JP6207012B2 - Method for enhancing plant cold tolerance by reducing abscisic acid action - Google Patents

Description

  The present invention relates to a method for enhancing the cold resistance of plants.

  Climate change is expanding with global warming, and not only abnormally high temperatures but also extremely low temperatures occur frequently in cold and cold regions, threatening the stable production of crops. For this reason, the development of varieties with enhanced cold resistance has been promoted, but cultivars that can overcome summer low temperatures such as those in the 1993 cold weather disaster have not been successfully developed. Thus, there is a limit to the low-temperature countermeasures that are only genetic improvements by breeding, and there is a demand for the development of cultivation methods and drugs that can enhance cold resistance.

  To date, abscisic acid (ABA) has been considered to be the most effective as a plant hormone that improves environmental stress tolerance (cold resistance, heat resistance, drought resistance, cold resistance, etc.) of plants. For example, ABA treatment is known to significantly increase seedling seedling resistance and drought tolerance (Lang et al., (1989) Theor. Appl. Genet., 77, p.729-734; Lu et al (2009) Plant Physiol. Biochem., 47, p. 132-138). On the other hand, ABA has been reported to have a strong inhibitory effect on plant growth and an inhibitory effect on pollen formation, and ABA treatment cannot increase the cold resistance during the booting period (pollen formation period). On the contrary, it is known that the cold resistance is weakened (Oliver et al., (2007) Plant Cell Physiol., 48, p.1319-1330). ABA is known to be synthesized and accumulated in plant bodies triggered by environmental stress. For example, when ABA encounters low temperatures during the booting period of rice, the ABA concentration in the plant body increases, which inhibits pollen formation. (Oliver et al., (2007) Plant Cell Physiol., 48, p. 1319-1330). Therefore, based on the idea that cold tolerance can be improved by improving rice so that the ABA concentration does not increase even at low temperatures, by overexpressing the ABA degrading enzyme gene, the ABA concentration can be lowered and the cold tolerance can be enhanced. Successful cases have been reported (Ji X, et al., (2011) Plant Physiol., 156, p.647-662). However, considering the merit that ABA generally improves environmental stress tolerance, genetically lowering ABA concentration is not necessarily good for cultivation. In addition, it is difficult to immediately utilize a method for enhancing cold tolerance using genetic recombination technology effectively in agricultural production in Japan where social acceptance of genetically modified foods and genetically modified crops has hardly progressed. For this reason, development of a new method for imparting cold resistance during the booting period is required.

  Plants have developed their own self-defense mechanisms, including not only abscisic acid synthesis induction against environmental stress, but also salicylic acid synthesis induction by pathogen infection and jasmonic acid synthesis induction by insect feeding damage. For example, when a plant is infected with a pathogen, it biosynthesizes salicylic acid or other antibacterial proteins and accumulates them in the body to induce systemic acquired resistance (SAR) and prevent secondary infection. The systemic acquired resistance can be artificially induced with a drug or the like, and the systemic acquired resistance inducing agent is already used for controlling pathogen infection in the agricultural field. For example, probenazole (trade name Orizemate (registered trademark)) developed as a pesticide that induces salicylic acid has been used as a rice blast control agent in rice fields in Japan, China, Korea, Taiwan, and Southeast Asian countries for more than 30 years. Yes. However, since the self-protection mechanism of plants is subject to complex control, the method of imparting resistance using the self-protection mechanism may not be effective enough, so effective pathogen infection control methods and environmental stress resistance There is still a need for the development of grant methods.

Lang et al., (1989) Theor. Appl. Genet., 77: 729-734 Lu et al., (2009) Plant Physiol. Biochem., 47 132-138 Oliver et al., (2007) Plant Cell Physiol., 48: 1319-1330 Ji X, et al., (2011) Plant Physiol., 156, p.647-662

  This invention makes it a subject to provide the reinforcement | strengthening method of cold tolerance at the booting stage of a Gramineae plant.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have remarkably suppressed the inhibitory action on plants by ABA, that is, the action of stopping the cell cycle (cell division) and the shoot elongation of seedlings by the action of salicylic acid. It has been found that it is possible to cancel the action and the like, and that the cold resistance of plants, particularly the grass family, can be enhanced, and the occurrence of pollen sterility can be suppressed, and the present invention has been completed.

That is, the present invention includes the following.
[1] A cold tolerance enhancer for the booting period for gramineous plants, comprising at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid derivative, and salts thereof.
[2] The cold tolerance enhancer of the booting stage according to the above [1], which is a pollen sterility inhibitor.
[3] The cold tolerance enhancer of the booting stage according to the above [1] or [2], which is applied to a young panicle formation stage or a booting stage.
[4] The cold resistance enhancer for the booting stage according to the above [1] to [3], wherein the salicylic acid inducer is probenazole.
[5] The cold tolerance enhancer of the booting stage according to the above [1] to [4], wherein the grass family is rice.
[6] A method for enhancing the cold resistance of a grass family at the booting stage, which comprises applying at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer and a salt thereof to the grass family.
[7] The method according to [6] above, wherein the enhancement of cold resistance during booting is suppression of pollen sterility.
[8] The method according to [6] or [7] above, wherein at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer, and a salt thereof is applied in the young panicle formation stage or the booting stage.
[9] The method of [6] to [8] above, wherein the salicylic acid inducer is probenazole.
[10] The method of [6] to [9] above, wherein the gramineous plant is rice.

  ADVANTAGE OF THE INVENTION According to this invention, the cold tolerance of the panaceae plant can be reinforce | strengthened and generation | occurrence | production of the pollen sterility by the low temperature of the booting phase can be suppressed.

FIG. 1 is a diagram showing the effect of salicylic acid (SA) on the shoot elongation inhibitory action of abscisic acid (ABA). ****, ***, and ** indicate that there was a significant difference at the 0.01%, 0.1%, and 1% levels for the SA 0 mM treatment group, respectively. FIG. 2 is a diagram showing the effect of salicylic acid (SA) on the expression of abscisic acid (ABA) cell cycle inhibitory factor gene group OsCDKs. * Indicates that there was a significant difference at the 5% level compared to the ABA single treatment group. FIG. 3 is a diagram showing the influence of salicylic acid (SA) on the expression of abscisic acid (ABA) cell cycle inhibitory factor gene group OsKRPs. *** and ** indicate that there were significant differences at the 0.1% and 1% levels, respectively, with respect to the ABA single treatment group. FIG. 4 is a photograph showing the results of a protein-protein interaction test by yeast two-hybrid analysis. FIG. 5 is a diagram showing the influence of abscisic acid (ABA) and salicylic acid (SA) on intranuclear doubling. ****, ***, and * indicate that there was a significant difference at the 0.01%, 0.1%, and 5% levels, respectively, with respect to the ABA alone treatment group. FIG. 6 is a diagram showing the influence of abscisic acid (ABA) and salicylic acid (SA) on new DNA synthesis. The results are shown for the untreated group that was not treated with either ABA or SA, the ABA single-treated group treated with ABA 2 μM, or the treated group treated with ABA 2 μM and SA 1 mM. FIG. 7 is a diagram showing the influence of abscisic acid (ABA) and salicylic acid (SA) on the expression of rice ABA-degrading enzyme gene. ****, ***, and * indicate that there was a significant difference at the 0.01%, 0.1%, and 5% levels, respectively, with respect to the ABA alone treatment group. Control: Untreated group not treated with either ABA or SA, ABA: ABA single treated group, SA-ABA: SA coexisting group treated with SA together with ABA, SA: SA single treated group. FIG. 7A shows changes over time in measured mRNA levels of OsABA8ox1, FIG. 7B shows OsABA8ox2, and FIG. 7C shows OsABA8ox3. FIG. 8 is a graph showing the effect of salicylic acid (SA) on the abscisic acid (ABA) concentration of shoots. *** and ** indicate that there were significant differences at the 0.1% and 1% levels, respectively, with respect to the ABA single treatment group. Control: Untreated group not treated with either ABA or SA, ABA: ABA single treated group, SA-ABA: SA coexisting group treated with SA together with ABA, SA: SA single treated group. FIG. 9 is a diagram showing the influence of probenazole on the shoot elongation inhibitory action of abscisic acid (ABA). ****, *** and * indicate that there were significant differences at the 0.01%, 0.1 and 5% levels, respectively, for the untreated group of probenazole (orizemate granule). FIG. 10 is a diagram showing the effect of suppressing the occurrence of pollen sterility at low temperatures by salicylic acid (SA). * Indicates that there was a significant difference at the 5% level relative to the SA untreated group. FIG. 11 is a diagram showing the effect of salicylic acid in enhancing the cold resistance during the booting period. *** indicates that there was a significant difference at the 0.1% level with respect to the SA untreated group. The untreated section is an average value of n = 16 individuals, and the treated section is an average value of n = 15 individuals. FIG. 12 is a diagram showing the effect of probenazole to enhance cold resistance during the booting period by suppressing pollen sterility. *** indicates that there was a significant difference at the 0.1% level compared to the plotenazole-untreated group. The untreated section is an average value of n = 20 individuals, and the treated section is an average value of n = 25 individuals. FIG. 13 is a diagram showing the effect of salicylic acid (SA) on the low temperature elongation of shoots. FIG. 14 is a diagram showing the effect of salicylic acid (SA) on the low temperature death resistance of young seedlings.

Hereinafter, the present invention will be described in detail.
Abscisic acid (ABA) is a hormone that brings about an increase in environmental stress tolerance, in which synthesis is induced by exposure to environmental stress (cold resistance, heat resistance, drought resistance, cold resistance, etc.) in plants. However, ABA synthesized in plants by encountering low temperatures during the booting period (pollen formation period) remarkably suppresses the growth of plants and inhibits pollen formation in the pods. Salicylic acid, on the other hand, is a signal molecule that is activated by the plant's self-protection mechanism against pathogen infection, and induces salicylic acid-responsive systemic acquired resistance (SAR) in plants. The identity of systemic mobility signaling substances in salicylic acid-responsive SAR has long been unknown, but in recent years, the signaling substance is methyl salicylate, and salicylic acid produced at the site of infection migrates as methyl esterified. It has been found that the volatile methyl salicylate is easily transferred to the whole plant and returns to salicylic acid by the action of esterase at the destination, thereby inducing resistance to various pathogen infections. It is also known that most salicylic acid is stored in vacuoles in the form of salicylic acid glucoside (SAG) in plants.

  This invention provides the method and chemical | medical agent which strengthen the cold tolerance of a plant based on salicylic acid (SA) being able to reduce the inhibitory effect | action with respect to a plant of abscisic acid. More specifically, the present invention relates to a panicle-time cold tolerance enhancer for gramineous plants comprising at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid derivative, and salts thereof, and salicylic acid. The present invention provides a method for enhancing the cold resistance of a grass family by applying at least one selected from a salicylic acid precursor, a salicylic acid derivative, and a salt thereof to the grass family.

  In the present invention, the term “salicylic acid precursor” means a salicylic acid derivative that can be metabolized in a plant to produce salicylic acid. Specifically, for example, a salicylic acid ester such as methyl salicylate, a salicylic acid glucoside or the like. Examples thereof include saccharides or derivatives thereof. In addition, although a salicylic acid and a salicylic acid precursor are not limited, For example, it can represent with the following formula | equation.

［式中、Ｒ_１及びＲ_２はそれぞれ独立して、水素原子、置換若しくは非置換の低級アルキル基（炭素数1〜6）又は糖残基である］

[Wherein, R ₁ and R ₂ are each independently a hydrogen atom, a substituted or unsubstituted lower alkyl group (having 1 to 6 carbon atoms) or a sugar residue]

Here, the substituted or unsubstituted lower alkyl group is, for example, a methyl group, and the sugar residue is, for example, a glucose residue. In one embodiment, when _one of R ₁ and R ₂ is a substituted or unsubstituted lower alkyl group (having 1 to 6 carbon atoms) or a sugar residue, the other is preferably a hydrogen atom. For example, R ₁ may be a substituted or unsubstituted lower alkyl group (having 1 to 6 carbon atoms) or a sugar residue, and R ₂ may be a hydrogen atom. For example, R ₁ may be a hydrogen atom, and R ₂ may be a substituted or unsubstituted lower alkyl group (having 1 to 6 carbon atoms) or a sugar residue.

  The “salicylic acid inducer” used in the present invention means an agent that promotes the synthesis or accumulation of salicylic acid in a plant and increases the endogenous salicylic acid content of the plant. In the agricultural field, drugs that promote biosynthesis and accumulation of salicylic acid and antibacterial proteins and induce systemic acquired resistance (SAR) (SAR inducers) have long been used as pathogen infection control agents such as rice blast control agents. It has been used. In the present invention, among salicylic acid-dependent systemic acquired resistance (SAR) inducers, SAR inducers that promote the biosynthesis and / or accumulation of salicylic acid can be suitably used as the salicylic acid inducer. Specific examples of salicylic acid inducers include probenazole, salicylic acid glycosyltransferase inhibitors implimins A and B, validamycin A, 1,2-benzisothiazol-3 (2H) -one 1,1-dioxide (BIT), theobroxide Examples of such SAR inducers include, but are not limited to, known substances and salts thereof. A particularly preferred example of a salicylic acid derivative is probenazole. By applying a salicylic acid inducer to a plant, the production of salicylic acid is induced and accumulated in the plant. Instead of the salicylic acid inducer, a salicylic acid-responsive systemic acquired resistance (SAR) inducer that activates the salicylic acid signaling pathway further downstream can be used.

  In the present invention, a salt of salicylic acid, a salicylic acid precursor, or a salicylic acid derivative may be used. Such a salt may be any salt that can be used in the agricultural chemical field, and is not particularly limited. For example, an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (magnesium salt, calcium salt) Etc.), ammonium salts (dimethylammonium salts, triethylammonium salts, etc.), inorganic acid salts (hydrochlorides, perchlorates, sulfates, nitrates, phosphates, etc.), organic acid salts (acetates, methanesulfonates, etc.) Citrate, lactate, tartrate, malate, etc.).

  In the present invention, the term “Gramineae plant” means any plant belonging to the family Gramineae, for example, rice (Oryza sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), corn ( Zea mays L., sorghum (Sorghum bicolor (L.) Moench), Erianthus spp, sugar cane (Saccharum officinarum L.), rye (Secale cereale L.), oat (Avena fatua), millet (Setaria italica) ), Gramineous crops such as Japanese millet (Echinochloa esculenta), but are not limited to these. Rice (Oryza sativa L.) is particularly preferable as the “Gramineae plant” to which the method and the drug of the present invention are applied. Arbitrary varieties of gramineous plants can be applied to the method and the drug of the present invention. For example, in the case of rice, “Kita Aoba”, “Obozuki”, “Tachijobu”, “Yukihikari”, “Nihonbare” , “Yumepirika”, “Nanatsuboshi”, “Milky Queen”, “Hoshino Yume”, “Kirara 397”, “Yukimaru”, “Honoka 224”, “Donto Koi”, “Koshihikari”, “Sasanishiki” , `` Hitomebore '', `` Akitakomachi '', `` Haenuki '', `` Tsutsuhime '', `` Nasuhikari '', `` Kinuhikari '', `` Mutsu Homare '', `` Hinohikari '', `` Tsugaru Otome '', `` Tsugaru Romance '', `` Yume Akari '' ”,“ Hanaetizen ”,“ Dream Tsukushi ”,“ Hatsushimo ”,“ Sababi Yori ”,“ Domannaka ”,“ Kakehashi ”,“ Iwa Tekko ”,“ Mana Musume ”,“ Menkoina ”,“ Chiyoshiki ”, Fukumirai "includes varieties such as" troubled ", but is not limited to these. These rice varieties can be obtained from, for example, the National Institute of Agrobiological Sciences, Genebank (Japan).

  In the present invention, at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer, and salts thereof is applied to the grass family plant to enhance the cold resistance during the booting period of the grass family plant. Can do.

  In the present invention, "to apply" to a grass plant means that the active ingredient used in the present invention, that is, at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer and salts thereof, Applying in an absorbable state (from roots or leaves). Specifically, at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid derivative, and salts thereof may be applied by, for example, spraying from above the planted gramineous plant, You may sprinkle on the surface of the soil where a family plant is cultivated, may mix in soil, and may add to hydroponic water of rice field water or hydroponics. In one embodiment, it is also preferable to apply salicylic acid, a salicylic acid precursor, a salicylic acid derivative or a salt thereof in the form of a liquid agent such as an aqueous solution. Such application can make the gramineous plant resorbable.

  Salicylic acid, a salicylic acid precursor, a salicylic acid derivative or a salt thereof is not limited, for example, the concentration in the environment is 0.05 μM to 10 mM, preferably 0.1 μM to 1 mM, more preferably 0.1 μM to 500 μM, for example 0.1. You may apply by the quantity used as (micro | micron | mu) -10micromol. Alternatively, salicylic acid, a salicylic acid precursor, a salicylic acid derivative or a salt thereof is 5 g to 10 kg, preferably 10 g to 1 kg, more preferably 10 g to 500 g, for example 10 g to 100 g, per 10 ares (10a) of land to be applied. It is also preferred to apply in quantities.

  Gramineae plants are in the seedling stage, tillering stage, young panicle formation stage where young panicles and spikelets differentiate, panicle blooming stage (pollen formation stage) until differentiated spikelets are completed, heading stage, flowering stage, climbing stage Fruits through each growing season. At least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer, and salts thereof can be applied to the grass family at any time from the earing stage to the ear stage. It is preferably carried out before the rose stage (pollen formation stage), more preferably from the late juvenile stage to the middle stage of the boot stage, and particularly preferably during the boot stage. As an example, in the case of rice, the ear formation period is 30 to 15 days before heading, of which the first part of the ear formation period is 30 to 25 days before heading, the second part of the ear formation stage is 25 to 15 days before heading, 15 to 1 days before heading, of which the first half of the heading period is 15 to 10 days before heading, the middle stage of heading is 10 to 5 days before heading, and the second half of the heading stage is 5 to 1 day before heading. In the present invention, for the purpose of enhancing the cold resistance during the booting period, it is preferable to apply before exposure to low temperatures during the booting period (pollen formation period). Also good. As long as salicylic acid, a salicylic acid precursor, a salicylic acid inducer, or a salt thereof is applied so that salicylic acid can act on the ears at the booting stage, it is applied before the booting stage (preferably at the young panicle formation stage). Alternatively, it may be applied both before the booting stage (preferably at the juvenile stage) and at the booting stage, or only during the booting stage. The application of salicylic acid, a salicylic acid precursor, a salicylic acid inducer or a salt thereof in the present invention (for example, administration to soil or water) may be performed only once, or may be repeated twice or more.

  The active ingredient is applied to the grass plant in the present invention in combination with other agents such as fungicides, fungicides, insecticides, acaricides, herbicides, fertilizers and the like. May be.

  The salicylic acid, salicylic acid precursor, salicylic acid inducer or salts thereof applied to the grass family are absorbed by the grass family. The salicylic acid precursor is converted into salicylic acid in the plant. Salicylic acid inducers induce the synthesis or accumulation of endogenous salicylic acid in plants. Salicylic acid is known to function as a signal molecule for triggering the self-defense mechanism in plants, but in the present invention, it has been shown to cause cold tolerance enhancement during the booting stage through inhibition of ABA transport rather than signal transduction. It was.

  Rice is a summer crop, and in the case of Japan's first crop, it is usually seeded (seeded) from April to May and harvested in late summer to autumn. Rice, which is originally a tropical crop, suffers from various obstacles by encountering cold temperatures (low temperatures above 0 ° C) during its growth, and yields are greatly reduced (occurrence of cold damage). For example, if you encounter temperature conditions of 12 ° C or less in the seedling stage, 19 ° C or less in the booting stage, and 15 ° C or less in the ripening stage, there is a high possibility that cold damage will occur. In particular, the cold temperature from the booting stage to the flowering stage causes pollen sterility, lowers the fertilization rate, and causes many sterility. In grasses exposed to low temperatures (typically cold), the synthesis of abscisic acid is induced, which causes growth retardation and pollen sterility during the booting stage.

  In the present invention, at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid derivative, and salts thereof is applied to a grass family as described above to reduce the action of abscisic acid of salicylic acid. Based on the above, it is possible to enhance the cold tolerance of gramineous plants, in particular, the cold tolerance during the booting period, thereby causing low temperature damage (cold temperature damage) caused by the low temperature (typically cold temperature) encountered during the booting period. ) Can be reduced. Therefore, application of the above-mentioned active ingredient to the grass family used in the present invention is particularly advantageously used under conditions where there is concern about low-temperature damage due to low temperature during the booting stage (for example, cold summer). Particularly in the case of rice, the “enhanced cold resistance during booting period” in the present invention usually means that the daily average temperature exceeds 0 ° C. and lower than 20 ° C., preferably 12 ° C. or higher and 18 ° C. or lower in the booting season. It means that cold injury is alleviated when exposed to cold temperatures.

  Low temperature damage caused by low temperatures encountered during the booting period is mainly pollen sterility and growth delay. The application of the active ingredient to the grass family in the present invention is not particularly limited, but is particularly effective in suppressing pollen sterility. In the present invention, “inhibition of pollen sterility” means to suppress the occurrence of pollen sterility and to reduce or prevent a decrease in the seedling rate due to pollen sterility. In the present invention, the suppression of pollen sterility is more specifically observed in the sterilized pollen rate in the section (treated section) where the active ingredient is applied to the gramineous plant compared to the control (non-treated section). This is indicated by a statistically significant reduction and a statistically significant reduction in the spikelet sterility rate.

  The sterile pollen rate is the average value of the percentage of sterile pollen (%) with respect to the total number of pollen contained in the camellia camellia immediately after heading, based on the fertility of the pollen immediately after heading, as determined by the iodine-iodokari method. Calculated. By applying the active ingredient to the grass family in the present invention, the sterile pollen rate is, for example, 15% or more, preferably 20% or more, more preferably 35% or more, compared to the control (non-treated area), More preferably, it can be reduced by 50% or more, particularly preferably by 60% or more.

  The spikelet sterility rate is calculated as an average value of the ratio (%) of sterile flowers with respect to the total number of spikelets of each ear, based on the ripening rate examined for spikelets 30 days after heading. By applying the above-mentioned active ingredient in the present invention to the gramineous plant, the sterility of the flower buds is, for example, 15% or more, preferably 20% or more, more preferably 35% or more, compared to the control (non-treated area). More preferably, it can be reduced by 50% or more.

  Accordingly, the present invention provides a method for enhancing the cold tolerance of the above-mentioned gramineous plants at the booting stage. Applying this method to Gramineae plants when there is concern about low temperature (typically cold) at the booting stage, strengthening the cold tolerance at the booting stage and preventing pollen sterility As a result, the yield reduction can be reduced or prevented.

  In addition, the method for enhancing the cold resistance of plants by reducing the action of abscisic acid in the present invention is also suitable for application to plants other than Gramineae plants.

  Furthermore, the present invention also provides a booting-time cold tolerance enhancer for gramineous plants based on the effect of salicylic acid as described above to enhance the cold-resistance of the grass family. That is, the present invention also relates to a cold tolerance enhancer for the booting season for gramineous plants comprising at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid inducer and salts thereof as described above. Although this booting-time cold-resistant reinforcement | strengthening agent is not limited, Especially, it can be used as a pollen sterility inhibitor. It is preferable that the cold tolerance enhancer for the booting stage according to the present invention be applied by the booting stage, in particular, in the young panicle forming stage or the booting stage. Application of the cold tolerance enhancer for the booting stage according to the present invention to a grass family plant can be carried out according to the above-described method for enhancing the cold tolerance of a plant of a grass family plant.

  The cold tolerance enhancer at the booting stage according to the present invention may contain a formulation adjuvant used in the agricultural chemical field in addition to at least one selected from salicylic acid, a salicylic acid precursor, a salicylic acid derivative, and salts thereof. Good. As formulation aids, diluents (mineral fine powder, buffer, etc.), surfactants, excipients, decomposition inhibitors, preservatives, binders, disintegrants, lubricants, colorants, flavoring agents, dissolution Adjuvants, suspending agents, coating agents and the like can be mentioned. The booting-time cold resistance enhancer according to the present invention may also contain other components such as fungicides, fungicides, insecticides, acaricides, herbicides and other disease control agents, fertilizer components, and the like. Good. The booting-time cold resistance enhancer according to the present invention may be formulated into an arbitrary dosage form, and such a dosage form is not limited to, for example, tablets, capsules, Examples thereof include granules, powders, powders, wettable powders, aqueous solvents, emulsions, liquids, oils, aerosols, and pastes.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Reduction effect of salicylic acid (SA) on growth inhibitory action of abscisic acid (ABA)-Influence on shoot elongation
Rice (Oryza sativa L.) seeds sprouting for 3 days in the dark at 28 ° C were prepared from abscisic acid (ABA) (0, 0.2, 1, or 5 μM) and salicylic acid (SA) (0, 0.2, 1, or 5 mM) ) Was sown on a net floating in a mixed aqueous solution and grown in the dark at 25 ° C. Five days after the start of treatment, the shoot length of 9 individuals was measured for each treatment group.

  The result is shown in FIG. The elongation of shoots was remarkably suppressed by ABA, and as the ABA concentration increased, the suppression of elongation became significant. On the other hand, when SA was applied together with ABA, the shoot elongation inhibitory action of ABA was significantly reduced by SA. In particular, in the 0.2 μM ABA group, the ABA elongation inhibitory action was almost completely released in the presence of 1.0 mM SA. Even in the 1 μM ABA group and the 5 μM ABA group, suppression of shoot elongation was significantly reduced in the presence of 1.0 mM and 5.0 mM SA.

  From this result, it was shown that the shoot elongation inhibitory action of abscisic acid (ABA) can be reduced by salicylic acid (SA) treatment.

[Example 2] Reduction effect of salicylic acid (SA) on growth inhibitory action of abscisic acid (ABA)-Effect on cell cycle (1) Quantitative PCR analysis
By examining changes in the expression level of factors involved in the cell cycle in seedling shoots grown in aqueous solutions in which ABA and SA coexisted, the molecular mechanism by which SA suppresses shoot elongation inhibition by ABA can be determined. investigated.

  Rice seeds germinated for 3 days in the dark at 28 ° C. were placed on a net on an aqueous solution of ABA 2 μM, ABA 2 μM + SA 1 mM, or SA 1 mM, cultured for 5 days in the dark at 25 ° C., and the shoots were elongated ( Processing zone). For the control group (ABA 0 μM + SA 0 mM), the shoot was extended in the same manner. RNA was extracted from the obtained shoots, and using this as a template, the expression levels of the OsCDK gene group and the OsKRP gene group were analyzed by quantitative PCR (Taqman probe method). As the TaqMan probe, an oligonucleotide modified with FAM at the 5 ′ end and TAMRA at the 3 ′ end was used (Roche Applied Science). The primer sequences and universal probe numbers used are as follows.

・ OsCDK gene group (15 types)
OsCDKA; 1 (GenBank accession number: AK120855)
Forward primer: 5'-cggcgtactaatgcactgaa-3 '(SEQ ID NO: 1)
Reverse primer: 5'-ctctggagctctataccacaagg-3 '(SEQ ID NO: 2)
Universal probe: # 37
OsCDKA; 2 (GenBank accession number: AK101344)
Forward primer: 5'-gatcggcgtaccaactcatt-3 '(SEQ ID NO: 3)
Reverse primer: 5'-tgctctataccacaatgtcacca-3 '(SEQ ID NO: 4)
Universal probe: # 37
OsCDKB; 1 (GenBank accession number: CI522617)
Forward primer: 5'-accggtgttgacatttggtt-3 '(SEQ ID NO: 5)
Reverse primer: 5'-tgccagtccctcaaatcagt-3 '(SEQ ID NO: 6)
Universal probe: # 68
OsCDKB; 2 (GenBank accession number: AK059682)
Forward primer: 5'-ctcaagaagtacacccacgaga-3 '(SEQ ID NO: 7)
Reverse primer: 5'-agatgtcaaccggagtggag-3 '(SEQ ID NO: 8)
Universal probe: # 142
OsCDKC; 1 (GenBank accession number: AK073808)
Forward primer: 5'-tttgcagagcttctcaatgg-3 '(SEQ ID NO: 9)
Reverse primer: 5'-catcaaatattttgctcagttgct-3 '(SEQ ID NO: 10)
Universal probe: # 72
OsCDKC; 2 (GenBank accession number: AK103469)
Forward primer: 5'-ggaaagccaatattgacagga-3 '(SEQ ID NO: 11)
Reverse primer: 5'-gcgtaccacaaagctcaaaaa-3 '(SEQ ID NO: 12)
Universal probe: # 106
OsCDKC; 3 (GenBank accession number: AK241084)
Forward primer: 5'-tcaacctggccgactacg-3 '(SEQ ID NO: 13)
Reverse primer: 5'-ccatagcctcgaatacttcacc-3 '(SEQ ID NO: 14)
Universal probe: # 124
OsCDKD; 1 (GenBank accession number: AK120162)
Forward primer: 5'-ttgctgaactgctgcttagg-3 '(SEQ ID NO: 15)
Reverse primer: 5'-ttcccaagttgatcaatgtcac-3 '(SEQ ID NO: 16)
Universal probe: # 80
OsCDKE; 1 (GenBank accession number: AK066824)
Forward primer: 5'- aagtgataacggggttgttgtt-3 '(SEQ ID NO: 17)
Reverse primer: 5'-ttcagcaaaaatgcaaccaa-3 '(SEQ ID NO: 18)
Universal probe: # 50
OsCDKF; 1 (GenBank accession number: AK059487)
Forward primer: 5'-cctgctcatctccgaggac-3 '(SEQ ID NO: 19)
Reverse primer: 5'-ttggtatgttcctgtctcctga-3 '(SEQ ID NO: 20)
Universal probe: # 162
OsCDKF; 2 (GenBank accession number: AL732536)
Forward primer: 5'-cgttcttgtccttgtgcttg-3 '(SEQ ID NO: 21)
Reverse primer: 5'-gaagcacgtcgcgaaact-3 '(SEQ ID NO: 22)
Universal probe: # 25
OsCDKF; 3 (GenBank accession number: AK098991)
Forward primer: 5'-tgatatgtgggctgttggtg-3 '(SEQ ID NO: 23)
Reverse primer: 5'-tatgatccggtgtcccaag-3 '(SEQ ID NO: 24)
Universal probe: # 102
OsCDKF; 4 (GenBank accession number: AK100966)
Forward primer: 5'-tgaagtttcagttccctcaggt-3 '(SEQ ID NO: 25)
Reverse primer: 5'-gtcccatgagcacagtgatg-3 '(SEQ ID NO: 26)
Universal probe: # 53
OsCDKG; 1 (GenBank accession number: AK111593)
Forward primer: 5′-ggtttcgcgaactacctctg-3 ′ (SEQ ID NO: 27)
Reverse primer: 5'-atgtttcttgaagcgcctgt-3 '(SEQ ID NO: 28)
Universal probe: # 15
OsCDKG; 2 (GenBank accession number: AK099095)
Forward primer: 5'-caccaggaaatcaatggaca-3 '(SEQ ID NO: 29)
Reverse primer: 5'-gggccatcaagcttcctatc-3 '(SEQ ID NO: 30)
Universal probe: # 5

・ OsKRP genes (6 types)
OsKRP1 (GenBank accession number: AK103084)
Forward primer: 5'-cgagatcgaggcgttctt-3 '(SEQ ID NO: 31)
Reverse primer: 5'-caccggagtccactcgaa-3 '(SEQ ID NO: 32)
Universal probe: # 30
OsKRP2 (GenBank accession number: DQ229363)
Forward primer: 5'-ggaattgttgggctgttctg-3 '(SEQ ID NO: 33)
Reverse primer: 5'-ctctcctcgtcgtgatcctc-3 '(SEQ ID NO: 34)
Universal probe: # 63
OsKRP3 (GenBank accession number: DQ229364)
Forward primer: 5'-gacgtcgtcagtggagaaaga-3 '(SEQ ID NO: 35)
Reverse primer: 5'-cagggttgtgcttgtgctc-3 '(SEQ ID NO: 36)
Universal probe: # 70
OsKRP4 (GenBank accession number: AK064173)
Forward primer: 5'-gccacaacattattccagca-3 '(SEQ ID NO: 37)
Reverse primer: 5'-ggcagtcattcacaggatca-3 '(SEQ ID NO: 38)
Universal probe: # 11
OsKRP5 (GenBank accession number: AK064723)
Forward primer: 5'-gcgacaacgttcttgacctc-3 '(SEQ ID NO: 39)
Reverse primer: 5'-aggggtgtgtctccctggt-3 '(SEQ ID NO: 40)
Universal probe: # 55
OsKRP6 (GenBank accession number: AK111010)
Forward primer: 5'-ccaggaaggcgaagaagg-3 '(SEQ ID NO: 41)
Reverse primer: 5'-ggagcgtccttgacgatg-3 '(SEQ ID NO: 42)
Universal probe: # 108

The results are shown in FIG. 2 (OsCDK gene group) and FIG. 3 (OsKRP gene group).
As shown in Fig. 2, among 15 types of rice CDK genes (OsCDK) having the function of advancing the cell cycle, the expression levels of OsCDKB; 1 and OsCDKB; 2 genes are significantly and significantly reduced by ABA treatment alone. However, treatment with SA along with ABA reduced the decrease. In addition, as shown in FIG. 3, among 6 types of rice KRP genes (OsKRP) having the function of stopping the cell cycle by inhibiting CDK, the expression level of OsKRP4 and OsKRP5 genes is significant when ABA alone is treated. However, the increase was suppressed by treatment with SA together with ABA.

(2) Yeast two-hybrid analysis
In order to identify OsCDK protein that interacts with OsKRP protein, a yeast two-hybrid analysis was performed. The Matchmaker ^™ Gold yeast two-hybrid system (Clontech, USA) was used for the analysis. The open reading frames of OsCDKA; 1, OsCDKA; 2, OsCDKB; 2, OsKRP4 and OsKRP5 genes were PCR amplified with cDNA cloning 1st PCR primers, and the amplified products were further PCR amplified with cDNA cloning 2nd PCR primers. Next, using the In-Fusion HD cloning kit (Clontech, USA), the PCR amplified fragments obtained for the OsCDKA1, OsCDKA2 and OsCDKB2 genes were transferred into the vector pGADT7-AD (Clontech, USA; prey) in the OsKRP4 And the PCR amplification fragment obtained for each gene of OsKRP5 was incorporated into the vector pGBKT7-BD (Clontech, USA; for bait) to prepare a vector expressing the fusion protein. About these final constructs, the base sequence was confirmed with the sequencer. Various combinations of bait and prey constructs were introduced into the yeast strain Y2HGold (Clontech, USA). For that yeast, minimal medium (DDO) lacking leucine and tryptophan and DDO with Xa-Gal and aureobasidin A added. The ability to grow in a medium (DDOXA) was examined at 30 ° C. The primer sequences used for PCR are as follows.

OsCDKA; 1
cDNA cloning 1st PCR
Forward primer: 5'-attgggcggcaaacggagg-3 '(SEQ ID NO: 43)
Reverse primer: 5'-aagggcacagcgttcacag-3 '(SEQ ID NO: 44)
cDNA cloning 2nd PCR
Forward primer: 5'-gaggccagtgaattcatggagcagtacgagaaggaggag-3 '(SEQ ID NO: 45)
Reverse primer: 5'-gagctcgatggatcctcattgtaccatctcaaggtcctt-3 '(SEQ ID NO: 46)
OsCDKA; 2
cDNA cloning 1st PCR
Forward primer: 5'-tcatccccaagtccaccac-3 '(SEQ ID NO: 47)
Reverse primer: 5'-ctcggctgagcaaacaatgc-3 '(SEQ ID NO: 48)
cDNA cloning 2nd PCR
Forward primer: 5'-gaggccagtgaattcatggagcagtacgagaaggtggag-3 '(SEQ ID NO: 49)
Reverse primer: 5'-gagctcgatggatccctacgccacttccaggtccttgaa-3 '(SEQ ID NO: 50)
OsCDKB; 2
cDNA cloning 1st PCR
Forward primer: 5'-caaaccctaaatccacgcgc-3 '(SEQ ID NO: 51)
Reverse primer: 5'-ttcagggcaggctagtcac-3 '(SEQ ID NO: 52)
cDNA cloning 2nd PCR
Forward primer: 5'-gaggccagtgaattcatggcggcgctccaccaccaggcg-3 '(SEQ ID NO: 53)
Reverse primer: 5'-gagctcgatggatcctcagtagagctccttgttcacgtc-3 '(SEQ ID NO: 54)
OsKRP4
cDNA cloning 1st PCR
Forward primer: 5'-ccttagccgctaatgctccg-3 '(SEQ ID NO: 55)
Reverse primer: 5'-tctgccacgctactactgtg-3 '(SEQ ID NO: 56)
cDNA cloning 2nd PCR
Forward primer: 5'-catggaggccgaattcatgggcaagtacatgcgcaaggcc-3 '(SEQ ID NO: 57)
Reverse primer: 5'-gcaggtcgacggatcctcagtctagcttgacccattcaaa-3 '(SEQ ID NO: 58)
OsKRP5
cDNA cloning 1st PCR
Forward primer: 5'-cgcgattctcctcccttcc-3 '(SEQ ID NO: 59)
Reverse primer: 5'-gaattgcattttgcctctgcc-3 '(SEQ ID NO: 60)
cDNA cloning 2nd PCR
Forward primer: 5'-catggaggccgaattcatggggaagtacatgcggaagggg-3 '(SEQ ID NO: 61)
Reverse primer: 5'-gcaggtcgacggatccctagcagtctagccttgtccattc-3 '(SEQ ID NO: 62)

  As shown in FIG. 4, it was found that OsKRP4 and OsKRP5 proteins interact with OsCDKA; 1 protein and OsCDKA; 2 protein. However, the interaction between OsKRP4 protein and OsCDKA; 2 protein was weak.

(3) Effect on the cell cycle As described above, ABA suppresses the expression of OsCDKB; 1 gene and OsCDKB; 2 gene, which progresses the cell cycle from G2 phase to M phase, but by treating with ABA with SA It was shown that the expression suppression action is reduced. In addition, the expression of OsKRP4 and OsKRP5 genes, which are inhibitors of CDKA; 1 and CDKA; 2 (both factors are involved in the progression from G1 to S), which form a complex with cyclin and advance the cell cycle, It was clarified that the expression promoting action is suppressed by treatment with SA together with ABA. That is, SA was shown to reduce the cell cycle progression inhibitory effect of ABA.

[Example 3] Reduction effect of salicylic acid (SA) on growth inhibitory action of abscisic acid (ABA)-Effect on endonucleodoubling and new DNA synthesis
Rice seeds germinated for 3 days in the dark at 28 ° C are placed in a net on an ABA 2μM, ABA 2μM + SA 0.4mM, ABA 2μM + SA 1mM, or SA 1mM solution and cultured in the dark at 25 ° C for 5 days Then, the chute was extended (processing section). For the control group (ABA 0 μM + SA 0 mM), the shoot was extended in the same manner. The tissue containing the shoot apex was cut out and the ratio of 4C cells (G2 phase tetraploid cells) doubling in the nucleus was measured by flow cytometry.

  As a result, compared to the control not treated with ABA and SA, the ratio of 4C cells decreased after 2 days of treatment in the ABA single treatment group, and became less than half after 5 days of treatment (FIG. 5). In contrast, in the coexistence treatment group using SA together with ABA (SA coexistence treatment group), the decrease in the ratio of 4C cells was alleviated, and in particular, the treatment group using 1 mM SA was almost the same as the ABA non-treatment group (control). The ratio was about 4C (Fig. 5). From these results, it was revealed that SA has a function of reducing the ABA intranuclear doubling inhibitory action.

  Furthermore, in order to investigate the effects of ABA and SA on new DNA synthesis in shoot shoot apical cells, we analyzed the uptake of new DNA in cells using thymidine analog BrdU (5-bromo-2'-deoxyuridine). did. First, rice seeds germinated for 3 days in the dark at 28 ° C are placed on a net on an ABA 2µM, ABA 2µM + SA 1mM, or SA 1mM aqueous solution, and cultured in the dark at 25 ° C for 24 hours to extend the shoots. Further, 50 μM BrdU and 1 μM 5-fluorodeoxyuridine were added, and cultured in the dark at 25 ° C. for 24 hours to incorporate BrdU into rice seedlings. Seedling tissues labeled with BrdU were fixed in 0.1 M phosphate buffer solution (pH 7.0) containing 4% (w / v) paraformaldehyde for 16 hours at 4 ° C. After fixation, the sample was washed with 0.1 M PBS containing 0.2 M sucrose, and dehydrated sequentially with an ethanol solution with increasing concentrations. The dehydrated sample was embedded with Technovit 7100 resin (Kulzer & Co., Werheim, Germany), and a quasi-ultra thin section (thickness 0.5 μm) of tissue containing shoot apical cells was prepared with an ultramicrotome. This section was subjected to fluorescence staining with a BrdU antibody labeled with Alexa 488, and the fluorescence of the nucleus was observed with a fluorescence microscope. Furthermore, staining using DAPI (4′6-diamidino-2-phenylindole dihydrochloride) that stains DNA was performed and observed.

  The results are shown in FIG. The DAPI staining level did not differ between the treatment sections. However, the ABA treatment group significantly reduced BrdU incorporation compared to the untreated group, indicating that ABA suppresses new DNA synthesis. It was done. In contrast, no decrease in BrdU incorporation was observed in the SA coexisting treatment group, indicating that SA reduces the suppression of new DNA synthesis by ABA.

[Example 4] Reduction effect of salicylic acid (SA) on growth inhibitory action of abscisic acid (ABA)-Expression analysis of ABA degrading enzyme gene and measurement of ABA concentration
In order to examine whether SA induces ABA degradation acceleration or migration inhibition, the expression level of the ABA degrading enzyme gene and the ABA concentration in the shoot were measured.

  First, in order to clarify the effect of SA on the expression of the ABA degrading enzyme gene, changes in the expression levels (mRNA levels) of the ABA degrading enzyme genes ABA8ox1, ABA8ox2, and ABA8ox3 were examined. Rice seeds germinated for 3 days in the dark at 28 ° C. were placed on a net on an aqueous solution of ABA 2 μM, ABA 2 μM + SA 1 mM, or SA 1 mM, cultured for 5 days in the dark at 25 ° C., and the shoots were elongated ( Processing zone). For the control group (ABA 0 μM + SA 0 mM), the shoot was extended in the same manner. RNA was extracted from the obtained shoots, and using this as a template, the expression levels of ABA8ox1, ABA8ox2 and ABA8ox3 genes were analyzed by quantitative PCR (Taqman probe method). As the TaqMan probe, an oligonucleotide modified with FAM at the 5 ′ end and TAMRA at the 3 ′ end was used (Roche Applied Science). The primer sequences and universal probe numbers used are as follows.

・ ABA8ox gene group (3 types)
ABA8ox1 (GenBank accession number: AK067007)
Forward primer: 5'-tcaacaccttccaagagatgaa-3 '(SEQ ID NO: 63)
Reverse primer: 5'-atctcctcctccccgaag-3 '(SEQ ID NO: 64)
Universal probe: # 56
ABA8ox2 (GenBank accession number: AK120757)
Forward primer: 5'-ggcgagcataatctccttca-3 '(SEQ ID NO: 65)
Reverse primer: 5'-ccctttggaatcaggaaacc-3 '(SEQ ID NO: 66)
Universal probe: # 34
ABA8ox3 (GenBank accession number: CI523426)
Forward primer: 5'-catggccctaacccacaa-3 '(SEQ ID NO: 67)
Reverse primer: 5'-gggataaggaaccctttgtactc-3 '(SEQ ID NO: 68)
Universal probe: # 159

  The result is shown in FIG. In the ABA single treatment group, the expression levels of ABA8ox1 increased significantly after 4 days of treatment, and the expression levels of ABA8ox2 and ABA8ox3 increased significantly after 1 day of treatment. These increases in gene expression are thought to have occurred in response to the increase in ABA concentration in the plant caused by ABA absorbed from the roots. In contrast, in the SA coexisting treatment group, no increase was observed in all three types of ABA-degrading enzymes (FIG. 7), so it is clear that SA does not induce the expression of ABA-degrading enzyme genes, and SA It was shown that the ABA concentration of shoots may not increase even if ABA is absorbed from roots.

  Then, in order to clarify the influence of SA on the ABA concentration, the ABA concentration of the shoot was measured. Rice seeds germinated for 3 days in the dark at 28 ° C. were placed on a net on an aqueous solution of ABA 2 μM, ABA 2 μM + SA 1 mM, or SA 1 mM, cultured for 5 days in the dark at 25 ° C., and the shoots were elongated ( Processing zone). For the control group (ABA 0 μM + SA 0 mM), the shoot was extended in the same manner. ABA contained in the ground extract of the obtained chute was quantified using an ABA Test Kit (Phytodetek).

  As a result, the ABA concentration in the shoot was significantly increased in the ABA alone treatment group, while it remained low in the SA coexistence treatment group (FIG. 8). ABA added to the medium is absorbed from the roots and moves to the shoots. Considering the above result that SA does not induce the expression of ABA-degrading enzyme gene, the cause of the ABA concentration not increasing in the shoots in the SA coexisting treatment area was considered to be inhibition of ABA movement (transport) by SA. That is, as shown in Examples 1-3, shoot elongation, cell cycle progression, nuclear doubling, and the inhibitory action of ABA on new DNA synthesis, SA can reduce the movement of ABA to the shoot apex. This was thought to be because SA inhibited.

[Example 5] Effect of reducing the growth inhibitory action of ABA with probenazole It is known that treatment with probenazole imparts resistance to pathogenic bacteria by inducing the production and accumulation of salicylic acid in plants (Yoshioka K, et al., (2001) Plant J., 25: 149-157; Midoh N., Iwata M., (1996) Plant Cell Physiol., 37: 9-18; Iwai T, Seo S., et al., ( 2007) Plant Cell Physiol., 48: 915-924).

  Therefore, instead of SA, the growth suppression effect by ABA is actually treated by treating seedlings immediately after germination with the bactericide Orizemate (registered trademark) granule (Meiji Seika Pharma Co., Ltd.) containing 48% of the SA inducer probenazole. It was investigated whether it was reduced. Specifically, rice seeds sprouting for 3 days in the dark at 28 ° C., ABA (0, 0.2, 1, or 5 μM) and oryzate granules (0, 1.25, 5, or 20 mg / 100 ml [provenazole 0, 26.9, 107.6, equivalent to 430.5 μM])) was sown on a net floated on a mixed aqueous solution and cultivated in the dark at 25 ° C. Five days after the start of treatment, the shoot lengths of 9 individuals in each treatment group were measured.

  The result is shown in FIG. As in the case of coexistence with SA in the medium, coexistence of probenazole significantly reduced the shoot growth inhibitory effect of ABA. Therefore, it was shown that the growth inhibitory effect of ABA can also be reduced by inducing SA accumulation in the plant body with probenazole or the like.

[Example 6] Cooling resistance enhancement effect by SA during booting period
The effect of SA treatment on cold resistance during the booting period was investigated. First of all, rice cultivar “Kita Aoba” was sown in 1 / 5000a pot and cultivated at 25 ℃ / 20 ℃ (15 hours in the day / 9 hours in the evening) with 20 round seeds, and one week before the low temperature treatment (early panicle formation period), 2 Add 100 mM SA stock solution so that the SA dose to the soil is 17.27 g / 10a (equivalent to SA concentration of 0.1 μM in the soil containing water) the day before (the booting period) and the day (the booting period) Stir well. It seems that an antagonistic effect can be obtained with a relatively low SA dose (SA concentration) of this level against the ABA concentration level induced by low-temperature treatment. The low-temperature treatment was carried out at 12 ° C (day and night) for 4 days at the time when the number of individuals 10 days before heading, which is the danger of cold damage of Kita Aoba, was the highest. After low-temperature treatment, the temperature was returned to 25 ° C./20° C. (day / night), and the seeds were formed by heading and flowering. The fertility of the pollen immediately after heading was investigated by the iodine-iodkari method. Specifically, 6 buds were collected from each spikelet immediately after heading and immersed in 100 μL of iodine-iodokari solution (containing 100 g of iodine, 2.0 g of iodine, 0.5 g of potassium iodide, and 10 g of glycerin). Inside, each wrinkle was cut with sharp tweezers and stirred to release pollen, and all the pollen contained in 1 μL was counted. Of all the pollen, pollen that is darkly stained with iodine / iodokari liquid is counted as fertile pollen, and the pollen that is not dyed is counted as sterile pollen, and the ratio of the number of sterile pollen to the total number is the sterile pollen rate . In addition, the fruit seedling rate (sterility) of spikelets 30 days after heading was investigated. The number of cocoon buds in each ear and the number of sterilized flowers were counted, and the ratio of the number of sterilized flowers to the total number was defined as the cocoon sterility rate.

  As a result, regarding fertility of pollen, 26.2% pollen was sterile in the SA-untreated area, whereas the sterility rate in the SA-treated area was as low as 6.8%. Significant differences were observed at the% level (FIG. 10). In addition, the spikelet sterility rate of individuals treated at 12 ° C for 4 days in the early microspore stage (10 days before heading), which is particularly sensitive to low temperatures during the booting period, was 36.7% in the SA untreated area. On the other hand, in the SA-treated group, it was as low as 19.4%, and a significant difference was observed between the two groups at the 0.01% level (FIG. 11). Therefore, it was shown that SA has the effect of suppressing the occurrence of pollen sterility due to low temperature and enhancing the cold tolerance during the booting stage.

[Example 7] Effect of probenazole to enhance cold resistance during booting period
When rice cultivar "Kita Aoba" is sown in 1 / 5000a pot and cultivated at 25 ° C / 20 ° C (15 hours in the day / 9 hours in the night) with 20 round seeds, the 8th leaf is extracted (June 3; young panicle formation) In the first half of the term; 30 days before heading), I moved to an outdoor aquarium in Sapporo. Since the temperature in June in Sapporo is too low for rice to form pollen, natural low temperatures can induce pollen sterility through ABA synthesis. Immediately after the transfer, orizemate granules (containing 48% probenazole) were administered to the aquarium so that the concentration was 667 g / 10a (equivalent to probenazole 320.16 g / 10a; converted to the concentration of probenazole with respect to the amount of water stored in the aquarium, 4.78 μM). The same amount of oryzate granules was administered on the 16th day (late stage of juvenile formation). We investigated the rate of ripening of spikelets 30 days after the ears that appeared in early July.

  As shown in FIG. 12, the spikelet sterility rate of individuals headed on July 2 was 49.7% in the SA-untreated group, whereas it was as low as 39.8% in the SA-treated group. There was a significant difference at the 0.1% level. Therefore, it was shown that probenazole which induces SA also has the effect of suppressing the occurrence of pollen sterility due to low temperature and enhancing the cold tolerance during the booting stage.

  Excessive accumulation of SA is known to reduce the freezing tolerance of Arabidopsis plants. This is because SA suppresses the expression of the transcription factor DREB1A / CBF3 gene necessary for improving freezing tolerance and inhibits low temperature signal transduction (Miura and Ohta, (2010) Journal of plant physiology 167: 555-560). In addition, SA binds to catalase and inhibits the metabolism of hydrogen peroxide to increase the intracellular hydrogen peroxide concentration (Chen et al, (1993) Science 262: 1883-1886). For this reason, it is considered that SA treatment may promote the low-temperature damage of plants, and it was predicted that the SA treatment would reduce the cold tolerance during the booting stage. However, as shown in the present example, it was actually found that the SA treatment has an effect of suppressing the occurrence of pollen sterility at low temperatures. This result suggests that for the development of pollen at low temperatures, it is more effective to suppress the negative effects of ABA on the cell cycle, etc. than to maintain the expression of transcription factors corresponding to DREB1A / CBF3 and the catalase activity. Conceivable. ABA is a hormone that enhances environmental stress tolerance in plants (Lang et al., (1989) Theor. Appl. Genet., 77, p.729-734; Lu et al., (2009) Plant Physiol. Biochem , 47, p.132-138), when rice encounters low temperatures during the booting period, the ABA concentration in the plant body increases, but high ABA significantly suppresses the growth of the plant as a side effect and causes pollen formation in the rice bran. (Oliver et al., (2007) Plant Cell Physiol., 48, p. 1319-1330). From the results of the above examples, it is shown that SA-inducing agents such as SA treatment or probenazole can suppress such negative effects of ABA, thereby suppressing the occurrence of pollen sterility at low temperatures. It was.

[Example 8] Effect of SA on seedling low temperature tolerance (1) Low temperature elongation Rice seeds were sown on a soil (pearl mat) and cultivated in a greenhouse at 25 ° C / 20 ° C for 10 days. For this rice, the aqueous solution of SA is such that the SA dose to the soil is 34.53g / 10a, 345.3 / 10a, or 3453g / 10a (corresponding to the SA concentration in the soil containing water of 0.2, 2, or 20 μM, respectively). And then left to stand for 2 days, transferred to a 17 ° C growth chamber (LED lighting), subjected to low temperature treatment for 4 days, returned to a 25 ° C / 20 ° C greenhouse, and measured the shoot length 12 days after completion of the low temperature treatment (SA treatment section). In the untreated section, the same low temperature treatment was performed without adding the SA aqueous solution (SA dose 0 g / 10a).

  The results are shown in FIG. The shoot length 12 days after 17 ° C treatment was 99.9 mm in the untreated group, whereas it was 97.4-109.7 mm in the SA-treated group, and there was a significant difference between the untreated group and the SA-treated group. I was not able to admit. Therefore, it was shown that SA has no effect of improving the low temperature elongation of seedlings.

(2) Resistance to low temperature withering Rice seeds were sown on a soil (pearl mat) and cultivated in a greenhouse at 25 ° C / 20 ° C for 10 days. For this rice, the SA dose to the soil is 34.53g / 10a, 345.3 / 10a, or 3453g / 10a (corresponding to SA concentration 0.2, 2, or 20μM in the soil (including water) containing water, respectively) After adding an aqueous SA solution and allowing it to stand for 2 days, it was transferred to a 3 ° C. growth chamber (LED lighting), subjected to low temperature treatment for 3 days, and returned to a 25 ° C./20° C. greenhouse. In the untreated section, the same low temperature treatment was performed without adding the SA aqueous solution (SA dose 0 g / 10a). Surviving individuals were counted one month after the low temperature treatment.

  As shown in FIG. 14, the survival rate after 1 month of treatment at 3 ° C. for 3 months was 30.0% in the untreated group, whereas it was 28.3 to 33.3% in the SA treated group. There was no significant difference between the treatment areas. Therefore, it was shown that SA has no effect of improving the low temperature mortality tolerance of seedlings.

  The present invention provides a method for enhancing cold tolerance during the booting period, which suppresses the occurrence of pollen sterility at low temperatures by reducing the inhibitory action of ABA with SA or an SA inducer, which is widely used in the agricultural field. Can be expected.

  Sequence number 1-68: Primer

Claims (6)

Salicylic acid, salicylic acid precursors, seen at least Tsuo含selected salicylic inducer and salts thereof, salicylic acid precursor is a glycoside or salicylic esters of salicylic acid, salicylic acid inducing agent is probenazole, for rice Cold resistance enhancer during booting. The cold tolerance enhancer of the booting stage according to claim 1 for suppressing pollen sterility.   The cold resistance enhancer for the booting stage according to claim 1 or 2, which is applied to the young panicle formation stage or the booting stage. A method for enhancing the booting-time cold resistance of rice , comprising applying the booting-time cooling resistance enhancer according to any one of claims 1 to 3 to rice . The method according to claim 4 , wherein the enhancement of cold resistance during booting is suppression of pollen sterility. The method according to claim 4 or 5 , wherein the cold-resistance enhancer for the booting period is applied during the young panicle formation period or the booting period.

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