Grafting improvement agent

Abstract

To provide a grafting improver.SOLUTION: A grafting improver includes: (A) cellulase; (B) a peptide which includes an amino acid sequence shown in the sequence number 1 or 2, or an amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in the sequence number 1 or 2, and has grafting improvement activity; (C) phenol amides or polyamine; and (D) at least one selected from the group consisting of an expression or generation accelerator of any one of (A) to (C).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a graft improvement agent and the like. [Background technology]

[0002] Grafting is the technique of surgically fusing two or more plants together. Grafting is generally performed using gymnosperm plants. Grafting is performed on plants and angiosperms, and is widely used in horticulture and agriculture. In general, grafting is a technique that uses a rootstock that forms the root and a scion that forms the above-ground part to create a plant that can utilize the excellent capabilities of both. There are many purposes and methods of grafting. For example, grafting is often used to clone and propagate new varieties of fruit trees in general. In addition, vegetable crops such as Solanaceae and Cucurbitaceae can acquire disease resistance and improve the quality and productivity of fruits by utilizing their useful root systems through grafting. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Simon, SV Jahrb. wiss. Bot., 1930. 72, 137-160. [Non-Patent Document 2] Nickell LG, Science, 1948. 108. 389. Summary of the Invention [Problem to be solved by the invention]

[0004] However, it is known that grafting can be hindered or rarely successful depending on the combination of plants, which is called graft incompatibility, and this has been a technical limitation. In general, grafting is more difficult between plants of the same species, then plants of the same genus, and finally plants of the same family. Although there have been reports of grafting between specific plants of different families (Non-Patent Documents 1 and 2), the combinations of plants of different families that can be grafted are limited.

[0005] In the course of research into the above-mentioned problems, the inventors have realized that the above-mentioned problems can be solved by a substance that has a grafting improving effect, such as improving grafting efficiency.

[0006] Therefore, an object of the present invention is to provide a graft improvement agent. [Means for solving the problem]

[0007] The present inventors have conducted intensive research into the grafting mechanism, although many aspects remain unclear. As a result, they have discovered that (A) cellulase, (B) the amino acid sequence shown in SEQ ID NO: 1 or 2, or the amino acid sequence shown in SEQ ID NO: 1 or 2 in which one or several amino acids have been substituted, deleted, added, or the like. (C) a peptide having an amino acid sequence inserted therein and having graft improvement activity; (D) expression or synthesis of any one of (A) to (C) If the grafting improvement agent contains at least one selected from the group consisting of growth promoters, The inventors have found that the above-mentioned problems can be solved. Based on this finding, the inventors have conducted further research and have completed the present invention.

[0008] That is, the present invention includes the following aspects.

[0009] Item 1. (A) Cellulase, (B) the amino acid sequence shown in SEQ ID NO: 1 or 2, or the amino acid sequence shown in SEQ ID NO: 1 or 2 The amino acid sequence has one or more amino acids substituted, deleted, added, or inserted. A peptide comprising an amino acid sequence and having graft improvement activity; (C) phenolamides or polyamines, and (D) an expression or production promoter of any one of (A) to (C) A grafting improvement agent containing at least one selected from the group consisting of:

[0010] Item 2. The graft improvement agent according to Item 1, which is used for interfamily grafts or intrafamily grafts.

[0011] Item 3. The expression or production promoter of (A) is the expression cassette of (A), The expression or production promoter of (B) is an expression cassette of (B), The expression or production promoter of (C) is an expression cassette of the biosynthesis-related gene of (C) or a biosynthesis-related factor of (C). Item 3. The grafting improvement agent according to item 1 or 2.

[0012] Item 4. Any of Items 1 to 3, wherein the polyamine is a polyamine containing four or more amino groups. A graft improvement agent as described above.

[0013] Item 5. The amino acid sequence shown in SEQ ID NO: 1 or 2, or the amino acid sequence shown in SEQ ID NO: 1 or 2 in which one or several amino acids have been substituted, deleted, added or inserted. A peptide comprising an amino acid sequence comprising the above structure and having graft improvement activity.

[0014] Item 6. A method for producing a plant substructure for grafting, comprising applying the grafting adhesive according to any one of Items 1 to 4 or the peptide according to claim 5 to a plant body or a plant substructure.

[0015] Item 7. A plant substructure for grafting obtained by the method according to Item 6.

[0016] Item 8. A method for producing a grafted plant body, comprising applying the graft improvement agent according to any one of Items 1 to 4 or the peptide according to claim 5 to a plant body after grafting, and / or grafting the plant partial structure for grafting according to Item 7 with another plant partial structure.

[0017] Item 9. A grafted plant obtained by the method according to Item 8. Effect of the Invention

[0018] According to the present invention, a grafting improvement agent can be provided. Furthermore, according to the present invention, it is also possible to provide a plant tissue for grafting and a method for producing the same, a grafted plant body and a method for producing the same, etc., which utilize the grafting improvement agent. [Brief description of the drawings]

[0019] [Figure 1] 1 shows the results of Test Example 1. A shows a schematic diagram of the grafted plant of Test Example 1. B is an example of a photograph of the grafted plant after the test. In B, ○ indicates successful grafting, and × indicates unsuccessful grafting. C is a graph showing the relative values ​​of the cellulase mRNA expression ratio ( / actin expression level) of each Nicotiana plant used in the test. D is a graph showing the grafting success rate. On the horizontal axis of C and D, "No graft" indicates an ungrafted wild-type Nicotiana plant, "No virus" indicates a grafted wild-type Nicotiana plant, "Vector control" indicates a grafted control vector (VIGS vector targeting the GFP gene)-introduced Nicotiana plant, and "VIGS for Cellulase" indicates a grafted cellulase knockdown (VIGS vector targeting the cellulase gene) Nicotiana plant. [Diagram 2] 1 shows the results of Test Example 2. A shows a schematic diagram of the grafted plant body of Test Example 2, and an example of a photograph of the grafting surface on which a necrotic layer has been formed. B is a graph showing the percentage of the adhesion area on which a necrotic layer has not been formed. In B, 1 shows a wild-type Nicotiana plant, 2 shows a Nicotiana plant into which a control vector (a VIGS vector targeting the GFP gene) has been introduced, and 3 shows a cellulase knockdown (a VIGS vector targeting the cellulase gene) Nicotiana plant. [Diagram 3] This shows the results of Test Example 3. The photograph shows a cross section of the grafted area. - indicates the case where cellulase was not administered. [Figure 4] The results of Test Example 4 are shown. The vertical axis indicates the success rate of grafting. On the horizontal axis, NT indicates the drug-untreated group, 2,4-D indicates the auxin-treated group, DMSO indicates the DMSO-treated group (negative control), and Grf006 and Grf010 indicate the Grf006-treated group and the Grf010-treated group, respectively. [Diagram 5]Shows the amount (relative value) of SIMYB14 mRNA in tomatoes used for grafting in Test Example 5. On the horizontal axis, Wild type indicates wild-type tomatoes, 2-4-1 indicates tomatoes in which SIMYB14 was knocked down by RNAi, and Escape 2-1-1 indicates tomatoes that were not transformed and developed during the process of producing RNAi transformants (i.e., tomatoes with the same characteristics as wild-type tomatoes). [Figure 6] The figures show the amounts (relative values) of phenolamides in the tomatoes used for grafting in Test Example 5. On the horizontal axis, Wild type indicates wild-type tomatoes, 2-4-1 indicates tomatoes in which SIMYB14 was knocked down by RNAi, and Escape 2-1-1 indicates tomatoes that were not transformed and were obtained during the process of producing RNAi transformants (i.e., tomatoes with the same characteristics as wild-type tomatoes). [Figure 7] The results of the evaluation of the universality of phenolamides in solanaceae plants in Test Example 5 are shown. The horizontal axis indicates the cultivar name of the solanaceae plant from which the sample was taken. "_A" indicates that the sample was taken from the cultivar in the untreated area (A), and "_B" indicates that the sample was taken from the cultivar in the wound-treated area (B). The vertical axis indicates the signal intensity value of LC-MS / MS. [Figure 8] The results of Test Example 5 are shown. Wild type indicates wild-type tomato, 2-4-1 indicates tomato in which SIMYB14 was knocked down by RNAi, and Escape 2-1-1 indicates a non-transformed tomato obtained during the process of producing an RNAi transformant (i.e., a tomato with the same characteristics as the wild-type tomato). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0021] In this specification, "and / or" is a term that includes both "and" and "or." That is, A and / or B includes the three meanings of A only, B only, and A and B.

[0022] As used herein, "identity" of an amino acid sequence refers to the degree of identity between two or more comparable amino acid sequences. It refers to the degree of identity of the amino acid sequences of two or more amino acid sequences. The higher the match between the sequences, the higher the identity or similarity between the sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87: 2264 -2268(1990), Karlin S, Altschul SF. “Applications and statistics for multiple "High-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). The programs called tblastn and tblastn have been developed. Specific methods for the method are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of base sequences is also defined in the same manner as above.

[0023] In this specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, a conservative substitution is a substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine. Other examples of conservative substitution include amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; and amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Conservative substitutions are also made between amino acid residues having acidic side chains; amino acid residues having beta-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0024] In the present specification, proteins and peptides such as cellulase may be chemically modified as long as their activity is not significantly impaired. Proteins and peptides have a carboxyl group (-COOH) or a carboxylate (-COOH) at the C-terminus. - ), amide (-CONH2) or ester (-COOR). Here, R in the ester is, for example, C methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alki C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups; for example , phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups; pivaloyloxymethyl groups, etc. are used. In proteins and peptides, carboxyl groups (or carboxylates) other than the C-terminus are amidated. In this case, the ester may be, for example, the C-terminal In addition, N-terminal esters are used for proteins and peptides. The amino group of the acid residue is protected by a protecting group (e.g., formyl group, acetyl group, etc.) 1-6 Alkanoyl C etc. 1-6 those protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups); those in which the N-terminal glutamine residue that may be cleaved in vivo is pyroglutamic-substituted; and those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups). 1-6 Alkanoyl group etc. 1-6 Also included are those protected by an acyl group or other suitable group, or conjugated proteins / peptides such as so-called glycoproteins / peptides bound to sugar chains.

[0025] In the present specification, proteins and peptides such as cellulase may have other amino acid sequences added thereto, so long as their activity is not significantly impaired. Examples of other sequences include various signal sequences (e.g., nuclear transport signals, nuclear export signals, etc.), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, etc.), and the like. Examples of such sequences include a tag, a V5 tag, a PA tag, etc.), a labeled protein sequence (e.g., a fluorescent protein, a luminescent enzyme protein, etc.), a protein destabilizing sequence (e.g., a PEST sequence, etc.), and a protein stabilizing sequence.

[0026] As used herein, the term "coding sequence" refers to a base sequence that codes for the amino acid sequence of a protein or peptide, and is not particularly limited thereto.

[0027] In the present specification, a polynucleotide may be single-stranded or double-stranded. It may also be linear or circular. Furthermore, the polynucleotide of the present invention includes not only DNA and RNA, but also those which have been subjected to known chemical modifications, as exemplified below. In order to prevent decomposition by hydrolases such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, etc. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). The base moiety (pyrimidine, purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a carbonyl group at the 2-position with a thiocarbonyl group. The phosphate moiety or hydroxyl moiety may be modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, or the like, but is not limited thereto. The term "polynucleotide" includes not only natural nucleic acids, but also any of BNA (bridged nucleic acid), LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.

[0028] In the present specification, proteins such as cellulase, peptides, and polynucleotides may be in the form of pharma- ceutically acceptable salts with acids or bases. The salts are not particularly limited as long as they are agriculturally acceptable salts. Both acid salts and basic salts can be used as the salts. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0029] In the present specification, proteins such as cellulase, peptides, and polynucleotides may be in the form of a solvate. The solvent is not particularly limited as long as it is agriculturally acceptable, and examples of the solvent include water, ethanol, glycerol, and acetic acid.

[0030] In the present specification, proteins such as cellulase, peptides, and polynucleotides may have a functional substance added thereto. The functional substance is not particularly limited and may be, for example, a labeling substance, a carrier, or the like. The functional substance is usually linked to the protein, peptide, or polynucleotide directly or indirectly (for example, via a linker). Examples of the labeling substance include fluorescent substances (e.g., fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark)). ), Cy3, Cy5, Alexa Fluor series, etc.), Examples of enzymes that can be used include enzymes such as β-galactosidase, alkaline phosphatase, glucose oxidase, peroxidase, and polyphenol oxidase. The carrier is not particularly limited as long as it can support the polypeptide of the present invention. The material of the carrier is not particularly limited, and examples thereof include organic materials such as various resins, and inorganic materials such as silicon materials and metals.

[0031] 2. Grafting improvement agent In one aspect, the present invention relates to a method for producing a cellulase comprising the steps of: (A) a cellulase; (B) an amino acid sequence shown in SEQ ID NO: 1 or 2, or one or several amino acids corresponding to the amino acid sequence shown in SEQ ID NO: 1 or 2; (C) a peptide having an amino acid sequence in which amino acids are substituted, deleted, added or inserted and having a graft improving activity, and (D) the above-mentioned (A) The present invention relates to a grafting improvement agent (sometimes referred to as "the grafting improvement agent of the present invention" in this specification) containing at least one selected from the group consisting of any one of the expression or production promoters of (A), (B), (C), and (D). This will be explained below. The components (A), (B), (C), and (D) may also be referred to as component A, component B, component C, and component D, respectively.

[0032] 2-1.About component A The cellulase is not particularly limited, and various cellulases can be used. Examples of cellulases include endoglucanase (EC3.2.1.4) and exoglucanase (EC.3.2.1.91). Examples of cellulases that can be used include various cellulases within a family, such as the glycosyl hydrolase family, and specifically, for example, glycosyl hydrolase 1, glycosyl hydrolase 2, glycosyl hydrolase 3, glycosyl hydrolase 4, glycosyl hydrolase 5, glycosyl hydrolase 6, glycosyl hydrolase 7, glycosyl hydrolase 8, glycosyl hydrolase 9, glycosyl hydrolase 10, glycosyl hydrolase 11, glycosyl hydrolase 12, glycosyl hydrolase 13, glycosyl hydrolase 14, glycosyl hydrolase 15, glycosyl hydrolase 16, glycosyl hydrolase 17, glycos Glycosyl hydrolase 4, Glycosyl hydrolase 5, Glycosyl hydrolase 6, Glycosyl Glycosyl hydrolase 7, glycosyl hydrolase 8, glycosyl hydrolase 9, glycosyl hydrolase Examples of glycosyl hydrolase include glycosyl hydrolase 10, glycosyl hydrolase 11, and glycosyl hydrolase 12. Among these, glycosyl hydrolase 9 (e.g., 9A, 9B, 9C, etc.) is particularly preferred. Among them, 9B is preferable, and 9B1 and 9B13 are more preferable.

[0033] The organism from which cellulase is derived is not particularly limited. Examples of the organism from which cellulase is derived include plants and microorganisms, and preferably include plants. Among plants, preferred are plants with high interfamily grafting ability, such as plants of the Solanaceae family, Brassicaceae family, Lamiaceae family, and Orobanchaceae family, but are not limited thereto.

[0034] Examples of the Solanaceae plant include, but are not limited to, Nicotiana tabacum, Anthocercis, Anthotroche, Crenidium, Cyperus spp. Cyphanthera, Duboisia, Grammosolen, Symonanthus, Petunia, Benthamiella , Bouchetia, Brunfelsia, Combera, Fabiana, Hunzikeria, Leptoglossis, Nierembergia, Pantacantha, Calibrachoa, Plowmania, Capsicum, Lycianthes, Solanum, Jaltomata, Datura, Kida Examples of the plant include plants belonging to the genera Jimson Weed, Physalis, Leptospermum, Naked Nightshade, Hyoscyamus, Belladonna, Mandragora, Lycium, and Calibrachoa. Among these, the plant of the genera Nicotiana, Anthocercis, and Anemone are particularly preferred. Anthotroche, Crenidium, Cyphanthera, Duboisia, Grammosolen, Symonanthus, Petunia, Benthamiella, Boquetia Genus Bouchetia, Brunfelsia, Combera, Fabiana, Hunzikeria, Leptoglossis, Preferred are the genera Nierembergia, Pantacantha, Calibrachoa, Plowmania, Capsicum, Lycianthes, Solanum, Jaltomata, and the like, more preferred are the genera Nicotiana, Petunia, Capsicum, Solanum, and the like, and even more preferred is the Nicotiana.

[0035] Examples of plants belonging to the genus Nicotiana include, but are not limited to, Nicotiana benthamiana, Nicotiana tabacum, Nicotiana umbratica, Nicotiana rustica, Nicotiana acuminata, Nicotiana alata, Nicotiana attenuata, Nicotiana clevelandii, and Nicotiana excelsior. , Nicotiana forgetiana, Nicotiana glauca, Nicotiana glutinosa, Nicotiana langsdorffii, Nicotiana longiflora, Nicotiana obtusifolia, Nicotiana paniculata, Nicotiana plumbagifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tomentosa etc. Among these, Nicotiana benthamiana, Nicotiana tabacum, Nicotiana umbratica, Nicotiana rustica etc. is preferred, and Nicotiana benthamiana is more preferred.

[0036] Plants belonging to the genus Petunia (Petunia genus) are not particularly limited, and examples thereof include Petunia × atkinsiana (petunia), Petunia alpicola, Petunia axillaris, Petunia bajeensis, Petunia bonjardinensis, Petunia exserta, Petunia guarapuavensis, Petunia inflata, Petunia integrifolia, Petunia interior, Petunia ledifolia, Petunia littoralis, Petunia mantiqueirensis, Petunia occidentalis, Petunia patagonica, Petunia reitzii, Petunia riograndensis, Petunia saxicola, and Petunia scheideana. , Petunia villadiana, etc. Among these, Petunia × atkinsiana is preferred. It is.

[0037] The plant belonging to the genus Capsicum is not particularly limited, and for example, Capsicum annuum L. For example, 'Grossum' (bell pepper), 'Abbreviatum', 'Acuminoum', 'Cerasiforme', 'Conoides', 'Fasciculatum', 'Longum', 'Nigrym', 'Parvo-acuminatum', etc.), Capsicum baccatum, Capsicum cardenasii, Capsicum chinense Jacq. Heser & Smith, Capsicum frutescens L., Capsicum pubescens Ruiz & Pav., etc. Among these, Capsicum annuum L. is preferred, and Capsicum annuum L. 'Grossum' (bell pepper) is more preferred.

[0038] Examples of plants belonging to the genus Solanum include, but are not limited to, Solanum lycopersicum L (tomato), Solanum melongena L (eggplant), Solanum tuberosum L, Solanum acaule Bitt, Solanum aethiopicum L, Solanum betaceum Cav, Solanum jasminoides Paxt, Solanum mammosum L, Solanum muricatum Aiton, Solanum nigrum L, Solanum pseudocapsicum L, Solanum ptychanthum Dunal, etc. Among these, Solanum lycopersicum L (tomato), Solanum melongena L (eggplant), etc. are preferred.

[0039] The Brassicaceae plant is not particularly limited, and examples thereof include the genus Arabidopsis, the genus Brassica, the genus Shepherd's purse, the genus Cardamine, the genus Melastomataceum, the genus Camellia, the genus Horseradish, the genus Variegata, the genus Watercress, the genus Brassica napus, the genus Shepherd's purse, the genus Lycopersicon gracilis, the genus Shepherd's purse, the genus Atractylodes gracilis ... Examples of plants that may be included include plants belonging to the genera Isolobus, Raphanus sativus, Isopsis radish, Brassica napus, Brassica napus, Isopsis radish, Scutellaria baicalensis, Isopsis quinqueradiata ... Among these, the genus Arabidopsis, the genus Camellia, the genus Brassica, the genus Edulis, the genus Acanthus, the genus Radish, the genus Prunus, the genus Scutellaria, the genus Oenothera, the genus Prunus japonica, the genus Cardamine, the genus Horseradish, the genus Variegata, the genus Nasturtium, and the genus Brassica napus are the most widely used. The genus Arabidopsis and the like are preferred, the genus Arabidopsis, the genus Brassica, the genus Capsella, the genus Cardamine and the like are more preferred, the genus Arabidopsis and the genus Brassica are even more preferred, and the genus Arabidopsis is even more preferred.

[0040] Plants belonging to the genus Arabidopsis are not particularly limited, and examples thereof include Arabidopsis thaliana, Arabidopsis arenicola, Arabidopsis arenosa, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pedemontana, Arabidopsis suecica, etc. Among these, Arabidopsis thaliana is preferred.

[0041] Examples of plants belonging to the genus Brassica include, but are not limited to, Brassica oleracea (e.g. Examples of such Brassica include broccoli, cauliflower, cabbage, etc.), Brassica Napus (e.g., rapeseed, etc.), Brassica Barrelieri, Brassica carinata, Brassica elongata, Brassica fruticulosa, Brassica juncea, Brassica narinosa, Brassica nigra, Brassica nipposinica, Brassica rapa, Brassica rupestris, Brassica Tournefortii, etc. Among these, Brassica Oleracea, Brassica Napus, etc. are preferred, Brassica Oleracea is more preferred, and broccoli is even more preferred.

[0042] Plants belonging to the genus Shelium are not particularly limited, and include, for example, Capsella rubella, Capsella abscissa, Capsella andreana, Capsella australis, Capsella austriaca, Capsella bursa-pastoris, Capsella divaricata, Capsella draboides, Capsella gracilis, Capsella grandiflora, Capsella humistrata, Capsella hybrida, Capsella hyrcana, Capsella integrifolia, Capsella lycia, Capsella mexicana, Capsella orientalis, Capsella pillosula, Capsella pubens, Capsella puberula, Capsella schaffneri, Capsella stellata, Capsella tasmanica, Capsella thomsoni, Capsella thrac ica, Capsella viguieri, Capsella villosula, etc. Among these, Capsella rubella is preferred.

[0043] Plants belonging to the Cardamine genus include, but are not limited to, Cardamine hirsuta (Michitanetsukibana), Cardamine anemonoides, Cardamine appendiculata, Cardamine arakiana, Cardamine dentipetala, Cardamine dentipetala var. longifructa, Cardamine fallax, Cardamine impatiens, Cardamine kiusiana, Cardamine leucantha, Cardamine lyrata, Cardamine niigatensis, Cardamine nipponica, Cardamine pratensis, Cardamine regeliana, Cardamine schinziana, Cardamine scutata, Cardamine tanakae, Cardamine torrentis, Cardamine valida, etc. Among these, Cardamine hirsuta (Michitanetsukibana) is preferred.

[0044] The Lamiaceae plant is not particularly limited, and examples thereof include the genus Perilla, the genus Lavender, the genus Bacillus, the genus Violaceus, the genus Teak, the genus Citrine, the genus Bacillus, the genus Ceruleanus, the genus Calendula, the genus Lycoris, the genus Malvaceae, the genus Spiraea, the genus Lamiaceae, the genus Lycoris, the genus Acanthus, the genus Lycoris, the genus Malvaceae, the genus Lycoris ... Examples of plants include plants belonging to the genera Corbicula, Lamium, Lamium amplexicaule, Thymus vulgare, Lysosmodium, Lamiaceae, Amplexicaule, Mesquite, Moniliforme, Ophiopogon, and Horehound. Among these, the genera Perilla, Lavandula, Caprifoliaceae, Lamiaceae, Leucanthemum vulgare, Zostera spp., Anemone spp., Melastoma spp., Oregano, Mint, Mentha spp., Ophiopogon spp., Salix spp., Prunella spp., Stye, Mentha spp., Common Mint, Monarda spp., Mentha spp., Brugmansia spp., Rosemary spp., Thymus spp., Montana spp., Mint spp., and Ocimum spp. are preferred, with the genera Perilla and Lavandula spp. being more preferred, and the genus Perilla being even more preferred.

[0045] Plants belonging to the genus Perilla are not particularly limited, and examples thereof include Perilla frutescens (e.g., perilla, perilla, etc.). Among these, Perilla frutescens is preferred, and perilla is more preferred.

[0046] Examples of plants belonging to the genus Lavender include, but are not limited to, Lavandula angustifolia (lavender), Lavandula latifolia, Lavandula stoechas, Lavandula multifida, and Lavandula × intermedia. Lavender) is preferred.

[0047] The Orobanchaceae plant is not particularly limited, and examples thereof include the genus Orobanchaceae, the genus Castilleja, the genus Orthocarpus, the genus Agalinis, the genus Aureolaria, the genus Esterhazya, the genus Seymeria, the genus Lamourouxia, the genus Cordylanthus, the genus Triphysaria, the genus Onyx, the genus Onyx, and the genus Hamabanchaceae. Examples of such plants include plants belonging to the genus Moray, Ophiopogon, Rattus, Oryza sativa, Scutellaria, Gardenia, Artemisia, Pedicularis, Artemisia, Striga, etc. Among these, plants belonging to the genus Pedicularis, Castilleja, Orthocarpus, Agalinis, Aureolaria, Esterhazya, Seymeria, Lamourouxia, Cordylanthus, Triphysaria, etc. are preferred, and the genus Pedicularis is more preferred.

[0048] Examples of plants belonging to the genus Phtheirospermum include, but are not limited to, Phtheirospermum japonicum, Phtheirospermum glandulosum, Phtheirospermum muliense, Phtheirospermum parishii, and Phtheirospermum tenuisectum. , Phtheirospermum japonicum (Koshiogama) is preferred.

[0049] Cellulase may have a mutation (e.g., substitution, deletion, insertion, addition, etc.) in the amino acid sequence so long as it has graft improvement activity. From this viewpoint, cellulase may be a protein consisting of an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more identity to the amino acid sequence of wild-type cellulase, and having graft improvement activity. Alternatively, from the same viewpoint, cellulase may be a protein consisting of one or more (e.g., 2 to 20, preferably 2 to 10, more preferably 2 to 5) amino acids in the amino acid sequence of wild-type cellulase. , more preferably 2 to 3, and even more preferably 2) amino acids are substituted, deleted, or added. or a protein having an amino acid sequence with an inserted (preferably conservative substitution) amino acid sequence and having graft improvement activity.

[0050] The presence or absence of graft improvement activity can be evaluated using an in vitro grafting test system such as Test Examples 3, 4, and 6 described below.

[0051] Specific examples of cellulases include proteins described in the following (i) or (ii): (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4, or (ii) a protein having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or 4 and having graft improvement activity; It is.

[0052] The protein consisting of the amino acid sequence shown in SEQ ID NO: 3 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 are both derived from Nicotiana benthamiana. Glycosyl hydrolase 9.

[0053] For the protein (ii) above, the degree of identity to the amino acid sequence shown in SEQ ID NO: 3 or 4 is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0054] The protein of (ii) above is preferably a protein of (iia) below: (iia) a protein having an amino acid sequence in which one or more amino acids have been mutated (e.g., substitution, deletion, addition, insertion, etc., preferably substitution, more preferably conservative substitution) with respect to the amino acid sequence shown in SEQ ID NO: 3 or 4, and having graft improvement activity; It is.

[0055] With respect to the protein (iia) above, the term "plural" refers to, for example, 2 to 20, preferably 2 to 10 More preferably, it is 2 to 5, even more preferably 2 to 3, and even more preferably 2.

[0056] The component A may be one type alone or a combination of two or more types.

[0057] 2-2. About component B The B component may be an amino acid sequence as shown in SEQ ID NO: 1 or 2, or an One or several amino acids are substituted, deleted, added or inserted in the amino acid sequence to be and having graft improving activity.

[0058] The term "several" refers to, for example, 2 to 3, preferably 2. The number of amino acids to be modified is, for example, 1 to 3, preferably 1 to 2, and more preferably 1.

[0059] The amino acid mutation is preferably a substitution, more preferably a conservative substitution.

[0060] The graft improvement activity is the same as that described for component A above.

[0061] The length of the peptide that is the B component is limited to the general length of an oligopeptide. The length is not particularly limited. For example, the length is 30 amino acid residues or less, preferably 20 amino acid residues or less, more preferably 15 amino acid residues or less, and even more preferably 12 amino acid residues or less. The length is, for example, 8 amino acid residues or more, preferably 9 amino acid residues or more, and more preferably 10 amino acid residues or more. The length ranges from, for example, 8 to 30 amino acids. The amino acid residue length is preferably 9 to 20 amino acid residues, more preferably 10 to 15 amino acid residues, and It is preferably 10 to 12 amino acid residues in length.

[0062] The component B is preferably endogenous to the plant. More preferably, the component B is endogenous to a plant having high interfamily grafting ability, such as a Solanaceae, a Brassicaceae plant, a Labiatae plant, or an Orobanchaceae plant. The explanation of the Solanaceae, the Brassicaceae, the Labiatae, and the Orobanchaceae plants is the same as that of the component A.

[0063] The component B may be one type alone or a combination of two or more types.

[0064] 2-3.About component C In the present invention, "phenolamides" as component C refer to a group of compounds in which an acyl group containing phenol (which may have two or more hydroxy groups or may have one or more substituents other than hydroxy groups (e.g., alkoxy groups)) is linked to a (poly)amine residue via one or more amide bonds. It is known that in plants, the acyl group of phenylpropanoids is transferred (bonded) to a polyamine to produce phenolamides (also known as hydroxycinnamic acid amides (HCAAs)). The phenolamides used in the present invention are The phenolamides include the naturally occurring phenolamides present in such plants.

[0065] An example of the phenolamide that can be used in the present invention is represented by the following formula (I).

[0066] [ka]

[0067] In the formula, R 1 is an n1-valent amine residue, where n1 is a number from 1 to 10. Preferably, n1 is 1 to 7, 1 to 5, 1 to 4, or 1 to 3. n2 is 0 to 5. R 2 represents a substituent (for example, a C1-5 alkoxy group), n3 is a number from 1 to 5, n4 is a number from 0 to 4, and the sum of n3 and n4 is 5 or less. When n1 is 2 or more, n2, n3, n4 and R 2may be the same or different from each other. When n2 is 2 or more, the hydrocarbon chain having n2 carbon atoms may contain a double bond.

[0068] n2 is preferably an integer of 1 to 4, n3 is preferably an integer of 1 to 2, and n4 is preferably an integer of 0 to 2.

[0069] R 1 The n1-valent amine residue represented by the formula (I) is bonded to C=O in the above formula through N. That is, the compound of formula (I) has n1 amide bonds in the molecule. More specifically, R 1 means a residue obtained by removing n1 hydrogen atoms bonded to one or more N atoms contained in a (poly)amine.

[0070] R 1 The n1-valent amine residue represented by the formula: is a polyamine residue having two or more amines. It is preferably a residue of a polyamine containing two or more primary amines, two or more secondary amines, or a combination of one or more pairs of primary amines and secondary amines. Of these, the residue of a polyamine having two or more primary amines and one or more secondary amines is preferred.

[0071] R 1 is a (poly)amine residue of C1 to C50 (preferably C2 to C40 or C2 to C30). is preferred. 1 Examples of the C1 to C50 hydrocarbon group include linear, branched, and cyclic hydrocarbon groups. Also, any of the above hydrocarbon groups may be used. The hydrocarbon group may also contain one or more double bonds or triple bonds. In addition, one or more of the above-mentioned hydrocarbon groups may contain an aromatic ring (such as a benzene ring). One of the carbon atoms in the carbon-carbon bond may be replaced by a heteroatom (such as a nitrogen atom or an oxygen atom), e.g., -C=N- or -CO-, or one or more hydrogen atoms bonded to the carbon may be , may be substituted with a substituent (for example, a hydroxyl group or a C1 to C5 alkoxy group).

[0072] R 1 Specific examples of R include residues obtained by removing n1 hydrogen atoms bonded to nitrogen atoms of agmatine, putrescine, spermidine, spermine, and thermospermine. However, R is not limited to these examples. 1 A preferred example of the polyamide is a polyamide having four or more amino groups. A methylamine residue, for example the residue of spermine.

[0073] Examples of the n1 acyl groups contained in the compound represented by formula (I) include the following acyl groups: In addition, it is known that natural phenolamides present in plants have, for example, 1 to 3, 1 to 2, or 2 of the following acyl groups.

[0074] [ka]

[0075] In the present invention, the term "polyamine" as component C refers to a group of compounds having two or more amino groups and one or more hydrocarbon groups located between them. The meaning of the hydrocarbon group, and Preferred examples thereof are the same as the C1 to C50 hydrocarbon groups contained in R1.

[0076] In addition, when the polyamine has two or more hydrocarbon groups located between two amino groups, the two or more hydrocarbon groups may be the same or different. The amino group contained therein may be any of primary, secondary and tertiary amino groups. It may be a combination.

[0077] Examples of polyamines as C components include agmatine, putrescine, spermidine, and spermidine. The polyamines include, but are not limited to, spermine and thermospermine. The polyamines preferably have four or more amino groups, and for example, spermine is preferred. .

[0078] The phenolamides (and phenylpropanoids that can become the acyl groups that constitute them) and polyamines are preferably endogenous to plants with high interfamily grafting ability, such as Solanaceae, Cruciferae, Lamiaceae, and Orobanchaceae plants. For Solanaceae, Cruciferae, Lamiaceae, and Orobanchaceae plants, the explanation for the above-mentioned component A is given. It is the same as Ming.

[0079] The component C may be one type alone or a combination of two or more types.

[0080] 2-4.D component The promoter for expression or production of component A is not particularly limited as long as it is capable of promoting the expression or production of component A. Examples of the promoter for expression or production of component A include an expression cassette for component A (cellulase), an expression cassette for a transcription activator of the component A (cellulase) gene, and a transcription repressor of the component A (cellulase) gene. Expression or function inhibitors (e.g., antibodies, siRNA expression cassettes, CRISPR / Cas system expression cassettes) Examples include

[0081] The expression or production promoter of component B is not particularly limited as long as it is capable of promoting the expression or production of component B. Examples of the promoter include an expression cassette for component B (peptide), an expression cassette for a transcription activator of the component B (peptide) gene, and an expression cassette for a transcription repressor of the component B (peptide) gene. or functional inhibitors (e.g. antibodies, siRNA expression cassettes, CRISPR / Cas system expression cassettes, etc.) ) etc.

[0082] The C component expression or production promoter is not particularly limited as long as it is capable of promoting the expression or production of the C component, and examples thereof include expression cassettes for C component biosynthesis-related genes, C component biosynthesis-related factors, and the like. Examples of biosynthesis-related genes include MYB family genes (e.g., NaMYB8, SIMYB14, etc.) that are master regulators of phenolamide biosynthesis. In addition to these, various enzyme genes involved in the biosynthesis and metabolism of polyamines and phenylpropanoids, which are precursors of phenolamides (e.g., arginase, ornithine decarboxylase, arginine decarboxylase, spermidine synthase, spermine synthase, 4-coumarate-CoA ligase, coumarate 3-hydroxylase, O-methyltransferase, ... Examples of the biosynthesis-related factors include jasmonic acid and the like.

[0083] The expression cassette is capable of expressing a desired protein or peptide (such as biosynthetic genes for component A, component B, or component C) in a plant or in a microorganism or virus viable in a plant. There is no particular limitation as long as it is a polynucleotide. A typical example of an expression cassette is a polynucleotide comprising a promoter and a coding sequence for a desired protein or peptide placed under the control of the promoter.

[0084] The promoter contained in the expression cassette is not particularly limited, and for example, RPS5A (ribo somal protein) promoter, UBQ (ubiquitin) promoter, ACT (actin) promoter, tubulin promoter, histone promoter, 35S promoter, NOS promoter, WOX promoter, LAT52 promoter, DUO promoter, plant virus Examples include subgenomic promoters.

[0085] The expression cassette may contain other base sequences in addition to those described above, such as a drug resistance gene, a reporter protein coding sequence, and their expression cassettes, a transcription termination signal, a protein tag coding sequence, a signal sequence coding sequence, a replication origin, and each element sequence of a binary vector used in the Agrobacterium method (e.g., a left border region, a right border region, etc.).

[0086] The expression cassette may constitute a vector. The type of vector is not particularly limited, and examples thereof include plasmid vectors; Agrobacterium vectors; tobacco mosaic virus, cucumber mosaic virus, African cassava mosaic virus, apple latent spherical virus, barley stripe mosaic virus, bean pod mottle virus, beet curly top virus, brome mosaic virus, cabbage leaf curl virus, cotton leaf crumple virus, cymbidium mosaic virus, grape A virus, pea early browning virus, poplar mosaic virus, potato virus X, rice tungro bacilliform virus, satella virus, and the like. Tobacco mosaic virus, Tobacco curly shoot virus, Tobacco rattle virus, etc. Plant virus vectors and the like. In addition, like the above vectors, In addition to vectors suitable for introduction into cells, examples of such vectors include vectors for transferring an expression cassette into such vectors (eg, Gateway (registered trademark) entry clone vector, etc.).

[0087] The component D may be one type alone or a combination of two or more types.

[0088] 2-5. Uses and other ingredients The grafting improvement agent of the present invention is selected from the group consisting of component A, component B, component C, and component DS. The grafting improvement agent of the present invention contains at least one of the following active ingredients: For example, it can be used to improve grafting efficiency, improve grafting success rate, improve graft compatibility, improve graft establishment rate, and reduce grafting failure rate.

[0089] In one embodiment of the present invention, the graft improvement agent of the present invention can be used for same-family grafts or different-family grafts.

[0090] The target plants of the graft improvement agent of the present invention are not particularly limited. For example, the agent can be widely applied to plants such as angiosperms (dicotyledons, monocotyledons, etc.), gymnosperms, and ferns.

[0091] From the viewpoint of plant classification, target plants include, for example, Malvaceae, Brassicaceae, Asteraceae, Salicaceae, Ranunculaceae, Lauraceae, Cranberryaceae, Houttuyniaceae, Araceae, Lamiaceae, Violetaceae, Apiaceae, Buxaceae, Ericaceae, Polygonaceae, Amaranthaceae, Convolvulaceae, Rosaceae, Santalaceae, Capranaceae, Geraniaceae, Vitaceae, Fagaceae, Caprifoliaceae, Diplocarponaceae, Fabaceae, Rutaceae, Sapindaceae, Proteaceae, Saxifragaceae, Apocynaceae, Gentianaceae, Dryopteris, Cupressaceae, Cucurbitaceae, Solanaceae, Pedunculaceae, Plantaginaceae, Orobanchaceae, Moniliaceae, Caprifoliaceae, Caliceraceae, Cesarean, Menyantheae, Examples of plants that may be used include plants belonging to the Styridiaceae family, Polygalaceae family, Surianaceae family, Muntingiaceae family, Quillinaceae family, Dipterocarpaceae family, Sarcolaenaceae family, Cistanaceae family, Baptaceae family, Sphaerocepalaceae family, Tetramelaceae family, Begoniaceae family, Dutiscaceae family, Berberidaceae family, Menispermaceae family, Hybridae family, Stilbaceae family, Scrophulariaceae family, Brachypodium spp., Bupleuraceae family, Thujoraceae family, Piperaceae family, Didymelaceae family, Brusselaceae family, Bulberry family, Solanaceae family, Polypodiaceae family, Cinnamon ...

[0092] From the viewpoint of another classification, for example, eggplants such as tomatoes, green peppers, chili peppers, eggplants, etc., melons such as cucumbers, pumpkins, melons, watermelons, etc., vegetables such as cabbage, broccoli, Chinese cabbage, etc., fresh or spicy vegetables such as celery, parsley, lettuce, etc., onions such as green onions, onions, garlic, etc., beans such as soybeans, peanuts, green beans, peas, adzuki beans, etc., other fruit vegetables such as strawberries, taproots such as radishes, turnips, carrots, burdock, etc., tubers such as taro, cassava, potato, sweet potato, Chinese yam, asparagus, spinach, etc. and soft lettuce such as mitsuba (Japanese laurel wreath), flowers such as lisianthus, stock, carnation, chrysanthemum, cereals such as rice, wheat, barley, oats, corn, turf grasses such as bentgrass and zoysiagrass, oil crops such as rapeseed and peanut, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, forage crops such as clover, sorghum, dent corn, deciduous fruit trees such as apples, pears, grapes, peaches, citrus fruits such as satsuma mandarins, lemons, grapefruit, and woody plants such as azalea, azalea, and cedar.

[0093] Furthermore, from the viewpoint of another classification, the graft improvement agent of the present invention can be suitably used in grafting of vegetables of the Solanaceae family, such as tomato, eggplant, green pepper, paprika, etc., and vegetables of the Cucurbitaceae family, such as cucumber, watermelon, melon, pumpkin, etc. However, as mentioned above, the target includes not only vegetables, but also woody plants such as rose family fruit trees.

[0094] The grafting improvement agent of the present invention may consist of only the active ingredient, but may also contain various other ingredients in addition to the active ingredient depending on the type of active ingredient contained, the dosage form described below, the application mode, etc. The content ratio of the active ingredient (dry weight) in the grafting improvement agent can be appropriately determined depending on the dosage form described below, the application mode, etc., and can be, for example, in the range of 0.0001 to 100 mass%. Other components include, for example, carriers, adhesives, dispersants, and auxiliary agents. Carriers include, for example, solid carriers such as talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, vermiculite, and silica sand; and liquid carriers such as water-soluble polymer compounds (polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, and the like), water, vegetable oil, and liquid animal oil. Adhesives include, for example, casein, gelatin, gum arabic, and alginic acid. Dispersants include, for example, alcohol sulfates, polyoxyethylene glycol ether, and the like. Auxiliaries include, for example, carboxymethylcellulose, starch, and lactose. The above carriers, adhesives, dispersants, and auxiliary agents can be used alone or in appropriate combination according to their respective purposes.

[0095] The formulation of the grafting improvement agent of the present invention is not particularly limited as long as it is an agriculturally acceptable formulation. For example, granules, 1 kilo granules, 500 gram granules, 250 gram granules, jumbo granules, etc.; powder; Examples of suitable formulations include powders such as DL powder, FD powder, etc.; powders such as granular powder, fine granular powder F, fine granular powder F, etc.; powders; hydrated formulations such as hydrated powder, flowable sol SC, granular hydrated powder, dry flowable, etc.; hydrated formulations such as water-soluble powder, water-soluble granular powder, etc.; emulsions such as emulsifiable concentrates, EW formulations, etc.; liquid formulations such as EW formulations, etc.; oil formulations such as oil agents, surf agents, etc.; aerosols; pastes; microcapsules; packs, etc.

[0096] By applying the grafting improvement agent of the present invention to a plant, it is possible to produce a plant partial structure for grafting that has improved grafting properties with other plants, and it is also possible to produce grafted plants more efficiently and improve grafting efficiency.

[0097] More specifically, for example, a plant substructure for grafting having improved graftability with other plants can be produced by a method comprising applying the grafting improvement agent of the present invention to a plant body or a plant substructure. Also, a grafted plant can be produced more efficiently by a method comprising applying the grafting improvement agent of the present invention to a plant body after grafting and / or grafting the plant substructure for grafting with another plant substructure.

[0098] A plant body includes all of the plant tissues, and is not particularly limited thereto. A plant substructure includes a plant body from which a portion of tissue / site has been removed, and is not particularly limited thereto.

[0099] The plant partial structure for grafting is not particularly limited as long as it is a plant partial structure that can be grafted. For example, it may be a structure consisting of the whole or a part of the underground part, a structure consisting of the whole or a part of the underground part and the whole or a part of the aboveground part, or a structure consisting of the whole or a part of the aboveground part. In this specification, the underground part is not particularly limited as long as it is a structure that can exist buried underground, and examples of such include roots and rhizomes. In this specification, the aboveground part is not particularly limited as long as it is a structure that can exist exposed above ground, and examples of such include stems, trunks, branches, petioles, leaves, buds, flowers, etc.

[0100] The shape of the plant partial structure for grafting is not particularly limited as long as it is a shape that allows grafting. From the viewpoint of enabling more efficient grafting, it is preferable that the plant partial structure for grafting has a surface suitable for grafting in a part thereof. The surface suitable for grafting may have various known shapes, for example, a flat shape, a V-shaped concave shape, a protruding convex shape, etc., so long as good contact is obtained as necessary. Examples of cut surfaces include:

[0101] In the present invention, grafting can be performed according to or in accordance with a known method. In the present invention, grafting typically includes a step of cutting any part of a plant (for example, any part on a stem) to obtain two or more plant substructures for grafting (for example, a rootstock, an intermediate rootstock, a scion, etc.). The grafting can be carried out by a method including a step (step 1), and a step (step 2) of cultivating the cut surfaces of the grafting part plant structure in contact with each other.

[0102] In step 1, the cut surfaces of the plant substructure for grafting have a larger contact area with each other. For example, a cut of about 1 to 2 cm is preferably made in the center of one of the cut surfaces. These processes include cutting the cut surface into a V-shape, and then cutting the cut surface into a V-shape. This can be done, for example, using a single-edged razor.

[0103] In step 2, the manner in which the cut surfaces are brought into contact with each other is not particularly limited. It is advisable to do this gently so as not to damage the structural components.

[0104] In step 2, after contact, the contact area is fixed so that it does not move as much as possible. Fixation can be achieved, for example, by wrapping the contact site with a sheet-like material such as parafilm.

[0105] It is desirable to keep the entire above-ground plant part structure for grafting (e.g., scion, intermediate stock, etc.) moist in the initial stage of cultivation in step 2 (e.g., 3 to 14 days after contact). Moisture retention can be achieved, for example, by covering the entire above-ground plant part structure for grafting with a plastic bag sprayed with water.

[0106] When moisture is applied during the initial stage of cultivation in step 2, it is preferable to cultivate without moisture application during the later stage of cultivation (for example, within 30 days after contact).

[0107] When three plant substructures for grafting are to be grafted, first two plant substructures for grafting are grafted according to the above steps 1 and 2, and then the plant substructure for grafting obtained from the obtained plant body is grafted with the third plant substructure for grafting. It is recommended to graft four or more plant substructures for grafting. When grafting, it is preferable to use a similar method.

[0108] The application is not particularly limited as long as the active ingredient can reach the grafting surface or the part that will become the grafting surface in the future. The application mode is not particularly limited as long as it is a known mode (or a mode to be developed in the future) as a mode of use of an agricultural chemical. For example, spraying, dripping, coating, mixing or dissolving in the plant growth environment (in soil, water, solid medium, liquid medium, etc.) can be mentioned. More specifically, for example, the mode of adding to a hydroponic solution, the mode of spraying on the soil surface, the mode of mixing with fertilizer, etc. can be mentioned. In addition, a preferred mode includes spraying, dripping, coating, etc. on the grafting surface or the grafting part.

[0109] Furthermore, when the active ingredient is a polynucleotide such as an expression cassette, its introduction into a plant is not particularly limited and can be carried out according to or in accordance with a known method.

[0110] The first example of the introduction method (Introduction Example 1) is a promoter (e.g., T7 promoter, T3 promoter, 35S promoter, etc.) and a promoter containing a sequence containing an expression cassette downstream of the promoter. The method can be carried out by a method including a step of preparing a plasmid (step a1), a step of obtaining genomic RNA of a plant virus from the plasmid obtained in step a1 by in vitro transcription (step b1), and a step of inoculating a plant with the genomic RNA (active ingredient) obtained in step b1 (for example, friction inoculation method, particle gun inoculation method, etc.) (step c1). Alternatively, the plasmid obtained in step a1 may be a Ti plasmid containing a promoter capable of activating transcription in plant cells, such as the 35S promoter. In the case of a plant that is a plant-derived plant, instead of the above steps b1 and c1, the method can be performed by a method including, for example, a step (step b2) of introducing the plasmid obtained in step a1 into Agrobacteria and culturing the same, and a step (step c2) of inoculating the culture solution obtained in step b2 (culture solution containing an active ingredient) into a plant (for example, infiltration method, toothpick inoculation method, suction injection method, etc.). Alternatively, instead of the above steps b1 and c1, the method can be performed by a method including, for example, a step (step c3) of inoculating the plasmid (active ingredient) obtained in step a1 into a plant (for example, grinding inoculation method, particle gun inoculation method, etc.). Alternatively, instead of the above step c2, the method can be performed by a method including, for example, a step (step c4) of performing the leaf disk method, inflorescence infiltration method, reduced pressure filtration method, etc. From the genomic RNA, plasmid, T-DNA, etc. introduced into the plant by these methods, the desired protein or peptide is produced.

[0111] As a second example of the introduction method (Introduction Example 2), (obtained, for example, by the above Introduction Example 1) An example of such a method includes a step (step d1) of collecting a plant virus from a plant containing the virus, and a step (step e1) of inoculating a plant with the virus collected in step d1 (a virus containing an active ingredient). The collection in step d1 can be carried out, for example, by grinding a part of a plant containing the plant virus (e.g., a leaf, etc.) to collect a virus liquid. The inoculation in step e1 can be carried out, for example, by using an abrasive such as silicon carbide to make a wound in the part of the plant to be inoculated (e.g., a leaf, etc.) and bringing the virus into contact with the wound.

[0112] 3. Partial plant structure for grafting, grafted plant body In one aspect, the present invention relates to a method for producing a plant substructure for grafting, which comprises applying the grafting adhesive of the present invention to a plant body or a plant substructure, and to a plant substructure for grafting obtained by said method.

[0113] In one aspect, the present invention relates to a method for producing a grafted plant, which comprises applying the graft improvement agent of the present invention to a plant body after grafting and / or grafting the plant part structure for grafting of the present invention with another plant part structure, and to the grafted plant obtained by the method.

[0114] For details on each term and specific aspects of the method, please refer to "2-5. Use and other ingredients" above. This is as explained in the explanation. EXAMPLES

[0115] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0116] Test Example 1. Evaluation of the grafting improvement effect of cellulase 1 Cellulase gene knockdown in Nicotiana benthamiana (scion) ) was grafted onto Arabidopsis thaliana (rootstock), and the degree of graft success was evaluated.

[0117] To investigate whether the cellulase gene contributes to the establishment of grafts, a virus-induced gene silencing (VIGS) construct was created by introducing a portion of the Niben101Scf01184g16001 gene (a 300 bp sequence from -154 to +156, with the A of ATG counted as +1), a cellulase gene, into a virus vector, and subjected to a knockdown test of the cellulase gene. As a negative control, a portion of the GFP gene was also introduced into the virus vector. A construct was created and used as the control group in the experiment.

[0118] In vitro RNA transcription was performed using the constructs targeting the cellulase gene or GFP gene as templates, and the transcribed RNA was inoculated onto the leaves of Nicotiana benthamiana plants grown about one month after germination to grow the viruses for knockdown tests.

[0119] Infected leaves of Nicotiana benthamiana where virus proliferation was confirmed were collected and the homogenate was prepared. The homogenate was then inoculated onto the leaves of another Nicotiana benthamiana plant that had been cultivated for about one month after germination to cause secondary infection with the virus, and these plants were then used in the grafting experiment.

[0120] One week after the virus inoculation, the Nicotiana benthamiana individual was used as a scion and grafted onto an Arabidopsis rootstock. The grafting was performed by making a 1.5 cm cut into the flower stalk of the Arabidopsis rootstock, inserting a V-shaped Nicotiana benthamiana stem into the cut, and fixing the two together. To prevent this, the grafting site was covered with Parafilm. Immediately after grafting, the scion was covered with a plastic bag that had been sprayed with water on the inside, and kept in this state for one week until the scion took root, if any, to prevent drying of the scion due to evaporation of water. At this time, non-inoculated Nicotiana benthamiana was also grafted in the same manner. , was designated as the target area.

[0121] The success of the grafting in each test plot was evaluated by checking whether the scion was alive or not two weeks after grafting. Checked the rate.

[0122] The knockdown of cellulase genes by VIGS was carried out on the stems of ungrafted Nicotiana benthamiana plants, on the grafting site of non-virus-inoculated Nicotiana benthamiana plants grafted onto Arabidopsis thaliana, and on the grafted sites of non-virus-inoculated Nicotiana benthamiana plants grafted onto Arabidopsis thaliana plants .... The grafting site of Arabidopsis thaliana was grafted onto Nicotiana benthamiana inoculated with The cellulase gene and actin gene were examined by qRT-PCR. qRT-PCR was performed using the primers 5'-GGCCAATCGAGAAAAGATGAC-3' (SEQ ID NO: 5) and 5'-AACTGTGTGGCTGACACCATC-3' (SEQ ID NO: 6) for amplifying the cellulase gene, and 5'-ATTGGGAGCCAATGATGTACC-3' (SEQ ID NO: 7) and 5'-TGTCATTTCCAACAACGCTTC-3' (SEQ ID NO: 8) for amplifying the actin gene.

[0123] The results are shown in Figure 1. It was found that knocking down the cellulase gene made grafting impossible. The decrease in graft viability correlated with the level of decrease in the expression amount of the cellulase gene in each experimental group, and it was also shown that the cellulase gene is an essential gene causally linked to interfamily grafting of Nicotiana plants. These findings demonstrated that cellulase has the effect of improving graft viability.

[0124] Test Example 2. Evaluation of the grafting improvement effect of cellulase 2 In the same manner as in Test Example 1, a grafting test was carried out between Nicotiana genus plants and Arabidopsis thaliana, and the frequency of necrotic layer regions was measured. Specifically, the following procedure was carried out.

[0125] Six grafts were performed for each grafting combination, and three manual sections were prepared from each. Each section had four areas of adhesion around the vascular bundles, and if there was an area of ​​adhesion of 100 μm or more (an area without necrotic layer) in each area, it was considered to be adhesion. In this way, for each of the six grafts, 3 sections x 4 survey areas = 12 areas were examined, and the results were compiled as data. The percentage of the adherent area in which no necrotic layer was formed was calculated.

[0126] The results are shown in Figure 2. It was found that knocking down the cellulase gene increased the frequency of necrotic layer formation at the grafting site, indicating that cellulase suppresses the formation of necrotic layer at the grafting site.

[0127] Test Example 3. Evaluation of the grafting improvement effect of cellulase 3 The effect of cellulase administration on grafting success was evaluated using an in vitro grafting test system. Specifically, the procedure was as follows.

[0128] In vitro grafting was performed using stems of Nicotiana benthamiana that had been cultivated for about one month after sowing. First, the main stem was prepared by removing the leaves, and then two pieces of cut stems were prepared by slicing them to a thickness of about 1 mm. These two pieces were placed on top of each other and buried in microcells that had been prepared in advance. The microcells were prepared by drilling holes of about 2 mm x 4 mm in polydimethylsiloxane resin that was about 4 mm thick. One set of two cut stem pieces was buried in one microcell. The two cut stem pieces could be accommodated in a microcell. The resin was placed on a nylon membrane on a plant growth agar medium (half strength Murashige and Skoog medium, 1% agar), with one side of the grafted part placed on the membrane. They were left in this state and cultured for 4-5 days. After culture, the two pieces of tissue were removed from the microcell while being pinched with tweezers on the outside to prevent them from coming apart. One side of the two removed cut stem pieces was then held down and the other side was tried to be pulled apart with the tweezers, and it was checked whether it came off easily or whether there was resistance and it could not be easily removed. Pieces that did not come off easily were judged to have been attached. This is the in vitro grafting test system.

[0129] Cellulase administration was performed by sprinkling a small amount of purified cellulase powder on one cut surface of a piece of cut stem, then overlapping the cut surface of the other cut stem to create a microcellulase. The tissue was then embedded in a graft and cultured, and an in vitro grafting test was performed.

[0130] The results are shown in Figure 3. It was found that administration of cellulase increased the grafting success rate.

[0131] Test Example 4. Evaluation of the grafting improvement effect of peptides The grafting improvement effect of the peptide (Grf006) consisting of the amino acid sequence (MVTDHVKNAT) of SEQ ID NO: 1 and the peptide (Grf010) consisting of the amino acid sequence (MALIVQMISA) of SEQ ID NO: 2 was evaluated in an in vitro grafting test system.

[0132] The various peptides to be tested were added at a concentration of 10 μM. The various peptides to be tested (Grf001, Grf002, Grf003, Grf004, Grf006, Grf010, Grf011, Grf020, Grf024) were adjusted to a concentration of 10 mM with dimethyl sulfoxide (DMSO) to prepare stock solutions. The stock solutions were diluted 1000-fold and dissolved in agar medium for plant growth used in the in vitro grafting test system. Culture media containing peptides at a concentration of 10 μM was prepared for each type of peptide. As a control, culture media containing DMSO diluted 1000 times was also prepared and used for the test. In addition, a non-additive medium was used as a negative control, and a medium containing artificial auxin 2,4-D at a concentration of 0.5 μM, which is known to enhance the adhesion effect of grafting, was also used for the test as a positive control. In vitro grafting tests were performed using these media.

[0133] The results are shown in Figure 4. It was found that Grf006 and Grf010 have a grafting improvement effect, and that this effect is as strong as that of auxin.

[0134] Test Example 5. Analysis of the effect of phenolamide biosynthetic pathway on grafting In Nicotiana attenuata, NaMYB8 regulates phenolamide biosynthesis. is known as the master regulator (Kaur et al., Plant Physiology, March 2010, Vol. 152, pp. 1731-1747, Onkokesung et al., Plant Physiology, January 2012, Vol. 158, pp. 389-407). In this experiment, SIMYB14, an ortholog of NaMYB8, was knocked down by the RNAi method in wild tomato plants to produce an RNAi line (2-4-1). The expression level of the SIMYB14 gene and the accumulation level of metabolites (phenolamides) in this 2-4-1 line were compared with those of the wild type, and the effect on grafting was evaluated.

[0135] <Production of 2-4-1 and 2-1-1 lines by the RNAi method> A partial sequence (323 bp) of the SIMYB14 gene was used as the RNAi trigger sequence, and a transformation vector (pANDA) that expresses the RNAi trigger sequence under the ubiquitin promoter was transformed into the cotyledons of tomato 'Ailsa Craig' by the Agrobacterium method. The obtained transformed shoots were rooted to obtain transformants (RNAi line (2-4-1)). Non-transformed individuals obtained during the process of producing transformants (i.e., individuals with the same traits as wild-type tomatoes) were used as Escape individuals (2-1-1 line) in the following experiments. <Measurement of SIMYB14 gene expression level>

[0136] Total RNA was extracted from wild-type plants, and the 2-4-1 and 2-1-1 lines produced above, respectively. Reverse transcription reaction was performed to obtain cDNA. Using the obtained cDNA as a template, primers for amplifying the partial length of SIMYB14 were used, and the expression level of the SIMYB14 gene was measured by quantitative PCR.

[0137] ​​The results are shown in Figure 5. From the results shown in Figure 5, it can be seen that in the 2-4-1 mutant, the expression of the SIMYB14 gene is significantly suppressed.

[0138] <Metabolite identification and quantification> A tube containing a frozen powder sample of SlMYB14RNAi (2-4-4) and one zirconia bead Add 5 μl of 80% methanol (containing 2.5 μM lidocaine as an internal standard) per 1 mg fresh weight. ) was added and extracted by stirring in a mixer mill. The tube was centrifuged to precipitate the residue, and the supernatant was transferred to a new tube to obtain an extract.

[0139] The wild type and Escape 2-1-1 were also extracted in the same manner as above. A sap was obtained.

[0140] Using each of these extracts, metabolites were identified and quantified by LC-MS / MS.

[0141] 100 μl of the extract was dispensed into a vial equipped with a glass insert for LC-MS / MS analysis.

[0142] For non-targeted metabolomic analysis, we performed MS and MS / MS analysis in positive and negative ion modes, using 1 μl of the extract for each analysis, using a Waters LC-QTOF / MS mass spectrometer (LC, Waters Acquity UPLC System; MS, Waters Xevo G2 Q-Tof).

[0143] The analysis conditions are as follows. Liquid chromatography (LC) section: column, Acquity bridged ethyl hybrid C18 (1.7 μm, 2.1 mm × 100 mm, Waters); solvent system: solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid); concentration gradient: 99.5% solvent A / 0.5% solvent B until 0 min, 99.5%A / 0.5%B until 0.1 min, 20%A / 80%B until 10 min, 0.5%A / 99.5%B until 10.1 min, 0.5%A / 99.5%B until 12.0 min, 99.5%A / 0.5%B until 12.1 min, 99.5%A / 0.5%B until 15.0 min; flow rate: 0.3 ml / min until 0 min, 0.3 ml / min until 10 min, 0.3 ml / min up to 10.1 min, 0.3 ml / ml up to 14.4 min, 0.3 ml / min up to 14.5 min: Column temperature: 40 degrees; Mass spectrometry (MS) section: polar, positive positive and negative ion modes; capillary voltage, +3.00 kV (positive) / -2.75 kV (negative); cone voltage: 25.0 V; ion source temperature: 120 °C; Solvation temperature: 450 °C; gas flow rate in the cone: 50 l / h; desolvation gas flow rate: 800 l / h; collision energy: 6 V; mass analysis range: m / z 50-1,500; scan duration: 0.1 s; inter-scan delay: 0.014 s; data acquisition method: centroid mode; rock spray (leucine enkephalin); scan duration: 1.0 s; inter-scan delay: 0.1 s.

[0144] MS / MS data was acquired as follows. (1) MS: polarity, positive and negative ion mode; mass analysis range, m / z 50-1,500 ;Scan duration, 0.1 s;Interscan delay, 0.014 s;Data acquisition method: Sen Toroid mode. (2) MS / MS: polarity, positive and negative ion mode; mass analysis range, m / z 50-1,500; scan duration, 0.02 s; inter-scan delay, 0.014 s. Data acquisition method: Android mode.

[0145] Several types of phenolamides were detected as metabolites, and their structures were determined based on LC-MS / MS data using standard substances, as necessary.

[0146] The MS / MS data of the above three types of extracts were collated to identify the phenolamides, and their quantitative values ​​were compared. The results are shown in Table 1. From the results shown in Table 1, it can be seen that the signal intensity of phenolamides is significantly reduced in the 2-4-4 form.

[0147] [Table 1]

[0148] As an example, Figure 6 shows the structure and quantitative analysis of phenolamides N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N19, N20, N21, N22, N23, N24, N25, N26, N27, N28, N2 10 The quantitative values ​​of -bisdihydrocaffeoyl spermidine are shown in the graph.

[0149] <Evaluation of the universality of phenolamides in Solanaceae plants> Next, the universality of phenolamides in solanaceae plants was evaluated using various varieties of solanaceae plants as samples.

[0150] Each solanaceae plant was divided into an untreated area (A) and a wound-treated area (B). , and sampled two days later. A was sampled in the same manner without any treatment. Metabolomic analysis was performed on each of the collected samples. The preparation of the samples for metabolomic analysis and the measurement conditions for LC-MS / MS were the same as those for the above-mentioned 2-4-1 and 2-4-2. The measurement conditions were the same as those for the phenolamides detected in each sample. The structures of the phenolamides were determined based on the LC-MS / MS data using standard substances as necessary.

[0151] Comparison of the untreated (A) and wounded (B) plots revealed that a group of phenolamides is universally present in Solanaceae plants and is wound-responsive. Examples of wound-induced phenolamides in Solanaceae include N1, N2, and N3. 10 The results for -bisdihydrocaffeoyl spermidine are shown in Figure 7. The name of the Solanaceae plant from which the sample was taken is written on the horizontal axis. "_A" indicates the non-treated area (A). "_B" indicates samples taken from the wound treatment area (B). This indicates that.

[0152] <Evaluation of induction of phenolamides in plants other than the Solanaceae family> Grafted plants of N. benthamiana (Arabidopsis thaliana) and A. thaliana (tobacco) were prepared, as well as wounded scions and rootstocks of N. benthamiana (Arabidopsis thaliana). The plants were grown for 28 days after grafting, and samples were taken 2 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, and 28 days later, and the metabolic rate was measured in the same manner as above. In addition, the wounded scions and rootstocks of Arabidopsis thaliana were grown for 7 days, and the structures of the phenolamides were determined and quantified after 2 hours, 1 day, 3 days, 5 days, and 6 days. After 12 days and 7 days later, samples were taken and metabolome analysis was performed in the same manner as above. The structures of the phenolamides were determined and quantified. The results were compared with the metabolome analysis results of samples (reference examples) taken from untreated Arabidopsis thaliana and tobacco, respectively, and it was confirmed that the amount of phenolamides clearly changed over time compared to the reference examples.

[0153] <Evaluation of impact on grafting> The wild type, and the 2-4-1 and 2-1-1 plants produced above were each self-grafted (the cut stem was grafted onto the original stem) and survival (growth of the scion) was evaluated.

[0154] The results are shown in Figure 8. The numbers under each photograph in Figure 8 indicate "number of successful grafts / number of grafts." The success of grafting was judged by visually observing the growth of the leaves of the scion and comparing it with the wild type. From the results shown in Figure 8, it can be seen that in the 2-4-4 plant, in which the amount of phenolamides was significantly reduced, the grafting It can be understood that the success rate of the grafts was significantly reduced. This indicates that the phenolamide biosynthetic pathway and phenolamides play an important role in the success of grafts.

[0155] Test Example 6. Evaluation of the grafting improvement effect of polyamines The effect of intermediates (polyamines) in the phenolamide biosynthetic pathway on grafting was investigated in vitro. The grafting test system was used to evaluate the effect of the grafting. Specifically, the test was performed as follows.

[0156] A pea stalk of 5 mm length was inserted into a mold made of PDMS (polydimethylsiloxane) resin, and the stem was cut into two pieces. The PDMS resin mold with the stem still inserted was then soaked in a solvent containing polyamines. The stem pieces were placed on MS medium containing no grafting agent (control), and the adhesion of the stem pieces after 3 to 5 days was evaluated to determine the grafting survival rate.

[0157] The results are shown in Table 2. It was found that polyamines, which are intermediates in the phenolamide biosynthetic pathway, have a grafting improving effect.

[0158]

Table 2

Claims

1. (C) a polyamine, and (D) Expression cassettes for MYB family genes Contains at least one selected from the group consisting of A graft improvement agent, wherein the polyamine is a compound consisting of two or more amino groups and one or more hydrocarbon groups located between them.

2. The graft improvement agent according to claim 1, which is used for interfamily grafts or intrafamily grafts.

3. The graft improvement agent according to claim 1 or 2, wherein the polyamine is a polyamine containing four or more amino groups.

4. The graft improvement agent according to any one of claims 1 to 3, wherein (C) is spermine.

5. A method for producing a plant substructure for grafting, which comprises applying the graft improvement agent according to any one of claims 1 to 4 to a plant body or a plant substructure.

6. A plant substructure for grafting obtained by the method according to claim 5.

7. A method for producing a grafted plant body, comprising applying the graft improvement agent according to any one of claims 1 to 4 to a plant body after grafting, and / or grafting the plant part structure for grafting according to claim 5 with another plant part structure.

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