JP4845579B2 - Rooting method of cutting - Google Patents

Description

  The present invention has made it possible to grow cherry trees, hypericum trees, or leguminous Paraceliantes trees, such as Yoshino cherry (cherry blossoms) or Japanese willow, which have been extremely difficult or impossible to grow by cuttings. The present invention relates to a method for rooting cuttings using a plant rooting inducer containing ketol unsaturated fatty acid as an active ingredient.

The propagation of plants is usually by seeds or cuttings, and in the case of propagation by the former seeds, the seeds are usually required to be pure.
This is because, if pure seeds are not used, the characteristics of the breeding plants will vary, but there are many plants that are difficult to obtain pure seeds in seed propagation.
Furthermore, in seed propagation, there is another problem that the propagation target is limited to plants from which seeds can be easily collected.

  For this reason, there is also breeding by cuttings as described above, which are widely practiced, but there are many plants that are difficult to root with cuttings, such as pine, bush, cypress, cedar, tea-buckthorn, lily-brush , Salmon, chestnuts, persimmons, anemone, walnuts, wild peaches. Therefore, when cutting these plants, it is normal to use auxin rooting inducers such as “Luton (active ingredient: 1-naphthylacetamide)”, “Oxyberon (active ingredient: indolebutyric acid)”, etc. It has become.

  As described above, even when an auxin-based rooting inducer is used, rooting of the aforementioned plants is often difficult. Therefore, there is a concern that the amount of use will inevitably increase and cause environmental pollution. Furthermore, the usage pattern is complicated, making simple mass processing difficult. That is, it is necessary to immerse the cuttings of cuttings and cuttings in a high concentration auxin solution for several hours, or attach auxin powders to the cuttings one by one, which cannot be used with a simple technique.

  And pre-treatment with silver nitrate, potassium permanganate, lime water, ethanol, etc. prior to treatment with auxin-based chemicals is also widely carried out, which not only makes the use of rooting inducers complicated. Furthermore, the problem is that it leads to environmental pollution. Moreover, there are many plants, especially trees, that are difficult to root even with such complicated processes. In addition, as a feature of trees, roots can be rooted in young trees, but the ability to form roots rapidly disappears as they grow. Because of these properties, the amount that can be used for cuttings is limited, which is also a major cause of difficulty in planting trees.

The present inventors, in order to overcome the current situation of rooting inducers as described above, are keenly aware of the social need for their development, and further focused on future research and development of rooting inducers. As a result, an indole derivative has already been developed and found to have rooting induction performance, and a patent application has been filed for the derivative (see Patent Document 1).
JP-A-10-77268 JP-A-11-29410 JP-A-10-324602

  The derivative developed by the present inventors can be used as a plant rooting inducer by a simple technique such as spraying. In this respect, the derivative is a disadvantage of the conventional auxin rooting inducer. This eliminates the complexity. However, the derivative is also the same indole compound as indole acetic acid, indole butyric acid and the like, which are representative compounds of auxin rooting inducers, and it is difficult to say that the rooting induction performance is sufficiently satisfactory. there were. In addition, auxin-based rooting inducers include naphthalene acetic acid, but compounds that exhibit rooting performance are of a limited range, and are based on aliphatic compounds, particularly fatty acids. The rooting-inducing action of substances is not known at all.

  For this reason, the present inventors have continued to research and develop a rooting inducer and a novel compound suitable therefor in order to find a superior rooting-inducing action from a wider range of compounds. . At that time, the present inventors made a hypothesis that rooting is less likely to be induced in the tree, and that the dormancy phenomenon was involved, and proceeded with research focusing on its control. As a result, it has been found that a specific ketol fatty acid (Patent Document 2) in which “flower bud formation promoting action”, “growth promoting action”, “dormancy inhibiting action” and the like are recognized surprisingly promotes rooting action. It was also found that it could be used by a simple method such as spraying, and the present invention was successfully developed.

  In this way, the present inventors have an excellent root-promoting or inducing ability for rooting of plants, particularly when rooting plants that are difficult to root such as pine, cedar, tea and walnuts are proliferated by cuttings. A plant rooting inducer that contains ketol unsaturated fatty acid, which is a compound that has a chemical structure much different from conventional auxin rooting inducer compounds, and can be used by simple methods such as spraying. It was.

  Furthermore, the present inventors have further compared to a cherry tree, a hypericum tree, or a leguminous paraceriantes tree, particularly a blue willow that is more difficult to root or a Yoshino cherry tree (cherry blossom) that is said to be impossible to root. It has been found that certain α-ketol unsaturated fatty acids among the ketol fatty acids have surprisingly rooting effects, and rooting of sakura, hypericum or leguminous paraceriantes It is an object of the present invention to provide a method.

  As described above, the present invention provides a method for rooting a cutting of a cherry tree, hypericum tree, or leguminous paraceriantes tree, which is a ketol fatty acid having 5 to 24 carbon atoms. A ketol unsaturated fatty acid having 1 to 6 carbon-carbon double bonds and an α-ketol structure or a γ-ketol structure, particularly preferably 9-hydroxy-10-oxo-12 (Z), 15 (Z) -The present invention relates to a method for rooting a cherry tree, a hypericum tree, or a leguminous paraceriantes tree using a solution containing octadecadienoic acid as an active ingredient.

In the present specification and claims, “Z” and “E” mean cis / trans isomers, of which Z represents a cis isomer and E represents a trans isomer.
The underline added below them indicates that “Z” and “E” should be originally written in italics.

  The present invention provides a plant rooting inducer that exhibits the excellent rooting performance necessary for growing plants by cuttings, especially for plants that are difficult to root, such as pine, cedar, tea, and walnuts. A plant rooting inducer that contains a ketol unsaturated fatty acid as an active ingredient, which has developed a path that can be propagated by cuttings, and can exhibit excellent rooting promotion or induction performance. It is.

  However, the present invention further relates to a cherry tree, a hypericum tree, or a leguminous paraceriantes tree, particularly a willow tree that is said to be more difficult to root and a Yoshino cherry tree (cherry tree) that is said to be impossible to root. The path that can be proliferated by cutting is pioneered. According to the present invention, a specific ketol unsaturated fatty acid is used as a rooting inducer for the plant, and the cuttings and cuttings are cut into a high concentration auxin solution for several hours like a conventional auxin rooting inducer. There is no need to immerse or attach a kixin powder one by one to the cut end, and use a simple method such as spraying, dripping or coating as a solution or emulsion to achieve the desired rooting. There are advantages that can be achieved.

  The active ingredient compound of the rooting inducer of the present invention is a derivative having a simple ketol structure in which a naturally occurring unsaturated fatty acid is a basic skeleton, and two oxygens and one hydrogen are added thereto. Moreover, since the predetermined performance can be expressed at a low concentration, the possibility of environmental contamination of the conventional auxin-based rooting inducer can also be reduced. Further, the ketol unsaturated fatty acid, particularly preferably 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid, which is an active ingredient, is a typical auxin rooting inducer. It is an aliphatic compound with an unsaturated fatty acid having a basic structure different from that of an indole compound or an aromatic compound such as naphthalene acetic acid, which is an active ingredient, and has a basic structure different from that of the basic structure. The fact that root inducing action was observed is completely outside the range of prediction. Therefore, the present invention opens up a new field in plant rooting inducers in particular in this respect, and provides an excellent technique.

In the following, the present invention will be described in detail with respect to embodiments of the invention including the best mode for carrying out the invention.
The active ingredient of the plant root-inducing agent used in the present invention is the ketol unsaturated fatty acid (preferably the ketol fatty acid having 5 to 24 carbon atoms and having 1 to 6 double bonds between carbons). And 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid) having an α-ketol structure or a γ-ketol structure. The ketol unsaturated fatty acid having the α-ketol structure or the γ-structure ketol structure is an unsaturated fatty acid in which the carbon atom constituting the carbonyl group and the carbon atom bonded to the hydroxyl group are located at the α-position or the γ-position.

  The ketol unsaturated fatty acid can also be represented by a general formula. In this case, the former ketol unsaturated fatty acid having an α-ketol structure is represented by the general formulas (1) and (2), and the latter ketol unsaturated fatty acid having a γ-ketol structure. Saturated fatty acids are represented by general formulas (3) and (4).

For the ketol unsaturated fatty acid having the α-ketol structure, in the general formulas (1) and (2), R ₁ is a linear alkyl group or a linear unsaturated hydrocarbon group having a double bond; ₂ is a linear alkylene or a linear unsaturated hydrocarbon chain having a double bond, and at least _one of R ₁ and R ₂ has one double bond, and all of ketol unsaturated fatty acids It is necessary that the number of carbon atoms is 5 to 24 and that the total double bond between carbons is 1 to 6.

For the ketol unsaturated fatty acid having a γ-ketol structure, in the general formulas (3) and (4), R ₃ is a linear alkyl group or a linear unsaturated hydrocarbon group having a double bond, R ₄ is a linear alkylene or a linear unsaturated hydrocarbon chain having a double bond, and the total carbon number of the ketol unsaturated fatty acid is 7 to 24, and the total double bond between carbons is 1 to 6 Needs to be selected.

  For these unsaturated ketol fatty acids, compounds having 18 carbon atoms and two double bonds between carbons are preferred as the active ingredient compounds of the rooting inducer of the present invention. Specific examples of the preferable ketol fatty acid include 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid [hereinafter referred to as specific ketol fatty acid (1a)] corresponding to the general formula (1). 13-hydroxy-12-oxo-9 (Z), 15 (Z) -octadecadienoic acid (hereinafter sometimes referred to as the specific ketol fatty acid (2a)) corresponding to the general formula (2), 13-hydroxy-10-oxo-11 (E), 15 (Z) -octadecadienoic acid (hereinafter sometimes referred to as specific ketol fatty acid (3a)) corresponding to general formula (3), general formula (4) 9-hydroxy-12-oxo-10 (E), 15 (Z) -octadecadienoic acid [hereinafter also referred to as specific ketol fatty acid (4a)] and the like. In the present invention, among them, 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid is used to root a Yoshino cherry tree or a Japanese willow cutting.

The chemical structural formulas of the specific ketol fatty acids (1a) to (4a) are described below.

1. About the manufacturing method of the active ingredient compound of this plant root induction agent Below, the specific ketol mentioned above is described about the manufacturing method of the ketol unsaturated fatty acid which has the alpha or gamma ketol structure which is an active ingredient of the plant root induction agent of this invention. This will be described in detail using fatty acids (1a) to (4a) as examples. In addition, in the rooting method of the genus Sakura tree, Hypericum tree or the Leguminosae Paraceriantes tree of the present invention, the ketol fatty acid having 5 to 24 carbon atoms, the number of double bonds between the carbon atoms is 1 to 6 ketol unsaturated fatty acids having an α-ketol structure or a γ-ketol structure are used as active ingredients.

The specific ketol fatty acid can be produced by a method according to the specific structure of the desired ketol fatty acid, which is as follows.
(1) The specific ketol fatty acid that is clearly contained in a natural product can be produced by extraction and purification from the natural product (hereinafter referred to as an extraction method).
(2) A specific ketol fatty acid can be obtained by causing an enzyme such as lipoxygenase to act on an unsaturated fatty acid according to the fatty acid metabolic pathway in the plant (hereinafter referred to as an enzymatic method).
(3) Depending on the specific structure of the desired specific ketol fatty acid, the specific ketol fatty acid can be obtained by using a known ordinary chemical synthesis method (hereinafter referred to as a chemical synthesis method).
These manufacturing methods will be specifically described below.

(1) Extraction method:
The specific ketol fatty acid (1a) can be obtained by extraction and purification from Lemna paucicostata, which is a kind of duckweed family. Duckweed ( Lemna paucicostata ), the raw material for this extraction method, is a small aquatic plant that floats on the surface of a pond or paddy field, and each foliage that floats on the surface of the water drops one root into the water. Known for being fast. The flower is formed on the body side of the frond, and two male flowers consisting of only one stamen and a female flower consisting of one pistil are wrapped in a common small fold.

  This crushed duckweed is subjected to centrifugation (8000 × g for about 10 minutes), and the supernatant and the precipitate obtained, excluding the supernatant, are used as a fraction containing the specific ketol fatty acid (1a). Can be used. Thus, the specific ketol fatty acid (1a) can be isolated and purified using the crushed material as a starting material. Furthermore, as a preferable starting material, there can be mentioned an aqueous solution from which the specific ketol fatty acid (1a) is eluted after duckweed is floated or immersed. By using this, an eluate having a high concentration of the specific ketol fatty acid (1a) can be obtained, and the specific ketol fatty acid (1a) can be efficiently prepared. In that case, as will be described later, it is preferable that an eluate having a higher concentration can be obtained by using a material subjected to stress such as drying stress.

Specific examples of the preparation of this aqueous solution are described in the examples described later.
The immersion time can be about 2 to 3 hours at room temperature, but is not particularly limited. When preparing the starting material of the specific ketol fatty acid (1a) by the above-described method, it can be induced to produce more specific ketol fatty acid (1a) in duckweed by applying a specific stress in advance. It is preferable in terms of production efficiency of the specific ketol fatty acid (1a).

  Specifically, a dry stress, a heat stress, an osmotic pressure stress, etc. can be mentioned as the specific stress. The drying stress can be applied, for example, by leaving duckweed spread on a dry filter paper at low humidity (preferably 50% or less relative humidity) at room temperature, preferably about 24 to 25 ° C. . The drying time in this case is approximately 20 seconds or more, preferably 5 minutes to 5 hours, although it depends on the arrangement density of duckweeds to be dried.

  The heat stress can be applied, for example, by immersing duckweed in warm water. The temperature of the hot water in this case should be selected according to the immersion time. For example, when immersed for about 5 minutes, it is possible at 40-65 degreeC, Preferably it is 45-60 degreeC, More preferably, it is 50-55 degreeC. Moreover, it is preferable to return the duckweed to room temperature water immediately after the heat stress treatment.

  The osmotic stress can be applied by bringing duckweed into contact with a high osmotic pressure solution such as a high concentration sugar solution. In this case, the sugar concentration is 0.3 M or more, preferably 0.5 to 0.7 M, for example, in the case of a mannitol solution. For example, when a 0.5 M mannitol solution is used, the treatment time is 1 minute or longer, preferably 2 to 5 minutes.

In this way, a starting material containing the desired specific ketol fatty acid (1a) can be efficiently prepared. In addition, although the kind of duckweed strain | stump | stock which becomes a basis of the above-mentioned various starting materials is not specifically limited, P441 strain | stump | stock is a strain especially preferable in manufacture of specific ketol fatty acid (1a).
The desired specific ketol fatty acid (1a) can be produced by subjecting the starting material prepared as described above to separation / purification means as described below.

  The separation means shown here is merely an example, and the separation means for producing the specific ketol fatty acid (1a) from the starting material is not limited to the exemplified means, and various types are particularly limited. It can be adopted without being done.

  It is preferable that the starting material prepared as described above is first subjected to solvent extraction to extract a component containing the specific ketol fatty acid (1a). The solvent used for such solvent extraction is not particularly limited, and for example, chloroform, ethyl acetate, ether and the like can be used. Among these solvents, chloroform is preferable in that impurities can be removed relatively easily.

  The oil layer fraction obtained by this solvent extraction is washed and concentrated using a known ordinary method, and then high-performance liquid chromatography (HPLC using a reverse phase partition column chromatography column such as an ODS (octadecylsilane) column. ) To identify and isolate the specific ketol fatty acid (1a), which is also a flower bud formation inducing activity fraction [It is already known that the specific ketol fatty acid has flower bud formation inducing activity ( See Patent Document 3)].

Of course, other known separation means such as ultrafiltration and gel filtration chromatography can be used in combination depending on the properties of the starting material.
As mentioned above, although the process which manufactures specific ketol fatty acid (1a) by the extraction method was demonstrated, when the specific ketol fatty acid of the desired aspect exists in plants other than a duckweed, the method according to the above, or said method It is possible to produce the specific ketol fatty acid by making full use of the modified method.

(2) About enzyme method:
Typical examples of the starting material for the enzymatic method include various unsaturated fatty acids having a double bond at a position corresponding to the structure of the desired specific ketol fatty acid and having 5 to 24 carbon atoms. . Examples of the unsaturated fatty acid include oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, 9,11-octadecadienoic acid, 10,12-octadecadienoic acid, 9,12,15- octadecatrienoic acid, 6,9,12,15-octadecatetraenoic acid, 11,14-eicosadienoic acid, 5,8,11-eicosatrienoic acid, 11,14,17-eicosatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid, 13,16-docosadienoic acid, 13,16,19-docosatrienoic acid, 7,10,13,16-docosatetraenoic acid, 7,10,13,16,19-docosapentaenoic acid, 4,7,10,13, Examples include 16,19-docosahexaenoic acid, but are not limited to these unsaturated fatty acids.

  These unsaturated fatty acids are mostly unsaturated fatty acids contained in animals, plants, etc., extracted and purified from these animals, plants, etc. through known ordinary methods, or chemically synthesized by known ordinary methods Of course, it is also possible to use a commercially available product. In this enzymatic method, lipoxygenase (LOX) is allowed to act using the unsaturated fatty acid as a substrate to introduce a hydroperoxy group (—OOH) into the carbon chain of these unsaturated fatty acids.

  The lipoxygenase is an oxidoreductase that introduces molecular oxygen into the carbon chain of unsaturated fatty acids as a hydroperoxy group. Its presence has been confirmed regardless of animals and plants, and in yeasts represented by Saccharomyces. The existence has also been confirmed. For example, in the case of plants, it is an enzyme whose presence has been confirmed in all angiosperms (specifically, dicotyledonous plants and monocotyledonous plants to which the present plant rooting inducer described later can be applied).

  Among these plants, especially soybean, flax, alfalfa, barley, broad bean, capsicum, lentil, pea, potato, wheat, apple, pan yeast, cotton, cucumber, currant, grape, pear, kidney bean, rice, strawberry, Sunflower or tea is preferred as the source of lipoxygenase. In addition, since chlorophyll has a strong tendency to inhibit the above-mentioned activity of lipoxygenase, it is preferable to select seeds, roots, fruits and the like that are free of chlorophyll in plants as much as possible as a raw material for lipoxygenase.

  In the present invention, the origin of the lipoxygenase is not particularly limited as long as it can introduce a hydroperoxy group at a desired position of the carbon chain of the unsaturated fatty acid, but the active ingredient of the rooting method of the present invention is not limited. In the case of the specific ketol fatty acid (1a), it is preferable to use a lipoxygenase that selectively oxidizes the double bond portion at the 9-position of linoleic acid or linolenic acid as much as possible. Representative examples of such selective lipoxygenase include lipoxygenase derived from rice germ (Yamamoto, A., Fuji, Y., Yasumoto, K., Mitsuda, H., Agric. Biol. Chem., 44, 443 (1980) etc.].

  And as unsaturated fatty acid selected as a substrate with respect to this selective lipoxygenase, it is preferable to use linoleic acid or (alpha)-linolenic acid. In addition, when performing lipoxygenase treatment using unsaturated fatty acid as a substrate, it is natural that the enzyme reaction is preferably allowed to proceed at the optimum temperature and pH of the lipoxygenase to be used. Further, impurities that are not intended to be produced and produced by the above lipoxygenase reaction step can be easily separated by using a known ordinary method, for example, HPLC described in the section (1) above. is there.

  As the lipoxygenase used here, those extracted and purified from the above-mentioned plants and the like by known ordinary methods can be used, or commercially available products can be used. In this way, hydroperoxy unsaturated fatty acids can be produced from the unsaturated fatty acids. This hydroperoxy unsaturated fatty acid can be positioned as an intermediate in the production process of a specific ketol fatty acid by an enzymatic method.

  As this hydroperoxy unsaturated fatty acid, for example, as an intermediate of the specific ketol fatty acid (1a), 9-hydroperoxy-10 (E), 12 (Z) can be obtained by allowing lipoxygenase to act on α-linolenic acid. , 15 (Z) -octadecatrienoic acid, and 13-hydroperoxy-9 (Z), 11 (E), 15 (Z) -octadeca as an intermediate of the specific ketol fatty acid (3a) Mention may be made of trienoic acid. Regarding these hydroperoxy fatty acids, the former 9-hydroperoxy-10 (E), 12 (Z), 15 (Z) -octadecatrienoic acid is used as the hydroperoxy fatty acid (1a ′) related to the present invention, and the latter 13- Hydroperoxy-9 (Z), 11 (E), 15 (Z) -octadecatrienoic acid is the present invention-related hydroperoxy fatty acid (3a ′), and the chemical structural formula is described below.

  The specific ketol fatty acid can be produced by allowing allenoxide synthase to act on a hydroperoxy unsaturated fatty acid as a substrate. This allene oxide synthase is an enzyme having an activity of converting a hydroperoxy group into a ketol form through epoxidation, and is an enzyme existing in plants, animals and yeasts as in the case of the lipoxygenase. [Specifically, it is an enzyme existing in dicotyledonous plants and monocotyledonous plants to which the plant rooting inducer described later can be applied]. In addition, this allen oxide synthase is recognized in barley, wheat, corn, cotton, eggplant, flax (seed etc.), chisha, oat, spinach, sunflower and the like.

With respect to the allene oxide synthase used for producing the specific ketol fatty acid in the present invention, for example, the 9-position of the above 9-hydroperoxy-10 (E), 12 (Z), 15 (Z) -octadecatrienoic acid There is no particular limitation as long as an epoxy group is formed by dehydrating the hydroperoxy group and a desired specific ketol fatty acid can be obtained as a result by nucleophilic reaction of OH ⁻ . As the allene oxide synthase used here, those extracted and purified from the above-mentioned plants and the like by known ordinary methods can be used, or commercially available products can be used.
The two-step enzyme reaction described above can be performed separately or continuously.

  When performing the treatment with the allene oxide synthase described above, it is naturally preferable to proceed the enzyme reaction at the optimum temperature and optimum pH of the allene oxide synthase to be used. Furthermore, for the enzyme, both a crude product and a purified product can be used, and the desired specific ketol fatty acid can be obtained by using the enzyme to advance the enzyme reaction. Further, the desired specific ketol fatty acid can be obtained by immobilizing the enzyme on a carrier, preparing these immobilized enzymes, and subjecting the substrate to column treatment or batch treatment.

  In addition, as a method for preparing the enzyme used in the above two steps, a genetic engineering technique can be used. That is, genes encoding these enzymes are extracted and obtained from plants or the like by a conventional method, or obtained by chemically synthesizing based on the gene sequence of the enzyme. A desired enzyme can be obtained by transforming cultured cells, plant cultured cells, etc., and expressing recombinant enzyme protein in these transformed cells.

Then, when the specific ketol fatty acid is to be obtained by the nucleophilic reaction of OH ⁻ after the formation of the epoxy group (above), depending on the mode of action of the nucleophilic substance in the vicinity of the epoxy group, α In addition to saturated fatty acids, γ-ketol compounds are produced.
This γ-ketol compound can be easily separated from the α-ketol compound by using known normal separation means such as HPLC described in the section (1) above.

(3) About chemical synthesis method:
The specific ketol fatty acid can also be produced by making full use of a known ordinary chemical synthesis method. For example, a saturated carbon chain having a reactive group such as an aldehyde group at one end and a carboxyl end to which a protecting group is bonded at the other end is synthesized by a known ordinary method. Using an unsaturated alcohol such as 3-hexen-1-ol as a starting material, an unsaturated carbon chain having a reactive end having an unsaturated group at a desired position is synthesized. Next, the saturated hydrocarbon chain and the unsaturated carbon chain can be reacted to produce a specific ketol fatty acid. In this series of reactions, the protecting group added to the terminal where the reaction is not intended and the catalyst for promoting the reaction can be appropriately selected and used according to the specific reaction mode.

Specifically, for example, the specific ketol fatty acid can be synthesized by the following procedure.
i) Synthesis of specific ketol fatty acid (1a) Specific ketol fatty acid (1a), which is an active ingredient of the rooting method of the present invention, can be produced by the following method.
Nonanedioic acid monoethyl ester is used as a starting material, reacted with N, N′-carbonyldiimidazole to form acid imidazolide, and then LiAlH _{4 is} reduced at low temperature to synthesize the corresponding aldehyde first.
It is also possible to synthesize a similar aldehyde using the above starting material as a diol such as 1,9-nonanediol.

Separately, cis-3-hexen-1-ol is reacted with triphenylphosphine and carbon tetrabromide, the resulting bromide compound is reacted with triphenyl phosphine, and n- A cis olefin is constructed by reacting with chloroacetaldehyde in the presence of BuLi, and this is further reacted with methylthiomethyl p-tolyl sulfone, and then the secondary alcohol derived by reacting with the above aldehyde in the presence of NaH is tert. The desired specific ketol fatty acid (1a) can be synthesized by protecting with -butyl diphenyl silyl chlorid (TBDPSCl), acid hydrolysis, and then deprotecting.
Below, a simple flowchart is shown about the synthetic | combination process of this specific ketol fatty acid (1a).

ii) Synthesis of specific ketol fatty acid (2a)
Nonanedioic acid monoethyl ester is used as a starting material to react with thionyl chloride to give acid chloride, and then NaBH ₄ reduction is performed to produce acid alcohol.
Next, after protecting the free carboxylic acid of this acid alcohol, it is reacted with triphenylphosphine and carbon tetrabromide, the resulting bromide compound is reacted with triphenylphosphine, and further reacted with chloroacetaldehyde in the presence of n-BuLi. Was further reacted with methylthiomethyl p-tolyl sulfone.
This reaction product is reacted with an aldehyde derived from the PCC oxidation of cis-3-hexen-1-ol separately in the presence of n-BuLi, and finally deprotected to give the desired specific ketol fatty acid ( 2a) can be synthesized.
Below, the simple flowchart of an example of the synthetic | combination process of this specific ketol fatty acid (2a) is shown.

iii) Synthesis of specific ketol fatty acid (3a)
Methyl vinyl ketone is used as a starting material, trimethylsilylchloride is reacted in the presence of LDA and DME, and MCPBA and trimethylamine hydrofluoric acid are added to the resulting silyl ether at low temperature (−70 ° C.) to prepare keto alcohol.
Then, after protecting the carbonyl group of the keto alcohol, the reaction is carried out using triphenylphosphine and trichloroacetone as reaction reagents without adding chloride to the olefin.

Next, this reaction product is reacted with formic acid in the presence of tributylarsine and K ₂ CO ₃ to construct a trans olefin to form a chloride. Thereafter, this chloride is reacted with an aldehyde derived from PCC oxidation of cis-3-hexen-1-ol, and this reaction product is combined with 6-heptenonic acid, and finally deprotected. The desired specific ketol fatty acid (3a) can be synthesized.
Below, a simple flowchart is shown about the synthetic | combination process of this specific ketol fatty acid (3a).

2. About the Plant Rooting Inducing Agent The active ingredient of the plant rooting inducing agent is a ketol fatty acid having 5 to 24 carbon atoms as described above, wherein the double bond between carbons is 1 to 6, and α A ketol unsaturated fatty acid having a ketol structure or a γ-ketol structure. Among them, the ketol unsaturated fatty acid having an α-ketol structure has 5 to 24 carbon atoms and 1 to 6 double bonds between carbon atoms, and is represented by the general formula (1) or (2). Can do. The ketol unsaturated fatty acid having a γ-ketol structure has 7 to 24 carbon atoms and 1 to 6 double bonds between carbon atoms, and is represented by the general formula (3) or (4). Can do.
In the general formulas, R ₁ , R ₂ , R ₃ and R ₄ are as described above.

  The rooting inducer can promote or induce the rooting of the plant by using it on the plant by a simple technique such as spraying. As its use mode, it is preferable to use the compound which is an active ingredient or the formulation which mix | blends it as an aqueous solution with simple methods, such as spraying, application | coating, and impregnation, to a plant. In the present invention, in particular, since the cuttings of Yoshino cherry or Japanese willow are rooted, the above-mentioned specific ketol fatty acid (1a) -containing solution is applied to or spread on the cut trees, or the cut trees are applied to the solution. The desired result can be obtained by impregnation.

  The upper limit of the dose of the specific ketol fatty acid to the plant is not particularly limited. That is, even if a large amount of the specific ketol fatty acid is administered with the plant rooting inducer, there are hardly any negative effects on the plant such as growth inhibition. This is because when plant hormones such as auxin and cytokinin, which have been used in the past, are excessively administered, a negative effect on the plant is prominent. When using these, special care is taken to prevent overdose. It can be said that this plant rooting inducer is very excellent compared with what must be done. In addition, the lower limit concentration of the specific ketol fatty acid to be administered to a plant varies depending on the kind and size of the plant individual, but is approximately 1 μmM or more per administration per one plant individual.

  The blending amount of the specific ketol fatty acid in the plant root inducing agent should be selected according to the use mode, the type of plant to be used, and the specific dosage form of the plant root inducing agent. Is possible. Although it is possible to use the specific ketol fatty acid as it is as an aspect of the plant root inducing agent, it is generally about 0.1 to 100 ppm with respect to the whole agent in consideration of the above-mentioned guideline for administration of the specific ketol fatty acid. Is more preferable, and more preferably about 1 to 50 ppm.

  Examples of the dosage form of the plant rooting inducer include dosage forms such as liquids, solids, powders, emulsions, and bottom floor additives. Depending on the dosage form, the dosage form can be applied. Such known carrier components, formulation adjuvants, and the like can be appropriately blended as long as the plant root induction action, which is the intended effect of the present invention, is not impaired. For example, as the carrier component, when the plant rooting inducer is a bottom floor additive or a solid agent, talc, clay, vermiculite, diatomaceous earth, kaolin, calcium carbonate, calcium hydroxide, clay, silica gel, etc. In the case of inorganic carriers, solid carriers such as wheat flour and starch, and liquid agents, water, aromatic hydrocarbons such as xylene, alcohols such as ethanol and ethylene glycol, ketones such as acetone, dioxane, tetrahydrofuran, etc. Liquid carriers such as ethers, dimethylformamide, dimethyl sulfoxide, acetonitrile and the like are used as the carrier component.

  Examples of the adjuvant for preparation include anionic surfactants such as alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, dialkyl sulfosuccinates, and cationic surfactants such as salts of higher aliphatic amines. Agent, polyoxyethylene glycol alkyl ether, polyoxyethylene glycol acyl ester, polyoxyethylene glycol polyhydric alcohol acyl ester, nonionic surfactant such as cellulose derivative, thickener such as gelatin, casein, gum arabic, thickener A binder or the like can be appropriately blended. Furthermore, if necessary, a general plant growth regulator, benzoic acid, nicotinic acid, nicotinic acid amide, pipecolic acid, etc. are used to induce rooting of the plant as long as the intended purpose of the present invention is not impaired. It can also mix | blend in an agent.

This plant root induction agent can be used for various plants by the method according to the dosage form.
The most characteristic feature is that it can be sprayed, dripped or applied as a solution or emulsion to not only the plant growth point but also part or all of the plant body including stems and leaves. Is significantly different from conventional auxin rooting inducers.
Auxin-based rooting inducers need to be soaked in high concentration auxin solution for several hours before cutting or cutting buds are inserted into the soil, or attached to auxin powder cuts one by one. It was difficult.

  This plant rooting inducer can insert the necessary number of cuttings and cuttings into the soil as it is, and then spray them together with a spreader etc., thus avoiding the difficulties and suitable for mass processing Rooting inducer. As an indispensable rooting inducer suitable for mass treatment, the indole lactone having the above-mentioned specific structure developed by the present inventors (see Patent Document 1) is probably the only one. The specific indole lactone form, like various auxins, has a weak effect on dormant plants, and the usage scene is limited. The plant root inducing agent has a great feature that it can also induce rooting of a dormant plant.

  Regarding the administration of the plant root-inducing agent to plants, the frequency varies depending on the type of plant individual, the purpose of administration, etc., but basically, a desired effect can be obtained even by a single administration. In the case of multiple administrations, it is efficient to leave an administration interval of one week or more.

  The types of plants to which the plant rooting inducer can be applied are not particularly limited, but also angiosperms (dicotyledonous and monocotyledonous plants), fungi, lichens, bryophytes, ferns and gymnosperms. Although it is effective, the specific plant rooting inducer used in the present invention is particularly effective for rooting of Japanese willow or cherry blossoms (cherry blossoms) which is said to be impossible to root. .

Example 1
In the following, specific examples of the production of the specific ketol fatty acid (1a) of the compound used in the plant rooting inducer of the present invention, the identification test example of the compound, and the rooting induction performance test example of the compound will be specifically described as Example 1. Although shown, this invention is not limited at all by this Example, and it cannot be overemphasized that it is specified by description of a claim.

Production example: Production example of specific ketol fatty acid (1a) [9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid] was produced by the enzymatic method as follows.
1. Preparation of rice germ-derived lipoxygenase 350 g of rice germ washed with petroleum ether, degreased and dried (250 g) was suspended in 1.25 L of 0.1 M acetate buffer (pH 4.5), and this suspension was homogenized. did.
Thereafter, the homogenized extract was centrifuged at 16000 rpm for 15 minutes to obtain a supernatant (0.8 L).

Next, 140.8 g (30% saturation) of ammonium sulfate was added to the obtained supernatant, left overnight at 4 ° C., centrifuged again at 9500 rpm for 30 minutes, and ammonium sulfate was added to the obtained supernatant (0.85 L). 232 g (70% saturation) was added and left at 4 ° C. for 5 hours.
Thereafter, centrifugation is carried out at 9500 rpm for 30 minutes, and the resulting precipitate (ammonium sulfate 30-70% saturated fraction of rice germ extract) is dissolved in 300 mL of pH 4.5 acetate buffer solution at 63 ° C. for 5 minutes. Heat treatment was performed.
Further, the produced precipitate was removed, and the resulting supernatant was desalted by dialysis (3 L × 3) using an RC dialysis tube (Spectrum pore 4: MWCO 12000-14000), and then the desired rice. A crude enzyme solution of embryo-derived lipoxygenase was obtained.

2. Preparation of allene oxide synthase derived from flax seed Flax seed was purchased from Ichimaru Falcos, 250 ml of acetone was added to 200 g of flax seed, and homogenized (20 s × 3). It was filtered and the solvent was removed.
Then, the precipitate was again suspended in 250 mL of acetone and homogenized (10 s × 3) to obtain the precipitate again.
The precipitate was washed with acetone and ethyl ether and then dried to obtain flaxseed acetone powder (150 g).

20 g of this flaxseed acetone powder was suspended in 400 mL of 50 mM phosphate buffer (pH 7.0) under ice cooling, and this was extracted by stirring with a stirrer at 4 ° C. for 1 hour.
The obtained extract was centrifuged at 11000 rpm for 30 minutes, and 105.3 g (0 to 45% saturation) of ammonium sulfate was added to the supernatant (380 mL) thus obtained, and the mixture was allowed to stand for 1 hour under ice-cooling. The precipitate obtained by centrifugation at 11000 rpm for 30 minutes was dissolved in 150 mL of 50 mM phosphate buffer (pH 7.0), desalted by dialysis (3 L × 3), and the desired allene oxide synthase derived from flax seed A crude enzyme solution was obtained.

3. Preparation of sodium salt of α-linolenic acid Since α-linolenic acid used as a starting material has a remarkably low solubility in water, α-linolenic acid was sodium-chlorinated in order to facilitate its function as an enzyme substrate.
That is, 530 mg of sodium carbonate was dissolved in 10 mL of purified water and heated to 55 ° C., and 278 mg of α-linolenic acid (Nacalai Tesque) was added dropwise thereto and stirred for 3 hours.
After completion of the reaction, a precipitate was formed by neutralization with an ion exchange resin [Dowex 50W-X8 (H ⁺ form) (manufactured by Dow Chemical Co.)].
This was filtered to separate the resin, dissolved in MeOH, and then the solvent was distilled off under reduced pressure.
The product thus obtained was recrystallized from isopropanol to obtain the desired sodium salt of α-linolenic acid (250 mg, 83%).

4). Production of Specific Ketol Fatty Acid (1a) The sodium salt of α-linolenic acid (15 mg: 50 μmol) obtained in the above 3 was dissolved in 30 mL of 0.1 M phosphate buffer (pH 7.0).
3.18 mL of the rice germ-derived lipoxygenase crude enzyme solution obtained in 1 above was added to the resulting solution at 25 ° C. in an oxygen stream, stirred for 30 minutes, and then the rice germ-derived lipoxygenase crude enzyme. 3.18 mL of liquid was added and stirred for 30 minutes.
After completion of this stirring, 34.5 mL of the crude enzyme solution of allene oxide synthase obtained in 2 above was added to the lipoxygenase reaction product under a nitrogen stream and stirred for 30 minutes, and then diluted hydrochloric acid was added under ice cooling to react. The pH of the solution was adjusted to 3.0.

Then, the reaction solution was extracted with CHCl ₃ -MeOH = 10: 1.
Magnesium sulfate was added to the organic layer obtained by the extraction to dehydrate, and the solvent was distilled off under reduced pressure to dry the organic layer.
The crude product thus obtained was subjected to HPLC, and a peak recognized as the specific ketol fatty acid (1a) (retention time: around 16 minutes) was collected.
Chloroform was added to the collected fraction, the chloroform layer was separated and washed with water, and the chloroform was distilled off with an evaporator to obtain a purified product.
In order to confirm the structure of the obtained purified product, ¹ H and ¹³ C-NMR spectra were measured with a deuterated methanol solution, and the measured spectra are shown in Table I.

As a result, in ¹ H-NMR, the terminal methyl group [δ 0.98 (t)], the two olefins [(δ 5.25, 5.40), (δ 5.55, 5.62)], the secondary hydroxyl group [δ 4.09 (dd)] and a number of methylene-based signals were observed, presumed to be the specific ketol fatty acid (1a).
Furthermore, the chemical shift value of ¹³ C-NMR in Table 1 that the measured, ¹³ C-NMR chemical shift values ([Patent Document 3, the first row from the column 11 under page 7, specific ketol fatty acid (1a) When compared with the chemical shift value of ¹³ C-NMR in “Production Example (Extraction Method)” described after the first (paragraph number 0054 / paragraph number 0055 from the third column on the left column of page 8) .
Therefore, the synthetic product obtained by the enzymatic method as described above is certainly 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid of the specific ketol fatty acid (1a). It could be confirmed.

Example 2
In this Example 2, the specific ketol fatty acid (1a), that is, 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid, which is an active ingredient compound of the rooting method of the present invention, is used. The rooting induction performance test of Yoshino cherry (cherry blossoms) was performed and the observation results are shown. However, the present invention is not limited in any way by this example, and is specified by the description of the claims. Needless to say. The evaluation test methods and test results are as follows.

[Rooting induction performance test of Yoshino cherry tree]
Using 15 kinds of experimental plots such as the rooting agent alone and a combination of the rooting agent and the existing rooting agent, a rooting induction performance test was conducted on Yoshino cherry. Existing rooting agents used in the test are indole derivatives (hereinafter abbreviated as “IBL”) and oxyberon (hereinafter abbreviated as “OX” or “Oxy”) represented by the following formula (5). In this test, the rooting agent is abbreviated as “KODA”.

[Rooting agent composition]
The rooting agent composition used in each experimental section is as follows.
Experimental Group (1) Water: Experimental Group (2) KODA 10 μM: Experimental Group (3) KODA 100 μM: Experimental Group (4) Oxy: Experimental Group (5) IBL 50 ppm: Experimental Group (6) IBL 100 ppm: Experimental Group (7) KODA 10 μM + Oxy: Experimental Group (8) KODA 100 μM + Oxy: Experimental group (9) KODA 10 μM + IBL 50 ppm: Experimental group (10) KODA 100 μM + IBL 50 ppm: Experimental group (11) KODA 10 μM + IBL 100 ppm: Experimental group (12) KODA 100 μM + IBL 100 ppm: Experimental group (13) KODA 10 μM + IBL 100 ppm (15) KODA 100 μM + IBL 100 ppm + Oxy.

[Rooting test procedure]
The Yoshino cherry branch used for the experiment was purchased from Sumitomo Forestry Greening Co., Ltd.
It was cut into a length of about 5 to 8 cm from the tip, and inserted into the mixed soil in a tray in which red jade earth and vermiculite were blended at a ratio of 7: 3.
At that time, 10 branches were used in each experimental section.
Since the experiment started in early March, the leaves were not yet developed.
The oxyberon (Oxy) used in the rooting test is an experimental group (4) (7) (8) (diluted 40-fold with oxyberon solution (containing 0.4% indolebutyric acid, Bayer Krupp Science). 13) The branches of Yoshino cherry for (14) and (15) were soaked for 3 hours and then inserted into the soil of each experimental section.

When the number of cuttings surviving 7 weeks after the start of the test was measured, the results were as follows.
Experimental group (1) 0%: Experimental group (2) 10%: Experimental group (3) 10%: Experimental group (4) 0%: Experimental group (5) 0%: Experimental group (6) 0%: Experimental group (7) 30%: experimental group (8) 10%: experimental group (9) 20%: experimental group (10) 30%: experimental group (11) 20%: experimental group (12) 30%: experimental group (13 ) 10%: experimental section (14) 20%: experimental section (15) 30%.
In addition, all the live cuttings were found to have active rooting.
Since it is as above, it turns out that this rooting agent has the rooting induction | guidance | derivation performance outstanding with respect to the Yoshino cherry tree which cutting cutting was impossible conventionally.

Example 3
In this Example 3, as in Example 2, the rooting induction performance test of Japanese willow was performed using the specific ketol fatty acid (1a) which is an active ingredient of the rooting method of the present invention.
The rooting agent composition and the rooting test procedure used in each experimental section were the same as in Example 2.
As a result of the test, it was found that when the rooting agent (10 μM) and IBL (50 ppm) were used, 100% of the willow cuttings survived.

  Further, the rooting performance test is as shown in FIG. 1. According to FIG. 1, the experimental group (2), the experimental group (3), the experimental group (8), the experimental group (11), the experimental group ( 13) In the experimental section (14) and the experimental section (15), the amount of rooting was clearly increased compared to the water treatment section (experimental section (1)).

From the above results, the use of the rooting agent is a necessary and sufficient condition for increasing roots. The symbol “*” arranged at the head of the bar graph in FIG. 1 indicates the risk rate in the significance test. One symbol is less than 5%, and two symbols are risk factors. 1% or less, three symbols indicate a risk factor of 0.5% or less.
That is, in the experimental section in which “*” is arranged at the head of the bar graph, the rooting amount is clearly increased as compared with the water treatment section (experiment group (1)).

Example 4
[Falkata rooting performance test]
Falcataria ( Parserianthes falcataria ), useful as a raw material for plywood, is a tropical tree, but as with other tree species, cuttings become impossible as the age of the tree advances. Using Falkata where cuttings became impossible, cutting propagation was attempted using KODA or the existing rooting inducer oxyberon (manufactured by Bayer CropScience). As the culture soil, small red balls were used. As shown in Table II, the results after 2 months, Falkata did not induce any rooting even with oxyberon, but a 44% rooting rate was obtained by spraying with 10 μM KODA.

It is drawing which shows the result of the willow rooting induction | guidance | derivation rooting performance test in Example 3. FIG.

Claims (4)

A cut tree of Yoshino cherry, Birch willow or Falkata, which is a ketol fatty acid having 5 to 24 carbon atoms, having 1 to 6 double bonds between carbon atoms, and an α-ketol structure or γ-ketol A rooting method for cuttings, which comprises applying or spraying a solution containing a ketol unsaturated fatty acid having a structure. A cut tree of Yoshino cherry tree, birch willow or falkata is a ketol fatty acid having 5 to 24 carbon atoms, having 1 to 6 double bonds between carbon atoms, and an α-ketol structure or a γ-ketol structure A method for rooting cuttings, characterized by impregnating a solution containing a ketol unsaturated fatty acid having the following formula:   The rooting method according to claim 1 or 2, wherein the tree is Yoshino cherry or Birch willow.   The rooting method according to any one of claims 1 to 3, wherein the ketol fatty acid is 9-hydroxy-10-oxo-12 (Z), 15 (Z) -octadecadienoic acid.

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