Transformed plants or parts thereof with enhanced dedifferentiation and regeneration abilities

Abstract

To produce and provide a plant in which dedifferentiation ability and redifferentiation ability are enhanced or a part thereof.SOLUTION: There is provided a genetically transformed plant in which expression or activity of SIZ1 (SUMO E3 ligase) is suppressed or inhibited, and in which dedifferentiation ability and redifferentiation ability are enhanced.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a transformed plant having enhanced dedifferentiation and redifferentiation abilities, and a method for producing dedifferentiated and redifferentiated plant cells. [Background technology]

[0002] Plants with totipotency can dedifferentiate differentiated somatic cells and form callus that has regained cell division ability and pluripotency. This callus can be redifferentiated into a tissue different from that before dedifferentiation or regenerated into a complete plant by performing a redifferentiation treatment and culturing under certain conditions (Non-Patent Document 1). Such techniques for inducing dedifferentiation and redifferentiation of plants are important techniques in the seed and floriculture industries for producing useful substances, molecular breeding, developing new varieties, mass-cultivating superior varieties, introducing genes into plants, regenerating transformants, and producing artificial seeds. Therefore, there is a demand for techniques for inducing dedifferentiation and redifferentiation in various plant species and methods for further improving the induction efficiency.

[0003] In general, dedifferentiation and redifferentiation of plant cells can be controlled by the concentration or concentration ratio of plant hormones such as auxin and cytokinin in the medium. For example, when the ratio of auxin in the medium is high, redifferentiation of adventitious roots from callus is promoted. In contrast, when the ratio of cytokinin is high, redifferentiation of adventitious shoots is promoted. On the other hand, it is known that the callus state is maintained at an appropriate concentration ratio of the two hormones (Non-Patent Document 1). Taking advantage of this property, a method of cutting a plant and culturing the tissue piece in a medium that can induce dedifferentiation, such as a callus induction medium containing plant hormones, is a common method for producing callus in this field. The callus obtained can be redifferentiated by culturing it with a different ratio of plant hormones in the medium.

[0004] For example, Non-Patent Document 2 discloses an exemplary method for redifferentiating Arabidopsis thaliana roots into stems and leaves. Specifically, the method involves culturing cut roots in a callus induction medium for four days, and then transferring the tissue pieces to a stem and leaf redifferentiation medium for culturing, thereby redifferentiating the stems and leaves. That is, in this method, dedifferentiation of plant cells is induced by cutting and plant hormone treatment, and then the cells that have acquired pluripotency are stimulated to redifferentiate, thereby regenerating the stems and leaves. This technique is still used as the standard method for Arabidopsis thaliana.

[0005] In addition, methods are also known that do not require cutting of tissues and culturing in a callus induction medium containing added plant hormones, etc. Patent Document 1 discloses a method for producing dedifferentiated cells by inducing dedifferentiation of plant cells by subjecting a plant body to a heat shock treatment.

[0006] On the other hand, there are still many plant species in which dedifferentiation and / or redifferentiation cannot be sufficiently induced by conventional methods. Therefore, there is a demand for a new method that can dramatically increase the efficiency of dedifferentiation and / or redifferentiation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2015-208256 [Non-patent literature]

[0008] [Non-Patent Document 1] Slater A, et al., 2003, Plant Tissue Culture (Chapter 2). in "Plant Biotechnology: The Genetic Manipulation of Plants" pp.35-53. [Non-Patent Document 2] Valvekens, D. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5536-5540. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to produce and provide a plant or a part thereof having enhanced dedifferentiation and regeneration capabilities. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors hypothesized that there exists a factor that negatively controls dedifferentiation and redifferentiation in plants, and came up with the idea that the efficiency of dedifferentiation and redifferentiation can be dramatically increased by suppressing the function of such a factor. As a result of intensive research based on this idea, the factor was found to be SIZ1, a SUMO E3 ligase. In a SIZ1 function-deficient mutant, the expression level of a regeneration-related factor that can promote dedifferentiation and redifferentiation was increased. Furthermore, it was found that the induction efficiency of dedifferentiation and redifferentiation is dramatically increased in a SIZ1 function-deficient mutant, and this led to the completion of the present invention. The present invention is based on this finding and provides the following.

[0011] (1) A transformed plant or a part thereof having enhanced dedifferentiation and redifferentiation capabilities, in which the expression or activity of SIZ1 (SUMO E3 ligase) is suppressed or inhibited. (2) The transformed plant or a part thereof according to (1), wherein the SIZ1 comprises any one of the amino acid sequences [1] to [3] below: [1] Amino acid sequence shown in SEQ ID NO:1 [2] An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:1. [3] An amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO:1. (3) A transformed plant or a part thereof according to (1) or (2), which is derived from a dicotyledonous plant or a monocotyledonous plant. (4) A method for producing dedifferentiated cells of a plant, comprising the steps of: A step of subjecting the transformed plant or a part thereof according to any one of (1) to (3) to a dedifferentiation induction treatment. The method comprising: (5) The method according to (4), wherein the dedifferentiation induction treatment uses auxin. (6) The method according to (4) or (5), wherein the dedifferentiation induction treatment is carried out at 15°C to 35°C. (7) A method for producing regenerated cells of a plant, comprising the steps of: A step of inducing redifferentiation of the transformed plant or a part of dedifferentiated cells thereof according to any one of (1) to (3). The method comprising: (8) The method according to (7), wherein the redifferentiation induction treatment uses a cytokinin. (9) The method according to (7) or (8), wherein the dedifferentiated cells are obtained by the method according to any one of (4) to (6). (10) The method according to any one of (7) to (9), wherein the redifferentiation induction treatment is carried out at 15°C to 35°C. (11) The method according to any one of (7) to (10), wherein the redifferentiated cells are stem and leaf cells. (12) A method for inducing dedifferentiation in a plant body, comprising the steps of: A step of enhancing the dedifferentiation ability of a plant body by suppressing or inhibiting the expression or activity of SIZ1; and A step of subjecting the plant body having enhanced dedifferentiation ability to a dedifferentiation induction treatment. The method comprising: (13) A method for inducing redifferentiation of a dedifferentiated cell of a plant, comprising the steps of: A step of enhancing the regeneration ability of dedifferentiated cells of a plant by suppressing or inhibiting the expression or activity of SIZ1; and A step of subjecting the dedifferentiated cells having enhanced redifferentiation ability to a redifferentiation induction treatment. The method comprising: (14) The method according to (12) or (13), wherein the plant body is derived from a dicotyledonous plant or a monocotyledonous plant. Effect of the Invention

[0012] According to the present invention, it is possible to provide a transformed plant or a part thereof with enhanced dedifferentiation and redifferentiation ability, as well as a method for producing dedifferentiated and redifferentiated plant cells using the transformed plant or a part thereof. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows the results of dedifferentiation induction for SIZ1 mutants. (a) Hypocotyl explants of wild type (WT), SIZ1 mutants (siz1-2 and siz1-3), and SIZ1 complementation line (siz1-2 pSIZ1::SIZ1) were cultured on CIM medium for 17 days at 22°C. The scale bar indicates 1 mm. (b) Callus weights (average values) of wild type (Col-0 (WT)), siz1-2 mutants, siz1-3 mutants, and siz1-2 pSIZ1::SIZ1 line (SIZ1 complementation line) after culturing on CIM medium for 17 days. Error bars indicate standard error, and * indicates P<0.05 (one-way ANOVA, post-hoc Tukey test). [Diagram 2] Figure 1 shows the results of regeneration induction for SIZ1 mutants. (a) Hypocotyl explants of wild type (WT), SIZ1 mutants (siz1-2 and siz1-3), and SIZ1 complemented strain (siz1-2 pSIZ1::SIZ1) were cultured in CIM medium for 4 days at 22°C, then transferred to SIM medium and cultured for 14 days. The scale bar indicates 1 mm. (b) The wild type (WT), siz1-2 mutant, siz1-3 mutant, and siz1-2 pSIZ1::SIZ1 strain (SIZ1 complemented strain) were cultured in CIM medium for 4 days, then transferred to SIM medium and cultured for 9 days. The number of shoots formed per explant in each group is shown. Error bars indicate standard error, and * indicates P<0.05 (one-way ANOVA, post-hoc Tukey HSD test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 1. Transformed plants or parts thereof 1-1. Overview A first aspect of the present invention is a transformed plant or a part thereof (often referred to herein as "transformed plant, etc.") with enhanced dedifferentiation and redifferentiation capabilities. In the transformed plant, etc. of the present invention, the expression or activity of an inhibitor of expression of a regeneration-related factor is suppressed or inhibited. The transformed plant, etc. of the present invention has enhanced dedifferentiation and redifferentiation capabilities, and is capable of inducing dedifferentiation and redifferentiation with high efficiency.

[0015] 1-2.Definition The following definitions are provided for terms frequently used in this specification. As used herein, "callus" refers to an amorphous cell mass that contains dedifferentiated plant cells that have regained pluripotency and cell division ability. Here, "differentiation" of plant cells refers to the division of one simple lineage or cell population into two or more mutually heterogeneous lineages or cell populations. In particular, redifferentiation from dedifferentiated cells is called "redifferentiation."

[0016] As used herein, "dedifferentiation" of plant cells refers to the return of differentiated tissue or organ cells to a highly undifferentiated state and regaining pluripotency. In other words, dedifferentiation is the reverse process of differentiation, and refers to the phenomenon in which cells that have completed functional or morphological differentiation of organs, tissues, etc. become capable of redifferentiating into various cell types. It is known that dedifferentiated cells often regain the ability to divide.

[0017] "SIZ1 (SAP-MIZ domain containing 1)" is a SUMO (Small Ubiquitin-like Modifier) ​​E3 ligase. Protein SUMOylation is a post-translational modification in which a SUMO protein consisting of approximately 100 amino acid residues is covalently attached to a target protein. SUMO E3 ligase catalyzes the final reaction of SUMOylation. In Arabidopsis, only two SUMO E3 ligases are known, in contrast to the more than 1400 ubiquitin E3 ligases.

[0018] In the present specification, SIZ1 refers to SIZ1 of a plant that is the subject of the present invention. SIZ1 of a plant that is the subject of the present invention is, for example, Arabidopsis SIZ1 consisting of the amino acid sequence shown in SEQ ID NO: 1, and an orthologue of Arabidopsis SIZ1. Examples of SIZ1 orthologues include an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and a SIZ1 consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 1. Examples of such SIZ1 include SIZ1 from apple (Malus domestica Borkh.) consisting of the amino acid sequence shown in SEQ ID NO: 3 (having 61.7% amino acid identity with the above-mentioned Arabidopsis SIZ1), SIZ1 from soybean (Glycine max) consisting of the amino acid sequence shown in SEQ ID NO: 5 (having 65.0% amino acid identity with the above-mentioned Arabidopsis SIZ1), SIZ1 from rapeseed (Brassica napus) consisting of the amino acid sequence shown in SEQ ID NO: 7 (having 81.2% amino acid identity with the above-mentioned Arabidopsis SIZ1), SIZ1 from rice (Oryza sativa) consisting of the amino acid sequence shown in SEQ ID NO: 9 (having 51.9% amino acid identity with the above-mentioned Arabidopsis SIZ1), and SIZ1 from tomato (Solanum lycopersicum) consisting of the amino acid sequence shown in SEQ ID NO: 11 (having 66.6% amino acid identity with the above-mentioned Arabidopsis SIZ1). Furthermore, the SIZ1 of a plant that is the subject of the present invention preferably contains a plant homeodomain-finger domain (PHD domain) and a Siz / PIAS-RING domain (SP-RING domain). The PHD domain and the SP-RING domain are highly conserved among SIZ1 of various species. The PHD domain of Arabidopsis thaliana SIZ1 corresponds to the amino acid region from positions 105 to 169 in the amino acid sequence shown in SEQ ID NO: 1, and has the amino acid sequence shown in SEQ ID NO: 13.The SIZ1 of the plant of the present invention preferably contains a PHD domain consisting of an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 13. The SP-RING domain of Arabidopsis thaliana SIZ1 corresponds to the amino acid region of positions 329 to 447 in the amino acid sequence shown in SEQ ID NO: 1 and has the amino acid sequence shown in SEQ ID NO: 14. The SIZ1 of the plant of the present invention preferably contains a SP-RING domain consisting of an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 14. The SIZ1 of the plant of the present invention has SIZ1 enzyme activity. In addition, the SIZ1 of the plant of the present invention has SUMO E3 ligase activity that catalyzes the final reaction of SUMOylation. It has been reported that Arabidopsis siz1 mutants are complemented by rice SIZ1 or tomato SIZ1, and the function of SIZ1 is conserved across plant species (Park, HC et al., 2010, Plant, Cell and Environment 33, 1923-1934; Zhang, S., 2017, JIPB 59(2), 102-117). Therefore, the function of SIZ1 is conserved in a wide range of plant species, including rice and tomato, whose SIZ1 orthologues have an amino acid identity of 50% or more to Arabidopsis SIZ1.

[0019] The "SIZ1 gene" is a gene encoding the SIZ1 protein. A specific example of the SIZ1 gene is a SIZ1 gene encoding a SIZ1 protein consisting of the amino acid sequence shown in SEQ ID NO: 1. More specifically, an Arabidopsis SIZ1 gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 is included. In addition, the SIZ1 gene includes SIZ1 protein variants and SIZ1 protein orthologs having an activity functionally equivalent to that of the SIZ1 protein encoded by the SIZ1 gene shown in SEQ ID NO: 2, i.e., SIZ1 enzyme activity. Specifically, the SIZ1 gene includes a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2, or a SIZ1 gene having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90%, 95% or more, 97% or more, 98% or more, or 99% or more of nucleotide identity with the nucleotide sequence shown in SEQ ID NO: 2. Alternatively, the term encompasses a gene that hybridizes under high stringency conditions with a nucleic acid fragment consisting of a part of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and encodes a protein having activity functionally equivalent to that of the SIZ1 protein. As used herein, "hybridizing under high stringency conditions" refers to hybridization and washing under low salt concentration and / or high temperature conditions. For example, incubation with a probe in 6xSSC, 5xDenhardt's reagent, 0.5% SDS, 100μg / ml denatured fragmented salmon sperm DNA at 65°C to 68°C is performed, followed by washing in a washing solution of 2xSSC, 0.1% SDS starting from room temperature, lowering the salt concentration in the washing solution to 0.1xSSC, and raising the temperature to 68°C until background signals are no longer detected. High stringency hybridization conditions can be found in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Examples of known SIZ1 gene orthologs include the SIZ1 gene of apple (Malus domestica Borkh.) consisting of the base sequence shown in SEQ ID NO: 4, the SIZ1 gene of soybean (Glycine max) consisting of the base sequence shown in SEQ ID NO: 6, the SIZ1 gene of rapeseed (Brassica napus) consisting of the base sequence shown in SEQ ID NO: 8, the SIZ1 gene of rice (Oryza sativa) consisting of the base sequence shown in SEQ ID NO: 10, and the SIZ1 gene of tomato (Solanum lycopersicum) consisting of the base sequence shown in SEQ ID NO: 12.

[0020] The plants that are the subject of the present specification are not particularly limited, and may be either angiosperms or gymnosperms. Angiosperms also include both dicotyledonous and monocotyledonous plants. Representative examples include plants that are important in agriculture, particularly in the seed and floriculture industries, such as crop plants such as grains, flowers, vegetables, and fruits. Specifically, dicotyledonous plants include species belonging to the Brassicaceae family (e.g., cabbage, radish, Chinese cabbage, and rapeseed), species belonging to the Fabaceae family (e.g., soybean, peanut, pea, kidney bean, adzuki bean, broad bean, and sweet pea), species belonging to the Solanaceae family (e.g., tomato, eggplant, potato, tobacco, bell pepper, capsicum, and petunia), species belonging to the Rosaceae family (e.g., strawberry, rose, apple, pear, peach, loquat, almond, plum, plum, and cherry), and species belonging to the Orchidaceae family (e.g., Cymbidium , Phalaenopsis, Cattleya, Dendrobium), species belonging to the Liliaceae family (e.g., lily, tulip, hyacinth, muscari, leek, onion, garlic), Rutaceae family (e.g., mandarin orange, orange, grapefruit, lemon, yuzu), species belonging to the Vitaceae family (e.g., grape), species belonging to the Asteraceae family (e.g., lettuce, chrysanthemum, dahlia, margaret, sunflower), species belonging to the Caryophyllaceae family (e.g., carnation, gypsophila), and species belonging to the Theaceae family (e.g., sasanqua, tea plant). In addition, as for monocotyledonous plants, species belonging to the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum) are included. In particular, plants that are difficult to cultivate from seed, such as species belonging to the Orchidaceae family, and plants that reproduce asexually from useful strains obtained by breeding are preferred as target plants of the present invention.

[0021] In this specification, the term "all" of a plant refers to all regions constituting a living plant. Also, the term "part" of a plant refers to a part of a region constituting a living plant, specifically, an organ (e.g., roots, stems, leaves, flowers, epidermis, or a combination thereof, or pollen, egg cells, seeds, etc.), a tissue or part thereof consisting of a group of morphologically and / or functionally differentiated cells, or a cell.

[0022] In the present specification, "multiple" refers to, for example, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. "Amino acid identity" refers to the percentage (%) of the number of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides to be compared are aligned by inserting appropriate gaps into one or both of them as necessary so that the number of identical amino acid residues is maximized. The alignment of two amino acid sequences to calculate the amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW. "Nucleotide identity" is calculated in the same manner.

[0023] As used herein, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties such as charge, side chain, polarity, and aromaticity among the 20 types of amino acids that constitute natural proteins. Examples include substitution within the uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) with a low polarity side chain, the branched chain amino acid group (Leu, Val, Ile), the neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), the neutral amino acid group with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), the acidic amino acid group (Asp, Glu), the basic amino acid group (Arg, Lys, His), and the aromatic amino acid group (Phe, Tyr, Trp). Amino acid substitution within these groups is preferred because it is known that it is unlikely to cause changes in the properties of the polypeptide.

[0024] 1-3.Configuration The construction of the transformed plant or the like according to the present invention in which the dedifferentiation and redifferentiation abilities are enhanced will be specifically described below. In the transformed plants etc. of the present invention with enhanced dedifferentiation and regeneration capabilities, the expression or activity of a factor that suppresses the expression of regeneration-associated factors is suppressed or inhibited.

[0025] As used herein, "regeneration-associated factor" refers to a factor that can function during plant regeneration. As used herein, the regeneration-associated factor is a peptide. Examples include factors that can function during callus formation and factors that can function during shoot formation. Specific examples of regeneration-associated factors that can function during callus formation or shoot formation include WIND1 (Wound Induced Dedifferentiation 1), WIND2, ESR1 (Enhancer of Shoot Regeneration 1), and ESR2.

[0026] As used herein, the term "factor that suppresses the expression of a regeneration-related factor" refers to a factor that has the activity of suppressively controlling the expression of one or more regeneration-related factors in a target plant (hereinafter referred to as "repressive regulator"). In other words, it refers to a factor that can increase the expression of a regeneration-related factor when the function of the factor is suppressed or inhibited in a target plant. The regeneration-related factor referred to here may be any of the above-mentioned regeneration-related factors (WIND1, WIND2, ESR1, ESR2, etc.). In the present invention, specifically, SIZ1 corresponds to the repressive regulator. In the SIZ1 mutant, the expression levels of regeneration-related factors such as the WIND1 gene, WIND2 gene, and ESR2 gene are increased.

[0027] The transformed plant of the present invention with enhanced dedifferentiation and redifferentiation abilities also includes progeny of the transformed plant in which the expression or activity of a factor that suppresses the expression of a regeneration-related factor is suppressed or inhibited. The progeny of the first generation of transformed plants can be obtained from the first generation of transformed plants by a known method. For example, the first generation can be allowed to bear fruit and its seeds can be obtained. The seeds and the plants that are germinated on an appropriate medium and then transplanted into a pot containing soil and grown therein become the first generation of progeny. The number of generations of the progeny is not important as long as the expression or activity of a factor that suppresses the expression of a regeneration-related factor is suppressed or inhibited. For the second and subsequent progeny, the same method as that for obtaining the first generation of progeny can be repeated.

[0028] Furthermore, the transformed plants, etc. of the present invention with enhanced dedifferentiation and redifferentiation abilities also include clones having the same genetic information, such as a part of a plant taken from a transformed plant, etc., which is taken by cutting, grafting or layering, a plant regenerated from callus after cell culture, or a new vegetative body produced from a vegetative propagation organ (e.g., rhizome, tuberous root, corm, runner, etc.) obtained by asexual reproduction from a transformed plant, etc.

[0029] As used herein, "the expression or activity of a factor that suppresses the expression of a regeneration-associated factor is suppressed or inhibited" means that the expression or activity of a factor that suppresses the expression of a regeneration-associated factor (repressive regulatory factor) is reduced compared to that of a wild-type strain of the target plant. The degree to which the expression or activity of the suppressive regulatory factor is reduced in the transformed plant of the present invention is not particularly limited, as long as the dedifferentiation ability and redifferentiation ability are enhanced in the transformed plant. For example, the expression or activity of the suppressive regulatory factor may be suppressed by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or inhibited 100%, compared to the wild-type strain of the target plant.

[0030] In the transformed plant of the present invention, any method known in the art can be used as a specific method for suppressing or inhibiting the expression or activity of the negative regulator. Without limitation, the expression or activity of the negative regulator can be suppressed or inhibited by using an existing mutant, mutagen treatment, gene knockdown, gene knockout, and / or introduction of an inhibitor, or any combination thereof. Each method will be described below.

[0031] (1) Use of existing mutants As used herein, "use of an existing mutant" means use of a mutant that already exists in the technical field. The "mutant" here refers to a plant body in which a mutation has occurred in a target gene, i.e., a gene encoding a suppressive regulator. The mutation includes addition, deletion, and / or substitution in the wild-type gene of the target gene. The mutant is preferably one in which the function of the target gene, including the mutation, is deleted, inhibited, or suppressed. The "existing mutant" is a mutant that has been collected in the past, and has been collected, stored, and provided by various distribution organizations, and can be used as appropriate. It may be any existing mutant that has been artificially created.

[0032] (2) Mutagen treatment In this specification, the term "mutagenization treatment" refers to a treatment that induces mutations in a target plant. The treatment method is not particularly limited. For example, it can be a treatment of irradiating electromagnetic waves or radiation such as X-rays or ultraviolet rays, a treatment of contacting a mutagen such as nitrosoguanidine, nitrosamine, bromodeoxyuridine, N-ethyl-N-nitrosourea, methyl ethanesulfonate, benzopyrene, or ethidium bromide, or a treatment of introducing nucleic acid into a random position in a genome using a virus or transposon. Mutagen treatment induces mutations such as point mutations, deletion mutations, and insertion mutations. After mutagen treatment, candidate mutants that have mutated the target gene due to phenotypes or the like are isolated, gene fragments are amplified by PCR, and the introduction of mutations into the target gene is confirmed by base sequencing to obtain the desired mutant of the target gene.

[0033] (3) Gene knockdown As used herein, "gene knockdown" refers to a procedure for reducing the expression level of a target gene. Examples of such methods include the antisense method, which uses antisense DNA capable of hybridizing to RNA transcribed from a target gene to degrade the mRNA of the target gene with RNase H, and the RNA interference method (RNAi), which uses siRNA or shRNA to degrade the target mRNA and thereby suppress gene expression after transcription. Examples of such methods include a method of introducing an expression vector for antisense nucleic acid or shRNA into cells, and a method of using antisense nucleic acid or shRNA including artificial nucleic acid.

[0034] (4) Gene knockout As used herein, "gene knockout" refers to disrupting the function of a gene by adding an insertion or deletion to a target gene on a chromosome. For gene knockout, a gene targeting method that uses homologous recombination to modify an endogenous gene, or a genome editing technique that uses an artificial DNA cleavage enzyme such as ZFN or TALEN, or a site-specific nuclease such as CRISPR / Cas can be used. The gene knockout in the present invention may be a conditional gene knockout. As used herein, "conditional gene knockout" refers to site-specific and / or time-specific gene knockout. The conditional gene knockout can be performed, for example, by introducing a recognition sequence for a DNA recombinase before or after a target gene, expressing the DNA recombinase under the control of a promoter having site-specific and / or time-specific activity, and inducing a site-specific recombination reaction. In the transformed plant of the present invention, the expression or activity of the negative control factor can be suppressed or inhibited by, for example, quantifying the amount of mRNA using real-time PCR. Those skilled in the art can appropriately select suitable primers and PCR conditions for use in real-time PCR.

[0035] (5) Introduction of inhibitors In the transformed plant of the present invention, an inhibitor can be introduced to suppress / inhibit the expression / activity of the suppressive regulator. The inhibitor is not limited as long as it can directly or indirectly suppress / inhibit the expression / activity of SIZ1. Examples of inhibitors that can indirectly suppress / inhibit the expression / activity of SIZ1 include inhibitors that can inhibit the activity of a complex formed by SIZ1 and inhibitors that can inhibit a catalytic reaction involving SIZ1. For example, a sumoylation inhibitor can be used to directly or indirectly suppress / inhibit the activity of SIZ1. The sumoylation inhibitor may be any of a SUMO E1 inhibitor, a SUMO E2 inhibitor, or a SUMO E3 inhibitor. Examples of sumoylation inhibitors include Anacardic Acid (WAKO 019-24491) and 2-D08 (Sigma SML1052).

[0036] 1-4.Effects The transformed plant etc. of the present invention can provide a transformed plant etc. with enhanced dedifferentiation and redifferentiation abilities. When dedifferentiation and / or redifferentiation is induced in the transformed plant etc. of the present invention by a plant hormone, cutting treatment, and / or temperature stimulation, etc., it is possible to induce dedifferentiation and / or redifferentiation more efficiently than in a wild-type strain.

[0037] The transformed plants of the present invention can induce dedifferentiation with high efficiency in plant species that have been difficult to dedifferentiate by conventional methods, such as woody plants. For example, it is possible to efficiently induce callus from all or a part of the plant body.

[0038] Furthermore, the transformed plants of the present invention can efficiently redifferentiate dedifferentiated cells from plants that have been difficult to regenerate using conventional methods, such as plants belonging to the family Poaceae, cruciferous plants such as rapeseed, grain plants, and medicinal plants. For example, it is possible to efficiently regenerate stems, leaves, and roots from callus, and to regenerate individual plants through the differentiation of adventitious embryos, adventitious buds, and adventitious roots.

[0039] As described above, the function of SIZ1 is conserved across plant species, and therefore the effects of the present invention can be achieved in a wide range of plant species.

[0040] The transformed plants etc. of the present invention can be used to produce dedifferentiated and redifferentiated plant cells, to induce dedifferentiation of a plant body, and to induce redifferentiation of dedifferentiated plant cells.

[0041] Furthermore, the transformed plant etc. of the present invention may be a transformed plant etc. for producing dedifferentiated plant cells and / or redifferentiated plant cells.

[0042] The transformed plants of the present invention can contribute to the improvement of efficient mass production techniques for useful varieties and molecular breeding techniques in specific plant industries such as the seed and seedling industry and the floriculture and horticulture industry.

[0043] 2. Method for Producing Dedifferentiated Plant Cells 2-1. Overview A second aspect of the present invention is a method for producing dedifferentiated plant cells. The method of the present invention includes, as an essential step, subjecting a transformed plant or the like in which the function of a suppressive regulatory factor has been suppressed or inhibited to a dedifferentiation induction treatment. According to the method for producing dedifferentiated cells of the present invention, it is possible to produce dedifferentiated plant cells more efficiently than conventional methods. Furthermore, it is possible to efficiently produce dedifferentiated cells even in plant species in which dedifferentiation was difficult using conventional methods.

[0044] 2-2. Method The method for producing dedifferentiated cells of the present invention includes a dedifferentiation induction treatment step as an essential step and a recovery step as a selection step. Each step will be specifically described below.

[0045] 2-2-1. Dedifferentiation induction treatment process In this embodiment, the "dedifferentiation induction treatment step" refers to a step of performing a dedifferentiation induction treatment on a transformed plant or a part thereof in which the function of a negative control factor has been suppressed or inhibited. The dedifferentiation induction in this step may be callus induction.

[0046] In this embodiment, the transformed plant etc. that is the target of the dedifferentiation induction treatment is similar to the transformed plant etc. described in the first embodiment. The target of the dedifferentiation induction treatment may be either the whole or a part of the plant body.

[0047] The method of dedifferentiation induction treatment in this step can be any method known in the art. It is not limited to the above, but can include plant hormones, temperature stimulation such as heat shock, injury such as cutting treatment, infection with viruses or bacteria, etc. Each method will be described below.

[0048] (1) Plant hormones The plant hormone used in this process is not limited as long as it can induce dedifferentiation. Examples include auxin, cytokinin, gibberellin, ethylene, abscisic acid, jasmonic acid, brassinosteroids, peptide hormones, etc. Auxins include 2,4-dichlorophenoxyacetic acid (2,4-D), phenylacetic acid, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), dicamba, picloram, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 1-naphthaleneacetic acid (NAA), and naphthoxyacetic acid. Cytokinins include kinetin, benzylaminopurine (BAP), isopentyl adenine (2-iPA), thidiazuron, benzyladenine, and zeatin. The plant hormone used in this step is preferably an auxin or a cytokinin, for example, 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), kinetin, or benzylaminopurine.

[0049] In this step, one type of plant hormone may be used alone, or two or more types of plant hormones may be used in combination. Those skilled in the art can appropriately select the plant hormone used in this step according to the target plant species, etc. When used alone, the plant hormone is not limited. For example, auxin may be used alone. When two or more types of plant hormones are used in combination, the combination of plant hormones is not limited. For example, auxin and cytokinin can be used in combination. In this case, the concentration of cytokinin may be 5 times or less, 4 times or less, 3 times or less, 2 times or less, 1 time or less, or 0.5 times or less of the auxin concentration in terms of molar concentration ratio.

[0050] Plant hormones can usually be added to callus induction media. In this specification, "callus induction media (CIM)" means a medium prepared so that callus can be induced from a plant body. Those skilled in the art can easily prepare callus induction media according to the method described in, for example, "Transformation Protocol [Plant Edition], Kagaku Dojin (2012)." Callus induction media generally contain a basal medium and a plant hormone.

[0051] As the basal medium for the callus induction medium, any medium such as MS medium, LS medium, N6 medium, B5 medium, R2 medium, and CC medium, preferably MS medium, can be used. The medium can be prepared, for example, by mixing a composition containing the salts of these media, for example, commercially available MS Salt (Duchefa Biochemie), with components other than salts, for example, vitamins such as myo-inositol or thiamine HCl, and / or additional salts such as CuSO4. The amount of each component constituting the MS medium can be easily determined by one skilled in the art. In addition, the callus induction medium may contain additional components, for example sugars such as glucose or sucrose at up to 100 g / L or up to 50 g / L, or 10-100 g / L, 20-80 g / L, 30-70 g / L, or 40-60 g / L, and / or factors that may increase stress tolerance, such as L-proline at, for example, 600-800 mg / L, 650-750 mg / L, 680-700 mg / L, or about 690 mg / L.

[0052] The callus induction medium preferably contains an auxin such as 2,4-D at, for example, 0.5 to 4.0 mg / L, 1.0 to 3.0 mg / L, 1.5 to 2.5 mg / L, 1.8 to 2.2 mg / L, 1.9 to 2.1 mg / L, or 2.0 mg / L. In addition to the auxin, the callus induction medium may optionally contain kinetin at, for example, 0.2 to 2.0 mg / L, 0.3 to 1.0 mg / L, 0.4 to 0.6 mg / L, or 0.5 mg / L.

[0053] The pH of the callus induction medium may be, for example, 5.0 to 7.0, 5.5 to 6.5, 5.6 to 6.0, 5.7 to 5.9, or about 5.8. The medium may be made into a solid medium by adding a gelling agent such as agar, gellan gum, gelsan, or agarose.

[0054] The temperature at which dedifferentiation induction treatment is performed using a plant hormone is not limited as long as dedifferentiation and plant growth are possible. For example, the temperature may be 15 to 35° C., and is preferably 22 to 32° C., 23 to 31° C., 24 to 30° C., 25 to 29° C., or 26 to 28° C., such as 27° C. In particular, within the range of 25 to 29° C. or 26 to 28° C., dedifferentiation can be induced with high efficiency.

[0055] The period for which the dedifferentiation induction treatment is performed may be 1 day or more, 2 days or more, 3 days or more, 5 days or more, or 1 week or more, and may be 1 month or less, 3 weeks or less, or 2 weeks or less.

[0056] (2) Temperature stimulation such as heat shock It is also possible to induce dedifferentiation of plant cells by applying a temperature stimulus such as a heat shock to all or a part of a plant body (JP Patent Publication No. 2015-208256). The heat shock treatment is not limited. For example, a treatment in which a target plant is heated at a predetermined temperature for a predetermined time to apply heat stress to the plant body is exemplified. The predetermined temperature is, for example, 28°C to 42°C, preferably 30°C to 41°C, 33°C to 40°C, or 35°C to 40°C. The predetermined time is, for example, 10 minutes to 5 days (120 hours), 1 hour to 100 hours, 3 hours to 4 days (96 hours), 5 hours to 85 hours, or 10 hours to 3 days (72 hours).

[0057] The method of heat shock treatment is not particularly limited as long as it can give a heat shock to the target plant. For example, the plant body may be placed in an incubator set at a predetermined temperature, or the whole or part of the plant body may be immersed in a solvent such as water adjusted to a predetermined temperature. A heat source such as a hand warmer may be brought into contact with a part of the plant body. Light may be irradiated from a light source that emits heat such as an incandescent bulb.

[0058] (3) Amputation and other injuries In this step, it is also possible to induce dedifferentiation by injuring the plant. For example, the plant to be subjected to the dedifferentiation induction treatment may be cut as a physical injury. "Cutting" here refers to cutting the plant body or a part thereof using a blade or the like, and / or scratching the surface of the plant body or a part thereof. The cutting step makes it possible to induce dedifferentiation of cells at the cut surface or wound exposed to the outside world. Other damage that can induce dedifferentiation include UV irradiation and X-ray irradiation.

[0059] (4) Viral or bacterial infection It is also possible to induce dedifferentiation of plant cells by infection with viruses or bacteria. Examples of viruses that induce dedifferentiation of plant cells include wound tumor virus (WTV) and rice gall dwarf virus. Examples of bacteria that induce dedifferentiation of plant cells include Rhizobium rhizogenes (Agrobacterium tumefaciens), Pantoea agglomerans pv. gypsophilae, and agglomerans pv. Betae.

[0060] 2-2-2. Recovery process The "recovery step" is a step of collecting dedifferentiated cells generated in a plant body, etc. after the dedifferentiation induction treatment step, or callus containing the dedifferentiated cells, from the plant body, etc. This step is a selection step and may be performed as necessary.

[0061] The method for harvesting dedifferentiated cells or callus from a plant body, etc. is not particularly limited. Only the dedifferentiated cells or callus may be extracted, or unnecessary parts may be cut off from the plant body, etc. in which the dedifferentiated cells or callus have developed. The harvested parts may contain parts other than the dedifferentiated cells or callus.

[0062] 3. Methods for Producing Plant Regenerated Cells 3-1. Overview A third aspect of the present invention is a method for producing redifferentiated cells of a plant. The method of the present invention includes, as an essential step, subjecting a transformed plant or the like in which the function of a suppressive control factor has been suppressed or inhibited to a redifferentiation induction treatment. According to the method for producing redifferentiated cells of the present invention, it is possible to produce redifferentiated cells of a plant more efficiently than conventional methods. Furthermore, it is possible to efficiently produce redifferentiated cells from dedifferentiated cells even in plants in which redifferentiation was difficult using conventional methods. It is possible to regenerate a plant body from the redifferentiated cells obtained in the redifferentiation induction treatment step.

[0063] 3-2. Method The method for producing redifferentiated cells of the present invention includes a redifferentiation induction treatment step as an essential step. The redifferentiation induction treatment step will be specifically described below.

[0064] 3-2-1. Redifferentiation induction treatment process The "redifferentiation induction treatment step" in this embodiment refers to a step of performing a redifferentiation induction treatment on a transformed plant in which the function of a negative control factor has been suppressed or inhibited, or on some of its dedifferentiated cells.

[0065] The "de-differentiated cells of a transformed plant" that are the subject of re-differentiation induction treatment in this step may be de-differentiated cells of a plant produced by the method described in the second aspect, i.e., de-differentiated cells obtained by performing de-differentiation induction treatment on the transformed plant etc. described in the first aspect, or cells obtained by suppressing or inhibiting the expression or activity of a factor that suppresses the expression of a regeneration-related factor in de-differentiated cells of a plant according to the method of suppressing or inhibiting expression or activity described in the first aspect. The "de-differentiated cells of a transformed plant or a part thereof" that are the subject of re-differentiation induction treatment in this step may be de-differentiated cells contained in a callus.

[0066] In this step, the redifferentiation induction treatment of the dedifferentiated cells may be carried out based on a method known in the art. For example, redifferentiation can be induced in the dedifferentiated cells or callus using a plant hormone.

[0067] The plant hormone used in this step is not limited as long as it can induce regeneration. Specific examples of plant hormones are as described in the second embodiment.

[0068] The plant hormones used in this step include, for example, cytokinin and auxin. For example, kinetin, NAA, etc. may also be used.

[0069] In this step, one type of plant hormone may be used alone, or two or more types of plant hormones may be used in combination. Those skilled in the art can appropriately select the plant hormone used in this step according to the target plant species, etc. When used alone, the plant hormone is not limited. For example, cytokinin may be used alone. When two or more types of plant hormones are used in combination, the combination of plant hormones is not limited. For example, cytokinin and auxin can be used in combination. In this case, the concentration of cytokinin may be 1-fold or more, 10-fold or more, 100-fold or more, or 1000-fold or more, relative to the concentration of auxin, in terms of molar concentration ratio. Plant hormones can usually be used by adding them to the regeneration-inducing medium.

[0070] In the present specification, the term "redifferentiation induction medium" refers to a medium prepared so that dedifferentiated cells or callus containing dedifferentiated cells can be redifferentiated into shoots or the like, and a person skilled in the art can easily prepare the medium according to the method described in, for example, "Transformation Protocol [Plant Edition], Kagaku Dojin (2012)". The redifferentiation induction medium is, for example, shoot induction media (SIM). The redifferentiation induction medium generally contains a basal medium, preferably an MS medium, and a plant hormone. The redifferentiation induction medium may optionally contain a factor capable of increasing stress resistance, such as proline, and / or a factor inhibiting necrosis of callus, such as PVP (polyvinylpyrrolidone) (Transformation Protocol [Plant Edition], Kagaku Dojin (2012)). The basal medium, additional components, and pH of the redifferentiation induction medium are similar to those of the "callus induction medium" described in the second embodiment.

[0071] The regeneration-inducing medium used in this step may contain, for example, a plant hormone such as auxin and / or cytokinin, etc. The regeneration-inducing medium preferably contains at least one of zeatin, indole acetic acid, NAA, BA, thidiazuron, and abscisic acid, and preferably two, three, or all of these. The concentration of the plant hormone in the regeneration-inducing medium can be appropriately determined by a person skilled in the art, and may be, for example, 0.1 to 2.0 mg / L, 0.2 to 1.0 mg / L, 0.4 to 0.6 mg / L, or about 0.5 mg / L for zeatin, 1.0 to 3.0 mg / L, 1.5 to 2.5 mg / L, 0.9 to 1.1 mg / L, or about 1.0 mg / L for indole acetic acid, 5 to 50 μg / L, 10 to 40 μg / L, 20 to 30 μg / L, or about 26.4 μg / L (0.1 μM) for abscisic acid, and 0.02 to 0.4 mg / L, 0.05 to 0.2 mg / L, 0.09 to 0.11 mg / L, or about 0.1 mg / L for thidiazuron.

[0072] In this step, the temperature at which the redifferentiation induction treatment is carried out is not limited as long as redifferentiation and plant growth are possible. For example, the temperature may be 15 to 35° C., and is preferably 22 to 32° C., 23 to 31° C., 24 to 30° C., 25 to 29° C., or 26 to 28° C., such as 27° C. In particular, within the range of 25 to 29° C. or 26 to 28° C., redifferentiation can be induced with high efficiency.

[0073] The period for which the redifferentiation induction treatment is performed may be 1 day or more, 2 days or more, 3 days or more, 5 days or more, or 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more, or may be 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or 2 weeks or less.

[0074] In one embodiment, the regenerated cells obtained in this step are root, stem, leaf, flower cells, or stem and leaf cells.

[0075] 4. Method for inducing dedifferentiation of plant bodies 4-1. Overview The fourth aspect of the present invention is a method for inducing dedifferentiation in a plant body. According to the method for inducing dedifferentiation of the present invention, it is possible to induce dedifferentiation in a plant body more efficiently than conventional methods. Furthermore, it is possible to induce dedifferentiation in plants that were difficult to dedifferentiate using conventional methods.

[0076] 4-2. Method The method for inducing dedifferentiation of a plant body of the present invention comprises, as essential steps, a suppression / inhibition step and a dedifferentiation induction treatment step.

[0077] In this embodiment, the plant body to be subjected to dedifferentiation induction is the whole or part of the transformed plant described in embodiment 1. The plant species is similar to the target plant described in embodiment 1. For example, the plant is derived from a dicotyledonous plant or a monocotyledonous plant.

[0078] In this embodiment, the "suppression / inhibition step" refers to a step of enhancing the dedifferentiation ability of a plant body by suppressing or inhibiting the expression or activity of a factor that suppresses the expression of a regeneration-related factor. Specifically, it is a step of suppressing or inhibiting the expression or activity of SIZ1. This step is similar to the method of suppressing or inhibiting the expression or activity of a negative control factor in the first embodiment.

[0079] The dedifferentiation induction treatment step in this embodiment is a step of performing a dedifferentiation induction treatment on the plant body with enhanced dedifferentiation ability obtained in the suppression / inhibition step. This step corresponds to "2-2-1. Dedifferentiation induction treatment step" of the second embodiment.

[0080] 5. Method for inducing redifferentiation of dedifferentiated plant cells 5-1. Overview The fifth aspect of the present invention is a method for inducing redifferentiation of dedifferentiated cells of a plant. According to the redifferentiation induction method of the present invention, it is possible to induce redifferentiation of dedifferentiated cells of a plant more efficiently than conventional methods. Furthermore, it is possible to induce redifferentiation of dedifferentiated cells of a plant that were difficult to redifferentiate by conventional methods.

[0081] 5-2. Method The method for inducing redifferentiation of a plant body of the present invention comprises, as essential steps, a suppression / inhibition step and a redifferentiation induction treatment step.

[0082] In this embodiment, the dedifferentiated cells to be induced to redifferentiate may be dedifferentiated cells contained in a callus.

[0083] In this embodiment, the plant species from which the dedifferentiated cells to be induced to redifferentiate are derived is similar to the target plants described in the first embodiment, for example, derived from dicotyledonous plants or monocotyledonous plants.

[0084] The suppression / inhibition step in this embodiment is a step of enhancing the regeneration ability of dedifferentiated plant cells by suppressing or inhibiting the expression or activity of a factor that suppresses the expression of a regeneration-related factor. Specifically, it is a step of enhancing the regeneration ability of dedifferentiated plant cells by suppressing or inhibiting the expression or activity of SIZ1. This step is similar to the method of suppressing or inhibiting the expression or activity of a negative control factor in the first embodiment.

[0085] The redifferentiation induction treatment step in this embodiment is a step of performing a redifferentiation induction treatment on the dedifferentiated cells of a plant with enhanced redifferentiation ability obtained in the suppression / inhibition step. This step corresponds to "3-2-1. Redifferentiation induction treatment step" of the third embodiment. EXAMPLES

[0086] <Example 1: Induction of dedifferentiation in SIZ1 mutant strain> (the purpose) We will verify that SIZ1 mutations enhance dedifferentiation ability. (method) All Arabidopsis thaliana strains used in the examples were strains on the Columbia strain (Col-0) background. Specifically, the following mutant strains were used: siz1-2 mutant strain (SALK_065397) and siz1-3 mutant strain (SALK_034008), which are loss-of-function mutants of SIZ1, and a siz1 complementation strain (siz1-2 pSIZ1::SIZ1) (Jin et al., 2008, Plant J 53: 530-540.; Miura et al., 2005, Proc Natl Acad Sci USA, 102: 7760-5.). The siz1 complementation strain (siz1-2 pSIZ1::SIZ1) is a strain in which wild-type SIZ1 is expressed under the control of an endogenous promoter by introducing pSIZ1::SIZ1 into the siz1-2 mutant strain.

[0087] The seeds were subjected to a cold and wet treatment at 4°C for 3 days, and then planted on MS plates. -2 s -1 Seedlings were incubated at 22°C under constant light. Seedlings were grown in the dark for 7 days to induce etiolation. Hypocotyl explants approximately 10 mm long were excised using a razor blade and incubated on CIM medium (Gamborg's B5 medium containing 0.25% Gelsang, 0.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg / L kinetin) for 17 days at 22°C under constant light.

[0088] The weight of the callus grown on the CIM medium was quantified as follows: the medium was removed from the resulting callus using Kimwipe (Nippon Paper Crecia Co., Ltd.), and the weight was measured using an XS104 balance (Mettler Toledo).

[0089] (result) Hypocotyl explants from wild-type (n = 25), siz1-2 mutant (n = 26), siz1-3 mutant (n = 27), and siz1 complemented (siz1-2 pSIZ1::SIZ1) (n = 19) were incubated on CIM medium at 22°C for 17 days to induce dedifferentiation. The results are shown in Fig. 1a. The callus weights after incubation on CIM medium for 17 days are shown in Fig. 1b. The callus formation ability of the siz1-2 and siz1-3 mutants was significantly higher than that of the wild-type. In the SIZ1 complemented (siz1-2 pSIZ1::SIZ1) line, the callus formation ability was reduced to the same level as that of the wild-type. These results indicate that SIZ1 mutation increases callus formation ability and enhances dedifferentiation ability.

[0090] <Example 2: Redifferentiation induction for SIZ1 mutant strain> (the purpose) We will verify that SIZ1 mutations enhance regeneration ability. (method) Following incubation on CIM medium as in Example 1, hypocotyl explants were transferred onto SIM medium (Gamborg B5 medium containing 0.25% Gelsang, 0.15 μg / ml IAA, and 0.5 μg / ml 2-IPA) and incubated at 22° C. for 14 days to induce shoot formation. Shoot formation was quantified by measuring the number of shoots formed per explant. Shoots were defined as areas bearing trichomes arising from a single meristem and were visualized from above using an Olympus SZX7 microscope.

[0091] (result) The wild-type strain, the siz1-2 mutant strain, the siz1-3 mutant strain, and the siz1-2 pSIZ1::SIZ1 strain (SIZ1 complementation strain) were cultured in CIM medium for 4 days, then transferred to SIM medium and cultured for 14 days. The results are shown in FIG. 2a.

[0092] The wild-type strain (n = 68), siz1-2 mutant strains (n ​​= 103), siz1-3 mutant strains (n ​​= 99), and siz1-2 pSIZ1::SIZ1 strain (SIZ1 complementation strain; n = 47) were cultured on CIM medium for 4 days and then transferred to SIM medium. The number of shoots formed per explant (average) in each group after 9 days of culture is shown in Figure 2b.

[0093] Wild-type explants began to form shoots about 9 days after transfer to SIM medium (Fig. 2a, b). Furthermore, an average of five shoots were formed per explant by 14 days after transfer. In contrast, siz1-2 and siz1-3 mutants began to form shoots 7 to 8 days after transfer to SIM medium, and the number of shoots formed was dramatically increased compared to the wild-type (Fig. 2a, b). In the siz1-2 and siz1-3 mutants, the number of shoots formed per explant exceeded 12 by 14 days after transfer to SIM medium (data not shown). In the SIZ1 complementation strain (siz1-2 pSIZ1::SIZ1), the shoot formation ability was reduced to a level similar to that of the wild-type.

[0094] These results indicate that the SIZ1 mutation enhances shoot formation and plant regeneration ability.

Claims

1. 1. A method for producing dedifferentiated cells of a plant, comprising: A step of subjecting a transformed plant or a part thereof in which the expression or activity of SIZ1 (SUMO E3 ligase) is suppressed or inhibited to a dedifferentiation induction treatment. The method comprising:

2. The method described in claim 1, wherein the SIZ1 consists of any one of the following amino acid sequences (1) to (3). (1) The amino acid sequence shown in SEQ ID NO:1 (2) An amino acid sequence in which 1 or 2 to 40 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:

1. (3) an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO:1

3. The method described in claim 1 or 2, wherein the transformed plant is derived from a dicotyledonous plant or a monocotyledonous plant.

4. The method according to any one of claims 1 to 3, wherein the dedifferentiation induction treatment uses auxin.

5. The method according to any one of claims 1 to 4, wherein the dedifferentiation induction treatment is carried out at 15°C to 35°C.

6. 1. A method for producing regenerated cells of a plant, comprising the steps of: A step of subjecting a transformed plant or a part of its dedifferentiated cells in which the expression or activity of SIZ1 (SUMO E3 ligase) is suppressed or inhibited to a redifferentiation induction treatment. The method comprising:

7. The method described in claim 6, wherein the SIZ1 consists of any one of the following amino acid sequences (1) to (3). (1) The amino acid sequence shown in SEQ ID NO:1 (2) An amino acid sequence in which 1 or 2 to 40 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:

1. (3) an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO:1

8. The method described in claim 6 or 7, wherein the transformed plant is derived from a dicotyledonous plant or a monocotyledonous plant.

9. The method according to any one of claims 6 to 8, wherein the redifferentiation-inducing treatment uses a cytokinin.

10. The method according to any one of claims 6 to 9, wherein the dedifferentiated cells are obtainable by the method according to any one of claims 1 to 5.

11. The method according to any one of claims 6 to 10, wherein the redifferentiation induction treatment is carried out at 15°C to 35°C.

12. The method according to any one of claims 6 to 11, wherein the regenerated cells are stem and leaf cells.

13. A method for inducing dedifferentiation in a plant body, comprising the steps of: A step of enhancing the dedifferentiation ability of a plant body by suppressing or inhibiting the expression or activity of SIZ1; and A step of subjecting the plant body having enhanced dedifferentiation ability to a dedifferentiation induction treatment. The method comprising:

14. A method for inducing redifferentiation of dedifferentiated cells of a plant, comprising the steps of: A step of enhancing the regeneration ability of dedifferentiated cells of a plant by suppressing or inhibiting the expression or activity of SIZ1; and A step of subjecting the dedifferentiated cells having enhanced redifferentiation ability to a redifferentiation induction treatment. The method comprising:

15. 15. The method according to claim 13 or 14, wherein the plant is of dicotyledonous or monocotyledonous origin.

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