JP6533037B2 - Flowering control method of citrus - Google Patents

Description

  BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a fruit set management technique aimed at stable annual production of citrus fruits. More specifically, the present invention relates to a flowering control technique focusing on abscisic acid acceptance signal in a developmental branch of a citrus plant and a fruit control technique using the same.

・ A problem of the biennial result of citrus Citrus fruit is a fruit tree with a strong biennial result, repeating good harvest and poor crop. The problem of this biennial result is a phenomenon that is caused because the fruiting conditions of the present year affect the number of flowers of the next year.
Therefore, in a citrus cultivation site, it is important to stably produce high-quality fruits over successive years by suppressing alternate annual fruiting.

In order to avoid the problem of biennial fruiting in citrus fruits, it is essential to properly manage the number of fruits, but in actual production sites, it is necessary to manually attach flowers by manual flower cutting and picking operations. Currently, the control of the number of fruits is being performed.
The labor of such farmers requires a lot of labor, and there is a problem that the burden on the production site is too large. This problem is a serious factor that impedes the expansion of the scale of business for citrus production and farmers.

Therefore, from the viewpoint of alleviating the burden of such human work, the development of citrus fruit picking agents using chemical substances is carried out, and the fruit pickers such as ethychlozate emulsion and 1-naphthaleneacetic acid sodium water solvent are put to practical use .
However, there is a risk that management technology using a 'fruit removal agent' causes excessive fruit loss due to environmental conditions such as high temperature, resulting in significant damage to production.
In addition, since management by a fruiting agent is a long-term treatment that is performed to avoid suppression of the decrease in fruiting amount in the next year, direct and efficient management control at earlier stages becomes possible. Control technology of quantity has been required at production sites.

-Conventional flowering control technology Based on the problems of the citrus cultivation site as described above, Gibberellin (GA) is currently registered as a pesticide as a drug that can be used for flowering control technology of citrus fruits.
When excessive flowering is expected, it is used to maintain flowering by suppressing flowering amount by spraying gibberellin during flower bud differentiation. This makes it possible to manage the fruiting amount.
However, as a fundamental problem of the gibberellin scattering technology, only the control for suppressing the amount of flowering can be directly controlled by gibberellin, there is a problem that "the control for increasing the amount of flowering" can not be directly performed. .

About the FT gene The FT (Flowing Locus T) gene is known as a flowering induction gene that promotes flowering (flowering induction and flowering), and in Arabidopsis thaliana, research on flowering mechanisms related to the FT gene is active. It has been done. "CiFT (Citrus FT) gene", which is a homologous gene of the FT gene, has also been reported from citrus plants, and is known to be involved in the induction of flowering (see non-patent documents 1 and 2).
However, in order to induce CiFT gene expression in citrus plants, it is necessary to perform forced expression by the gene introduction method, but establishment of the gene introduction method is not sufficient for various citrus plant species. In addition, when a gene is introduced into a citrus (tree) in a usual cultivation site, it is necessary to replace the currently grown tree with a transgenic individual for cultivation. In addition, harvested fruits are transgenic crops, which are not consistent with consumers' natural preferences.
Therefore, an effective means for inducing the expression of the CiFT gene at a citrus cultivation site to promote flowering is not easy to introduce the method itself and can not be said to be a more realistic method.

  Also, a method has been reported in which the CiFT protein itself prepared as a recombinant protein is artificially administered from outside the plant instead of the endogenous expression increase of the CiFT gene (see Patent Document 1: WO 2011/059051). The said technology is a report of the experimental stage and has not yet come to practical use. In addition, artificial preparation of FT protein requires preparation using recombinant protein technology, and therefore, it is recognized that there are problems in terms of manufacturing cost, storage management of products, and the like.

Thus, at present, as a technology that can be applied at a citrus cultivation site, a technology that directly and efficiently increases the amount of flowering has not been established.

WO 2011/059051 (flowering inducer) JP 2013-515501 gazette (suppression every other year)

Endo et al. (2005); Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.); Transgenic Research 14: p703-712 Nishikawa et al. (2007); Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.); Journal of Experimental Botany, Vol. 58, No. 14, p3915-3927

  An object of the present invention is to solve the above-mentioned problems and to provide a direct and efficient novel flowering control technique which can be applied at a citrus cultivation site. Specifically, the present invention is a novel flowering that enables direct acceleration control of flowering (direct acceleration control, not indirect flower amount maintenance by maintaining a tree trend) in addition to suppression control of flowering. It aims to provide control technology.

  As a result of intensive studies, the present inventors in Citrus plants increase the abscisic acid acceptance signal of the developing branch to induce the expression of the CiFT gene and promote the induction of flowering and flowering (flowering). I found it possible. On the other hand, by reducing the abscisic acid acceptance signal of the developmental branch, it was found that the expression of the CiFT gene was not induced and that flowering induction and flowering (flowering) could be suppressed.

The present invention is conceived based on the above-mentioned findings newly found in citrus plants.
That is, the invention according to [Claim 1] controls the number of flowering of plants belonging to the genus Citrus using any of the means described in the following (A) or (B): The present invention relates to a method for controlling the flowering of citrus which is characterized.
(A): A means for controlling the increase in flowering number by increasing the abscisic acid acceptance signal of the developing branch.
(B): A means for controlling the decrease in the number of flowers by decreasing or eliminating the abscisic acid receiving signal of the developing branch.
Moreover, the invention which concerns on [Claim 2] controls a flowering number with respect to the plant which belongs to a citrus genus (Citrus) using either means as described in the following (C) or (D). The present invention relates to a method for controlling the flowering of citrus which is characterized.
(C): A means for controlling the increase in the number of flowering by exogenous use of abscisic acid or abscisic acid receptor agonist on the developmental branch.
(D): A control means for decreasing the number of flowers by exogenous use of an abscisic acid receptor antagonist to the developmental branch.
Moreover, the invention which concerns on [Claim 3] controls a flowering number with respect to the plant which belongs to citrus genus (Citrus) using either means as described in the following (E) or (F). The present invention relates to a method for controlling the flowering of citrus which is characterized.
(E): Means for controlling the increase in the number of flowering by increasing the abscisic acid content of the developing branch.
(F): A control means for decreasing the number of flowering by decreasing the abscisic acid content of the developing branch.
Moreover, the invention which concerns on [Claim 4] controls a flowering number with respect to the plant which belongs to citrus genus (Citrus) using either means as described in the following (G) or (H). The present invention relates to a method for controlling the flowering of citrus which is characterized.
(G): A means for controlling the increase in the number of flowering by exogenous use of an abscisic acid decomposing enzyme inhibitor to the developmental branch.
(H): A control means for decreasing the number of flowers by exogenous use of an abscisic acid synthetase inhibitor to the developmental branch.
Moreover, the invention which concerns on [Claim 5] controls a flowering number with respect to the plant which belongs to a citrus genus (Citrus) using either means as described in the following (I) or (J). The present invention relates to a method for controlling the flowering of citrus which is characterized.
(I): A means for controlling the increase in the number of flowers by exogenous use of an abscisic acid-8'-hydroxylase inhibitor to the developmental branch.
(J): A control means for decreasing the number of flowers by exogenous use of 9-cis-epoxycarotenoid dioxygenase inhibitor in the developmental branch.
Moreover, the invention which concerns on [Claim 6] is abscissin of the growth branch in a citrus plant characterized by substantially cultivating a plant belonging to a citrus genus (Citrus) under a temperature condition of 4 to 20 ° C. It relates to a method for improving the acid content.
The invention according to [claim 7] is characterized in that it comprises abscisic acid, abscisic acid receptor agonist, or a compound having abscisic acid-8'-hydroxylase inhibitory activity as an active ingredient. And a flowering promoter of a plant belonging to the genus Citrus.
The invention according to [Claim 8] is characterized by comprising an abscisic acid receptor antagonist or a compound having a 9-cis-epoxycarotenoid dioxygenase inhibitory activity as an active ingredient. The present invention relates to an agent for suppressing flowering of a plant belonging to (Citrus).
The invention according to [claim 9] is characterized by using the method according to any one of claims 1 to 5 for a plant belonging to the genus Citrus, wherein the fruit set of citrus fruit is characterized. It relates to a control method.

Patent Document 2 Here, Patent Document 2 (Japanese Patent Application Publication No. 2013-515501) will be described. Patent Document 2 describes a method for suppressing biennial fruiting in perennial crops by increasing the flowering degree using nucleic acid-related natural metabolites such as 'adenosine etc.'. Here, as perennial crops, there are mosses, mosses, walnuts, pistachios, citrus (listing such as navel oranges), avocados, wild moths, peaches, plums, nectarines, cherries, plums , Apricots, peaches (* duplicate description), pears, pecans, apples, mangoes, and almonds are listed (see Patent Document 2, paragraph [0032]).

However, in the outline of the invention of Patent Document 2, “in 'Mandarin (a kind of citrus),' accumulation 'of abscisic acid or the like causes sprouting suppression” (paragraph [0039]); “abscisic acid or the like When the ratio (to cytokinin) is 'decreased', the flowering pathway gene is also affected, and the flowering degree (flower shoot and number of flowers) is increased ”(paragraph [0039]);“ ratio of abscisic acid etc. That 'increase' (to cytokinin) suppresses sprouting ”(paragraph [0040]);“ 'accumulation' of abscisic acid etc. (and reduction of cytokinin concentration) causes suppression of spring flower bud sprouting (Paragraph 0042) and the like are described.
These descriptions are the findings completely opposite to the features of the present invention with respect to plant hormones related to flower bud formation of citrus species. That is, in the patent document 2, the "inhibition reason" for creativity such as "the abscisic acid content increase in citrus growth branches directly promotes flowering induction", which is a technical feature of the present invention, is the whole text. Is clearly stated.
Therefore, Reference Document 2 is recognized as a publication that can not be a cited document that denies the novelty and inventive step of the present invention.

The present invention provides a direct and efficient novel flowering control technology that can be applied at a citrus cultivation site. Specifically, the present invention is a novel flowering that enables direct acceleration control of flowering (direct acceleration control, not indirect flower amount maintenance by maintaining a tree trend) in addition to suppression control of flowering. Provide control technology.

The results of evaluation of the relationship between the ABA content in the stem and the CiFT gene expression level and the number of flowering of the seedlings when the low-temperature cultivation treatment of Satsuma mandarin oranges was conducted in fiscal 2011 in Test Example 1 are shown. . (A) The figure which measured the time-dependent change of the ABA content in a stem, and the CiFT gene expression amount after low-temperature cultivation start. (B) After low temperature cultivation start, a sapling is collect | recovered, and it is a constant temperature cultivation at 25 degreeC, and the figure which shows the time-dependent change of the number of flowerings per node where germination was observed.

The results of evaluation of the relationship between ABA content in the stem and CiFT gene expression level and the number of flowering of the seedlings when experimenting with low temperature cultivation treatment of Satsuma mandarin orange in 2013 are shown in Test Example 2 . (A) The figure which measured the time-dependent change of the ABA content in a stem, and the CiFT gene expression amount after low-temperature cultivation start. (B) After low temperature cultivation start, a sapling is collect | recovered, and it is a constant temperature cultivation at 25 degreeC, and the figure which shows the time-dependent change of the number of flowerings per node where germination was observed. (C) After low-temperature cultivation starts, seedlings are collected and temperature-cultivated at 25 ° C., and the temporal change in the number of flowering per node on the whole spring branch of the seedlings is shown.

The results of evaluation of the relationship between the ABA content in the stem and the amount of CiFT gene expression and the number of planted seedlings when the low-temperature cultivation treatment was performed on the completely defoliated Satsuma mandarin saplings in Test Example 3 are shown in FIG. . (A) The figure which measured the time-dependent change of the ABA content in a stem, and the CiFT gene expression amount after low-temperature cultivation start. (B) After low temperature cultivation start, a sapling is collect | recovered, and it is a constant temperature cultivation at 25 degreeC, and the figure which shows the time-dependent change of the number of flowerings per node where germination was observed.

(A) The results of examining the correlation between the “ABA content in stem” and the “CiFT gene expression level in stem” in Test Example 4 are shown in the diagram. (B) The results of examining the correlation between "the content of ABA in the stem" and "the number of flowerings per sprouting node when the seedlings are cultured at 25 ° C under constant temperature" in Test Example 4 are shown in the figure.

The result of having measured the expression level of CiFT gene in the stem after an exogenous ABA spraying process in Experiment 5 is shown in a figure. (A) Result of analysis of CiFT gene expression in stem. (B) Results of analysis of CiFT gene expression in leaves.

In Experiment 6, it is a figure of a result of expression analysis of an enzyme gene involved in ABA metabolism of Satsuma Mandarin seedlings. (A) The result of having measured the time-dependent change of ABA content in a stem after low temperature cultivation start. (B) The figure which shows the result of having measured the time-dependent change of the ABA content in a stalk after low temperature cultivation start with respect to the seedlings which carried out whole leaf removal. (C) The figure which measured the time-dependent change of NCED3 gene expression amount in a stem after low temperature cultivation start, and the figure which shows it. (D) The figure which shows the result of having measured the time-dependent change of NCED3 gene expression amount in a stem after low temperature cultivation start with respect to the sapling which carried out whole leaf removal. (E) The figure which shows the result of having measured the time-dependent change of A8OX gene expression amount in a stem after low temperature cultivation start. (F) The figure which shows the result of measuring the time-dependent change of the A8OX gene expression amount in a stem after low temperature cultivation start with respect to the sapling which carried out whole leaf removal.

It is the figure which showed the outline of the flowering mechanism of the citrus species which flowering induction was induced by low temperature which became clear from the result of this test example.

It is the figure which showed the outline of the flower induction period in a season in the citrus family plant of citrus genus and its relative genus. (A) Citrus genus. (B) Kinkan. (C) Genus Genus.

It is a photographic image figure which image | photographed the branch of the sapling of citrus genus (Satsuma mandarin orange). 1. Developmental branch (branch in developmental stage). 2. Germinated leafy flowers. 3. Germinated leaf buds.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to a flowering control technique and a fruit setting control technique focusing on an abscisic acid acceptance signal in a developmental branch of a citrus plant.

[Citrus plants]
Citrus plants to which the technology according to the present invention is applied specifically refer to plants belonging to the genus Citrus.
Here, "Citrus (aka Citrus genus)" is a genus of citrus fruits belonging to the family Ritaceae, and is a taxonomic group consisting of a group of species branched from a single common ancestor in evolution.
Species belonging to the genus genus Citrus are sometimes classified as 'species', but there are many cases where the distribution as a cultivar has been expanded by vegetative reproduction from a mutagenized single tree. Genetic differences often occur only at the level of variation and breed. In recent research comparing genotypes, it is strongly suggested that only about four citrus species are native species, and most other species are hybrids.
In this respect, citrus genus is a taxonomic group which is relatively easy for interspecific hybrid (crossing) and grafting, and is a taxonomic group in which genetic background (basic property as a plant species) is composed of species close to each other. is there.

Citrus fruits are often an important agricultural resource for food and food. The characteristic of the fruit is that the inside of the peel is a structure in which the sachets filled with pulp are arranged in a row.
Plants of the genus Citrus are evergreen shrubs (trees) and do not fall off leaves. It originates from the tropics to the subtropics and has properties suitable for cultivation in warm regions.

In addition, among the species belonging to the genus Citrus, a particularly important feature is the property that inducing flowering of trees is induced by experiencing 'low temperature'. This property is known to be a common phenomenon in many citrus species such as Satsuma mandarin orange, lime and the like.
Given the closeness of genetics among species belonging to the genus Citrus, Citrus has a common floral induction mechanism that "flowering is induced by low temperature cultivation", and it is generally accepted in the art that It is recognized and reported in a comprehensive book on citrus (Spiegel-Roy, P. and Goldschmidt, EE (1996). Biology of Citrus. Cambridge University Press, New York).

On the other hand, Citrusaceae plants such as Fortunella and Poncirus are considered to be relatively close to citrus species, but induction of flowers occurs under high temperature conditions (early summer) (Nishikawa et al. al. (2011), Bull. Natl. Inst. Fruit Tree Sci. 12: p27-32).
Specifically, in Kumquat, flower bud induction occurs in 'early summer' and flower buds flower in summer. In addition, flower bud induction occurs in 'early summer' in trifoliate orange, but the formed flower buds flower in the next spring over winter (see FIG. 8).
Therefore, it is recognized that there is a flowering induction mechanism by induction control different from that of citrus in Citrus plants such as Quercus and Trifoliata.
The above findings are considered to indicate that the "upstream control mechanism of low temperature flowering induction system" in Citrus genus is a unique flowering control mechanism in citrus species.

As a species belonging to Citrus (Citrus), according to the classification of Nagasaburo Tanaka, it is said that classification of around 160 is possible, but in recent studies comparing genotypes, these citrus Most species are genetically related, and the theory is that they are born by hybridization. In particular, it is recognized that the species considered to be true citrus subgenus are closely related.
In any case, they are closely related so that they are capable of hybridizing to each other, and are not considered to be classified and all are considered to be plants belonging to the genus Citrus.

  Specifically, Satsuma mandarin orange, Ponkan, mandarin orange, tachibana, pomegranate mandarin orange, cherry zami mandarin orange, karamanshi, color mandarin, mandarin orange, Valencia orange, navel orange, navel orange, sour orange, blood orange, clementine, grapefruit, orange, orange , Lemon, lime, bergamot, sudachi, kabosu, sambokan, shikuwasa (shikuwasha, lice lemon), yuzu, hanayu, daidai, citron, bush can, nut cane, hassatsu, hyuga natsu, deukopon, cocktail fruit, sweetie, jabala, kinot, yoyokan , Tangan, tangor, seminol, tan zero, buntan, etc. are all belonging to the genus Citrus.

[Flow control by abscisic acid (ABA)]
The present invention is a flowering control technology focusing on abscisic acid acceptance signals in the developmental branch of citrus plants.

  Here, “abscisic acid” is a type of plant hormone denoted as ABA (abscisic acid). Also known as abscisic acid or abscisic acid. The CAS registration number is 21293-29-8. Structurally, it belongs to sesquiterpenes. The structural formula of the compound is shown in the following formula (i).

Abscisic acid functions from the research of many plant species including trees such as birch and maple etc. including model plants such as Arabidopsis thaliana so far, and induction of plant dormancy, growth suppression and germination suppression by stimulation such as low temperature drying It is known that it is a function to induce and the like.
Although there are few reports on flower bud formation, for example, Patent Document 2 describes that accumulation of abscisic acid 'suppresses' flower bud formation.

The flowering control technology of the present invention is a control technology based on a novel finding that is completely contrary to the findings from known plant species so far reported. Specifically, the present inventors elucidated that "in the case of citrus plants, the expression of the FT gene is induced and the induction of flowering (flowering) is promoted by increasing the abscisic acid acceptance signal". This technology was developed focusing on the flowering induction mechanism (see Figure 7).
The phenomenon of abscisic acid receptive signal in citrus plants has not been reported at all from other plants, as well as from Arabidopsis thaliana and rice, for which molecular level flowering mechanisms have been extensively studied. Based on this point, it is recognized that the phenomenon is a unique phenomenon in citrus species.

[Control means of the number of flowering]
In the present invention, attention is directed to the abscisic acid acceptance signal of the developmental branch, thereby promoting or suppressing flowering and controlling the number of flowering. This makes it possible to directly control the increase and decrease in the number of citrus flowers.

FT gene The control technology according to the present invention is a technology achieved through endogenous FT gene expression control. The abscisic acid acceptance signal of the present invention needs to be controlled in the developmental branch (developmental branch). The increase in abscisic acid acceptance signal in the developmental branch induces the expression of the FT gene in stem cells and promotes the induction of flowering.

Here, the “FT (Flowering locus T) gene” is a gene responsible for induction of flowering found in the study of the flowering mechanism in Arabidopsis thaliana (Kobayashi et al. (1999); Science 286 p1960-1962).
In citrus, there are homologous genes of three kinds of FT genes with extremely high sequence similarities of CiFT1 to 3. These common ancestor genes and the FT gene of Arabidopsis thaliana have an orthologous relationship with each other. All three types of genes of CiFT1 to 3 exert a function to promote flowering induction of whole citrus trees (Non-patent document 1: Endo et al. 2005, Non-patent document 2: Nishikawa et al. 2007).

The mechanism of induction of flowering in the whole citrus tree (plant body) can be explained by FT gene research in Arabidopsis thaliana. Specifically, after 'FT protein' is produced by the expression of 'FT gene' in stem and leaf vasculature cells, the FT protein moves through the vascular bundle throughout the plant body and is apical bud or lateral bud. It is thought that flower induction is performed in cooperation with proteins of other flower induction genes (FD protein) (Abe el al. (2005); Science 309 (5737): 1052-1056, etc.).
The finding of Arabidopsis thaliana is consistent with the finding that flowering is induced in the entire citrus tree (plant body) after the expression of the CiFT gene in the citrus developmental branch is increased. In this respect, it is recognized that a molecular mechanism common to Arabidopsis thaliana exists in citrus plants for the control of flowering 'from downstream of the FT gene'.

-Target plant body Further, according to the technique, an individual is juvenile phase (5 years or more, preferably 8 years or more, more preferably 10 years or more, still more preferably 12 years or more, particularly preferably 15 years or more). Apply to trees in the mature phase. Even if the technology is applied at an early age stage, floral induction does not occur unless the individual has reached the mature phase.
In addition, the technology can also be applied to the saplings of citrus fruits obtained by grafting a branch in mature stage to a stock.

Regulation of the abscisic acid receptor signal in the art is preferably performed at the developmental branch. This is because when the abscisic acid acceptance signal in the developmental branch is increased, it is possible to increase 'FT gene expression' and induce flowering induction of the whole tree.
Here, "developmental branch" refers to a branch including vascular phloem cells (tissue in which the FT gene is highly expressed) in a developmental stage (growth and growth stage).
In particular, it is preferable that it is a one-year-old vegetative branch during germination and growth from dormant sprouts. (※ Here, “1 year-old vegetative branch” is a horticultural term that indicates that the shoot is a branch of the first year during germination and development. From the next year onwards, the next branch from the branch concerned The buds germinate, not a branch that withers in one year.)
Also, in order to efficiently induce FT gene expression, it is desirable that the branches be after sprouting and elongation, and in a developmental stage. Particularly preferably, a branch which is sprouted after cold dormancy and in which the elongated branch (i.e., the spring branch) hardens and is in a developmental stage is preferred.

Exogenous Use In addition, as a means for realizing the control in the present invention, a means of exogenously using the target substance may be adopted.
Here, "use exogenously" refers to an action of administering an exogenous substance (from outside the plant to which the substance is to be applied) to a plant and using it.
Specifically, it refers to an action of absorbing and ingesting a target substance in a citrus plant to be applied. Here, the act of absorbing and ingesting refers to the act of ingesting the liquid containing the target substance into the plant body by absorption from the surface (or a damaged part) such as stems, leaves, and roots.

(1) As a specific example, there can be mentioned, for example, an act of artificially spraying, spraying, spraying, applying, etc. a target substance. In the action, a liquid containing a target substance is formed into fine droplets and attached to the surface of a plant by an operation such as spraying. In this case, the target substance can be taken into the plant from the surface of the plant.
(2) In addition, use the absorbent cotton, cloth, sponge, etc. containing the liquid containing the target substance to also act to absorb and ingest the liquid containing the target substance from the surface (or the damaged part) of the plant. Can. In addition, it is also preferable to scratch the surface of the plant (with a razor or the like) to more positively introduce a solution containing the target substance into the plant.
(3) In addition, it is also possible to carry out continuous uptake while maintaining a long-term contact from the plant surface by using a gelling agent, a viscosity agent, a disintegrant and the like.
(4) In addition, it can be used to impregnate a liquid containing the target substance into absorbent cotton, cloth, sponge or the like and apply it to the surface of a plant. In this case, by using together with the abrasive, it is possible to positively take in from the wound of the plant surface.
(5) In addition, it is also possible to cite an action of injecting a solution containing the target substance into the plant body more positively by using a syringe needle or the like and taking in the solution.
(6) It is also possible to take up the target substance by water absorption from the root.

  The exogenous use act can be performed on any part of the plant body, but preferably, it is absorbed as it is by the act directly performed on the branch (growth branch) at the above developmental stage. It is preferable to be ingested.

  In addition, it is effective to carry out the exogenous use continuously or continuously. For example, it is preferable that the solution be sprayed every several hours to several days, or that the plants be ingested and absorbed in such a manner that they can be absorbed continuously.

[Means for increasing abscisic acid acceptance signal]
In the present invention, the means specifically shown below can be used as a means for increasing the abscisic acid receptor signal (means for enhancing, means for increasing) in the control technique.

(1) Exogenous use of abscisic acid In the control technique according to the present invention, it is possible to artificially increase the abscisic acid receptive signal during the developmental branch by using abscisic acid exogenously .
The exogenous use makes it possible to incorporate abscisic acid into tissues and cells of the developmental branch to artificially increase the abscisic acid receptive signal.
The increased receptor signal strongly induces the expression of the FT gene and promotes the induction of flowering.

  In this means, it is preferable to use natural S-(+)-abscisic acid as the 'abscisic acid' (the compound of the above formula (i)), although it may contain optical isomers. good. Also, they can be used as their salts.

In the use of abscisic acid, it may be used at such a concentration that an adverse effect on plants (trees, fruits, etc.) is not generated and a desired effect can be expected. For example, it is desirable to use an aqueous solution prepared to have a concentration of abscisic acid of 1 μM to 10 mM, preferably 10 μM to 5 mM, more preferably 50 μM to 2 mM, more preferably 100 to 1 mM, and particularly preferably 200 to 800 mM.
The solution is preferably prepared as an aqueous solution, but it is also possible to include low concentration salts, buffer components and the like.

(2) Exogenous Use of Abscisic Acid Receptor Agonist In the control technology according to the present invention, the abscisic acid receptor signal during developmental branch can be obtained by using abscisic acid receptor agonist (activator) exogenously. It is possible to artificially increase.
The exogenous use makes it possible to incorporate the agonist into tissues and cells of the developmental branch to artificially increase the abscisic acid receptive signal.
The increased receptor signal strongly induces the expression of the FT gene and promotes the induction of flowering.

  Here, as the 'abscisic acid receptor agonist' (agonist), it binds to "PYR (Pyrabactin resistance) protein and / or its homologous protein group," which is the abscisic acid receptor (receptor), to receive abscisic acid Compounds that function to increase the signal can be suitably used.

As such a compound, specifically, 'a derivative of abscisic acid' which exerts the same physiological function as abscisic acid (action to induce an abscisic acid receiving signal) can be used. The said abscisic acid derivative is what substituted the one functional group of abscisic acid by the other functional group, Comprising: The thing by which the physiological function of abscisic acid is ensured can be mentioned.
As the abscisic acid derivative, there can be mentioned specifically a derivative having a structure capable of avoiding the degradation of ABA-8′-hydroxylase to improve the persistence of the ABA receptor signal. Specifically, 8'-methoxylated abscisic acid, 8'-alkylated abscisic acid, 8'-acetylated abscisic acid, 8'-methoxyalkylated abscisic acid, 8'-methoxyacetylated abscisic acid, 8'- Examples thereof include fluorinated abscisic acid and the like (see, for example, JP-A-2000-502107 and JP-A-7-300443). Here, it is preferable that the alkylation or acetylation be a lower one having 2 or less carbon atoms (methylation, ethylation, methylenation, ethylation).
Other examples include 2 ', 3'-dihydroxylated abscisic acid, 4'-deoxy abscisic acid, and the like.
These derivatives preferably have the same stereoisomeric relationship of optical isomers as natural abscisic acid, but may contain those optical isomers. Moreover, it is also possible to use the salt for the said abscisic acid derivative.

Moreover, as an abscisic acid agonist, the compound which does not have an analogous structure with abscisic acid can also be used suitably. For example, quinabactin (Quinabactin), pyrabactin (Pyrabactin), tetralone abscisic acid (tetralone abscisic acid), etc. can be used. In addition, derivatives of these compounds (having some of the functional groups of the compound replaced with other functional groups), which have similar physiological functions, can also be used.
In particular, it is preferable to use quinabactin from the viewpoint of the effect of the working property and the non-degradable property (Okamoto et al., 2013, PNAS vol. 110 no. 29, p12132-12137).
Moreover, these compounds can also use the salt.

In the use of these agonist compounds, they may be used at such a concentration that an adverse effect on plants (trees, fruits, etc.) is not generated and a desired effect can be expected. For example, it can be prepared and used as an aqueous solution prepared to have a compound concentration of 0.1 μM to 1 mM, preferably 0.5 to 500 μM, more preferably 1 to 200 μM, and particularly preferably 10 to 100 μM.
The solution is preferably prepared as an aqueous solution, but it is also possible to include low concentration salts, buffer components and the like.

(3) Exogenous use of abscisic acid decomposing enzyme inhibitor In the control technology according to the present invention, the abscisic acid receptive signal during developmental branch is artificially used by using abscisic acid decomposing enzyme inhibitor exogenously It is possible to increase
The exogenous use makes it possible to incorporate the abscisic acidase inhibitor into the tissues and cells of the developmental branch to increase the endogenous abscisic acid content of the plant body, thereby abscisic acid receptive signal. Can be artificially increased.
The increased receptor signal strongly induces the expression of the FT gene and promotes the induction of flowering.

  As the 'abscisic acid decomposing enzyme inhibitor', any compound can be used as long as it is a compound having an activity of inhibiting an enzyme involved in the decomposition of abscisic acid in citrus. Specifically, a compound having an inhibitory action on abscisic acid-8'-hydroxylase (A8OX) directly involved in the decomposition of abscisic acid can be suitably used (see FIG. 7). .

Here, as a compound having the inhibitory action of abscisic acid-8'-hydroxylase (an active ingredient of ABA-8'-hydroxylase inhibitor), an azole-based P450 inhibitor can be used. (Note that this description does not exclude non-azoles.)
Specifically, there may be mentioned abcinazole E2B (Abscinazole-E2B), abcinazole E1 (Abscinazole-E1), abcinazole F1 (Abscinazole-F1), diniconazole (Diniconazole), uniconazole (Uniconazole), asymidol (Ancymidol), etc. . In addition, it is also possible to use a compound of these derivatives (one having some functional group of the compound substituted by another functional group), in which the same physiological function is secured.
In particular, abcinazole E2B (Japanese Patent Laid-Open No. 2013-231014), which is highly selective for ABA 8'-hydroxylase and less likely to adversely affect plant growth, abcinazole E1 (Okazaki et al. 2011, Bioorg. Med. Chem., vol. 19 1 p406-413), abcinazole F 1 (Todoroki et al. 2009, Bioorg. Med. Chem., Vol. 17 15 p 6620-6630)), etc. are preferably used. In particular, abcinazole E2B is preferred.

In the use of the inhibitor, it may be used at such a concentration that an adverse effect on plants (trees, fruits, etc.) is not generated and a desired effect can be expected. For example, it can be prepared and used as an aqueous solution prepared to have a compound concentration of 0.1 μM to 1 mM, preferably 0.5 to 500 μM, more preferably 1 to 200 μM, and particularly preferably 10 to 100 μM.
The solution is preferably prepared as an aqueous solution, but it is also possible to include low concentration salts, buffer components and the like.

(4) Low temperature cultivation treatment In the control technique according to the present invention, it is possible to artificially increase the endogenous abscisic acid content in the growing branch by low temperature cultivation of citrus plants. By carrying out the low temperature cultivation, the abscisic acid content of the developing branch becomes remarkably high, and it becomes possible to increase the abscisic acid acceptance signal.

The low temperature cultivation is matured after the individual passes juvenile phase (5 years or more, preferably 8 years or more, more preferably 10 years or more, still more preferably 12 years or more, particularly preferably 15 years or more). It is preferable to carry out on the trees in phase. In particular, it is preferable that the method is performed on an individual in a state in which the spring branch (branch expanded by germination after cold dormancy) hardens and is in a developmental stage.
In addition, the technology can also be applied to a sapling of a citrus in which a branch in a mature phase is grafted on a rootstock.
Moreover, it is preferable that the said low temperature cultivation is performed in the state which the tree of application object attached enough leaves.

Here, "low temperature cultivation" refers to cultivation at a temperature that promotes endogenous abscisic acid synthesis of citrus. Specifically, it may be cultivated so that the temperature of the top part of the citrus is in the following temperature range.
The upper limit of the low temperature is preferably 20 ° C. or less, preferably 19 ° C. or less, more preferably 18 ° C. or less, still more preferably 17 ° C. or less, particularly preferably 16 ° C. or less. On the other hand, the lower limit may be such that the plant does not cause cultivation problems, for example, 4 ° C. or more, preferably 5 ° C. or more, more preferably 6 ° C. or more, still more preferably 7 ° C. or more, particularly preferably Of 8 ° C. or more, more preferably 9 ° C. or more, more preferably 10 ° C. or more.
In the low temperature cultivation, if the cultivation of the temperature zone which belongs to the “substantially” temperature range is carried out, the effect of the low temperature cultivation within the range which belongs to the present invention is secured. For example, if the daily average temperature is a temperature within the range, the effect of low temperature cultivation is secured even if there is a time zone exceeding the range.

  The low temperature cultivation is performed for 5 days or more, preferably 10 days or more, more preferably 12 days or more, and further preferably 15 days or more, whereby the effect of promoting the abscisic acid content is exhibited. In addition, even if low temperature cultivation is interrupted for several days (for example, 3 days or less, preferably 2 days or less, more preferably 1 day or less) during the low temperature cultivation period, the cultivation time is integrated and the cultivation period concerned Low temperature cultivation can be expected to increase the abscisic acid content.

[Means for reducing abscisic acid acceptance signal]
In the present invention, the means specifically shown below can be used as a means for reducing the abscisic acid acceptance signal in the control technique. It is also possible to eliminate the receptor signal completely or temporarily by taking strong measures.

(1) Exogenous use of abscisic acid synthetase inhibitor In the control technique according to the present invention, the abscisic acid acceptor signal during development is artificially used by using abscisic acid synthetase inhibitor exogenously. It is possible to reduce
It is possible to artificially reduce the abscisic acid receptor signal by incorporating the abscisic acidase inhibitor into the tissues and cells of the developing branch and reducing the endogenous abscisic acid content of the plant body. . The decreased receptor signal suppresses the expression of the FT gene and suppresses the induction of flowering.

Here, as the 'abscisic acid synthetase inhibitor', any compound can be used as long as it is a compound having an action of inhibiting an enzyme involved in the synthesis of abscisic acid in citrus. Specifically, a compound having the inhibitory action of 9-cis-epoxycarotenoid dioxygenase (NCED, neoxanthine oxidative cleavage enzyme) involved in the rate-limiting step of abscisic acid synthesis system can be suitably used (see FIG. 7). ).
The NCED is an enzyme involved in the synthesis of xanthotoxin (ABA precursor) by cleaving two molecules of violaxanthin (carotenoid), and is a rate-limiting enzyme of abscisic acid biosynthetic system.

  Here, specific examples of the compound having an inhibitory action on 9-cis-epoxycarotenoid dioxygenase (an active ingredient of the NCED inhibitor) include nordihydroguayretic acid (NDGA) and abamine. be able to. In addition, it is also possible to use a compound of these derivatives (one having some functional group of the compound substituted by another functional group), in which the same physiological function is secured.

In the use of the compound, it may be used at such a concentration that an adverse effect on plants (trees, fruits, etc.) is not generated and a desired effect can be expected. For example, it can be prepared and used as an aqueous solution prepared to have a compound concentration of 0.1 μM to 1 mM, preferably 0.5 to 500 μM, more preferably 1 to 200 μM, and particularly preferably 10 to 100 μM.
The solution is preferably prepared as an aqueous solution, but it is also possible to include low concentration salts, buffer components and the like.

(2) Exogenous use of abscisic acid receptor antagonist In the control technology according to the present invention, the abscisic acid receptor signal in the developmental branch is obtained by using abscisic acid receptor antagonist (antagonist) exogenously. It is possible to reduce artificially.
The exogenous use makes it possible to incorporate the antagonist into tissues and cells of the developmental branch and artificially reduce the abscisic acid receptive signal.
The decreased receptor signal suppresses the expression of the FT gene and suppresses the induction of flowering.

  Here, as the 'abscisic acid receptor antagonist' (antagonist), it binds to "PYR (Pyrabactin resistance) protein and / or its homologous protein group," which is the abscisic acid receptor (receptor), to receive abscisic acid Functional compounds that reduce or eliminate the signal can be suitably used.

As such a compound, although it may be an abscisic acid derivative or a compound which is not structurally similar, an ASn compound shown in the following structural formula (ii) can be specifically mentioned. (* N in Structural Formula (ii) represents an integer of n = 5 to 12)
Here, the ASn compound is a kind of abscisic acid derivative. Preferably, AS5 to AS12 (Structural formula (ii) n = 5 to 12) in which the number of carbon atoms ahead of the sulfur atom of the 3 'side chain of the compound is 5 to 12 are preferably used as an abscisic acid receptor antagonist Can.
In particular, AS 6 to 10 (structural formula (ii) n = 6 to 10), further AS 6 to 8 (structural formula (ii) n = 6 to 8), and further AS 6 to 7 (structural formula (ii) n = 6 ~ 7), and further AS6 (Structural formula (ii) n = 6), are preferred (Takeuchi et al. Nature Chemical Biology 10, 477-482 (2014)).
The AS compound is preferably one having the same stereoisomer relationship of optical isomers as natural abscisic acid, but may contain the optical isomers. Moreover, it is also possible to use the salt for the said compound.

In the use of these antagonist compounds, they may be used at such a concentration that an adverse effect on plants (trees, fruits, etc.) is not caused and a desired effect can be expected. For example, it can be prepared and used as an aqueous solution prepared to have a compound concentration of 0.1 μM to 1 mM, preferably 0.5 to 500 μM, more preferably 1 to 200 μM, and particularly preferably 10 to 100 μM.
The solution is preferably prepared as an aqueous solution, but it is also possible to include low concentration salts, buffer components and the like.

[Flow control agent]
In the present invention, each compound that can be used for the above-mentioned "exogenous use" can be used as a flowering control agent containing each as an active ingredient.
Specifically, “abscisic acid”, “abscisic acid receptor agonist”, or “compound having abscisic acid degrading enzyme inhibitory activity” can be used as an active ingredient of a flowering promoter. Moreover, it can also be set as the mixing agent which contains two or more compounds which belong to these.
On the other hand, “abscisic acid receptor antagonist” and “compound having abscisic acid synthetase inhibitory activity” can be used as an active ingredient of a flowering inhibitor. Moreover, it can also be set as the mixing agent which contains two or more compounds which belong to these.

As the agent form, any form can be adopted as long as it is an agent form suitable for the above-mentioned 'exogenous use'. However, it is preferable that it is an agent form which can be put into a solution state at the time of final use.
For example, liquid agent forms such as liquid, ampoule, and concentrated liquid can be mentioned. In addition, solid or semi-solid forms such as powder, granules, clays, pastes, and the like can be mentioned.
Moreover, the form solidified by mixing with an excipient etc., the form filled with a capsule, etc. are employable.
Moreover, it is also possible to make it the form containing a gelling agent, a viscosity agent, a disintegrant, an emulsifier, a pigment, an antioxidant, a buffer component and the like.

[Control of fruit set, every year result control]
In the present invention, by using the above-mentioned flowering control means, it becomes possible to directly control the amount of fruit setting (the number of fruit setting) of the fruits which the tree to which the technology is applied carries.
Specifically, the control to reduce the number of flowering allows control to reduce the amount of fruit set on the fruit and maintain the vigor. In addition, by controlling the increase in the number of flowering, it is possible to control to directly increase the amount of fruit setting of fruits.

In the present invention, by directly controlling the fruiting amount using the flowering control technology, it becomes possible to solve the problem of the result of every two years of citrus and to realize the continuous annual production of citrus.

  Hereinafter, the present invention will be described by way of examples, but the scope of the present invention is not limited by these.

[Test Example 1] "Low temperature cultivation treatment experiment"
When low-temperature cultivation was applied to seedlings of Satsuma mandarin orange, the relationship between the ABA content in the stem and the amount of CiFT gene expression, and the number of flowering seedlings was evaluated.

・ 1-1) "Experimental procedure and conditions"
(1) Low temperature cultivation treatment (flowering induction treatment)
A year-round grafted tree seedling (planted individual with 10 or more side branches: 1 plant 3 x 5 samplings per sampling) of Satsuma mandarin orange (variety: Okitsu early life), 2011 after the spring branch hardens "Low temperature cultivation processing" was started on the 22nd. The low temperature treatment was carried out by transferring the seedlings to a glass room capable of natural light, and cultivating them under a constant temperature condition adjusted to a room temperature of 15 ° C. (※ Citrus plants to which Satsuma mandarin orange (C. unshiu) belongs flowering is induced by cultivating at a low temperature of about 15 ° C.)

(2) Sampling Three seedlings were collected every 0.5 months from the start of the low temperature treatment. Then, as samples for RNA extraction and ABA content measurement, 2 to 3 side branches are collected and cut out from each seedling in total, side branches for a total of 3 seedlings are collected together, frozen with liquid nitrogen, and crushed at -80 ° C. Saved with.

(3) Evaluation of 25 ° C. constant temperature cultivation treatment (flower bud formation) and flowering inducibility All three seedlings of the seedlings after cutting the side branches are defoliated (all the leaves are picked) under constant temperature conditions of room temperature 25 ° C. The plants were cultivated for about 4 weeks (※ period in which the number of sprouts reached a plateau and no longer sprouting), and the sprouting date, number of sprouts and type of sprouts (leaf sprouts, straight flowers, leafy flowers) for each node of branches were recorded.
And based on these observation results, the average value of "the number of flower bud formation (number of flowerings = number of direct flowers + number of leafed flowers)" per one node where germination was observed was calculated.

(4) Measurement of ABA content Acetone obtained by adding deuterium labeled ABA (D-ABA, [2H6] ABA) as an internal standard to the frozen sample collected in (2) above is used as an extraction solvent, Acetone extraction was performed. Next, to the residue obtained by vacuum concentration with a rotary evaporator, equal amounts of ultrapure water and an ethyl acetate-hexane mixed solution (1: 1) were added to dissolve the solid content.
After concentration under reduced pressure, hydrochloric acid and water-saturated ethyl acetate were added and suspended, and centrifugation was performed to separate the organic layer. The organic layer was concentrated under reduced pressure and dissolved in methanol.
After the methylation treatment, the ABA content (ng / gFW) per 1 g wet weight was measured based on an internal standard (D-ABA) using a GC-MS instrument (QP2010, manufactured by Shimadzu Corporation).

(5) Determination of CiFT gene expression (quantitative RT-PCR)
From the frozen sample obtained in (2) above, total RNA (total RNA) was extracted using ISOGEN (manufactured by Wako) and RNeasy Plant (manufactured by QIAGEN). CDNA (complementary strand DNA) was synthesized using the obtained RNA as a template and using Quantitect Reverse Transcription kit (manufactured by QIAGEN).

Quantitative RT-PCR of CiFT gene was performed by real-time PCR using cDNA from each sample.
As an amplification primer of the CiFT gene, a DNA primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 (CiFT-f) and SEQ ID NO: 2 (CiFT-r) in the sequence listing was used. The primer set for the CiFT gene used here is three similar CiFT1 to 3 (CiFT1 SEQ ID NO: 23 accession number AB027456; and CiFT2 SEQ ID NO: 24 accession number AB301934; CiFT3 is a primer set targeting all common sequences of SEQ ID NO: 25 accession number AB 301935) (see Non-Patent Document 2: Nishikawa et al. 2007).
In addition, the EF1α gene is adopted as an internal standard gene, and as an amplification primer thereof, a DNA primer set consisting of the nucleotide sequences shown in SEQ ID NO: 3 (QTEF1a-f) and SEQ ID NO: 4 (QTEF1a-r) in the sequence listing is used It was.
Real-time PCR was performed using Applied Biosystems 7300 Real-Time PCR system (manufactured by Applied Biosystems) as an analyzer. The PCR reaction was performed at an annealing temperature of 60 ° C. using Power SYBR Green PCR Master Mix (Applied Biosystems), using the template cDNA and the primer set. The fluorescence intensity of the amplification product in the PCR reaction was measured over time in real time.
The reaction is performed in triplicate, and "measurement value (average value) of PCR reaction using CiFT amplification primer" to be measured is measured value of PCR reaction using internal standard "EF1α amplification primer (average value The value obtained by dividing by “)” was determined and calculated as the expression level of the CiFT gene.

・ 1-2) "Result"
As a result, as shown in FIG. 1, by starting low temperature cultivation treatment (flowering induction treatment) at 15 ° C. for Satsuma mandarin orange saplings, the ABA content in the stem increases remarkably, and approximately 0.5 from the start of the low temperature cultivation treatment. After the month, it was shown that the ABA content became maximum (see FIG. 1 (A)).
In addition, the expression level of the CiFT gene in the stem also increases, correlating with the increase in the ABA content in the stem, and it is shown that the gene expression level is maximized 1 to 1.5 months after the start of the low temperature cultivation treatment. (See Figure 1 (A)).
And, the flower bud formation ability of the seedling is also increased in correlation with the rise of the FT gene in the stem, and in the seedling 1.5 to 2 months after the start of the low temperature cultivation treatment, 0.7 to 1 attachment per node where germination was observed It has been shown that the flower is ready (flower induced) (see FIG. 1 (B)).

[Test Example 2] "Low-temperature cultivation treatment experiment (additional test of Test Example 1)"
In a year (2013) different from the experimental year (2011) of Test Example 1, a repeat test of the low temperature cultivation treatment experiment was conducted to confirm the reproducibility.

・ 2-1) "Experimental procedure and conditions"
We prepare year-round grafted tree seedling (plant planted individual with 10 or more side branches: 1 plant 3 × 5 sampling per sampling) of Satsuma mandarin orange (variety: Okitsu early life), and it is after curing the spring branch 7 2013 On March 3, (1) "low temperature cultivation treatment" (flowering induction treatment) was started. Thereafter, (2) sampling, (3) 25 ° C. constant temperature cultivation treatment and evaluation of flowering ability, (4) measurement of ABA content, and (5) quantification of expression of CiFT gene (quantitative RT-PCR) were performed. (※ Basic experimental operation etc. were carried out in the same manner as the method described in Test Example 1.)
(2) Regarding the evaluation of flowering inducibility, in order to evaluate how many flower buds were formed in the whole seedling, the “number of flower buds formed per one node on the whole spring branch of the seedling (number of flowering = The average value of "the number of direct flowers + the number of leafy flowers)" was also calculated.

・ 2-2) "Result"
As a result, as shown in FIG. 2, by starting low temperature cultivation treatment (flowering induction treatment) at 15 ° C. for Satsuma mandarin orange saplings, the ABA content in the stem increases remarkably, and approximately 0.5 from the start of the low temperature cultivation treatment. After the month, it was shown that the ABA content became maximum (see FIG. 2 (A)).
In addition, the expression level of the CiFT gene in the stem is also increased in correlation with the increase in the ABA content in the stem, and the gene expression level is rapidly increased from 1.5 months to 2 months after the start of the low temperature cultivation treatment. Was shown (see FIG. 2 (A)).
And, the flower bud formation ability of the seedling is also increased in correlation with the rise of the FT gene in the stem, and in the seedling two months after the start of the low temperature cultivation treatment, about 0.7 flowers can be formed per node where germination was observed. It was shown that it was in the normal state (the state in which flowering was induced) (see FIG. 2 (B)).
In addition, in the sapling after 2 months concerned, even when it is seen in "the whole seedling (when both sprouted nodes and ungerminated nodes are included)", about 0.3 flowers can be set per one node. Was shown (see FIG. 2 (C)).

From the results of the test example, it was confirmed that the results of Test Example 1 (low temperature cultivation of citrus seedlings ⇒ ABA content increase 現象 phenomenon in which FT gene expression increase and flowering induction occur) are reproducible.
In addition, it was confirmed that the flowering amount of flower buds induced here is a sufficient amount even when seen in the whole seedling.

[Test Example 3] "Low-temperature cultivation treatment experiment with totally defoliated seedlings (comparative experiment)"
The effects of low temperature cultivation treatments were investigated when whole seedlings of Satsuma mandarin orange were defoliated.

・ 3-1) "Experimental procedure and conditions"
A year-round grafted tree seedling (planted individual with 10 or more side branches: 1 plant 3 x 5 samplings per sampling) of Satsuma mandarin orange (variety: Okitsu early life), 2011 after the spring branch hardens The work (total defoliation) of picking all the leaves of all individuals was performed on the 22nd. Then, on the same day, (1) "low temperature cultivation treatment" (flowering induction treatment) was started. Thereafter, (2) sampling, (3) 25 ° C. constant temperature cultivation treatment and evaluation of flowering ability, (4) measurement of ABA content, and (5) quantification of expression of CiFT gene (quantitative RT-PCR) were performed. (※ Basic experimental operation etc. were carried out in the same manner as the method described in Test Example 1.)

3-2) "Result"
As a result, as shown in FIG. 3, even in the case where the low temperature cultivation treatment (flowering induction treatment) was started at 15 ° C., the increase in the ABA content in the stem occurred at all in the seedlings whose leaves were completely defoliated. There was no (see FIG. 3 (A)). In addition, the expression level of the CiFT gene in the stem did not increase at all (see Fig. 3 (A)), and it was shown that the seedling "is not a seedling capable of flowering (in a state where flowering is induced)". (See Figure 3 (B)).
As the result shows, it was shown that the leaf is necessary for the flower induction signal generation by the low temperature cultivation treatment.

[Test Example 4] "Correlation between ABA content and flower inducibility"
It was verified whether the ABA content in the stem had a correlation with the expression level of the CiFT gene and the ability to induce flower formation of the seedlings.

・ 4-1) “Correlation between ABA content in stem and CiFT gene expression level”
Using the data of the cultivation experiments of Experiment Examples 1 to 3 (and the same experiment performed in 2012), the correlation between the ABA content in the stem and the CiFT gene expression level was examined.
Plot the “maximum value of ABA content in stem” after the start of low-temperature cultivation treatment on the horizontal axis of the graph and “maximum value of CiFT gene expression level in the stem” on the vertical axis of the graph, The correlation between the data groups in was examined to calculate the contribution rate (R ² ).
(* Here, 'contribution factor' is a value indicating the square of the correlation coefficient “R”, and the maximum value is 1.0. When the contribution factor is in the range of 0.7 to 1.0, two data to be compared There is a strong correlation between the groups.)

As a result, as shown in FIG. 4 (A), the contribution rate (R ² ) becomes a high value of 0.8973, and “the maximum value of ABA content in the stem (horizontal axis)” and “the expression amount of CiFT gene in the stem It was shown that there is a strong correlation (positive correlation) between the “maximum values (vertical axis)”.

4-2) "Correlation between ABA content in stem and flower induction ability"
Similar to the above, the correlation between the ABA content in the stem and the number of flowerings per sprouting node was examined. On the horizontal axis of the graph, “maximum of ABA content in stem after start of low temperature cultivation treatment”, on the vertical axis of the graph, “number of flowering per sprouting node when seedlings are subjected to constant temperature cultivation at 25 ° C. The values representing inducibility were plotted to examine the correlation between the two data groups in the scatter plot.

As a result, as shown in FIG. 4 (B), the contribution rate (R ² ) is a high value of 0.9498, and “maximum value of ABA content in stem (horizontal axis)” and “temperature-controlled cultivation of seedlings at 25 ° C. It was shown that there is a strong correlation (positive correlation) between the number of flowerings per sprouting node (vertical axis) at the time of germination.

[Discussion from Test Examples 1 to 4]
As the results of Test Examples 1 to 4 indicate, in the case of citrus species (plant species belonging to the genus Citrus) in which flowering induction is induced by low temperature, the low temperature cultivation process senses that the low temperature state is a signal. It became clear that the ABA content in the stem of the developmental branch was significantly increased, which led to an increase in FT gene expression.
In addition, it was revealed that there is a strong correlation (positive correlation) between the ABA content in the stem and the FT gene expression level, and between the ABA content in the stem and the flowering inducibility of the seedlings.
From the above, it has become clear that in the citrus species (plant species belonging to the genus Citrus) in which flowering induction is induced by low temperature, there is a "flowering induction mechanism in which an increase in ABA content promotes flowering induction".

[Test Example 5] "Control of CiFT gene expression level by exogenous ABA treatment"
By performing exogenous ABA treatment, it was examined whether control of CiFT gene expression level in the stem was possible.
・ 5-1) "Experimental procedure and conditions"
Prepare annual freshwood graft (seed planted individuals with 10 or more side branches: 3 individuals × 2 treatment areas per treatment area) of Satsuma mandarin orange (variety: Okitsu early life), in a glass room where natural light can be collected It moved and cultivated under constant temperature conditions (conditions which do not become low temperature: conditions which avoid an intrinsic flowering induction) adjusted to room temperature 25 ° C.
An aqueous solution of 500 μM ABA ((±) -ABA) was sprayed on the seedlings, and side branches and leaves were separately collected and cut immediately after spraying (0 hour) and 24 hours after spraying. These were separately freeze-crushed for each sapling and stored at -80 ° C.
As a control, pure water was sprayed instead of 500 μM ABA aqueous solution, and after 24 hours similarly, the side branch was collected, cut, frozen and stored at -80 ° C.
Thereafter, each sample was used to quantify the amount of CiFT gene expression. (※ The basic experimental procedure was the same as described in Test Example 1.)

・ 5-2) "Result"
As a result, as shown in FIG. 5, it was shown that the expression amount of CiFT gene in stems and leaves was rapidly increased in the seedlings of the treated section sprayed with exogenous ABA. In particular, in the “stem”, it was shown to rise sharply to about 6.5 times that before treatment (see FIG. 5 (A)).
From the results, it was experimentally confirmed that ABA is a substance having an action of increasing the amount of FT gene expression in stems.
Also, it is important to note that from the results, it is possible to artificially control the expression level of the FT gene by controlling the ABA content in the stem by the addition of exogenous ABA.

[Test Example 6] "enzyme involved in ABA content"
We searched for enzymes that could control ABA content in stems.

· 6-1) "Experimental procedure and conditions"
The frozen samples prepared in Test Example 1 were subjected to quantitative RT-PCR expression analysis for each gene of enzymes involved in the synthetic degradation metabolism of ABA.
In the said experiment, it becomes possible by grasping | ascertaining transition of the expression level of each metabolic enzyme gene to grasp | ascertain "transition of each metabolic enzyme content" produced | generated by those gene expression. (* It has been reported that the expression level of the enzyme gene involved in the synthesis system is correlated with the amount of reaction product produced by the activity of the enzyme that is the gene product (Kato et al. (2004); Plant Physiology 134: p824-837, Kato et al. (2006); Journal of Experimental Botany 57: p2153-2164).

As an experimental procedure, specifically, as enzymes related to carotenoid metabolism, PSY (phytoene synthase), PDS (phytoene desaturase), ZDS (ζ-carotene desturase), LCYe (lycopene ε-cyclase), LCYb (lycopene β-cyclase) The amount of each gene expression was quantified focusing on HYb (β-ring hydroxylase) and ZEP (zeaxanthin epoxidase).
In addition, NCED3 (9-cis-epoxycarotenoid dioxygenase 3: rate-limiting enzyme of ABA synthetic system) involved in the synthesis of xanthotoxin (ABA precursor) from violaxanthin (carotenoid) and A8OX (ABA 8'-) directly involved in ABA degradation The amount of gene expression was quantified for hydroxylase (ABA degrading enzyme).
As a primer used for RT-PCR, each primer set of the combination shown in Table 2 was used. In the table, "-f" indicates a forward primer and "-r" indicates a reverse primer. (※ Basic experimental operation etc. were carried out in the same manner as the method described in Test Example 1.)

・ 6-2) "Result"
As a result, as shown in FIG. 6, a positive correlation was recognized between the transition of “ABA content” and the transition of “NCED3 content”, which is an ABA synthetase. Moreover, a negative correlation was recognized with the transition of "A8OX content" which is an ABA degrading enzyme.

(1) Transition of NCED3 (ABA synthetase) In the stems of seedlings (0.5 months after the start of low temperature cultivation treatment), the content of ABA is increased by the start of low temperature cultivation treatment (* experiment example 1 experiment of cultivation). It has been shown that the expression level of is also increased. And it was shown that the expression amount of "NCED3" also turned to a decrease in the seedlings having a decrease in ABA content (one to two months after the start of the low temperature cultivation treatment) (Fig. 6 (A), ( C) see).
Moreover, when low temperature cultivation treatment (* experiment of cultivation of * experiment example 3) was performed to the whole-leaved sapling, the ABA content showed the tendency to decrease, but the expression amount of "NCED3" also has the tendency to decrease similarly. (FIG. 6 (B), (D)).
As these results show, it was confirmed that the gene expression pattern of “NCED3”, which is an ABA synthetase, shows a behavior that completely matches the transition of ABA content.
From the above, it was recognized that the transition of the “ABA content” in the stem had a positive correlation with the transition of the “content of the NCED3 enzyme” which is an ABA synthetase.

(2) Transition of A8OX (ABA degrading enzyme) On the other hand, the expression of "A8OX" shows that the ABA content is rising when the low temperature cultivation treatment is started (cultivation experiment of * Experiment example 1) (low temperature cultivation The expression level was low at the stem after 0.5 months of treatment initiation. However, in the seedlings whose ABA content turned to decrease (1 to 2 months after the start of low-temperature cultivation treatment), it was shown that the expression level of "A8OX" turned to increase (Fig. 6 (A), ( E) see).
Moreover, when low temperature cultivation treatment (* experiment of cultivation of * Experiment example 3) was performed to the defoliated sapling, the ABA content showed the tendency to decrease gradually, and the expression level of "A8OX" may rise moderately. It is shown (see FIG. 6 (B), (F)).
As these results show, it was confirmed that the gene expression pattern of "A8OX", which is an ABA degrading enzyme, completely reverses the behavior of ABA content.
From the above, it was recognized that the transition of "ABA content" in the stem was negatively correlated with the transition of "A8OX enzyme content", which is an ABA degrading enzyme.

(3) Changes in other carotenoid metabolism enzymes
Carotenoid metabolism enzyme genes other than NCED3 and A8OX did not show an expression pattern that was found to be related to the transition of ABA content (see Table 3).
That is, in the enzymes involved in the biosynthesis system (carotenoid metabolism) upstream of the ABA precursor, the presence of an enzyme correlated with the ABA content could not be confirmed.

[Discussion from Test Examples 5 and 6]
From the results of Test Example 5, it was shown that control of the FT gene expression level (number of flowering of seedlings by induction of flowering) is artificially possible by adjusting the increase or decrease of ABA content in the stem.
Also, from the results of Test Example 6, it was shown that control of the ABA content in the stem becomes possible by controlling the "NCED3 (ABA synthetase) content" and / or the "A8OX (ABA degrading enzyme) content". .

From the above, it is possible to control the increase of FT gene expression level by using “ABA” exogenously to the seedlings of citrus species (plant species belonging to citrus genus) in which flowering induction is induced by low temperature It was shown that
In addition, it was shown that by using "an inhibitor of NCED3 (ABA synthetase)" exogenously, it is possible to control the decrease in the number of flowering via reduction of FT gene expression level.
In addition, it was shown that by using "an inhibitor of A8OX (ABA degrading enzyme)" exogenously, it is possible to control the increase in the number of flowering via an increase in FT gene expression level.

According to the present invention, it is possible to efficiently perform fruit set management of a citrus and avoid biennial results, and it is expected that it will be a fruit set management technique aiming at the continuous stable production of citrus.

1. Developmental branch (branch in developmental stage)
2. Germinated leafy flowers 3. Sprouted leaf bud

Claims (3)

For plants belonging to the genus Citrus of the property that induction of flowering is induced by the increase in the expression of the FT gene by low temperature cultivation at 15 ° C.
A method for controlling flowering of a citrus, characterized by controlling the number of flowering using the means described in the following (A) , wherein the plant is an orange mandarin orange :
(A): It is a means for controlling the increase in the number of flowering by increasing the abscisic acid acceptance signal in the stem of the developmental branch of the first year after germination and elongation and hardening of the dormant bud,
The increase control means, abscisic acid to the development branch, abscisic acid receptor agonists, abscisic acid degrading enzyme inhibitor, or abscisic acid 8'Ri exogenous use der hydroxide enzyme inhibitors,
The developmental branch is a developmental branch including vascular phloem cells in a developmental stage . For plants belonging to the genus Citrus of the property that induction of flowering is induced by the increase in the expression of the FT gene by low temperature cultivation at 15 ° C.
A method for controlling flowering of a citrus, characterized in that the number of flowering is controlled using the means described in (B) below , wherein the plant is an orange mandarin orange :
(B): A control means for controlling the decrease in the number of flowers by decreasing or eliminating the abscisic acid reception signal in the stem of the developmental branch of the first year after germination and elongation and hardening of the dormant bud,
The reduction control means, abscisic acid receptor antagonist to the growth branch, abscisic acid synthase inhibitor, or 9-cis - Ri exogenous use der epoxy dioxygenase inhibitor,
The developmental branch is a developmental branch including vascular phloem cells in a developmental stage . Wherein the plant belonging to the citrus (Citrus), characterized in that it uses a method according to claim 1 or 2, fruit set control method for citrus fruits.