Method for modifying plant trait

Abstract

The present invention improves the efficiency of substance introduction when a particle bombardment method is used to introduce, into a plant-derived tissue, at least one substance selected from nucleic acids and proteins for modifying a trait. A method for modifying a plant trait according to the present disclosure comprises: a step for washing a plant-derived tissue with water; a step for introducing, by using a particle bombardment method, particles retaining at least one substance selected from nucleic acids and proteins into the tissue washed with water; a step for obtaining plant bodies by growing the tissue into which the particles have been introduced; and a step for selecting a plant body of which the trait has been modified from the obtained plant bodies.

Description

Methods for modifying plant traits

[0001] The present invention relates to a method for modifying plant traits using particle bombardment.

[0002] Patent Document 1 discloses an in plant transformation method using particle bombardment, which includes the steps of coating microparticles having a diameter of 0.3 μm to 0.9 μm with at least one type of nucleic acid, shooting the coated microparticles into the shoot apex of a mature seed embryo or the shoot apex of a tuber bud using a gene gun, growing the shoot apex bombarded with the coated microparticles to obtain a plant, and selecting a transformed plant from the plant.

[0003] Patent Document 2 discloses genome editing of plants using the method described in Patent Document 1, except that instead of the microparticles, microparticles coated with at least one type of nucleic acid (e.g., guide RNA) and / or at least one type of protein (e.g., Cas nuclease) are used.

[0004] JP 2017-205103 A JP 2017-205104 A

[0005] An object of the present invention is to improve the efficiency of introducing one or more substances selected from nucleic acids and proteins for trait modification into plant-derived tissues by particle bombardment.

[0006] The present inventors have found that when one or more substances selected from nucleic acids and proteins for trait modification are introduced into plant-derived tissue by particle bombardment, washing the tissue with water beforehand can efficiently introduce the substances and improve the efficiency of trait modification. Specifically, the present disclosure includes the following aspects.

[0007] [1] A method for modifying the characteristics of a plant, comprising: a step of washing tissue derived from the plant with water; a step of introducing particles carrying one or more substances selected from nucleic acids and proteins into the tissue washed with water by particle bombardment; a step of growing the tissue into which the particles have been introduced to obtain a plant; and a step of selecting a plant whose characteristics have been modified by the substances from the plant.

[0008] [2] The method according to [1], wherein the step of washing the tissue with water includes shaking the water in which the tissue is immersed.

[0009] [3] The method according to [1] or [2], further comprising a step of drying the tissue washed with water prior to the step of introducing the particles.

[0010] [4] The method according to any one of [1] to [3], wherein the plant belongs to the Allium genus.

[0011] [5] The method according to [4], wherein the plant is an onion.

[0012] [6] The method according to any one of [1] to [5], wherein the tissue contains cells capable of differentiating into germ cells.

[0013] [7] The method according to [6], wherein the tissue containing cells capable of differentiating into germ cells is a tissue containing an exposed shoot apex of the plant.

[0014] [8] The method according to any one of [1] to [7], wherein the method for modifying a trait is a genome editing method, and the substance comprises one or more selected from the group consisting of a protein that functions as a genome editing tool, a nucleic acid comprising a base sequence that encodes a protein that functions as a genome editing tool, a nucleic acid that functions as a genome editing tool, and a nucleic acid comprising a base sequence that encodes a nucleic acid that functions as a genome editing tool.

[0015] [9] The method according to any one of [1] to [8], wherein the plant whose trait has been modified by the substance is a plant containing cells having genomic DNA containing a partial base sequence modified by the substance.

[0016]

[10] The method according to [9], further comprising a step of obtaining, from the plant body containing cells having genomic DNA containing the partial base sequence modified by the substance, a progeny plant containing cells having genomic DNA containing the partial base sequence.

[0017] This specification includes the disclosure of Japanese Patent Application No. 2004-109157, from which the present application claims priority. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

[0018] According to the present invention, it is possible to efficiently introduce one or more substances selected from nucleic acids and proteins for modifying traits into tissues derived from plants.

[0019] Figure 1 shows the procedure for collecting tissue containing the shoot apex from the disc stalk of an onion bulb. Figure 2 shows the results of fluorescence microscopy of tissue containing the onion shoot apex transformed by bombarding it with gold particles carrying a plasmid expressing the fluorescent protein GFP using a particle gun. The left image shows the results of Experiment 1, Treatment 4, in which the tissue was bombarded without washing after preparation. The right image shows the results of Experiment 1, Treatment 5, in which the tissue was bombarded after preparation and washing. The circled tissues are those in which fluorescence was observed at the shoot apex.

[0020] 1. Plants to be modified in traits Plants to be modified in traits by the method of the present disclosure are not particularly limited. The plant may be, for example, a seed plant including angiosperms and gymnosperms, with angiosperms being particularly preferred. The angiosperm may be one or more selected from monocotyledonous plants and dicotyledonous plants, with monocotyledonous plants being particularly preferred.

[0021] Examples of monocotyledonous plants include Amaryllidaceae plants, Poaceae plants, Musaceae plants, Bromeliaceae plants, and Orchidaceae plants, with Amaryllidaceae plants being particularly preferred. Examples of Amaryllidaceae plants include Allium plants, with Allium plants including onion (Allium cepa), leeks (Allium fistulosum), and garlic (Allium sativum), with onions being particularly preferred. Examples of Poaceae plants include rice, wheat, barley, corn, oats, turfgrass, sorghum, rye, foxtail millet, and sugarcane. Examples of Musaceae plants include bananas. Examples of Bromeliaceae plants include pineapples. Examples of Orchidaceae plants include orchids.

[0022] 2. Plant-Derived Tissues Plant-derived tissues used in the methods of the present disclosure may be any tissue that can produce a plant body by growing after the introduction of particles carrying one or more substances selected from nucleic acids and proteins, as described below. Such tissues are preferably tissues containing cells capable of differentiating into germ cells. As used herein, "germ cells" is a general term for germ cells ranging from primordial germ cells to the final products, egg cells and sperm cells. By modifying the traits of tissues containing cells capable of differentiating into germ cells, traits that can be inherited by progeny plants can be introduced.

[0023] Examples of tissues containing cells capable of differentiating into germ cells include tissues containing meristems and calli. In particular, modifying meristem-containing tissues using the method of the present disclosure is preferable because, unlike the modification of calli, a plant with modified traits can be obtained by plant growth without tissue culture manipulation. The method of modifying meristem-containing tissues can be referred to as the in planta trait modification method (see Patent Documents 1 and 2).

[0024] Tissues containing a meristem include, in particular, tissues containing the shoot apex. The shoot apex is tissue containing the meristem of the stem (shoot apical meristem). In the present disclosure, it is particularly preferred to use tissue containing the exposed shoot apex, in order to facilitate the introduction of particles containing one or more substances selected from nucleic acids and proteins into cells of the shoot apex (particularly cells of the L2 layer of the shoot apex). Here, the "L2 layer" refers to the second outer cell layer of the shoot apical meristem. It is believed that the germ cells of a plant body mainly differentiate from cells of the L2 layer.

[0025] 3. One or more substances selected from nucleic acids and proteins for modifying traits The method of the present disclosure includes introducing particles carrying one or more substances selected from nucleic acids and proteins into washed plant tissues by particle bombardment, thereby modifying the traits of plant cells.

[0026] In the present disclosure, "modifying a trait" broadly refers to modifying the traits of plant cells. That is, in addition to conventional transformation in which a nucleic acid containing a gene of interest is introduced into a cell to modify the traits of the cell, "modifying a trait" in the present disclosure is a concept that encompasses genome editing in which a nucleic acid containing a base sequence encoding a protein or nucleic acid that functions as a gene editing tool, or a protein or nucleic acid that functions as a gene editing tool, is introduced into a cell and the gene editing tool is allowed to act in the cell to partially modify the base sequence of the genomic DNA of the cell, thereby modifying the traits of the cell.

[0027] As used herein, nucleic acid refers to a DNA molecule, an RNA molecule, or a DNA / RNA hybrid molecule. Nucleic acids used as vectors are generally DNA molecules. DNA molecules are preferably double-stranded. Nucleic acids may be circular or linear.

[0028] Examples of nucleic acids for modifying traits include vectors containing the base sequence of a gene of interest. The gene of interest is not particularly limited as long as expression or inhibition of expression of the gene is desired, and may be an endogenous gene of the target plant or a foreign gene. Foreign genes may be from different biological species, and for example, genes from animals, plants, microorganisms, viruses, etc. can be used. Examples of such genes include genes for producing useful substances. In addition to the sense strand, the gene of interest may be introduced so as to express antisense, ribozymes, RNAi, etc., depending on the purpose of gene introduction. Furthermore, the gene of interest may be a gene containing a base sequence encoding a protein or nucleic acid that functions as a gene editing tool. Genome editing can be achieved by transformation using a vector containing a gene of interest that contains a base sequence encoding a protein or nucleic acid that functions as a gene editing tool.

[0029] The vector into which the base sequence of the target gene is inserted is not particularly limited, and examples that can be used include pAL series (pAL51, pAL156, etc.), pUC series (pUC18, pUC19, pUC9, etc.), pBI series (pBI121, pBI101, pBI221, pBI2113, pBI101.2, etc.), pPZP series, pSMA series, and intermediate vector series (pLGV23Neo, pNCAT, etc.).

[0030] A vector containing the nucleotide sequence of a gene of interest can be prepared, for example, as follows. To insert a gene of interest into a vector, for example, purified DNA can be cleaved with an appropriate restriction enzyme, and inserted into a restriction enzyme site or multicloning site of an appropriate vector DNA, followed by ligation to the vector. Alternatively, the gene of interest can be inserted into an intermediate vector by double cross-over recombination, or TA cloning, in-fusion cloning, or the like can be used.

[0031] In addition to the gene of interest, for example, a promoter, an enhancer, an insulator, an intron, a terminator, a poly A addition signal, a selection marker gene, etc. can be ligated to the vector.

[0032] Multiple types of target genes may be inserted into one vector, and multiple types of vectors may be coated onto one particle. For example, a vector containing the base sequence of a target gene and a vector containing a drug resistance gene may be prepared separately, mixed together, and then coated onto particles for injection into plant tissue.

[0033] The promoter does not have to be derived from a plant, as long as it is DNA that functions in a plant body or plant cells and is constitutively expressed or can induce expression in specific tissues or at specific developmental stages of the plant. Specific examples include the cauliflower mosaic virus (CaMV) 35S promoter, the El2-35S omega promoter, the nopaline synthase gene promoter (Pnos), the maize ubiquitin promoter, the rice actin promoter, the tobacco PR protein promoter, the ADH promoter, and the RuBisco promoter.

[0034] The terminator may be a sequence having a poly A addition signal and capable of terminating transcription of a gene transcribed by the promoter, such as the CaMV 35S terminator, the nopaline synthase (NOS) gene terminator, or the octopine synthase (OCS) gene terminator.

[0035] Examples of selectable marker genes include drug resistance genes and enzyme genes such as fluorescent or luminescent reporter genes (such as the green fluorescent protein (GFP) gene).

[0036] Next, nucleic acids and proteins for genome editing, which are used in embodiments in which the method for modifying a trait is a genome editing method, will be described.

[0037] Known genome editing techniques include methods that involve cutting genomic DNA using a nuclease capable of targeting a cutting site, or a guide RNA and a nuclease as genome editing tools.

[0038] Genome editing using a nuclease capable of targeting a cleavage site or a guide RNA and a nuclease can also introduce mutations by inserting or substituting the base sequence of a foreign gene into the site of genomic DNA cleaved by the nuclease through recombination. In this case, a nucleic acid (donor template) containing the foreign gene of interest and the base sequence required for recombination is also used.

[0039] Nucleases that can target cleavage sites include zinc finger nucleases, TAL effector nucleases (TALENs), and the like.

[0040] An example of a combination of a guide RNA and a nuclease is the CRISPR-Cas system. The guide RNA and Cas protein used in the CRISPR-Cas system may be a combination that occurs in nature or a combination that does not occur in nature.

[0041] The guide RNA may be a single guide RNA in which tracrRNA and crRNA are fused together, or may be a combination of tracrRNA and crRNA.

[0042] The length of the guide RNA is at least 15, 16, 17, 18, 19, or 20 nucleotides. The upper limit of the nucleotide length is preferably 30 or less, more preferably 25 or less, even more preferably 22 or less, and most preferably 20 or less.

[0043] CRISPR-Cas systems are classified into Class 1, which uses multiple types of proteins, and Class 2, which uses only one type of protein. In Class 1 CRISPR-Cas systems, in addition to the Cas protein, one or more other proteins such as cascade component proteins may be further used. Known examples of Class 1 CRISPR-Cas systems include the CRISPR-Cas3 system. Known examples of Class 2 CRISPR-Cas systems include the CRISPR-Cas9 system and the CRISPR-Cas12 system.

[0044] The Cas protein may be naturally derived or may have an artificial mutation introduced therein. The Cas9 protein is preferably Cas9 derived from Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus, and may also include mutant Cas9 derived from these organisms. The Cas9 protein may be a Cas9 homolog or ortholog.

[0045] Genome editing techniques include not only the above-mentioned techniques using nucleases, but also various techniques using other proteins and / or nucleic acids as genome editing tools have been developed in recent years. For example, a method called Target-AID is known, which uses a complex of a guide RNA and a fusion protein of a nuclease protein with deactivated nuclease activity and a deaminase instead of the Cas protein in the CRISPR-Cas system. Another example is a method using a fusion protein of a PPR (Pentatricopeptide repeat) protein domain that specifically binds to a target base sequence and a nucleic acid metabolic activation domain such as a nuclease or deaminase as a genome editing tool. In addition, a method called the PODiR (Partially Overlapped Direct Repeat) system has also been developed, which uses only nucleic acids as a genome editing tool.

[0046] In an embodiment in which the purpose of trait modification using the method of the present disclosure is genome editing, particles carrying one or more substances selected from the group consisting of a protein that functions as a genome editing tool, a nucleic acid containing a base sequence encoding a protein that functions as a genome editing tool, a nucleic acid that functions as a genome editing tool, and a nucleic acid containing a base sequence encoding a nucleic acid that functions as a genome editing tool are introduced into tissue derived from a washed plant by particle bombardment. The one or more substances can be selected appropriately depending on the genome editing method used, and two or more substances can also be combined. Examples of proteins that function as genome editing tools include nucleases, cascade-constituting proteins, fusion proteins of nuclease proteins with inactivated nuclease activity and deaminases, and fusion proteins of PPR protein domains and nucleic acid metabolic activation domains, as described above. Examples of nucleic acids that function as genome editing tools include guide RNAs, donor templates, and the like, as described above. Here, nucleic acids containing a base sequence encoding a protein that functions as a genome editing tool or nucleic acids containing a base sequence encoding a nucleic acid that functions as a genome editing tool can be carried in particles as vectors containing the target gene. The configuration of the vectors is as described above. The protein and / or nucleic acid that functions as a genome editing tool can be retained in a particle and introduced into a cell. The protein and / or nucleic acid that functions as a genome editing tool can also be pre-formed into a complex and then retained in the particle.

[0047] 4. Method for modifying traits of plants The method for modifying traits of plants according to the present disclosure relates to a method comprising: a step of washing tissue derived from the plant with water; a step of introducing particles carrying one or more substances selected from nucleic acids and proteins into the tissue washed with water by particle bombardment; a step of growing the tissue into which the particles have been introduced to obtain a plant; and a step of selecting, from the plant, a plant whose trait has been modified by the substance.

[0048] The present inventors have found that when particles carrying one or more substances selected from nucleic acids and proteins are introduced into plant-derived tissue by particle bombardment without washing with water, the efficiency of trait modification by the substances may be low, whereas washing the plant-derived tissue with water significantly improves the efficiency. The reason why washing with water improves the efficiency of trait modification is not entirely clear, but it is assumed that high molecular weight compounds such as polysaccharides are normally present in plant cells, such as fructans present in the vacuoles of cells of Allium plants such as onions, and that cutting the cell tissue may cause secretions to coat the tissue and inhibit the introduction of the particles. However, it is assumed that washing with water removes the high molecular weight compounds coating the tissue, thereby improving the efficiency of particle introduction.

[0049] The method for washing plant-derived tissue with water is not particularly limited. For example, the tissue can be gently washed by immersing the tissue in water filled in a container such as a culture dish and shaking the water, or by washing the tissue under running water. The amount of water used for washing is not particularly limited, and an excess amount relative to the tissue can be used. The temperature of the water used for washing is not particularly limited, and can be, for example, between 4°C and 25°C. The washing time is not particularly limited, and can be, for example, between 10 and 20 minutes. The water used for washing can be distilled water, deionized water, or the like, and can also be an aqueous solution or suspension in which components other than water are dissolved or suspended. For example, a buffer solution in which a buffer agent is dissolved in water can be used as the "water" in the present disclosure. It is preferable that the water used for washing is sterilized in advance.

[0050] After washing with water, the plant-derived tissue is preferably dried to remove excess water adhering to the surface before being bombarded with particles. This drying does not need to dry the inside of the cells of the plant-derived tissue; it is sufficient to remove excess water from the surface of the cells. The drying method is not particularly limited, but examples include a method in which the water on the surface of the tissue after washing with water is wiped off with a paper wiper or the like, and the tissue is dried in air at a temperature of 4°C to 25°C for 10 to 20 minutes.

[0051] Next, particles carrying one or more substances selected from nucleic acids and proteins are introduced into the plant-derived tissue, which has been washed with water and further dried as necessary, by particle bombardment, a method in which the particles are shot into cells using a device called a gene gun or particle gun (hereinafter referred to as "particle gun") to deliver the substances into the cells.

[0052] The particles that hold the substance can be metal fine particles such as gold particles, tungsten particles, etc. The particle size (diameter) of the particles is preferably 0.3 μm or more and 1.5 μm or less, more preferably 0.4 μm or more and 1.2 μm or less, and most preferably 0.5 μm or more and 1.0 μm or less.

[0053] The method for retaining one or more substances selected from nucleic acids and proteins on particles is not particularly limited as long as it is a method that can coat and retain the substance on the surface of the particles. For example, after sterilizing the particles, the particles, the substance, CaCl ２ The particles are then mixed with the cellulose acetate solution and spermidine under stirring to coat and retain the substance on the surface of the particles. The particles retaining the substance are washed with a washing solution such as ethanol or phosphate buffered saline, as needed.

[0054] The particles carrying the substance are applied as uniformly as possible to a macrocarrier film and then dried in a sterile environment such as a clean bench. In the case of particles carrying proteins, it is preferable to use a hydrophilic macrocarrier film.

[0055] The macrocarrier film coated with the substance-holding particles and a plate bearing the target tissue derived from the cleaned plant are then placed in a particle gun device, and high-pressure helium gas is fired from the gas acceleration tube toward the macrocarrier film. The macrocarrier film is stopped by a stopping plate, but the particles pass through the stopping plate and penetrate the target placed below, becoming introduced into the cells that make up the plant-derived tissue.

[0056] The distance between the stopping plate and the target tissue may vary depending on the particle size, but may be, for example, 2 cm to 9 cm, preferably 3 cm to 8 cm, and more preferably 4 cm to 7 cm. The optimum value of the distance between the stopping plate and the target can be determined appropriately depending on the type of microparticle, particle size, gas pressure, etc.

[0057] The gas pressure may vary depending on the type of particles and the distance to the target, but may be, for example, 1,100 psi to 1,600 psi, preferably 1,200 psi to 1,500 psi. The optimum gas pressure can also be determined appropriately depending on the type of particles, the type of target, the distance between the target and the stopping plate, etc.

[0058] The number of times that the particles carrying the substance are shot into the washed plant-derived tissue using a particle gun can be, for example, from 1 to 20 times, preferably from 2 to 5 times. The optimal number of shots can be determined as appropriate.

[0059] In cells bombarded with particles, nucleic acids and / or proteins are released from the particles and act within the cells. For example, a nucleic acid encoding a nuclease introduced as a genome editing tool translocates to the nucleus and expresses the nuclease, cleaving genomic DNA, and mutations occur during the repair process, resulting in a genome-edited cell. Furthermore, when a nuclease protein is introduced, the nuclease protein translocates to the target nucleus or organelle via a nuclear localization signal or organelle localization signal, and genome editing occurs by cleaving the genomic DNA of the nucleus or organelle. Genome editing can be applied not only to genomic DNA in the nucleus, but also to genomic DNA in organelles (intracellular organelles such as chloroplasts and mitochondria). In this case, a localization signal to the target organelle may be linked to the nuclease and introduced, or a promoter that is expressed only in the target organelle may be used as a promoter linked to a base sequence encoding the nuclease.

[0060] The tissue into which the particles have been introduced is then grown to obtain a plant. It is preferable that the tissue into which the particles have been introduced is grown on a medium for, for example, about one month, and then transplanted into soil and further grown to obtain a plant. During initial growth on the medium, selective pressure may be applied to selectively grow only plants into which the desired trait has been introduced. For example, when particles carrying both a nucleic acid and / or protein as a genome editing tool and an expression vector for a drug resistance gene are introduced into a tissue, initial culture of the tissue on a medium containing a drug can selectively culture tissue containing cells into which the genome editing tool has been introduced.

[0061] Next, from the grown plants, plants whose traits have been modified by one or more substances selected from the introduced nucleic acid and protein are selected. For example, the traits of the grown plants can be examined and plants having the modified traits can be selected. Alternatively, genomic DNA or mRNA can be extracted from the grown plants, and if genomic DNA or mRNA containing a partial base sequence modified by the substance is detected, the plant can be selected as one that has been modified to the desired trait.

[0062] When the trait modification in the method of the present disclosure is genome editing, the trait-modified plant contains cells having genomic DNA containing a partial base sequence that has been modified (i.e., genome-edited) by one or more substances selected from an introduced nucleic acid and a protein. When the trait-modified plant contains germ cells having genomic DNA containing the partial base sequence, the partial base sequence may be inherited by progeny plants.

[0063] Therefore, in the method of the present disclosure, a plant (E) containing cells having genomic DNA containing a partial base sequence modified by one or more substances selected from nucleic acids and proteins introduced by particle bombardment is further provided. ０ The method can include a step of obtaining a progeny plant containing cells having genomic DNA containing the partial base sequence from the first generation (hereinafter referred to as "first generation"). ０Seeds can be obtained from the plants of the previous generation by self-pollination or by crossbreeding with other plants, or seeds can be obtained from plants grown from the obtained seeds by self-pollination or by crossbreeding with other plants. Whether the progeny plant has a specific genomic DNA can be identified by confirming the traits of the progeny plant or by confirming genomic DNA or RNA extracted from the progeny plant.

[0064] [Experiment 1] Transformation by introduction of a plasmid vector

[0065] (1) Preparation of onion shoot apex-containing tissues Onion bulbs including disc stalks of onion (Allium cepa) (variety: Sharm) were sterilized by immersing the bulbs in a 1% aqueous solution of sodium hypochlorite for 1 hour and then air-drying at 24°C.

[0066] The scale leaves were removed from the bulb portion of a sterilized onion, leaving the disc stalk, and tissue including the exposed shoot apex and its surrounding area was excised from the disc stalk (see FIG. 1).

[0067] (2) Washing and drying of shoot apex-containing tissues The shoot apex-containing tissues prepared in (1) above were immersed in sterilized water in a 50 mL culture dish at a rate of 25 pieces per dish to float them. The culture dish was then shaken for 15 minutes to wash the tissues. The temperature of the sterilized water was 24°C, and the ambient temperature during shaking was 24°C.

[0068] After 15 minutes of washing, the tissue was removed from the sterile water, the water adhering to the tissue was wiped off with a paper wiper, and the tissue was allowed to stand in a clean bench set at 24°C for 15 minutes to dry naturally.

[0069] The dried tissues were plated on MS-sucrose medium (4.3 g / L MS salt, MS vitamin, 30 g / L sucrose, 0.98 g / L MES, 3% PPM (plant preservative mixture, Nacalai Tesque), 7.0 g / L Phytagel (registered trademark, Sigma-Aldrich), pH 5.8) at several to several tens of cells per plate.

[0070] For comparison, in Treatments 1 and 2 described below, the onion shoot apex-containing tissue prepared by the above procedure was directly plated on MS-sucrose medium without washing or drying. In Treatment 4 described below, the onion shoot apex-containing tissue prepared by the above procedure was air-dried overnight in a clean bench set at 24°C without washing and then plated on MS-sucrose medium.

[0071] (3) Gene Introduction Gene introduction into the shoot apex of onion was carried out using the particle gun method as follows.

[0072] Plasmid DNA (pUC-based plasmid) containing the fluorescent reporter gene GFP (S65T) was used. This gene was designed to be expressed under the control of the 35S promoter of cauliflower mosaic virus (CaMV). The NOS terminator was added as a terminator.

[0073] 30 mg of gold particles with a particle size of 0.6 μm were weighed, 500 μL of 70% ethanol was added, and the mixture was thoroughly suspended using a vortex mixer. The gold particles were then precipitated by centrifugation, and the ethanol was removed. 500 μL of 50% glycerol was then added to prepare a sterilized gold particle suspension.

[0074] Plasmid DNA solution (1 μg / μL) purified using a Qiagen Maxi Kit (Qiagen) was placed in a 1.5 mL tube so that the concentration was 5 μg per 750 μg of gold particles. Before use, the sterilized gold particle suspension was thoroughly suspended using an ultrasonic generator (US-1KS ultrasonic cleaner manufactured by SND Corporation), and an appropriate amount was added to the tube and stirred by pipetting. Next, the tube was filled with 2.5 M CaCl per 750 μg of gold particles. ２ 25 μL of 0.1 M Spermidine (Nacalai Tesque) and 10 μL of 0.1 M Spermidine (Nacalai Tesque) were added. Immediately after mixing, the mixture was vigorously suspended by vortexing for 5 minutes. After leaving the mixture to stand at room temperature for 10 minutes, it was centrifuged at 9,100 × g for 2 seconds. The supernatant was removed, and the mixture was washed with 70% ethanol and 99.5% ethanol. Finally, the supernatant was removed, and 24 μL of 99.5% ethanol was added and the mixture was thoroughly suspended. 6 μL of the mixture was poured into the center of each macrocarrier in a clean bench and allowed to air dry.

[0075] The particle gun used was a Biolistic (registered trademark) PDS-1000 / He Particle Delivery System (BIO-RAD), with an injection pressure of 1,350 psi and a distance of 6 cm to the target tissue. Each dish was injected three or four times. After injection, the dish was left to stand overnight at 22°C in a dark place.

[0076] (4) Investigation of transient expression efficiency of GFP protein The efficiency of GFP gene introduction in the shoot apex was calculated by observing GFP fluorescence (excitation: 470 / 40, absorption: 525 / 50) in the shoot apex under a stereoscopic fluorescence microscope (Leica MZFL III) (Figure 2).

[0077] Among the transformed onion tissues, those in which GFP fluorescence was observed in the shoot apex were designated as gene-introduced tissues, and the gene introduction efficiency (number of gene-introduced tissues / number of treated tissues×100) was calculated.

[0078] The results are shown in the table below. When gene transfer was performed by particle gun gun method without washing the onion shoot apex-containing tissue (treatments 1, 2, and 4), the gene transfer efficiency was extremely low. On the other hand, when gene transfer was performed after washing and drying the onion shoot apex-containing tissue (treatments 3, 5, 6, and 7), it was confirmed that the gene transfer efficiency was significantly increased.

[0079]

[0080] [Experiment 2] Genome editing by introducing gRNA and Cas9 protein

[0081] (1) Preparation of onion shoot apex-containing tissues Onion shoot apex-containing tissues were prepared by the method described in Experiment 1(1). Charm (Takii Seed Co., Ltd.) and Sapporo Yellow were used as onion varieties.

[0082] (2) Washing and drying of tissue containing shoot apex According to the method described in Experiment 1(2), tissue containing onion shoot apex was washed with sterilized water for 15 minutes, then dried for 15 minutes, and then placed on MS-sucrose medium.

[0083] (3) Gene Introduction Three types of crRNA that selectively recognize three regions contained in the base sequence of the Lachrymatory Factor Synthase gene (LFS gene) in onion genomic DNA were synthesized using a custom synthesis service. Each of the three types of crRNA is an RNA molecule consisting of 20 bases.

[0084] Furthermore, a tracrRNA consisting of 80 bases was synthesized using a custom synthesis service, which can be linked to any of the three types of crRNA to form gRNA and serve as a scaffold for binding of the Cas9 protein.

[0085] spCas9 was used as the Cas9 protein.

[0086] Preparation of gold particles carrying gRNA and Cas9 protein: 4.0 μg tracrRNA, 1.67 μg each of three types of crRNA, and 40 μg spCas9 were mixed to form a ribonucleotide protein complex (RNP), and then 0.6 μm gold particles were added and left to stand at 24 ° C for 10 minutes to attach the RNP to the gold particles. The mixture was centrifuged at 6,000 rpm for 5 minutes to remove the supernatant, and the precipitate was suspended in 24 μl of water. 6 μl of the suspension was applied to a total of four macrocarriers per sheet, air-dried in a clean bench at 24 ° C, and then subjected to a bombardment experiment.

[0087] RNP introduction by particle gun method was carried out by the method described in Experiment 1(3).

[0088] (4) Growth of RNP-introduced plants After RNP introduction, the plants were cultured on MS-sucrose medium for approximately two months, then transplanted into culture soil and grown for another two months. After growth, the fourth and subsequent scale leaves were sampled to confirm the introduction of the target gene mutation.

[0089] (5) E ０ Confirmation of target gene mutation introduction in the next generation ０In the next generation of plant individuals, whether mutations had been introduced into the target gene in the genomic DNA was investigated using the following method. 100 μl of DNA extraction buffer (50 mM Tris-HCl (pH = 9.5), 1 M KCl, 10 mM EDTA) was added to 100 mg of sampled scale leaves, and the scale leaves were crushed using a BioMasher (Nippi Co., Ltd.). The crushed solution was centrifuged at 15,000 rpm for 15 minutes, and PCR was performed using the supernatant as a template. PCR was performed using KOD FX DNA polymerase (Toyobo Co., Ltd.) and a primer set consisting of a forward primer consisting of 21 bases of DNA and a reverse primer consisting of 24 bases of DNA, which can selectively amplify a partial sequence containing three target regions by the CRISPR-Cas9 system within the base sequence of the LFS gene in the genomic DNA. The resulting PCR amplification product was treated with the restriction enzyme XcmI (New England Biolabs), and the presence or absence of cleavage fragments of the target band was detected using an electrophoresis apparatus MutiNA (Shimadzu Science East Japan Co., Ltd.) to confirm whether the mutagenesis was successful.

[0090] (6) E １ Confirmation of target gene mutation introduction in the next generation E in which mutation was confirmed in (5) above ０ Seeds obtained by self-pollination were collected from the individual plants of the first generation. １ In the plant lines of the next generation, it was investigated by the method described in (5) above whether a mutation had been introduced into the target gene in the genomic DNA.

[0091] E ０ Generation plant individuals and E １ The results of the investigation into the introduction of targeted gene mutations into plant lines of various generations are shown in the table below. It was confirmed that the method of the present disclosure can introduce mutations into genomic DNA. ０ The mutations introduced into the genomic DNA of the plant individuals of the next generation are １ Since this is inherited through generations, it was confirmed that the method disclosed herein makes it possible to edit the genome of shoot apical stem cells contained in the shoot apex and which differentiate into germ cells.

[0092]

Claims

1. A method for modifying the characteristics of a plant, comprising the steps of: washing tissue derived from the plant with water; introducing particles carrying one or more substances selected from nucleic acids and proteins into the tissue washed with water by particle bombardment; growing the tissue into which the particles have been introduced to obtain a plant; and selecting from the plant a plant whose characteristics have been modified by the substance.

2. The method of claim 1, wherein the step of washing the tissue with water includes shaking the water in which the tissue is immersed.

3. The method of claim 1 or 2, further comprising the step of drying the tissue washed with water prior to the step of introducing the particles.

4. The method according to any one of claims 1 to 3, wherein the plant belongs to the Allium genus.

5. The method of claim 4, wherein the plant is an onion.

6. The method according to any one of claims 1 to 5, wherein the tissue contains cells capable of differentiating into germ cells.

7. The method according to claim 6, wherein the tissue containing cells capable of differentiating into germ cells is a tissue containing an exposed shoot apex of the plant.

8. The method according to any one of claims 1 to 7, wherein the method for modifying a trait is a genome editing method, and the substance comprises one or more selected from the group consisting of a protein that functions as a genome editing tool, a nucleic acid comprising a base sequence that encodes a protein that functions as a genome editing tool, a nucleic acid that functions as a genome editing tool, and a nucleic acid comprising a base sequence that encodes a nucleic acid that functions as a genome editing tool.

9. The method according to any one of claims 1 to 8, wherein the plant whose trait has been modified by the substance is a plant containing cells having genomic DNA containing a partial base sequence modified by the substance.

10. The method according to claim 9, further comprising the step of obtaining, from the plant body containing cells having genomic DNA containing the partial base sequence modified by the substance, a progeny plant containing cells having genomic DNA containing the partial base sequence.

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