Agar, pH 5.8: Ishida et al., 2007, NATURE) The cultured tissues were placed on a bed (PROTOCOLS, Vol. 2, No. 7, pp. 1614-1621) and cultured for 10 days at 25°C in the dark. Next, each cultured tissue was treated with AgNO ３Modified LSD1.5B medium without (LS inorganic salt, 0.5 mg / L; Nicotinic acid, 0.5 mg / L; Pyridoxine hydrochloride, 1 mg / L; Thiamine hydrochloride, 100 mg / L; Myo-inositol, 1.5 mg / L; 2,4-D, 20 g / L; Sucrose, 0.7 g / L; Proline, 0.5 g / L; MES, 250 mg / L; Cefotaxime, 100 mg / L; Carbenicillin, 10 mg / L; Phosphinothricin, 8 g / L; Agar, pH 5.8: Ishida et al., 2007, NATURE) The samples were transferred to PROTOCOLS, Vol. 2, No. 7, pp. 1614-1621 and cultured at 25°C in the dark for 3 weeks.

[0087] The proliferated callus is cut apart, and new AgNO is formed. ３ The tissues were transferred to a modified LSD1.5B medium that did not contain PPT and cultured at 25°C in the dark for 3 weeks. The proliferated callus was dissected and placed on LSZ (Ishida et al., 2007, NATURE PROTOCOLS, Vol.2, No.7, pp.1614-1621) redifferentiation medium containing 5 mg / L PPT, and redifferentiated and cultured at 25°C under continuous illumination of 5,000 lx for 2 weeks to obtain transformed and redifferentiated individuals. The number of tissues used (test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0088]

[0089] (Example 1: Effect of PVP in Maize Transformation) An experiment was conducted to verify the effect of PVP in maize transformation. Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 10,000 was added at a concentration of 0.01% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. Table 1 shows the results of counting the number of tissues used (test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (tissues from which transformed and redifferentiated individuals were produced).

[0090] (Example 2: Effect of PVP in maize transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 10,000 was added at a concentration of 0.10% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0091] (Example 3: Effect of PVP in maize transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 10,000 was added at a concentration of 1% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0092] (Example 4: Effect of PVP in Maize Transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 40,000 was added at a concentration of 0.01% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0093] (Example 5: Effect of PVP in maize transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 40,000 was added at a concentration of 0.10% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0094] (Example 6: Effect of PVP in maize transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 40,000 was added at a concentration of 1% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0095] (Example 7: Effect of PVP in Maize Transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 360,000 was added at a concentration of 0.01% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0096] (Example 8: Effect of PVP in maize transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 360,000 was added at a concentration of 0.10% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0097] (Example 9: Effect of PVP in Maize Transformation) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 360,000 was added at a concentration of 1% (w / v) to LS-inf medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 1.

[0098] When PVP was not included in the Agrobacterium suspension (Comparative Example 1), the rate of tissues producing transformed and redifferentiated individuals was 25.0%. When PVP was included (Examples 1 to 9), in all examples with weight-average molecular weights ranging from 10,000 to 360,000 and PVP concentrations from 0.01 to 1% (w / v), the rate of tissues producing transformed and redifferentiated individuals was significantly higher than that of Comparative Example 1.

[0099] In the GUS staining test, no difference in results was observed between Comparative Example 1 and Examples 1 to 9.

[0100] Browning of callus and culture medium due to phenolic compounds was not observed in any of the groups in Comparative Example 1 or Examples 1 to 9. Furthermore, the degree of callus growth was similar in all of the groups in Comparative Example 1 and Examples 1 to 9.

[0101] <Production of Wheat Transformants> (Comparative Example 2: Wheat Transformation) Transformation of immature wheat embryos was carried out according to the Agrobacterium method described in Ishida et al., 2015 Methods Mol Biol 1223 189-198.

[0102] Specifically, immature wheat embryos (1.5 mm–2.5 mm in size: Cronox) 14 days post-flowering, cultivated in a greenhouse, were aseptically collected and washed once in LS-inf liquid medium (Ishida et al., 2015 Methods Mol Biol 1223 189–198). A pretreatment (centrifugation at 20,000 × g for 10 minutes) was performed to enhance gene transfer efficiency.

[0103] For the Agrobacterium strain used, EHA105 (pLC41 GUS- / Bar) containing the vector pLC41 GUS-Bar described in Comparative Example 1 was used. This Agrobacterium strain was inoculated into MG / L medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198), cultured overnight with shaking at 28°C, and then suspended in WLS-inf liquid medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198) to an absorbance of 0.4 at 600 nm to prepare an Agrobacterium suspension.

[0104] The Agrobacterium suspension was added to the centrifuged immature embryos, stirred for 30 seconds, and then allowed to stand at room temperature for 5 minutes. Next, immature embryos inoculated with Agrobacterium were placed in WLS-AS co-culture medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198) with the blastodisc facing upwards. After culturing for 7 days in the dark at 23°C, four of the immature embryos cultured in co-culture were subjected to GUS staining.

[0105] Two days after the start of co-culture, the radicle, bud, and hypocotyl were removed from the immature embryos using a scalpel and tweezers, and the embryos were placed on WLS-Res medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198) and cultured at 25°C in the dark for 5 days. Subsequently, the immature embryos were placed on WLS-P5 primary selection medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198). After culturing under the same conditions for two weeks, immature embryos were placed on WLS-P10 secondary selection medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198) and cultured under the same conditions for three weeks. Subsequently, they were placed on LSZ-P5 redifferentiation medium (Ishida et al., 2015 Methods Mol Biol 1223 189-198) and cultured at 25°C under illumination for two weeks. Table 2 shows the results of counting the number of tissues used (test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (tissues from which transformed and redifferentiated individuals were produced).

[0106]

[0107] (Example 10: Effect of PVP in Wheat Transformation) An experiment was conducted to verify the effect of PVP in wheat transformation. Transformation was carried out in the same manner as in Comparative Example 2, except that PVP with a weight-average molecular weight of 40,000 was added to WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.10% (w / v). Table 2 shows the results of counting the number of tissues used (test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (tissues from which transformed and redifferentiated individuals were produced).

[0108] (Comparative Example 3: Wheat Transformation) Transformation was carried out in the same manner as in Comparative Example 2, except that the wheat variety was changed from Cronox to Paragon. Table 2 shows the results of counting the number of tissues used in the experiment (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced).

[0109] (Example 11: Effect of PVP in wheat transformation) Transformation was carried out in the same manner as in Comparative Example 3, except that PVP with a weight-average molecular weight of 40,000 was added to WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.10% (w / v). The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 2.

[0110] (Comparative Example 4: Wheat Transformation) Transformation was carried out in the same manner as in Comparative Example 2, except that the wheat variety was changed from Cronox to Mace. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) are shown in Table 2.

[0111] (Example 12: Effect of PVP in wheat transformation) Transformation was carried out in the same manner as in Comparative Example 4, except that PVP with a weight-average molecular weight of 40,000 was added to the WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.10% (w / v). The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 2.

[0112] (Comparative Example 5: Wheat Transformation) Transformation was carried out in the same manner as in Comparative Example 2, except that the wheat variety was changed from Cronox to Fielder. Table 2 shows the results of counting the number of tissues used (test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (tissues from which transformed and redifferentiated individuals were produced).

[0113] (Example 13: Effect of PVP in wheat transformation) Transformation was carried out in the same manner as in Comparative Example 5, except that PVP with a weight-average molecular weight of 40,000 was added to WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.10% (w / v). The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 2.

[0114] (Comparative Example 6: Wheat Transformation) Transformation was carried out in the same manner as in Comparative Example 5. The number of tissues used in the experiment (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) are shown in Table 2.

[0115] (Example 14: Effect of PVP in wheat transformation) Transformation was carried out in the same manner as in Comparative Example 5, except that PVP with a weight-average molecular weight of 360,000 was added to WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.01% (w / v). The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 2.

[0116] (Example 15: Effect of PVP in wheat transformation) Transformation was carried out in the same manner as in Comparative Example 5, except that PVP with a weight-average molecular weight of 360,000 was added to WLS-inf liquid medium for suspending the Agrobacterium strain at a concentration of 0.10% (w / v). The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 2.

[0117] Compared to cases where PVP was not included in the Agrobacterium suspension (Comparative Examples 2 to 6), the rate of tissues producing transformed and redifferentiated individuals was significantly higher in all varieties when PVP was included (Examples 10 to 15).

[0118] In the GUS staining test, no difference in results was observed between Comparative Example 2 and Examples 10 to 15.

[0119] Browning of callus and culture medium due to phenolic compounds was not observed in any of the groups in Comparative Example 2 or Examples 10 to 15. Furthermore, the degree of callus growth was similar in all of the groups in Comparative Example 2 and Examples 10 to 15.

[0120] <Production of Potato Transformants> (Comparative Example 7: Potato Transformation) Transformation of potatoes was carried out according to the Agrobacterium method described in Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp. 33-41, with some modifications.

[0121] Specifically, the area is 0.3 cm². ２ -0.5cm ２ To achieve this, leaf fragments of the potato variety Sassy were aseptically cut from the leaves of cultured seedlings that had been cultured at the shoot tip in a culture room (25°C, 16-hour photoperiod).

[0122] LBA4404 was used as the Agrobacterium strain for gene transfer. This strain was introduced into the vector pTOK233 (described in Hiei et al., The Plant Journal, 1994, Volume 6, Issue 2, pp. 271-282), which contains an HPT gene (P35S-HPT-T35S) driven by the cauliflower mosaic virus 35S promoter in the T-DNA region, and a GUS gene (P35S-IGUS-Tnos) regulated by the cauliflower mosaic virus 35S promoter and mediated by a castor catalase intron.

[0123] The Agrobacterium strain was inoculated onto YP medium containing 50 mg / L kanamycin (Yeast Extract 5 g / L, Bacto Peptone 10 g / L, NaCl 5 g / L), incubated at 28°C for 1 day, and then an Agrobacterium suspension was prepared in liquid medium PCM containing 100 μM acetosyringone (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, p. 33-41) so that the absorbance at 600 nm was 1.9.

[0124] The leaf fragments were immersed in the Agrobacterium suspension and allowed to stand for 10 minutes. Next, the leaf fragments were transferred to filter paper to remove excess suspension, and the leaf fragments were placed with their surface facing upwards in coexistence medium PCM containing 140 μM acetosyringone (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, p. 33-41). After incubation for 3-4 days under 25°C illumination, four leaf fragments from those cultured in coexistence medium were subjected to GUS staining tests.

[0125] In the GUS staining test, leaf samples were immersed in 1M phosphate buffer (pH 6.8) containing 0.1% (w / v) Triton X-100 and 1.0 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) and reacted at 37°C for 24 hours. After that, the leaf samples were decolorized by immersion in 99.5% ethanol at 65°C for 4 hours, and the presence or absence of color change was observed.

[0126] Leaf fragments other than those used in the GUS staining test were placed on selection callus medium PCM containing 20 mg / L hygromycin (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp. 33-41) on the same day as GUS staining, and cultured for 7 days under 25°C lighting. Next, each cultured tissue was placed on selection redifferentiation medium PCM containing 20 mg / L hygromycin (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp. 33-41), and cultured for 14 days under 25°C lighting. This procedure was repeated twice. Table 3 shows the results of counting the number of tissues used in the experiment (test tissues) and the number of tissues that produced transformed and redifferentiated individuals (tissues that produced transformed and redifferentiated individuals).

[0127]

[0128] (Example 16: Effect of PVP in potato transformation) An experiment was conducted to verify the effect of PVP in potato transformation. Transformation was carried out in the same manner as in Comparative Example 7, except that 1% (w / v) of PVP with a weight-average molecular weight of 360,000 was added to a liquid medium PCM containing 140 μM acetosyringone for suspending the Agrobacterium strain. Table 3 shows the results of counting the number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced).

[0129] (Comparative Example 8: Potato Transformation) Transformation was carried out in the same manner as in Comparative Example 7, except that the potato variety was changed from Sassy to Konafubuki. Table 3 shows the results of counting the number of tissues used in the experiment (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced).

[0130] (Example 17: Effect of PVP in potato transformation) An experiment was conducted to verify the effect of PVP in potato transformation. The experiment was conducted in the same manner as in Example 16, except that the potato variety Konafubuki was used. Table 3 shows the results of counting the number of tissues used (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced).

[0131] When PVP was not included in the Agrobacterium suspension, the percentage of tissues that produced transformed and redifferentiated individuals was 27.8% for Sassy (Comparative Example 7) and 9.1% for Konafubuki (Comparative Example 8). In contrast, when PVP was included, the percentages increased to 50.0% for Sassy (Example 16) and 13.6% for Konafubuki (Example 17), showing improved efficiency in both varieties.

[0132] In the GUS staining test, no difference in results was observed under any of the conditions.

[0133] Browning of callus or culture medium due to phenolic compounds was not observed under any conditions. Furthermore, the degree of callus growth was similar under all conditions.

[0134] <Production of Corn Transformants (2)> (Comparative Example 9: Corn Transformation) The rate of tissues that produced transformed and redifferentiated individuals in corn transformation was compared for cases where PVP was not used, a solid medium containing PVP was used, a PVP solution was used, and both a solid medium containing PVP and a PVP solution were used. The case where PVP was not used was carried out in the same manner as in Comparative Example 1. The number of tissues tested (number of test tissues) and the number of tissues that were able to produce transformed and redifferentiated individuals (number of tissues that produced transformed and redifferentiated individuals) are shown in Table 4.

[0135]

[0136] (Comparative Example 10: Effect of PVP solid medium in corn transformation) Co-existing medium LS-As, AgNO ３ Modified LSD1.5A medium that does not contain AgNO ３ Transformation was carried out in the same manner as in Comparative Example 1, except that a modified LSD1.5B medium without PPT and an LSZ medium containing 5 mg / L PPT were used, and PVP with a weight-average molecular weight of 40,000 was added at a concentration of 0.10% (w / v). Examples of PVP concentrations added to solid media include 1% for sorghum (Gurel, et al., (2009) Plant cell rep 28:429-444), 0.02-0.05% for tobacco (Chinese Patent Application Publication No. 107996402), and 0.18% for kiwifruit (Chinese Patent Application Publication No. 108522277), but there are no examples of PVP being added to corn. Therefore, in this example, 0.10%, which is the average concentration added to solid media for plant culture, was adopted as the PVP concentration used in the test. Table 4 shows the results of counting the number of tissues used in the experiment (test tissues) and the number of tissues that produced transformed and redifferentiated individuals (tissues that produced transformed and redifferentiated individuals).

[0137] (Example 18: Effect of PVP solution on maize transformation) Transformation was performed in the same manner as in Comparative Example 1, except that PVP with a weight average molecular weight of 40,000 was added at a concentration of 0.10% (w / v) to LS-inf liquid medium containing 100 µM acetosyringone for suspending Agrobacterium strains. Table 4 shows the results of counting the number of tested tissues (number of test tissues) and the number of tissues from which transformed redifferentiated individuals could be produced (number of tissues producing transformed redifferentiated individuals).

[0138] (Example 19: Effect of PVP solution and PVP solid medium on maize transformation) PVP with a weight average molecular weight of 40,000 was added at a concentration of 0.10% (w / v) to LS-inf liquid medium containing 100 µM acetosyringone for suspending Agrobacterium strains, and to co-culture medium LS-As, AgNO ３ modified LSD1.5A medium not containing, AgNO ３ modified LSD1.5B medium not containing, and LSZ medium containing 5 mg / L PPT, transformation was performed in the same manner as in Comparative Example 1, except that PVP with a weight average molecular weight of 40,000 was added at a concentration of 0.10% (w / v).

[0139] When PVP was not included in either the Agrobacterium suspension or the solid medium, the rate of tissues producing transformed redifferentiated individuals was 23% (Comparative Example 9), whereas when PVP was included in the Agrobacterium suspension, the efficiency improved to 39% (Example 18). On the other hand, when PVP was included in the solid medium, the rate of tissues producing transformed redifferentiated individuals was 32% (Comparative Example 10), which was improved compared to Comparative Example 9 but did not reach the effect of Example 18. Furthermore, when PVP was included in both the Agrobacterium suspension and the solid medium, the rate of tissues producing transformed redifferentiated individuals was 44% (Example 19), which was the highest efficiency.

[0140] (Comparative Example 11: Maize culture) In order to verify the effect of PVP solution in a plant culture method that does not include the step of introducing an exogenous gene, the rate of tissues producing redifferentiated individuals in maize culture was compared.

[0141] Use the B73 maize variety, add 0.10% (w / v) PVP with a weight-average molecular weight of 40,000 to the LS-inf liquid medium for collecting immature embryos, do not allow immature embryos to infiltrate the Agrobacterium suspension, and use AgNO ３ The procedure was the same as in Comparative Example 1, except that a modified LSD1.5A medium that did not contain AgNO3, a modified LSD1.5B medium that did not contain AgNO3, and LSZ medium did not contain PPT. The number of tissues tested (number of test tissues) and the number of tissues that produced redifferentiated individuals (number of tissues that produced redifferentiated individuals) were counted and the results are shown in Table 5.

[0142]

[0143] (Example 20: Effect of PVP in Corn Culture) Culture was carried out in the same manner as in Comparative Example 11, except that PVP with a weight-average molecular weight of 40,000 was added at a concentration of 0.10% (w / v) to LS-inf liquid medium containing 100 μM acetosyringone for suspending the Agrobacterium strain.

[0144] When PVP was not included in the liquid culture medium LS-inf, the rate of tissues that produced redifferentiated individuals was 10% (Comparative Example 11), whereas when PVP was included, the rate increased significantly to 50% (Example 20).

[0145] The contact process, which involves bringing the PVP solution into contact with plant cells, was found to not only increase the efficiency of producing transformed and redifferentiated individuals, but also to improve the efficiency of obtaining redifferentiated individuals from plant tissues that have not been introduced with foreign genes.

[0146] (Comparative Example 12: Corn Culture) While PVP is a non-crosslinked linear polymer of N-vinyl-2-pyrrolidone (NVP), polyvinylpolypyrrolidone (PVPP) is a crosslinked polymer of NVP. Similar to PVP, PVPP has been reported to improve plant culture efficiency through its phenolic substance adsorption function (Saxena and Gill (1986) Brief Communications 28 313-315). In the present invention, in order to verify whether PVPP exhibits the same effect as PVP, the percentage of tissues that produced transformed and redifferentiated individuals in corn culture was compared.

[0147] If PVPP or PVP was not used, the procedure was the same as in Comparative Example 1. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) are counted and shown in Table 6.

[0148]

[0149] (Comparative Example 13: Effect of PVPP in Corn Culture) Transformation was carried out in the same manner as in Comparative Example 1, except that PVPP was added at a concentration of 0.01% (w / v) to LS-inf liquid medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 6.

[0150] (Comparative Example 14: Effect of PVPP in Corn Culture) Transformation was carried out in the same manner as in Comparative Example 1, except that PVPP was added at a concentration of 0.10% (w / v) to LS-inf liquid medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 6.

[0151] (Comparative Example 15: Effect of PVPP in Corn Culture) Transformation was carried out in the same manner as in Comparative Example 1, except that PVPP was added at a concentration of 1.00% (w / v) to LS-inf liquid medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 6.

[0152] (Example 21: Effect of PVP in Corn Culture) Transformation was carried out in the same manner as in Comparative Example 1, except that PVP with a weight-average molecular weight of 40,000 was added at a concentration of 0.10% (w / v) to LS-inf liquid medium containing 100 μM acetosyringone for suspending the Agrobacterium strain. The number of tissues tested (number of test tissues) and the number of tissues from which transformed and redifferentiated individuals were produced (number of tissues from which transformed and redifferentiated individuals were produced) were counted and the results are shown in Table 6.

[0153] When PVPP or PVP was not included in the Agrobacterium suspension, the percentage of tissues producing transformed and redifferentiated individuals was 2.5% (Comparative Example 12), whereas when PVP was included, it was 4.9% (Example 21). On the other hand, when PVPP was included in the Agrobacterium suspension, the percentages at concentrations of 0.01%, 0.10%, and 1.00% were 2.5% (Comparative Example 13), 0.0% (Comparative Example 14), and 2.5% (Comparative Example 15), respectively, and did not exceed the results when PVP was not included. These results indicate that although both PVP and PVPP are structures made of NVP, PVPP does not have the same effect as PVP.

[0154] Examples of the present invention include the following: <1> A method for introducing a substance into plant cells, characterized by comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells. <2> The method for introducing a substance into plant cells according to <1>, wherein the substance is a nucleic acid, a peptide, or a compound. <3> The method for introducing a substance into plant cells according to <1> or <2>, wherein the contact time between the plant cells and the polyvinylpyrrolidone solution is 24 hours or less. <4> The method for introducing a substance into plant cells according to any one of <1> to <3>, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 1,000 or more. <5> The method for introducing a substance into plant cells according to any one of <1> to <4>, wherein the concentration of the polyvinylpyrrolidone solution is 0.001% (w / v) or more. <6> The method for introducing a substance into plant cells according to any one of <1> to <5>, wherein the introduction is by the Agrobacterium method. <7> A method for culturing plant cells, characterized by including a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution. <8> The method for culturing plant cells according to <7>, wherein the contact time between the plant cells and the polyvinylpyrrolidone solution is 24 hours or less. <9> The method for culturing plant cells according to <7> or <8>, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 1,000 or more. <10> The method for culturing plant cells according to any one of <7> to <9>, wherein the concentration of the polyvinylpyrrolidone solution is 0.001% (w / v) or more. <11> The method for culturing plant cells according to any one of <7> to <10>, wherein the plant cells are transformed plants or genome-edited plant cells. <12> A method for producing transformed plants or genome-edited plants, characterized by including a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into the plant cells. <13> A method for producing a transformed plant or genome-edited plant according to <12>, wherein the substance is a nucleic acid, a peptide, or a compound.<14> A method for culturing plant cells according to <12> or <13>, wherein the introduction is by the Agrobacterium method. <15> A method for producing a plant transformant or genome-edited plant according to any one of <12> to <14>, wherein the contact time between the plant cells and the polyvinylpyrrolidone solution is 24 hours or less. <16> A method for producing a plant transformant or genome-edited plant according to any one of <12> to <15>, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 1,000 or more. <17> A method for producing a plant transformant or genome-edited plant according to any one of <12> to <16>, wherein the concentration of the polyvinylpyrrolidone solution is 0.001% (w / v) or more. <18> The method according to any one of <1> to <17> above, further comprising at least one of the following gene transfer efficiency improvement treatments: a) centrifugation; b) addition of silver nitrate and / or copper sulfate to the co-culture medium; c) heat treatment; d) heat and centrifugation; e) pressurization; f) inoculation with Agrobacterium in the presence of powder; g) addition of cysteine ​​to the co-culture medium; h) physical / chemical damage to one or more selected sites from the radicle, pupae, and hypocotyl simultaneously with and / or after the co-culture step. All publications, patents, and patent applications referenced herein are incorporated herein by direct reference.

Claims

1. A method for introducing a substance into plant cells, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution, and an introduction step of introducing the substance into the plant cells.

2. The method for introducing a substance into plant cells according to claim 1, wherein the substance is a nucleic acid, a peptide, or a compound.

3. The method for introducing a substance into plant cells according to claim 1 or 2, wherein the contact time between the plant cells and the polyvinylpyrrolidone solution is 24 hours or less.

4. The method for introducing a substance into plant cells according to claim 1 or 2, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 1,000 or more.

5. The method for introducing substances into plant cells according to claim 1 or 2, wherein the concentration of the polyvinylpyrrolidone solution is 0.001% (w / v) or higher.

6. The method for introducing a substance into plant cells according to claim 1 or 2, wherein the introduction is by the Agrobacterium method.

7. A method for culturing plant cells, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution.

8. The method for culturing plant cells according to claim 7, wherein the contact time between the plant cells and the polyvinylpyrrolidone solution is 24 hours or less.

9. The method for culturing plant cells according to claim 7 or 8, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 1,000 or more.

10. The method for culturing plant cells according to claim 7 or 8, wherein the concentration of the polyvinylpyrrolidone solution is 0.001% (w / v) or higher.

11. The method for culturing plant cells according to claim 7 or 8, wherein the plant cells are transformed plants or genome-edited plant cells.

12. A method for producing a transformed plant or a genome-edited plant, comprising a contact step of bringing plant cells into contact with a polyvinylpyrrolidone solution and an introduction step of introducing a substance into plant cells.

13. A method for producing a transformed or genome-edited plant according to claim 12, wherein the substance is a nucleic acid, a peptide, or a compound.

14. A method for producing a transformed plant or a genome-edited plant according to claim 12 or 13, wherein the introduction is by the Agrobacterium method.

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