JP6830591B1 - How to enhance the properties of plants - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new technique for enhancing the characteristics of a plant without using a gene recombination technique. By immersing another plant tissue in an extract of one plant tissue that has undergone a freezing step, the same effect as applying the freeze-thaw awakening method to the other plant tissue can be imparted. .. [Selection diagram] Fig. 2

Description

The present invention relates to a method of enhancing plant properties without resorting to genetic manipulation.

Since ancient times, human beings have produced plants with advantageous properties by breeding methods. Conventional breeding methods require a long time to fix certain characteristics, but with the advent of generation promotion technology, it has become possible to shorten the time required for fixing. However, there is a problem that it takes several years to fix even with the generation promotion technology. Therefore, biotechnology such as anther culture that does not require fixing work has been developed.

In addition, a gene recombination technique is known as a method for producing a plant having advantageous properties. Genetically modified technology has produced herbicide-tolerant crops, pest-resistant crops, disease-resistant crops, and crops with increased shelf life.

On the other hand, a method of inducing mutation and enhancing the characteristics of plants by applying a specific treatment has been proposed. For example, Patent Document 1 discloses a breed improvement method for imparting cold resistance, which includes a step of performing gamma ray irradiation and chromosome doubling treatment.

In addition, a method for controlling the characteristics of plants without changing the gene sequence has been devised. For example, Patent Document 2 describes salt stress, illuminating stress, strong light stress, dry stress, excessive humidity stress, high temperature stress, low temperature stress, nutritional stress, heavy metal stress, etc. caused in the cultivation environment during the nutritional growth period of plants. A method of controlling the flowering time in the next generation of plants by applying stress treatment such as disease stress, oxygen deficiency stress, ozone stress, CO ₂ stress, and strong wind stress is disclosed.

By the way, most regions of Japan belong to the temperate zone, and Hokkaido and the Tohoku region belong to the subarctic zone (cold zone). Therefore, we are relying on imports for crops that are not suitable for cultivation in the Japanese climate, such as those cultivated in the subtropical to tropical regions.
As an epoch-making technique for solving this problem, a technique called "freeze-thaw awakening method" (Patent Document 3) has been developed by the present inventor and has achieved many excellent achievements so far. For example, in Okayama Prefecture, domestically produced pesticide-free bananas to which the freeze-thaw awakening method is applied are sold under the name of Miracle Banana (registered trademark).

The freeze-thaw awakening method is to cultivate a frozen and thawed plant tissue to adapt to the environment such as the characteristics of the plant, specifically, growth rate, cold resistance, heat resistance, highland adaptation characteristics and lowland adaptation characteristics. It is a method for enhancing characteristics, amount and size of fruits and seeds, sweetness, pest resistance, drought resistance, and the like. The freeze-thaw awakening method is not limited to plants of a certain family, genus, or species, and is applicable to all plants. To date, more than 230 varieties have been successfully cultivated.

Japanese Unexamined Patent Publication No. 2006-25632 Japanese Unexamined Patent Publication No. 2016-182094 JP-A-2018-183112

An object to be solved by the present invention is to provide a new technique for enhancing the characteristics of a plant without using genetic modification.

In the process of studying the further development of the freeze-thaw awakening method, the present inventor immerses another plant tissue in the extract of one plant tissue that has undergone the freezing step, thereby treating the other plant tissue. It was discovered that the same effect as that of applying the freeze-thaw awakening method can be imparted, and the present invention has been completed. Specifically, the present invention provides the following.

[1] A freezing step of freezing the plant tissues, and selection step of selecting a living plant tissue from the plant tissue through said freezing step, look including an extraction step of obtaining an extract from the screening step a through plant tissue, The minimum temperature during freezing in the freezing step is −20 ° C. or lower, the rate of temperature drop in the freezing step is 0.8 ° C./day or less, and the period of the freezing step is 100 days or more. A method for producing an extract for enhancing the growth characteristics and / or cold resistance of a plant .
[2] The production method according to [1] , wherein in the freezing step, the plant tissue is frozen while being immersed in an aqueous saccharide solution.
[3] The production method according to [2], wherein the saccharide is trehalose.
[4] The production method according to any one of [1] to [3] , wherein the selection step performs a fermentation treatment on the plant tissue that has undergone the freezing step.
[5] The production method according to [4], wherein the fermentation treatment is performed by leaving the plant tissue that has undergone the freezing step in the open air.
[6] The production method according to [5], wherein the leaving is carried out at 0 ° C to 40 ° C.
[7] The item according to any one of [4] to [6] , wherein in the selection step, a dead plant tissue and a living plant tissue are separated and treated after the fermentation treatment. Production method.
[8] The production method according to [7], wherein the separation treatment is performed by washing the fermented plant tissue.
[9] The production method according to [8], wherein the washing is washing with water.
[10] The production method according to any one of [1] to [9] , wherein the extraction step performs a crushing treatment on a living plant tissue.
[11] The production method according to [10], wherein the crushing treatment is a mashing treatment.
[12] The production method according to [11], wherein the grinding process is performed over several tens of seconds to several hours.
[13] An extract produced by the production method according to any one of [1] to [12] for enhancing the growth characteristics and / or cold resistance of plants .
[14] The extract is characterized in that it comprises a sugar or sugar alcohol, extract according to [13].
[15] The extract according to [14], wherein the sugar or sugar alcohol is sucralose and / or trehalose.
[16] The extract according to any one of [13] to [15] , wherein the extract is diluted.
[17] An extracted dried product obtained by drying the extract according to any one of [13] to [16] .
[18] The growth characteristics and / or cold resistance are enhanced in the extract according to any one of [13] to [16] or the extract obtained by dissolving the dry extract according to [17]. A method for enhancing the growth characteristics and / or cold resistance of a plant tissue, which comprises an immersion step of immersing the desired plant tissue.
[19] wherein the plant tissue to be enhanced growth characteristics and / or cold resistance is dried prior to the immersion step, enhanced growth characteristics and / or cold tolerance of plant tissue according to [18] Method.
[20] The method for enhancing growth characteristics and / or cold resistance according to [18] or [19] , wherein the immersion time in the immersion step is 1 to 100 hours.
[21] By applying the method for enhancing growth characteristics and / or cold resistance according to any one of [18] to [20] , a plant tissue having enhanced growth characteristics and / or cold resistance is produced. Method.
[22] A plant tissue having enhanced growth characteristics and / or cold hardiness obtained by applying the method according to [21].
[23] A method for producing a plant having enhanced growth characteristics and / or cold resistance , which comprises the step of cultivating the plant tissue according to [22].
[24] A plant having enhanced growth characteristics and / or cold resistance produced by the production method according to [23].
[25] A method for producing fruits or seeds of the plant by cultivating the plant according to [24].
[26] Fruits or seeds produced by the method according to [25].
[27] A method for searching for genes involved in enhancing plant growth characteristics and / or cold resistance .
A step of treating a plant by the method according to any one of [18] to [20] , and
(I) A step of identifying a gene that exhibits a higher expression level in the treated plant as compared to the untreated plant and / or (ii) compared to the untreated plant. , A step of identifying a gene showing a low expression level in the treated plant,
A search method characterized by including.
[28] An extract produced by the production method according to any one of [1] to [12] is prepared as an analysis target.
Extracts extracted from plant tissues that have not undergone the freezing step are prepared for comparison.
By comparatively analyzing the extract to be analyzed and the extract to be compared,
Identification that identifies components that are contained in the extract to be analyzed but not in the extract to be compared, or components that are contained more or less in the extract to be analyzed than in the extract to be compared. A method for analyzing an extract, which comprises a process.
[29] An extract produced by the production method according to any one of [1] to [12] is prepared as an analysis target.
Extracts extracted from plant tissues that have not undergone the freezing step are prepared for comparison.
By comparatively analyzing the extract to be analyzed and the extract to be compared,
An identification step for identifying components that are contained in the extract to be analyzed but not in the extract to be compared, or components that are contained in the extract to be analyzed more than the extract to be compared. A method for searching for factors that enhance plant growth characteristics and / or cold resistance , which are characterized by being provided.
[30] A dipping step of immersing a plant tissue or a plant cell in a solution containing one or more kinds of components identified by the identification step.
When the plant tissue or plant cell that has undergone the immersion step has enhanced plant growth characteristics and / or cold resistance as compared with the plant tissue or plant cell that has not undergone the immersion step , the component is added to the plant growth characteristic. And / or a determination step for determining that it is a factor for enhancing cold resistance ,
[29] The search method according to [29].
[31] A step of carrying out the search method according to [30] and
The growth characteristics and / or the discriminated said component and a factor of cold resistance enhancing plant by the searching method characterized by comprising the step of adding to the aqueous medium, the growth characteristics and / or cold tolerance of plants A method of making a solution for augmentation.
[32] the solution prepared by the method according to [31], growth characteristics and / or a dipping step of dipping the plant tissue to be enhanced cold resistance, a method enhancing the growth characteristics and / or cold tolerance of plant tissue ..

According to the present invention, it is possible to obtain a plant having enhanced characteristics without using a breeding method or a gene recombination method for several years. Further, in the freeze-thaw awakening method, individual plant tissues must be subjected to a predetermined process of freezing and thawing, but in the present invention, a plant tissue having characteristics once enhanced by the freeze-thaw awakening method can be obtained. Once done, there is no need to perform the time-consuming processes of freezing and thawing. Therefore, by simply obtaining an extract from the plant tissue and immersing it in the extract, it is possible to impart excellent properties to a large amount of another plant tissue at one time at no cost and in a short time. There is an advantage. As a result, the propagation speed of the technique for enhancing the characteristics of plants by the freeze-thaw awakening method is further accelerated, and it can be further spread to various plants or regions around the world.

Since grains such as rice, wheat, wheat, and soybean are consumed in large quantities as staple foods or feeds, they are cultivated in large quantities with a huge acreage. In other words, grain cultivation requires a huge amount of seeds to supply a sufficient amount of crops to meet the demand.
Here, the property enhancing method of the present invention has an advantage that excellent properties can be imparted to a large amount of plant tissues at one time. Therefore, the property-enhancing method of the present invention can be extremely suitably used for grain seeds that require a huge amount of planting.

It is a photograph which shows the result of Test Example 1. The right is the wheat of the example, and the left is the wheat of the comparative example. It is a photograph which shows the result of Test Example 1. The wheat planted in the right column is the wheat of the example, and the wheat planted in the left column is the wheat of the comparative example. It is a photograph which shows the result of Test Example 2. The wheat planted in the upper row is the wheat of the example, and the wheat planted in the lower row is the wheat of the comparative example. It is a photograph which shows the wheat ear of the Example of Test Example 2. It is a photograph which shows the result of Test Example 2. The left is the wheat of the example, and the right is the wheat of the comparative example. It is a photograph showing the result of Test Example 4. The one planted in the right column is the corn of the example, and the one planted in the left column is the corn of the comparative example. It is a photograph showing the result of Test Example 4. The left is the corn of the example, and the right is the corn of the comparative example.

[Manufacturing method of extract]
The method for producing an extract of the present invention includes a freezing step of freezing a plant tissue.
The plant tissue to be subjected to the freezing step may be obtained from any plant species. For example, Caricaceae, Bromeliad, Musaceae, Cucurbitaceae, Myrtaceae, Myrtaceae, Oxalidaceae, Moraceae, Moraceae, Moraceae, Moraceae, Moraceae Rubiaceae, Laureaceae, Pasifloraceae, Sapindaceae, Guttiferae, Ebenaceae, Legume, Rutaceae, Legume) , Cactaceae, Rosaceae, Legumes, Poaceae, and the like can be exemplified.

More specifically, the genus Papaya (Carica), the genus Ananas (Ananas), the genus Basho (Musa), the genus Lacanca Siraita, the genus Annona (Psidium), the genus Annona (Averhoroa), the genus Ficus, the genus Persimmon (Theobroma). ), Coffee tree (Coffea), Cinnamomum, Hylocereus (Passiflora), Reishi (Litchi), Fukugi (Garcinia), Persimmon (Diospyros), Kashiromia (Casimiroa), Annona Illustrate plants belonging to the genus Phoenix, Hylocereus, Cerasus, Glycine, Hordeum, Tricium, Zea, etc. Can be done.

The plant tissue to be subjected to the freezing step is not limited, and examples thereof include plant seeds, roots, buds, stems, leaves, and petals. These tissues may be frozen as they are when subjected to the freezing step, or may be partially excised and frozen in the form of sections.

In the freezing step, it is preferable to freeze the plant tissue in a state of being immersed in a liquid. As the liquid for immersing the plant tissue, it is preferable to use a frost damage protective agent composed of an aqueous solution of DMSO (dimethyl sulfoxide), glycerin, ethylene glycol, sugar or the like. Of these, it is preferable to use an aqueous solution of saccharides, particularly an aqueous solution of trehalose.

The upper limit of the minimum freezing temperature in the freezing step is preferably -20 ° C or lower, more preferably -30 ° C or lower, still more preferably -40 ° C or lower, still more preferably -50 ° C or lower, still more preferably -55 ° C or lower. is there.
The lower limit of the minimum freezing temperature is preferably −200 ° C. or higher, more preferably −150 ° C. or higher, still more preferably −100 ° C. or higher, still more preferably −80 ° C. or higher, still more preferably −70 ° C. or higher, and further. It is preferably −65 ° C. or higher.

In the freezing step, it is preferable to slowly lower the temperature rather than rapidly lowering the temperature to the minimum temperature at the time of freezing. The rate of temperature drop is preferably 0.8 ° C./day or less, more preferably 0.6 ° C./day or less, more preferably 0.5 ° C./day or less, still more preferably, from the viewpoint of survival rate after thawing. It is 0.3 ° C./day or less, more preferably 0.2 ° C./day, still more preferably 0.1 ° C./day.
When the temperature is slowly lowered in this way, it is preferable to use a program freezer in the freezing step.

The lower limit of the freezing step period is preferably 100 days or longer, more preferably 120 days or longer, still more preferably 150 days or longer, still more preferably 160 days or longer, still more preferably 180 / day or longer.

The "freezing process period" is a period from the time when the temperature of the plant tissue is lowered to the time when the thawing process is started.

The plant tissue that has undergone the freezing step may be subjected to the next extraction step in a frozen state, but is preferably thawed before being subjected to the extraction step. The defrosting method is not particularly limited. The frozen plant tissue may be naturally thawed by leaving it at room temperature, or the frozen plant tissue may be thawed while rinsing with running water. It is preferable to thaw naturally at room temperature.

It is preferable to include a selection step between the freezing step and the extraction step described later. The selection step is a step of selecting a living plant tissue from the frozen plant tissue.
The specific embodiment of the selection process is not limited as long as the living plant tissue can be selected.

A preferred form of the selection step includes a method including fermenting a plant tissue that has undergone a freezing step. This is a method that utilizes the difference in resistance between living and dead plant tissues to fermentation by microorganisms.
Living plant tissues maintain their tangible state without being decomposed by microorganisms. On the other hand, dead plant tissue is decomposed by microorganisms and softened or liquefied. Therefore, after the fermentation treatment, the living plant tissue and the dead plant tissue can be easily sorted.

The method of fermentation treatment is not particularly limited. A method performed by leaving the plant tissue that has undergone the freezing step in the open air is preferably exemplified.
In this case, the lower limit of the temperature of the environment to be left is preferably 0 ° C. or higher, more preferably 10 ° C. or higher, still more preferably 20 ° C. or higher. The upper limit is preferably 50 ° C. or lower, more preferably 40 ° C. or lower.

The period of leaving in the open air is not particularly limited as long as it can be fermented, and preferably several days to several weeks, specifically 1 to 4 weeks can be mentioned as a guide.

After the fermentation treatment, it is preferable to separate the dead plant tissue from the living plant tissue. The specific mode of the separation treatment is not particularly limited as long as the living plant tissue can be separated from the mixture of dead plant tissue and living plant tissue.
As mentioned above, after the fermentation process, the dead plant tissue is decomposed by microorganisms and softened or liquefied. Therefore, by washing the fermented plant tissue, the dead plant tissue can be easily washed away and removed. As the washing, washing with water can be preferably exemplified.

The present invention includes an extraction step of obtaining an extract from a plant tissue that has undergone a freezing step. The extraction method is not particularly limited. The extractant used for extraction is preferably an aqueous solvent, more preferably water or an aqueous solution.

The extractant preferably contains a sugar or a sugar alcohol. More specifically, monosaccharides (dextrose, lactose, treose, arabinose, xylose, galactose, ribose, glucose, sorbose, fructose, mannose), disaccharides (sucrose (sucrose), lactose (lactose), maltose (maltose)). , Trehalose, cellobiose, isomaltose, isotorehaloth, neotrehalose, neolactose, turanose, palatinose) and other polysaccharides (trisaccharides: raffinose, meregitos, maltotriose, tetrasaccharides: acarbose, stachiose, glycogen, solubilized starch , Amyrose, dextrin, glucan, β1,3-glucan, fructan, N-acetylglucosamine, chitin, chitosan), sugar alcohols (xylitol, sorbitol, erythritol, mannitol, martitol), oligosaccharides (rafinose, panose, maltotri) Extract containing one or more saccharides or sugar alcohols selected from aus, meregitos, gentianose, sucrose, cyclodextrin, xylooligosaccharides, cellulose oligosaccharides, lactose oligosaccharides, fructo-oligosaccharides, galactooligosaccharides, mannan oligosaccharides) Is preferably used.
More preferably, as the sugar or sugar alcohol, an extractant containing one or a combination of sucralose and trehalose is used.
When a drawing agent containing no sugar or sugar alcohol is used, the sugar or sugar alcohol may be added to the extract obtained after the extraction step.

It is preferable to carry out a crushing treatment for crushing the plant tissue that has undergone the freezing step, more specifically, the living plant tissue that has undergone the freezing step. As a method of crushing treatment, mashing treatment can be preferably exemplified.
The mashing treatment may be carried out by using a crusher such as a mixer or a ball mill, but it is preferably crushed by mashing with a mortar.

The stress applied to the plant tissue in the crushing treatment is not particularly limited, but it is preferable to gently crush the plant tissue without applying too much stress. Particularly preferably, it is gently ground with a mortar and a pestle.

The time required for the crushing treatment is not particularly limited, but is preferably several tens of seconds to several hours. Specifically, the lower limit is preferably 10 seconds or longer, more preferably 30 seconds or longer. The upper limit is preferably 10 hours or less, more preferably 5 hours or less, and further preferably 3 hours or less.

The crushing treatment of the plant tissue may be performed in a state where the plant tissue is immersed in the extractant, but is preferably performed before the extractant is added. That is, it is preferable to extract the plant tissue that has undergone the crushing treatment by bringing it into contact with the extractant.

By contacting the plant tissue that has undergone the freezing step with the extractant, and more specifically, immersing the plant tissue in the extractant, the components contained in the plant tissue are transferred to the extractant to obtain an extract.
After the extraction step, a step of filtering the extract to remove the residue of the plant tissue may be provided.

The amount of the extractant used in the extraction step is not particularly limited, but is preferably 0.5 parts by mass or more, more preferably 1 part by mass or more, still more preferably 3 parts by mass or more, based on 1 part by mass of the plant tissue. More preferably, it is 5 parts by mass or more.
The upper limit is not particularly limited, and the amount of the extractant is preferably 100 parts by mass or less, more preferably 50 parts by mass or less, and further preferably 20 parts by mass or less with respect to 1 part by mass of the plant tissue.

The immersion time in the extractant is not particularly limited. The lower limit of the immersion time is not limited, but is preferably 1 minute or longer, more preferably 10 minutes or longer, and even more preferably 30 minutes or longer. The upper limit of the soaking time is not limited, but it can be preferably 2 days or less, more preferably 1 day or less, further preferably 12 hours or less, still more preferably 6 hours or less.

The temperature of the extractant at the time of immersion is also not particularly limited. The lower limit is preferably 0 ° C. or higher, more preferably 10 ° C. or higher. The upper limit is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, still more preferably 45 ° C. or lower, still more preferably 40 ° C. or lower.

When a liquid or paste in which the components contained in the plant tissue are dissolved or dispersed is obtained by the crushing treatment, the liquid or paste is also included in the "extract".
In this case, the crushing process itself constitutes the extraction process.

[Extract and dried extract]
The extract of the present invention is produced by the above production method.
The present invention also includes a dried extract obtained by drying the extract to remove the solvent. The method for obtaining the extracted dried product is not particularly limited, and examples thereof include spray drying and freeze drying.

In the present specification, "extract" is not a term that refers only to an extract that is primarily obtained through an extraction step. The "extract" also includes a diluted solution obtained by diluting the originally obtained extract with an arbitrary liquid, and a concentrated solution obtained by concentrating the primarily obtained extract. Further, the "extract" also includes a solution obtained by dissolving the dried extract in an arbitrary solution, in other words, a solution obtained by exchanging a solvent with the originally obtained extract.
Further, as described above, when a liquid or paste in which the components contained in the plant tissue are dissolved or dispersed is obtained by the crushing treatment, the liquid or paste is also included in the "extract".

The extract and dried extract of the present invention can be used for enhancing the characteristics of plants. In addition, the "characteristics of plants" in the present specification include environmental adaptation characteristics such as cold resistance, heat resistance, highland adaptation characteristics and lowland adaptation characteristics, growth rate, amount and size of fruits and seeds, sweetness, and pest resistance. It is a concept that broadly includes the characteristics of plants such as drought resistance.

[Characteristic enhancement method]
The property enhancing method of the present invention includes a dipping step of immersing a plant tissue whose characteristics are desired to be enhanced in the above-mentioned extract.
The plant tissue to be subjected to the dipping step may be obtained from any plant species. For example, Caricaceae, Bromeliad, Musaceae, Cucurbitaceae, Myrtaceae, Myrtaceae, Oxalidaceae, Moraceae, Moraceae, Moraceae, Moraceae, Moraceae Rubiaceae, Laureaceae, Pasifloraceae, Sapindaceae, Guttiferae, Ebenaceae, Legume, Rutaceae, Legume) , Cactaceae, Rosaceae, Legumes, Poaceae, and the like can be exemplified.

More specifically, the genus Papaya (Carica), the genus Ananas (Ananas), the genus Basho (Musa), the genus Lacanca Siraita, the genus Annona (Psidium), the genus Annona (Averhoroa), the genus Ficus, the genus Persimmon (Theobroma). ), Coffee tree (Coffea), Cinnamomum, Hylocereus (Passiflora), Reishi (Litchi), Fukugi (Garcinia), Persimmon (Diospyros), Kashiromia (Casimiroa), Annona Plants belonging to the genus Phoneix, the genus Hylocereus, the genus Cerasus, the genus Glycine, the genus Hordeum, the genus Annona, and the like can be exemplified.

The plant species of the plant tissue from which the extract used in the dipping step is derived and the plant species of the plant tissue used in the dipping step may be the same or different. That is, the plant tissue of a plant species other than the specific plant species may be immersed in the extract obtained by freezing and extracting the plant tissue of a specific plant species. Even in such a heterogeneous application, a desired effect can be obtained according to the property enhancing method of the present invention.

The plant tissue to be subjected to the dipping step is not limited, and examples thereof include plant seeds, roots, buds, stems, leaves, and petals. These tissues may be immersed as they are when subjected to the immersion step, or may be partially excised and immersed in the form of sections.

It is also preferable that the plant tissue whose properties are desired to be enhanced is dried before the dipping step. Since it is sufficient to dry the surface of the plant tissue, for example, the plant tissue may be dried in the sun for a few days. In addition, drying may be performed using a dryer such as a dryer. This makes it possible to increase the permeation efficiency of the extract into the plant tissue in the dipping step.

When the seeds having seed coats are subjected to the dipping step, the thickness of the seed coats is preferably 3 cm or less, more preferably 1 cm or less, more preferably 5 mm or less, and more preferably 3 mm or less. When the thickness of the seed coat is within the above range, the permeation efficiency of the extract is good, and the extract can be permeated into the seeds in a short time.
As such seeds, seeds of grains such as barley, wheat, soybean, and rice are mentioned as preferable examples of seeds to which the present invention is applied.

The immersion time is not particularly limited, but is preferably 30 minutes or longer, more preferably 1 hour or longer, more preferably 6 hours or longer, more preferably 12 hours or longer, more preferably 24 hours or longer, still more preferably 48 hours or longer, and further. It is preferably 60 hours or more. The upper limit of the immersion time can be preferably 300 hours or less, more preferably 200 hours or less, and further preferably 100 hours or less.

The temperature of the extract in the dipping step is preferably set to a temperature at which germs do not grow during the same step. The temperature is preferably 0 ° C. or higher, more preferably 5 ° C. or higher, and even more preferably 10 ° C. or higher. As the upper limit, preferably 50 ° C. or lower, more preferably 45 ° C. or lower, still more preferably 40 ° C. or lower, still more preferably 35 ° C. or lower, still more preferably 30 ° C. or lower can be mentioned as a guide.

The extract used in the dipping step is preferably a diluted solution obtained by diluting the extract primary obtained in the extraction step with an arbitrary liquid. By diluting the extract primary obtained by the extraction step, many plant tissues can be subjected to the dipping step at one time, and the production efficiency can be improved.

The dilution ratio is not particularly limited. The volume of the diluted solution after dilution can be preferably 100 times or more, more preferably 1000 times or more, still more preferably 5000 times or more, still more preferably 8000 times or more the volume of the plant tissue used for extraction. Even if diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
The upper limit of the dilution ratio is not particularly limited, but is preferably 100,000 times or less, more preferably 50,000 times or less, still more preferably 20,000 times or less, still more preferably 10,000 times or less.

Further, when a diluted solution is obtained by diluting the liquid or paste obtained by crushing the plant tissue without adding an extractant, the dilution ratio is preferably 100 times or more, more preferably. It can be 1000 times or more, more preferably 5000 times or more, still more preferably 8000 times or more. Even if diluted at such a high dilution rate, the effect of the present invention can be sufficiently obtained.
In this case, the upper limit of the dilution ratio is not particularly limited, but preferably 100,000 times or less, more preferably 50,000 times or less, still more preferably 20,000 times or less, still more preferably 10,000 times or less.

The liquid used for dilution is preferably a liquid containing sugars or sugar alcohols, like the extractant. More specifically, monosaccharides (dextrose, lactose, treose, arabinose, xylose, galactose, ribose, glucose, sorbose, fructose, mannose), disaccharides (sucrose (sucrose), lactose (lactose), maltose (maltose)). , Trehalose, cellobiose, isomaltose, isotorehaloth, neotrehalose, neolactose, turanose, palatinose) and other polysaccharides (trisaccharides: raffinose, meregitos, maltotriose, tetrasaccharides: acarbose, stachiose, glycogen, solubilized starch , Amyrose, dextrin, glucan, β1,3-glucan, fructan, N-acetylglucosamine, chitin, chitosan), sugar alcohols (xylitol, sorbitol, erythritol, mannitol, martitol), oligosaccharides (rafinose, panose, maltotri) A liquid containing one or more saccharides or sugar alcohols selected from aus, meregitos, gentianose, sucrose, cyclodextrin, xylooligosaccharides, cellulose oligosaccharides, lactose oligosaccharides, fructo-oligosaccharides, galactooligosaccharides, mannan oligosaccharides). It is preferably used for dilution.
More preferably, as the sugar or sugar alcohol, a liquid containing one or a combination of sucralose and trehalose is used for dilution.

In the dipping step, it is preferable to immerse the plant tissue in an amount of preferably 0.5 kg or more, more preferably 1 kg or more, still more preferably 1.5 kg or more per liter of the extract.
The upper limit of the weight of the plant tissue immersed in 1 liter of the extract is not particularly limited, but is preferably 3 kg or less, more preferably 2.5 kg or less.

In the dipping step, it is preferable that the entire plant tissue is immersed in the extract. When the entire plant tissue is not immersed in the extract at one time, the extract is brought into contact with the entire plant tissue by rolling or stirring the plant tissue in the extract during the immersion step. It doesn't matter.

The plant tissue subjected to the dipping step is enhanced in the characteristics of the plant as in the case of being subjected to the freeze-thaw awakening method. As described above, the "property of the plant" that can be enhanced by the property enhancing method of the present invention includes the characteristics of the plant without limitation.
Specifically, according to the property enhancing method of the present invention, environmental adaptation characteristics such as cold resistance, heat resistance, highland adaptation characteristics and lowland adaptation characteristics, growth rate, amount and size of fruits and seeds, sweetness, and resistance. It is possible to enhance the properties of one or more selected from pest resistance and drought resistance.

By cultivating a plant tissue whose characteristics have been enhanced by the property enhancing method of the present invention, a plant having enhanced characteristics can be obtained. In addition, the plant tissue after the dipping step can be sown as it is without any treatment.
The cultivation method is not particularly limited. When the plant tissue subjected to the dipping step is a plant seed, it can be sown according to a conventional method to generate an individual plant and cultivated according to a conventional method.

When the plant tissue subjected to the dipping step is a plant part other than seeds, it may be transferred to soil or a medium as it is and germinated, or it may be finely chopped and cell culture is carried out according to a conventional method to induce callus. By inducing adventitious embryos and inducing adventitious buds, individual plants can be generated and cultivated.

Next-generation plants obtained by methods other than sexual reproduction from plants to which the property-enhancing method of the present invention has been applied inherit the enhanced properties. Therefore, if a plant having enhanced properties can be obtained by the property enhancing method of the present invention, a plant individual independent of the plant can be generated from a plant tissue (offspring, etc.) other than seeds, which is obtained from the plant. The offspring of the next generation and beyond also have enhanced properties.

In addition, the plant to which the property enhancing method of the present invention is applied exhibits enhanced properties even when used as a scion of a grafted tree.

By applying the property enhancing method of the present invention, it is possible to improve the size and yield of fruits and seeds of a plant that bears fruits and seeds. Therefore, the merit of applying the present invention as a method for producing fruits or seeds is very large.
In the fruits or seeds produced in this way, nutritional components such as sweetness are enhanced, and the superiority in the agricultural industry is very high.

[Search method (1)]
Plants treated by the characterization method of the present invention have significant variations in gene expression profile. It can be said that an increase or decrease in the expression level of a specific gene is a factor for enhancing the characteristics. That is, it can be said that a gene whose expression level increases or a gene whose expression level decreases in a plant cell by applying the characteristic enhancement method of the present invention is a gene involved in the enhancement of plant characteristics.

Therefore, it is possible to search for a gene that enhances the characteristics of a plant by analyzing and identifying a gene whose expression level increases or decreases in a plant cell by applying the property enhancing method of the present invention.

That is, the present invention comprises a step of treating a plant by the above-mentioned property enhancing method and
It also relates to a method for searching for a gene involved in enhancing the characteristics of a plant, which identifies a gene whose expression level is different in the treated plant as compared with the untreated plant.

Specifically, the search method of the present invention includes a step of treating a plant by the above-mentioned method of enhancing the characteristics of the present invention, and the following steps (i) and / or (ii).
(I) Step of identifying a gene showing a high expression level in the treated plant as compared with the untreated plant (ii) The treatment as compared with the untreated plant Steps to identify genes with low expression levels in plants

The steps (i) and (ii) in the search method of the present invention can be performed by a conventional method. For example, transcriptome analysis of microarrays, RNA sequences, and the like can identify genes whose expression levels vary in plants treated by the property-enhancing method of the present invention.

[Screening method]
As described above, a gene whose expression level fluctuates in a plant to which the trait enhancement method of the present invention is applied is a factor of trait enhancement. Therefore, if the expression level of the gene is used as an index, the trait enhancement factor of the plant can be used. Can be screened.
That is, the present invention also relates to a method for screening a factor that enhances the characteristics of a plant, using the fluctuation of the expression level of the gene in the plant to which the test substance is applied as an index. Here, the "plant to which the test substance is applied" includes a plant into which the test substance has been introduced, a plant treated with the test substance, a plant that has been in contact with or exposed to the test substance, and the like.

Specifically, the following genes (i) and / or (ii) are used as indexes as compared with the plants that have not been treated by the above-mentioned property enhancing method of the present invention.
(I) Gene showing high expression level in the treated plant (ii) Gene showing low expression level in the treated plant

Then, when the expression level of the gene (i) in the plant to which the test substance is applied is higher than the expression level of the gene in the plant to which the test substance is not applied, the test substance is used as a characteristic of the plant. Screen as an enhancer.
Further, when the expression level of the gene (ii) in the plant to which the test substance is applied is lower than the expression level of the gene in the plant to which the test substance is not applied, the test substance is used as a characteristic of the plant. Screen as an enhancer.

The expression level of the gene can be confirmed by a conventional method such as Northern blotting or real-time PCR.

[Analysis method]
The present invention also relates to a method for analyzing an extract.
Specifically, the extract produced by the method for producing the extract of the present invention described above is prepared as an analysis target. In addition, as a comparison target, an extract extracted from a plant tissue that has not undergone a freezing step is prepared.
In order to realize highly accurate analysis, it is preferable that the production conditions of the extract to be compared match the production conditions of the extract to be analyzed and the conditions other than the presence or absence of the freezing step.

The prepared extract to be analyzed and the extract to be compared are compared and analyzed.
The method of comparative analysis can be performed by a conventional method, and a mass spectrometer (LC-MS, GC-MS, etc.) can be preferably exemplified.

By comparative analysis, components contained in the extract to be analyzed but not included in the extract to be compared, or components contained in the extract to be analyzed more or less than the extract to be compared. To identify.
The identification method is not particularly limited. For example, when a mass spectrometer is used in comparative analysis, there is a method of comparing mass spectra and identifying components corresponding to characteristic peaks. In this case, a database capable of searching for a compound from the m / z value may be used, or the component corresponding to the peak may be isolated and subjected to NMR measurement to identify the component.

By performing a component comparative analysis of the extract to be compared and the extract to be analyzed using the analysis method of the present invention, it is possible to grasp the changes brought about by the freezing step on the plant tissue, and by extension, the characteristics of the present invention. The details of the mechanism of action of the enhancement method can be clarified.

[Search method (2)]
The present invention also relates to a method for searching for factors that enhance the characteristics of plants. Here, the "factor for enhancing the characteristics of plants" is a component contained in the extract of the present invention, which acts on plant tissues, plant cells or plants generated from them to enhance the characteristics. It is an ingredient that can be derived.
Specifically, the extract produced by the method for producing the extract of the present invention described above is prepared as an analysis target. In addition, as a comparison target, an extract extracted from a plant tissue that has not undergone a freezing step is prepared.
In order to realize highly accurate analysis, it is preferable that the production conditions of the extract to be compared match the production conditions of the extract to be analyzed and the conditions other than the presence or absence of the freezing step.

The prepared extract to be analyzed and the extract to be compared are compared and analyzed.
The method of comparative analysis can be performed by a conventional method, and a mass spectrometer (LC-MS, GC-MS, etc.) can be preferably exemplified.

By comparative analysis, components contained in the extract to be analyzed but not included in the extract to be compared, or components contained in the extract to be analyzed more or less than the extract to be compared. To identify.
The identification method is not particularly limited. For example, when a mass spectrometer is used in comparative analysis, there is a method of comparing mass spectra and identifying components corresponding to characteristic peaks. In this case, a database capable of searching for a compound from the m / z value may be used, or the component corresponding to the peak may be isolated and subjected to NMR measurement to identify the component.

A preferred embodiment of the present invention may further include a dipping step and a discriminating step.
In the immersion step, the plant tissue or plant cell is immersed in a solution containing one or more components identified by the identification step.
As for the embodiment of immersing the plant tissue, the above-mentioned description of the immersing step regarding the method for enhancing the characteristics of the present invention is valid as it is.
When the plant cells are immersed, the plant cells may be seeded in a culture vessel and cultured in a culture solution containing the above components.

In the discrimination step, in the plant tissue or plant cell that has undergone the immersion step, when the characteristic of the plant is enhanced by the plant tissue or plant cell that has not undergone the immersion step, the component is a factor for enhancing the characteristic of the plant. To determine.

As a specific embodiment of the discrimination step, there is a form of observing a plant generated from a plant tissue or a plant cell that has undergone the immersion step and a plant that has been generated from the plant tissue or the plant cell that has not undergone the immersion step.
Specifically, in the discrimination step, the characteristics of the plant generated from the plant tissue or plant cell that has undergone the immersion step are observed, and the plant is more than the plant generated from the plant tissue or plant cell that has not undergone the immersion step. When enhancement of characteristics is observed, the component is determined to be a factor of enhancement of plant characteristics.
The characteristics of the plants observed here include environmental adaptation characteristics such as cold resistance, heat resistance, highland adaptation characteristics and lowland adaptation characteristics, growth rate, amount and size of fruits and seeds, sweetness, pest resistance, and drought resistance. It is possible to adopt the characteristics of plants such as sex without limitation.

Further, in the discrimination step, it may be an embodiment in which discrimination is performed by a gene analysis method.
Specifically, the expression level of the gene identified by the method described in the above [Search method (1)] was analyzed, and an improvement in the expression level of the gene identified in (i) described in the same section was observed. In this case, or when a decrease in the expression level of the gene identified in (ii) described in the same section is observed, the component is determined to be a factor for enhancing the characteristics of the plant.
The increase or decrease in gene expression can be easily confirmed by Northern blot, real-time PCR, or the like.

[Solutions for enhancing plant properties, methods for producing them, and methods for enhancing properties]
The present invention also relates to a solution for enhancing the properties of a plant and a method for producing the same.
The solution for enhancing the characteristics of plants is prepared by adding a component determined to be a factor for enhancing the characteristics of plants to an aqueous medium by the search method of the present invention described in the above [Search method (2)]. Can be manufactured.

As the aqueous medium, the “liquid used for dilution” of the extract described in the above section [Characteristic enhancement method] is preferably used.

The component added to the aqueous medium may be obtained by extracting from the plant tissue. More preferably, the above components are obtained by isolation and purification from a plant extract.
Moreover, the said component may be obtained by artificially synthesizing. The synthetic method may be either a chemical method or a molecular biological method.
When the component is a protein, it can be obtained by introducing an expression vector of the protein into an appropriate cell / bacterium to express the protein and extracting the protein. When the component is RNA, it can be chemically synthesized by an appropriate nucleic acid synthesis method according to a conventional method. When the component is a polysaccharide, it can be chemically synthesized by a method such as a reverse hydrolysis reaction method, a melting method, or a solvent method. When the component is a low molecular weight compound, it can be synthesized by an appropriate organic chemical synthesis method.

By immersing the plant tissue whose characteristics are desired to be enhanced in the solution produced in this manner, the characteristics of the plant tissue can be enhanced. As for the embodiment of the characteristic enhancement method, the above description of the characteristic enhancement method is valid as it is.

<Test Example 1> Enhancement of wheat characteristics using papaya extract A commercially available papaya seed was immersed in a trehalose aqueous solution and allowed to stand in a program freezer to freeze. Freezing was carried out slowly over 180 days at a temperature drop rate of 0.5 ° C./day so that the minimum freezing temperature was −60 ° C.

Frozen papaya seeds were naturally thawed at room temperature (25 ° C). This was left in the outside air (25 ° C.) for one week. Seeds that died in the freezing process were fermented by being left in the open air and softened or liquefied. The fermented seeds (ie dead seeds) were washed away by placing the seeds in a colander and washing with water, and only live seeds were selected.

The live seeds remaining in the colander were gently ground with a mortar and pestle and crushed to obtain a paste. 1 cc of the paste was diluted to 10 L with an aqueous solution of sucralose and trehalose (diluted about 8000 to 10000 times) to prepare a diluted extract.

20 kg of wheat "Fukuhonoka" seeds were immersed in 10 L of the diluted extract and left for 72 hours. The soaked seeds used were naturally dried in the sun for a few days before soaking.

The seeds that had undergone this dipping process were sown on September 30, 2019 in the field of Kamiukan, Ukancho, Takahashi City, Okayama Prefecture. As a comparative example, untreated seeds of the same variety were also sown. The cultivated area is located in an area where it is extremely cold, which will be below freezing in December.

FIG. 1 shows photographs of wheat of Comparative Examples and Examples as of November 2, 2019. In addition, FIG. 2 shows photographs of the wheat cultivation conditions of the comparative examples and the examples as of November 13, 2019.
As shown in FIGS. 1 and 2, the growth rate of the wheat of the example is remarkably improved as compared with the wheat of the comparative example. The wheat of the example buds 3 to 4 days after sowing, the growth rate is uniform, and sparse growth is not observed as compared with the wheat of the comparative example. The roots were also firm, and the green color of the leaves was darker than that of the wheat in the comparative example. Probably the amount of chlorophyll is different. In addition, such a high growth rate makes it difficult for weeds to grow, so there is a possibility that a herbicide is not required. Moreover, in light of the fact that the cultivated area is a severely cold region, it can be observed that the wheat of the example has improved cold resistance (see FIG. 2).
The above results show that by immersing the plant tissue in the extract extracted from the plant tissue that has undergone the freezing step, the soaked plant tissue and the plants generated from the plant tissue have an effect of enhancing the growth characteristics and cold resistance. It shows that it can be done.

In addition, the wheat of the example and the comparative example extracted from the soil was left in the open air for a while. When the texture of the wheat of the comparative example was confirmed for the wheat of the example after being left to stand, the wheat of the example was clearly moist as compared with the comparative example. That is, the wheat of the example clearly retained the water content as compared with the wheat of the comparative example (see FIG. 1).
This result indicates that the application of the present invention can also improve the drought resistance of plants.

<Test Example 2> Enhancement of wheat characteristics using papaya extract Wheat seeds soaked in papaya seed extract were obtained by the same procedure as in Test Example 1. This was sown and cultivated on a farm in Miyazaki City, Miyazaki Prefecture in November 2019. As a comparative example, untreated seeds of the same variety were also sown.

FIG. 3 shows photographs of wheat in Examples and Comparative Examples at the end of January 2020 (about 90 days later). Further, FIG. 4 shows a photograph of wheat ears of an example taken on the same day. Further, FIG. 5 shows a photograph when wheats of Examples and Comparative Examples were collected on March 5, 2020.
A remarkable improvement in growth rate (about 3 times that of the comparative example) was observed in the wheat of the example (see FIG. 3), and fruiting was observed in spite of the cold weather at the end of January (see FIG. 4). Then, as in Test Example 1, improvement in uniformity of growth rate (see FIG. 3) and strengthening of root tension were observed. In addition, despite the fact that the wheat was cultivated in the winter season from November to January, it grew at a remarkable rate, and in light of the fruiting, the wheat of the example was also found to have improved cold resistance (Fig. 3). And FIG. 4). In the early morning, the wheat of the comparative example had a large amount of frost, but the wheat of the example had no frost at all. Frost causes the death of plants and usually requires measures such as frost protection, but the wheat of the example does not require frost protection and is very suitable for cultivation in cold regions. It is recognized as a thing.
In addition, in the situation when wheat was collected on March 5, the comparative example on the right side had no ears at all, whereas the example on the left side grew more than three times as long and had many ears. It was attached (see Fig. 5). Surprisingly, each of these was grown from a single seed, and the examples had more stems and more ears than the comparative examples. Such results appear to suggest that double cropping of wheat is possible. No fertilizer was given in both the examples and the comparative examples.
The above results show that by immersing the plant tissue in the extract extracted from the plant tissue that has undergone the freezing step, the soaked plant tissue and the plants generated from the plant tissue have an effect of enhancing the growth characteristics and cold resistance. It shows that it can be done.

<Test Example 3> Enhancement of wheat characteristics using wheat extract The test was carried out in the same procedure as in Test Example 1 except that wheat seeds were used instead of papaya seeds in the preparation of the extract. That is, an extract was prepared from wheat seeds that had undergone a freezing step, wheat seeds were immersed in the extract, and the seeds were sown in a field for cultivation.
As a result, similar to the results of Test Example 1 and Test Example 2, the treated wheat was found to have significantly improved growth characteristics and cold resistance as compared with the untreated wheat.
The results of Test Examples 1 to 3 show that the plant species of the plant tissue from which the extract used in the dipping step is derived and the plant species of the plant tissue used in the dipping step may be the same or different. ing. In other words, the present invention shows that both homologous application and heterogeneous application are possible.

<Test Example 4> Enhancement of characteristics of corn using papaya extract An extract is prepared from papaya seeds by the same method as in Test Example 1, and the corn seeds are immersed in the extract by the same method as in Test Example 1. did. When this corn seed was cultivated in Hainan Province, China (Hainan base) at the end of 2019 (sown on November 29), the growth characteristics and cold resistance were compared to the case where untreated corn seeds were cultivated. (See FIGS. 6 and 7). Like wheat, it was dark in color and had strong roots. In addition, the treated corn yielded about four times as much as the untreated corn. The cultivated area is also an area with many typhoons, but since the roots are growing firmly, it is thought that this will lead to good yields without collapsing due to the wind.

<Test Example 5> Enhancement of characteristics of soybean using papaya extract An extract is prepared from papaya seeds by the same method as in Test Example 1, and soybean seeds are immersed in the extract by the same method as in Test Example 1. did. When this soybean seed was cultivated in a cold region of China, a remarkable improvement in growth characteristics and cold resistance was observed as compared with the case where untreated soybean seed was cultivated.

<Test Example 6> Enhancement of wheat characteristics using papaya extract An extract is prepared from papaya seeds by the same method as in Test Example 1, and the wheat seeds are immersed in the extract by the same method as in Test Example 1. did. When the wheat seeds were cultivated in the cold regions of China, significant improvements in growth characteristics and cold resistance were observed as compared with the case where untreated wheat seeds were cultivated.

<Test Example 7> Enhancement of wheat characteristics using papaya extract An extract is prepared from papaya seeds by the same method as in Test Example 1, and the wheat seeds are immersed in the extract by the same method as in Test Example 1. did. When the wheat seeds were sown in Russian permafrost, they were harvested only two months after sowing. In addition, the yield was 13 tons per hectare, which was four times the normal yield. Since the cultivated area is permafrost, weeds do not take root. In addition, because of its high growth rate, it absorbs all the nutrients in the soil. Therefore, the use of herbicides was not necessary for cultivation. Moreover, when the ground was dug up and the state of the wheat roots of the examples was observed, the roots were found to be rooted in the completely frozen permafrost. The amount of precipitation at the time when Test Example 7 was carried out was insufficient to cover the amount of water required for wheat cultivation, but the wheat of Example was growing by absorbing more water than the ice contained in the permafrost. It is presumed to be. In addition, if the land is cultivated together with the remaining leaves and stems after cutting the fruits, the nutrients can be returned to the soil, so it is thought that agriculture with less soil load can be achieved.
This result indicates that the application of the present invention can significantly improve the growth characteristics, cold resistance, and fertility of plants.

<Test Example 8> Other plants The seeds of the plants listed below were treated with an extract extracted from papaya seeds by the same method as in Test Example 1, and sown and cultivated. Cultivation was carried out in Okayama prefecture, Japan.
Coffee, chili hazelnuts, golden egg fruit, bananas, dwarf coconuts, cacao, lychee, palm palm, sansho, dorian, cashew nuts, carob, popo mango, acacia, hinoki, pineapple, guava, acai, dates, bakupari, daniel

As a result, it was confirmed that the growth characteristics and cold resistance of all the plant species listed above were enhanced by the treatment of the extract.
This result shows that the property enhancing method of the present invention is effective for all plant species.

<Discussion>
By applying the method of freezing the plant tissue, that is, the freeze-thaw awakening method (Patent Document 3), the characteristics of the plant, specifically, growth rate, cold resistance, heat resistance, highland adaptation characteristics and lowland adaptation Environmental adaptability such as characteristics, amount and size of fruits and seeds, sweetness, pest resistance, drought resistance, etc. can be enhanced. The plant species that can enhance the characteristics are not particularly limited, and papaya, pineapple, banana, coffee, Luo Han Guo, Guava, star fruit, strawberry, cacao, ceylon cinnamon, passion fruit, lychee, mangosteen, black sapote, white sapote, thorns. It has been confirmed that all plant species tested so far have the desired effect, such as leaf sugar apple, date palm, red dragon fruit, almond, soybean, wheat, barley, and corn. As is clear from the plant species listed here, the freeze-thaw awakening method is not a technique applicable only to plants of a specific lineage, but a universal technique applicable to all plant species.

It is said that 97% of plant genes are sleeping. When expression analysis was performed using De novo RNA-seq for plants whose characteristics were enhanced by treatment with the freeze-thaw awakening method, the expression levels of thousands of genes in the treated group were different from those in the untreated group. Has recently become clear.
As a result of the analysis, increased expression of genes related to growth such as plant hormones and increased expression of genes related to various environmental stress responses such as salt, high temperature, low temperature and drought are observed.
The fact that the properties enhanced by freeze-thaw arousal are inherited without loss during growth or straining indicates that the gene expression profile described above is not lost during cell division.

As described above, changes and maintenance of the gene expression profile are observed, and it is clear that epigenetic changes are involved in the characteristic enhancement by the freeze-thaw awakening method.
That is, in the region where the gene encoding the above-mentioned enhancement of plant characteristics is triggered by the freezing step, the transcription of the gene is activated by the addition of an epigenetic marker that positively controls the expression. Can be understood. On the contrary, in the region in which the gene that acts in the direction of controlling the characteristics of the plant described above is encoded, it can be understood that the transcription of the gene is suppressed by the addition of the epigenetic marker that suppresses the expression. ..

Based on this finding, the results of the test examples herein will be considered. As shown in Test Examples 1 to 8, in the plant tissue immersed in the extract of the plant tissue that has undergone the freezing step, the same characteristic enhancement as when the freeze-thaw awakening method is applied is observed. That is, it can be understood that the epigenetic gene expression profile is changed in the plant tissue to which the present invention is applied as in the plant tissue to which the freeze-thaw awakening method is applied.
Expression analysis was performed using De novo RNA-seq using several types of plants to which the property enhancing method of the present invention was actually applied. As a result, the plants to which the present invention was applied and the plants to which the freeze-thaw awakening method was applied were applied. Similar changes in gene expression profile were observed in and.

The common point between the present invention and the freeze-thaw awakening method is the freezing step. In light of this commonality, it is reasonably derived that the freezing process triggers some specific factor that triggers epigenetic changes and the resulting enhancement of properties.
In the freeze-thaw awakening method, it is considered that the specific factor generated through the freezing step induces epigenetic changes by acting on plant cells.
On the other hand, in the present invention, the specific factor generated by the freezing step is contained in the extract, and the specific factor acts on the cells constituting the plant tissue immersed in the extract. , It is understood that it brings about epigenetic changes similar to the freeze-thaw awakening method to plant cells.

That is, it is considered that the present invention exerts the same characteristic enhancing effect as the freeze-thaw awakening method through the above-mentioned specific factors. In other words, it can be understood that the characteristics of plants that can be enhanced by the freeze-thaw awakening method can also be enhanced by applying the present invention.
Further, in light of the fact that the freeze-thaw awakening method can be applied to all plant species without limitation, and that the effects of the present invention have been confirmed in various species of plants in Test Examples 1 to 8, the present invention also It can be understood that, as with the freeze-thaw awakening method, it is not a technique that can be applied only to plants of a specific lineage, but a universal technique that can be applied to all plant species.

Furthermore, the results of Test Examples 1, 2, 4 to 8 demonstrating that the characteristics can be enhanced by applying the papaya-derived extract to a large number of systematically distant plant species are notable. Looking at the base sequences of plant genes, amino acid sequences of proteins, and plant hormones, it is known that they have high homology even between different species systematically distant. It can be said that the results of these test examples prove the wide range of interspecies application of the present invention based on the high degree of homology between different plants.
That is, it can be reasonably understood that the specific factor is not only suitable for a specific plant species but also has a high heterogeneity compatibility that is widely compatible with plants in general. Therefore, in the present invention, it can be understood that the desired effect can be obtained regardless of the combination of the plant species from which the extract is derived and the plant species to be subjected to the dipping step.

The present invention can be applied to the production technique of agricultural products.

Claims (25)

The freezing step includes a freezing step of freezing the plant tissue, a selection step of selecting a living plant tissue from the plant tissue that has undergone the freezing step, and an extraction step of obtaining an extract from the plant tissue that has undergone the selection step. The minimum temperature at freezing is −20 ° C. or lower, the rate of temperature drop in the freezing step is 0.8 ° C./day or less, and the period of the freezing step is 100 days or more. A method for producing an extract for enhancing growth characteristics and / or cold resistance. The production method according to claim 1, wherein in the freezing step, the plant tissue is frozen while being immersed in an aqueous saccharide solution. The production method according to claim 2, wherein the saccharide is trehalose. The production method according to any one of claims 1 to 3, wherein the selection step performs a fermentation treatment on the plant tissue that has undergone the freezing step. The production method according to claim 4, wherein the fermentation treatment is performed by leaving the plant tissue that has undergone the freezing step in the open air. The production method according to claim 5, wherein the leaving is performed at 0 ° C to 40 ° C. The production method according to any one of claims 4 to 6, wherein in the selection step, a dead plant tissue and a living plant tissue are separated and treated after the fermentation treatment. The production method according to claim 7, wherein the separation treatment is performed by washing the fermented plant tissue. The manufacturing method according to claim 8, wherein the washing is washing with water. The production method according to any one of claims 1 to 9, wherein the extraction step performs a crushing treatment on a living plant tissue. The manufacturing method according to claim 10, wherein the crushing treatment is a mashing treatment. The manufacturing method according to claim 11, wherein the grinding process is performed over several tens of seconds to several hours. An extract produced by the production method according to any one of claims 1 to 12 for enhancing the growth characteristics and / or cold resistance of the plant. The extract according to claim 13, wherein the extract contains a sugar or a sugar alcohol. The extract according to claim 14, wherein the sugar or sugar alcohol is sucralose and / or trehalose. The extract according to any one of claims 13 to 15, wherein the extract is diluted. An extracted dried product obtained by drying the extract according to any one of claims 13 to 16. Immerse the plant tissue whose growth characteristics and / or cold resistance are desired to be enhanced in the extract according to any one of claims 13 to 16 or the extract obtained by dissolving the dry extract according to claim 17. A method for enhancing the growth characteristics and / or cold resistance of a plant tissue, which comprises a dipping step. The method for enhancing the growth characteristics and / or cold resistance of a plant tissue according to claim 18, wherein the plant tissue whose growth characteristics and / or cold resistance is desired to be enhanced is dried before the dipping step. The method for enhancing growth characteristics and / or cold resistance according to claim 18 or 19, wherein the immersion time in the immersion step is 1 to 100 hours. A method for producing a plant tissue having enhanced growth characteristics and / or cold resistance by applying the method for enhancing growth characteristics and / or cold resistance according to any one of claims 18 to 20. A method for searching for genes involved in enhancing plant growth characteristics and / or cold hardiness.
A step of treating a plant by the method according to any one of claims 18 to 20;
(I) A step of identifying a gene that exhibits a higher expression level in the treated plant as compared to the untreated plant and / or (ii) compared to the untreated plant. , A step of identifying a gene showing a low expression level in the treated plant,
A search method characterized by including. An extract produced by the production method according to any one of claims 1 to 12 is prepared as an analysis target.
Extracts extracted from plant tissues that have not undergone the freezing step are prepared for comparison.
By comparatively analyzing the extract to be analyzed and the extract to be compared,
Identification that identifies components that are contained in the extract to be analyzed but not in the extract to be compared, or components that are contained more or less in the extract to be analyzed than in the extract to be compared. A method for analyzing an extract, which comprises a process. An extract produced by the production method according to any one of claims 1 to 12 is prepared as an analysis target.
Extracts extracted from plant tissues that have not undergone the freezing step are prepared for comparison.
By comparatively analyzing the extract to be analyzed and the extract to be compared,
An identification step for identifying components that are contained in the extract to be analyzed but not in the extract to be compared, or components that are contained in the extract to be analyzed more than the extract to be compared. A method for searching for factors that enhance plant growth characteristics and / or cold resistance, which are characterized by being provided. A dipping step of immersing a plant tissue or a plant cell in a solution containing one or more kinds of components identified by the identification step, and a soaking step.
When the plant tissue or plant cell that has undergone the immersion step has enhanced plant growth characteristics and / or cold resistance as compared with the plant tissue or plant cell that has not undergone the immersion step, the component is added to the plant growth characteristic. And / or a determination step for determining that it is a factor for enhancing cold resistance,
24. The search method according to claim 24 .