JP4646571B2 - Adventitious root formation promoter - Google Patents

Description

  The present invention relates to a plant adventitious root formation promoter, and more particularly to a plant adventitious root formation promoter containing as an active ingredient a soybean protein degradation product obtained by allowing a specific enzyme to act on soybean protein.

  Vegetable oil-squeezed potatoes such as soybean meal (soybean meal) have been used for a long time as organic fertilizers, but these have the disadvantage of poor absorbability to plants due to their high molecular weight protein as the main component. Due to the development of cheaper and more effective chemical fertilizers such as ammonium sulfate and urea, they are hardly used at present, and are being landfilled or dumped into the sea except for being used as part of livestock feed. On the other hand, chemical fertilizers have a very good effect in the initial use, but as the number of use increases, accumulation in the soil decreases the type and amount of microorganisms in the soil, reducing the plant growth effect. There are drawbacks.

  As a result of intensive studies aimed at effective utilization of soybean meal, the present inventors previously obtained a strain belonging to the genus Bacillus that produces an enzyme capable of specifically and rapidly degrading and degrading the protein of soybean meal. In addition, the present inventors have found a proteolytic enzyme derived from the strain and succeeded in developing a plant growth fertilizer derived from soybean meal obtained by using these, and completed the inventions related thereto (see Patent Documents 1 and 2).

The fertilizer previously developed by the present inventors is based on the fact that it contains a large amount of soybean meal-degrading peptide together with the above-mentioned strain, for example, the effect of increasing the yield (weight) of Komatsuna and Kaiware radish and shortening the cultivation period The effect can be produced, and the effect of growing the root hairs of plants can be expected.
Japanese Unexamined Patent Publication No. 6-337760 Japanese Patent Laid-Open No. 14-362988

  The objective of this invention is providing the adventitious root formation promoter of a plant.

  The present inventors have promoted adventitious root formation in the soybean proteolysate contained in the fertilizer in the process of earnestly researching the previously developed plant growth fertilizer, particularly the various soybean proteolytic peptides contained therein. It was found that a substance having an action exists.

  Based on this new knowledge, the present invention has been completed as a result of further intensive studies, and provides the plant adventitious root formation promoter and the plant adventitious root formation promotion method described in Item 1-6 below.

  Item 1. A soy protein hydrolyzate having an action to promote adventitious root formation of a plant obtained by allowing Bacillus circulans HA12 (FERM P-13428) strain or a proteolytic enzyme derived from the strain to act on a soy protein raw material A plant adventitious root formation promoter characterized by containing as an active ingredient.

  Item 2. The adventitious root formation promoter according to Item 1, wherein the soybean protein raw material is soybean meal.

  Item 3. The adventitious root formation promoter according to Item 1, wherein the soybean protein degradation product having an adventitious root formation promoting action is a peptide having a molecular weight of 5000 or less.

  Item 4. The adventitious root formation promoter according to Item 1, wherein the plant is selected from Solanum, Leguminosae, Asteraceae and Nadesicoaceae.

  Item 5. A method for promoting adventitious root formation in a plant, comprising contacting the plant with the adventitious root formation promoter according to Item 1.

  Item 6. The method for promoting adventitious root formation according to Item 5, wherein the plant is selected from the family Solanum, Leguminosae, Asteraceae, and Nadesicoaceae.

  The adventitious root growth promoter active ingredient (soy protein breakdown product) of the present invention can exert the action of promoting the growth of adventitious roots of plants, and is rapidly absorbed by the plants to grow the adventitious roots. Increases the growth and growth of the body, increases the yield when the plant body is vegetables, fruits, etc., enables long-term growth when it is cut flowers, and promotes its differentiation and growth when it is callus To do. In addition, the active ingredient of the present invention promoter is obtained by using soybean meal, which is easily available at low cost, and can be used effectively, and there is no possibility of adversely affecting the environment and the human body.

  Hereinafter, the adventitious root formation promoter of the plant of this invention is explained in full detail.

  The plant adventitious root growth promoter according to the present invention is obtained by allowing a Bacillus circulans HA12 (FERM P-13428) strain itself or a proteolytic enzyme derived from the strain to act on a soybean protein raw material. It contains a soybean protein degradation product having a plant adventitious root formation promoting action as an active ingredient.

  Here, the soy protein raw material that acts on the enzyme may be a protein obtained by refining from soybean, but it is not necessary, for example, soybean itself containing this, its pulverized product, and extracting fat and oil from the soybean. The remaining soybean oil residue may be used. In particular, in the present invention, the soybean protein residue is a soybean oil cake, and among them, the oil and fat (edible oil material) is extracted from the soybean, then dried by heating to improve digestion, and the soybean meal is pulverized into granules. Meal (hereinafter referred to as “soybean cake”) can be used effectively. The yield of the soybean meal with respect to the raw soybean is about 80%, and the protein content reaches about 45%. In this respect, soybean meal can be suitably used in the present invention. When the enzyme is allowed to act on the soy protein raw material, the soy protein raw material is usually prepared in a solution form to which water is added.

  The Bacillus circulans HA12 (FERM P-13428) strain or the proteolytic enzyme derived from the strain that acts on soy protein raw material was developed by the present inventors (see Patent Document 1). . The proteolytic enzyme can also be used after being isolated and purified from a microorganism, but it is not necessary to isolate and purify it in particular. For example, as the strain itself or as a culture of the bacterial body It can also be used as a liquid. When using the microbial cells themselves, by culturing the microbial cells using an appropriate medium containing the soy protein raw material, the microbial cells can assimilate the soy protein raw material to obtain a desired soy protein degradation product. .

  The amount of enzyme or strain used for the raw soybean protein such as soybean meal is appropriately determined on the assumption that the raw material can be decomposed, and is not particularly limited. Generally, conditions for inoculating a 1 v / v% HA12 strain culture solution on a 10 w / v% soybean meal liquid medium are employed. The decomposition reaction of soybean protein raw material with an enzyme can usually be carried out under an appropriate condition in consideration of the optimum condition of the enzyme. More specifically, the temperature condition can include about 50 ° C., and the reaction is generally completed in about 48 hours under shaking culture conditions. It is not necessary to adjust the pH of the reaction system.

  The soy protein degradation product having a plant adventitious root formation promoting action as an active ingredient can be obtained from the culture solution obtained above according to a conventional method, and can be purified as necessary. More specifically, the proteolytic enzyme derived from the Bacillus circulans HA12 (FERM P-13428) strain is allowed to act on the soy protein raw material under the above conditions, and the resulting soy protein hydrolyzate is centrifuged. Can be obtained as the supernatant. The supernatant is treated with activated carbon, fractionated into an activated carbon adsorbed fraction and an activated carbon non-adsorbed fraction, and the activated charcoal adsorbed fraction is purified by gel filtration chromatography to obtain a purified soybean protein degradation product. be able to. This purified soy protein product can be obtained, for example, as a fraction (peptide) having a retention time of 8.5 to 9.9 minutes as shown in the Examples below, and its molecular weight is 5000 or less, usually about 500 to 2000.

  The adventitious root growth promoter of the present invention is not particularly limited as long as it contains the purified product of soybean protein degradation product, and may contain, for example. Specifically, as described above, an aqueous solution or a culture solution obtained by allowing a specific enzyme or a microorganism producing the same to act on a soybean protein raw material can be used as it is as the promoter of the present invention.

  The application of the promoter of the present invention to a plant is not particularly limited as long as the application form allows the plant to absorb the promoter. For example, what is necessary is just a form which a plant stem contacts with the liquid containing this invention promoter.

  The usage form of the promoter of the present invention is not particularly limited, and may be either liquid form or solid form (powder form). In the liquid form of the present accelerator, the active ingredient of the present invention is usually contained in the liquid in an amount of about 1 to 500 μg / mL, preferably about 30 to 400 μg / mL in terms of peptide / amino acid concentration. This concentration measurement will be described in detail in Example 3 below.

  The application to the plant can be performed by a general method such as spraying or spraying. Moreover, when a plant is a cut flower, callus, etc., it can also be based on the method of immersing these in this invention promoter solution.

  The plant to which the promoter of the present invention is applied is not particularly limited, and may be any of various plants suitable for cultivation, ornamental use, harvesting and the like. Specifically, the applicable plants include solanaceae (tomatoes, peppers, etc.), legumes (soybeans, green beans, peas, bean sprouts, etc.), asteraceae (chrysanthemum, lettuce, etc.), radish family (carnation, misty, etc.). Grass etc.).

The adventitious root formation promoter of the present invention can exert the action of promoting the formation of adventitious roots of various plants, and can be effectively used for various applications shown below, for example, using this action.
(1) Growing plants by promoting callus formation and growth,
(2) Store cut flowers for a long time,
(3) breed lawn,
(4) Increase sales of sweet potatoes,
(5) Reduce (decrease) pesticide organic cultivation by reducing the amount of pesticide,
(6) Improving the storage stability when transplanting garden trees, etc.
(7) Perform plant stocking (the same variety can be stocked in large quantities from the stem)
(8) Increase plant yields by plant factories (hydroponics) (promoting the formation of adventitious roots without the use of soil can be expected to shorten cultivation days and increase yields).
EXAMPLES Examples are given below to explain the present invention in more detail.

(1) Screening for soybean meal-degrading microorganisms
(1-1) Preparation of soybean meal liquid medium 100 ml of distilled water was added to 10 g of soybean meal and autoclaved (121 ° C./20 minutes) in a 500 ml shaking culture flask to prepare soybean meal liquid medium.

(1-2) Preparation of screening medium
Add 1 g of peptone, 0.5 g of yeast extract, 0.5 g of sodium chloride, 0.5 g of agar, 2 g of casein, and 100 mL of distilled water to a 300 mL Erlenmeyer flask, cap and sterilize by autoclaving (121 ° C / 20 min), and complete the autoclave. Then, after the medium has cooled to 60-70 ° C, dispense the LB + agar + casein plate (medium) by shaking it into a petri dish sterilized with dry heat while thoroughly shaking the agar at the bottom of the Erlenmeyer flask. did.

(1-3) Preparation of LB liquid medium for preculture 1 g of peptone, 0.5 g of yeast extract and 0.5 g of sodium chloride were added to 100 mL of distilled water to prepare LB liquid medium for preculture, and this was sterilized in an autoclave. 5 mL each was dispensed into 10 sterilized test tubes.

1 g of a soil sample collected from nature was added to the soybean meal liquid medium and cultured with shaking (150 rotations / minute, 50 ° C., 24 hours). After shaking culture, 100 μL of the obtained culture solution is dispensed and diluted in a test tube containing 10 mL of sterile water for dilution. Further, 100 μL of the diluted culture solution is added to a test tube containing 10 mL of sterile water for dilution. The operation of aliquoting was repeated twice to dilute the culture solution 10 ⁶ times.

  Distribute 100 μL of the diluted culture solution to the screening medium plate prepared in (2) above, spread it with a conage rod, and culture at 50 ° C. for 12 hours. The number, color, morphology, and halo of grown colonies Observed the situation.

  A single colony in which halo was observed was inoculated into a new screening medium plate with a sterilized toothpick and cultured at 50 ° C. for 12 hours. Thereafter, one platinum loop was collected from each grown single colony, added to the test tube containing the LB liquid medium for preculture prepared in (3) above, and cultured at 50 ° C. for 12 hours (preculture).

  Next, add 1 mL of the obtained preculture solution to 100 mL of soybean meal liquid medium prepared in (1) above, and shake culture at 50 ° C. for 24-72 hours in a shaking incubator (150 rpm). did. When soybean meal in the osmotic culture vessel became muddy, the bacteria were determined to be positive (+), and the others were determined to be negative (-). In addition, it considered except the hard part of the epidermis.

(2) Soybean assimilation and growth temperature of each strain
Add 300 mL of distilled water together with 1 g of peptone, 0.5 g of yeast extract, 0.5 g of sodium chloride and 2 g of agar to a 300 mL Erlenmeyer flask, sterilize in an autoclave, dispense into a petri dish sterilized by dry heat, and plate of LB + agar (medium) Was made.

  Bacteria determined to be positive (+) by screening for soybean meal-degrading microorganisms are collected with a sterilized toothpick, inoculated into the above LB + agar plate (medium), 30 ° C or lower, 37.5 ° C, 50 ° C, 70 ° C and The cells were cultured for 12 hours in a thermostat set to 75 ° C.

  Bacteria in which a halo producing protease (protease) by degrading casein was observed were determined to be positive (+), and other bacteria were determined to be negative (−). As a result, a total of 20 strains grown on soybean meal medium were obtained and designated as HA1 to HA20, respectively.

  The obtained 5 strains of soybean meal-degrading bacteria and their culture conditions (temperature) are shown in Table 1 below.

(3) Identification of strains In order to examine the bacteriological properties of the soybean meal-degrading bacteria that were separated and selected, various confirmation tests including Gram staining were conducted by the following methods.

1. Gram staining
According to the modified Hucker method, the bacterial solution smeared on a slide glass, dried and fixed with flame was stained with Hucker's solution (1.5% crystal violet-ethanol solution) for 30 seconds. Immediately after dyeing, it was washed with water and mordanted with Lugol solution for 1 minute. After the mordanting, it was washed with water, further decolored with alcohol for 30 seconds, and immediately washed with water. After thoroughly draining the water, it was stained with safranin for 30 seconds. The stained specimen was observed with a microscope, and the bacteria stained purple were regarded as Gram positive, and the bacteria stained red were regarded as Gram negative. Moreover, the form of the fungus was observed with a microscope.

2. Growth temperature Liquid thioglycolate medium having the following composition was sterilized at 121 ° C. for 15 minutes, and 5 mL was dispensed into a test tube. Bacteria collected from the colonies were planted in this medium, statically cultured at each temperature, and it was judged that the medium had become turbid after 12 or 24 hours.

<Composition of liquid thioglycolate medium>
L-cystine 0.75g
NaCl 2.5g
Glucose 5.0g
Yeast extract 5.0g
Agar 0.75g
Casein digest 15.0g
Sodium thioglycolate 0.5g
0.1% resazurin 1.0mL
Distilled water 1000mL
pH 7.2
3. Oxidase Tested using Bactlab oxidase test from Terumo Corporation. That is, microbial cells were attached to a cotton swab containing N, N, N ′, N′-tetramethyl-p-phenylenediamine dichloride, and a blue-purple color produced by oxidase activity was produced.

4. After bacteria were attached to one end of the catalase capillary tube, 3% hydrogen peroxide solution was sucked up from one end of the capillary tube to mix the cells and hydrogen peroxide solution in the capillary tube. Catalase was positive when oxygen bubbles were generated in the capillary tube due to hydrogen peroxide decomposition.

5.Nitrate reduction ability
Fungus was inoculated into a 0.1% sodium nitrate-containing broth medium and cultured at the optimum growth temperature for 5 days. In the obtained culture broth, α-naphthylamine solution (0.1 g of α-naphthylamine dissolved in 200 mL of 30% acetic acid solution) and 1.0 mL of sulfanilic acid solution (0.5 g of sulfanilic acid dissolved in 150 mL of 30% acetic acid solution) After adding 1.0 mL and mixing well, a case where a pinkish red color due to nitrous acid produced by nitric acid reduction was observed within 30 minutes was regarded as positive.

6. Crigler medium Crigler medium having the following composition was dispensed into test tubes, and then autoclaved at 121 ° C for 15 minutes to harden on a high slope. The test bacteria were planted on the slope part and the upper part, and cultured at the growth temperature of each fungus for 18 to 24 hours. After culturing, when the slope portion turned yellow, the slope portion was positive (lactose was decomposed to produce acid), and when it turned yellow at the high portion, the high layer portion was positive (glucose was broken to produce acid).

<Composition of Krigler medium>
Meat extract 4.0g
Peptone 15.0g
Lactose 10.0g
Glucose 1.0g
Sodium chloride 5.0g
Sodium thiosulfate 0.08g
Sodium sulfite 0.4g
Ferrous sulfate 0.2g
Phenol red 0.02g
Agar powder 15.0g
Purified water 1000mL
pH 7.2
7. SC Medium A medium having the following composition was dissolved in distilled water and dispensed into a test tube at about 2 mL, and autoclaved at 121 ° C. for 15 minutes and solidified on all slopes. Test bacteria were planted on the slope and cultured at the growth temperature of each fungus for 18-24 hours. The case where it grew using the citric acid which is the only carbon source contained in this culture medium was made positive. When positive, brom thymol contained in the medium changed from green to deep blue.

<Composition of SC medium>
Sodium chloride 5.0g
Magnesium sulfate 0.2g
Ammonium dihydrogen phosphate 1.0 g
Potassium dihydrogen phosphate 1.0g
Sodium citrate 2.0g
Brom Timor Blue 0.08g
Agar powder 15.0g
Purified water 1000mL
pH 6.8
8. SIM medium A medium having the following composition was dissolved by heating in 1000 mL of distilled water, dispensed into test tubes approximately 3 mL at a time, autoclaved at 121 ° C for 15 minutes, and then solidified into a high layer. The test bacteria were punctured into the upper layer and cultured at the growth temperature of each bacteria for 18-24 hours. When the medium turns black after culture, hydrogen sulfide is generated (+). When it grows from the puncture site, it grows (+). Further, 1 mL of chloroform is overlaid on the top of the medium, and 1 mL of Kovac reagent is added on top. Indole (+) was defined as the case where the chloroform layer turned red after layering.

<Composition of SIM medium>
Ehrlich Meat Extract 3.0g
Proteose peptone 10.0g
Polypeptone 20.0g
Sodium thiosulfate 0.2g
Ammonium iron citrate 0.2g
Agar powder 3.0g
Purified water 1000mL
pH 7.3
9. Methyl red 3 mL of glucose-phosphate-peptone water having the following composition was dispensed into a test tube, and sterilization at 100 ° C. for 30 minutes was repeated three times. The test bacteria were planted in this medium and cultured at the growth temperature of each bacteria for 3 days. 1 mL of the obtained culture solution was transferred to another test tube, methyl red was added dropwise, and the color of the medium turned red was regarded as positive.

<Composition of glucose-phosphate-peptone water>
Peptone 7.0g
Potassium dihydrogen phosphate 5.0g
Glucose 5.0g
Distilled water 1000mL
10. Foguez Proskawell (VP)
3 mL of the glucose-phosphate-peptone water was dispensed into a test tube, and the sterilization operation at 100 ° C. for 30 minutes was repeated three times. The test bacteria were planted in this medium and cultured at the growth temperature of each bacteria for 3 days. Transfer 1 mL of the obtained culture solution to another test tube, add 0.6 mL of α-naphthol solution (5% α-naphthol-anhydrous ethanol solution) and 0.2 mL of 40% KOH aqueous solution, and mix well. The tube was tilted and allowed to stand. Changes in the color of the medium were observed 15 minutes and 1 hour after standing, respectively, and those in which the medium color became dark red were considered positive.

11.OF (Oxidation or Fermentation)
According to the method of Hugh-Leifson, an OF medium of the following composition was autoclaved at 121 ° C for 15 minutes, and glucose sterilized by filtration to a final concentration of 1% when the medium was cooled to about 60 ° C was obtained. 3 mL of each medium was dispensed into two test tubes and hardened in a high layer. The test bacteria were punctured into this medium, and liquid paraffin was layered to a thickness of 1 to 2 cm in one test tube and cultured anaerobically, and the other test tube was cultured aerobically as it was. After culturing at the growth temperature of each microbial cell for 3-4 days, the bacterium that undergoes oxidative saccharolysis and cultured under aerobic conditions is shown as yellow (O) in the result and is expressed as fermentative sugar. Degraded bacteria whose medium color turned yellow under both aerobic and anaerobic conditions are shown as (F) in the results, and glycolysis was observed in the next 7 days after 4 days of culture Was written in the results as (W).

<Composition of OF medium>
Tryptone 2.0g
Sodium chloride 5.0g
Agar 2.5g
Potassium dihydrogen phosphate 0.3g
BTB 0.03g
Distilled water 1000mL
pH 7.1
12.Urease
Using a urease confirmation urea medium (sterilized) manufactured by Eiken Chemical Co., Ltd. having the following composition, 0.5 to 1.0 mL was dispensed into a sterilized test tube and inoculated with the test bacteria. This medium was cultured at the growth temperature of each test bacterium for 6 to 24 hours, and a case where the medium turned red due to the production of ammonia was regarded as positive. If red discoloration is weak, “W” is displayed.

<Composition of urea medium>
Peptone 2.0g
Urea 30.0g
Sodium chloride 5.0g
Monopotassium phosphate 9.0g
Disodium phosphate 3.0g
Phenol red 0.01g
pH 6.2
13.NaCl
Add various concentrations of NaCl to 1% peptone water, add about 3 mL of sterilized medium (pH 7.2) to the test tube, inoculate the test bacterium, and leave it at the growth temperature of each cell for 18 to 24 hours. Cultured. And the thing in which the culture solution became turbid was made positive.

14. Mannitol Salt (MS) Medium Using Eiken Chemical Co., Ltd. Mannite Salt Medium “Eiken” with the following composition, 112 g of this medium was dissolved in 1000 mL of distilled water and autoclaved at 121 ° C. for 15 minutes. did. About 20 mL each of the sterilized medium was dispensed into a sterilized petri dish and hardened on a flat plate. The test bacteria were inoculated into the obtained plate medium and cultured at the growth temperature of each cell for 24-48 hours. The mannit was decomposed and the area around the colony turned yellow was regarded as MS positive, and the one that grew without yellowing and formed a colony was regarded as NaCl positive.

<Composition of Mannite Salt Medium “Eiken”>
Meat Extract “Eiken” 2.5g
Peptone "Eiken" 10.0g
Mannit 10.0g
Sodium chloride 75.0g
Phenol red 0.025g
Agar 15.0g
15.Hemolysis
While dissolving 15 mL of ordinary agar and keeping it at 45 ° C., defibrinated blood was added thereto at a rate of 5%. A small amount of the test bacteria was planted in this medium, mixed well, poured into a sterile petri dish and hardened on a flat plate. The cells were cultured for 24 hours at the growth temperature of each bacterium, and judged by the presence or absence of hemolysis. Those that caused hemolysis and produced halos were considered positive.

16. Esculin
When the Barsiekow medium having the following composition, which had been autoclaved at 121 ° C. for 15 minutes, was cooled to 80 ° C., esculin (glycoside) sterilized by filtration was added at a rate of 0.5%, and 2 mL was dispensed into a sterile test tube. The test bacteria were inoculated into this medium, cultured at the growth temperature of each bacterial cell for 18 to 24 hours, and the case where the color of the medium changed from blue to yellow due to esculin decomposition was regarded as positive.

<Composition of Barsiekow medium>
Peptone water 100mL
0.2% BTB solution 1.2mL
17. Liquefaction of gelatin 100 g of purified gelatin was added to 1000 mL of ordinary bouillon, and 3 mL of a medium sterilized by autoclaving at 121 ° C. for 12 minutes was dispensed into a sterile test tube and solidified into a high layer. The test bacteria were punctured into the upper layer and cultured for 1 week at the growth temperature of each cell. The presence or absence of gelatin liquefaction along the puncture line was observed.

18.Decarboxylation test from ornithine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 mL), autoclaved at 121 ° C. for 15 minutes, and dispensed into a sterile test tube (3 mL). The test bacteria were inoculated into this medium and cultured at the growth temperature of each cell for 18 to 24 hours, and the medium color tone changed from red beans to yellow was regarded as positive.

19.Decarboxylation test from lysine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 mL), autoclaved at 121 ° C. for 15 minutes, and dispensed into a sterile test tube (3 mL). The test bacteria were inoculated into this medium and cultured at the growth temperature of each cell for 18 to 24 hours, and the medium color tone changed from reddish purple to yellow was regarded as positive.

20.Decarboxylation test from arginine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 mL), autoclaved at 121 ° C. for 15 minutes, and dispensed into a sterile test tube (3 mL). The test bacteria were inoculated into this medium and cultured at the growth temperature of each cell for 18 to 24 hours, and the medium color changed from brown to yellow was regarded as positive.

21. Determination of assimilation ability of sugar The OF medium is autoclaved at 121 ° C for 15 minutes, and after the medium temperature is cooled to about 60 ° C, 1% of sterilized sugar is added to the OF medium and mixed well. 3 mL was dispensed into a tube and hardened to a higher layer. The medium was punctured with the test bacteria and cultured at the growth temperature of each bacterial cell for 24 hours.

  The results of the above test are shown in Table 2.

  In addition, NC means slope part negative, Y means high part positive, R means high part negative.

  From the results of the various identification confirmation tests shown in Table 2 above, the strains separated and selected here are Gram-positive medium fungi, and have properties such as forming endospores and producing oxidase and catalase. Was confirmed. Both strains were found to belong to the genus Bacillus, and the species names were identified as HA12 as circulans and HA19 as stearothermophilus, respectively. It was named Lance (Bacillus circulans) HA12 and Bacillus stearothermophilus HA19. Hereinafter, these strains are referred to as HA12 and HA19, respectively. The inventors of the present invention acquired these new strains on February 12, 1993 at the former Institute of Microbial Industrial Technology (Independent Administrative Institution with an address in 1-1-6 Higashi 1-1-1 Tsukuba, Ibaraki, Japan) The deposit numbers are FERM P-13428 and FERM P-13429, respectively.

The behavior of soybean meal decomposition product using the new strain HA12 (FERM P-13428) will be described. Changes in protein concentration over time during the soybean meal decomposition process using the new strain HA12 (FERM P-13428) were measured using the BCA method as follows.
Preparation of soybean meal liquid medium and reagents < Preparation of soybean meal liquid medium>
Distilled water (150 mL) was added to soybean meal (1.5 g), and autoclaved (121 ° C./20 minutes) in a 500 mL shake culture flask to obtain a soybean meal liquid medium.
<Adjustment of BCA reagent>
Reagent A: 100 solutions + Reagent B: 2 solutions <Preparation of standard solution for calibration curve (bovine albumin)>
According to Table 3 below, standard solutions having various protein concentrations were prepared.

  The prepared standard curve albumin standard solution (0.1 mL) and BCA reagent (2.0 mL) were stirred in a test tube, and then left in a constant temperature bath at 37 ° C. for 30 minutes to carry out a BCA reaction. After completion of the reaction, each test tube was allowed to stand at room temperature, and the absorbance at 562 nm was measured using a solution of 0.1 mL water and 0.2 mL BCA reagent simultaneously measured as a blank to prepare a calibration curve.

  Next, aseptically collect 1mL from autoclaved 1w / v% soybean meal liquid medium (150mL), and measure the protein concentration by BCA method on the supernatant obtained by centrifugation at 10000 rpm for 15 minutes. The obtained protein concentration was defined as the protein concentration at culture time 0 (zero). Similarly, aseptically collect 1 mL from 1 w / v% soybean meal liquid medium, and inoculate 1.5 ml of HA12 (FERM P-13428) bacterial solution pre-cultured in soybean meal liquid medium in advance. Then, the culture was performed at 50 ° C. and 150 rpm for 15 minutes, the culture solution was aseptically collected from this culture solution over time, and the protein concentration at each culture time was measured. Fig. 1 (horizontal axis: time (hr), vertical axis: peptide (displayed on the left side) and protein (displayed on the right side) of protein concentration during the soybean soybean decomposition process using the new strain HA12 (FERM P-13428) Concentration (mg / mL)).

  As can be seen from FIG. 1, the new strain HA12 (FERM P-13428) decomposes soybean meal protein at low speed and efficiently. In FIG. 1, white circles indicate protein concentration (mg / mL), and black circles indicate peptide concentration (mg / mL).

  The effect of adventitious root formation promoter containing the soybean protein degradation product of the present invention as an active ingredient will be described.

(1) Example using cherry tomato (Pico) (Takii Seed Co., Ltd., Kyoto)
[Test plant]
A sample obtained by cutting a stem of cherry tomato grown for 3 weeks was used as a sample.
[Adjustment of test reagent]
In LB medium 5 mL, the strain HA12 prepared in Example 1 (FERM P-13428) ( 1 loopful or 1 × 10 ⁹ cells / ml to 5 × 10 ⁹ cells / mL) was inoculated 1% overnight, A preculture was prepared by shaking culture at 37 ° C. Add 1v / v% of the obtained pre-culture solution to 10w / v% soybean meal medium, shake culture (50 ° C, 120 rotations / min, 48 hours), and after culturing, the pH of the culture solution exceeds 9.0. The sample was used as a test reagent (DSP, Degradation Soybean meal Products).

  Subsequently, 1 L of ion-exchanged water was added to 10 g of activated carbon (Darco G-60), and the activated carbon was washed by stirring for 1 hour. After stirring, the mixture was allowed to stand until activated carbon precipitated. After standing, ion-exchanged water was discarded, and 100 mL each of ion-exchanged water and DSP was added to the resulting residue and stirred for 1 hour to adsorb DSP onto activated carbon. After stirring, the mixture was centrifuged (10000 rpm / 10 minutes) and separated into a supernatant and a precipitate (activated carbon). The supernatant was filtered with a filter paper to remove activated carbon (an activated carbon non-adsorbed fraction). For precipitation, 100 mL each of ion-exchanged water and 100% acetone was added and stirred for 1 hour. After stirring, the mixture was centrifuged (10000 rpm / minute, 10 minutes), separated into a supernatant and a precipitate (activated carbon), and the obtained supernatant was distilled under reduced pressure to remove acetone (activated carbon adsorption fraction).

  As a phenolic substance in DSP, 100 mL of diethyl ether was added to 20 mL of DSP (30 mg / mL), and the diethyl ether was extracted from the supernatant using a rotary evaporator (Rotavapor R-114, Shibata, Tokyo). I used something.

[Test method]
The cut stalk portion of the plant sample was immersed in a container containing each of the test reagents shown below and allowed to grow for 1 week in that state, and then the appearance of adventitious roots was visually observed.

Container A: Distilled water (H ₂ O) only (control)
Container B: DSP 30μg / mL
Container C: DSP 300μg / mL
Container D: DSP activated carbon adsorption fraction 30μg / mL
Container E: DSP activated carbon adsorption fraction 300μg / mL
Container F: DSP activated carbon non-adsorbed fraction 30 μg / mL
Container G: DSP activated carbon non-adsorbed fraction 300μg / mL
Container H: Phenolic substances in DSP The concentration of each test reagent was determined based on the ninhydrin reaction (hydrindantine method, Moore, S. and Stein, WH, "Amodified ninhydrin regent for the photometric determination of amino acids and related compounds. ", Biol. Chem., 211, pp.907-913 (1954))" shows peptide / amino acid concentrations (μg / mL). The concentration is calculated by y = 0.0176x (x: peptide / amino acid concentration, y: absorbance at 570 nm).

[result]
The obtained results are shown in FIG. 2 and FIG.

  FIG. 2 shows the results obtained using the above containers A to H (photos of cherry tomatoes (3 pieces) grown by immersing the stems in each container; Are displayed as A to H). In addition, FIG. 3 shows the results obtained using containers BE and G (magnified photographs of cut stem tips of cherry tomatoes (one) grown by immersing stems in each container; each photograph in the figure) Are labeled BE and G in correspondence with the containers used).

From the above figures, it is clear that adventitious root formation is observed when containers B, C, D, E, and G are used, and adventitious root formation is not observed with other containers (A and F). In particular, it was found that the number of adventitious roots formed was highest in the container C and then decreased in the order of E, B, D, and G.
[Discussion]
From the above results, it is clear that soybean degradation protein (DSP) used as an active ingredient in the present invention exhibits an adventitious root formation promoting effect and is effective as an adventitious root formation accelerator. In addition, it is clear that the main active ingredient substance (peptide) that exhibits the effect of promoting adventitious root formation exists in the activated carbon adsorption fractions indicated as D and E.

Callus culture
(1) Experimental Method A callus induction agar medium (MS medium) was prepared with the following composition, and callus induction was performed using carrots.

<Preparation of medium>
Preparation of stock solution of MS medium A stock solution of MS medium consisting of the following components A to D was prepared.

  A. Major inorganic nutrients

  After adjustment, store at 4 ° C.

  B. Trace components

  After adjustment, store at 4 ° C.

  C. Iron component

  After autoclaving for 20 minutes, measure up and store at 4 ° C.

  D. Organic nutrients

  After adjustment, aliquot and store at -20 ° C.

Preparation of MS medium An MS medium composed of each component described in Table 8 below was prepared.

  The adjusted MS medium was adjusted to pH 5.8 with 1N KOH, autoclaved, and a growth regulator (2,4-dichlorophenoxyacetic acid, 2,4-D) was added to this sample as a comparative sample. DSP, which is an active ingredient of a formation accelerator, was added to each of 100 μg / mL and 300 μg / mL to obtain a sample of the present invention. A control (control sample) was the one to which nothing was added.

  2,4-D is a synthetic auxin, and auxin is known to have a callus-growing action.

(2) Sterilization of carrots and callus induction Fresh carrots were washed well and the upper and lower parts were cut off. The remaining part was immersed in 70% ethanol for 10 seconds. Next, the carrot was sterilized by immersing in a 1% sodium hypochlorite aqueous solution in a clean bench for 15 minutes, and then washed with sterilized water three times or more. Then, the sterilized carrot was punched out with a cork borer and extruded with a large rod. The punched carrot formation layer was cut out to a thickness of about 1 mm, planted on a callus induction agar medium, and cultured in the dark at 25 ° C. for 7 days.

(3) Results FIG. 4 shows the results obtained using each of the control sample, the comparative sample, and the sample of the present invention (results of callus induction culture on day 0 and day 7).

  In FIG. 4, the left column is the result of the 0th day, and the right column is the result of the 7th day. Also, in order from the top, a control sample (control), a comparative sample (displayed as 2,4-D), and a sample of the present invention (using DSP 100 μg / mL and DSP 300 μg / mL) are shown.

  From the results shown in FIG. 4, there is almost no change in the control when comparing day 0 and day 7, but 2,4-D and DSP 100 μg / mL and 300 μg / mL are both on day 7 of culture. Callus hypertrophy was observed compared to 7 days of control culture.

  The present invention provides a drug that promotes the formation of adventitious roots of plants, which, based on the effect of promoting adventitious root formation of plants, improves the growth, growth, and harvest of plants, and plants are involved. Useful in the field.

6 is a graph showing changes over time in peptide / protein concentration during soybean meal decomposition by the HA12 strain according to Example 2. FIG. It is a photograph which clarifies that adventitious root formation is recognized in a cherry tomato in the test according to Example 3. It is a photograph which clarifies that adventitious root formation is recognized in a cherry tomato in the test according to Example 3. 4 is a photograph showing the results of carrot callus culture in the test according to Example 4. FIG.

Claims (6)

As an active ingredient, soybean protein raw material obtained by allowing Bacillus circulans HA12 (FERM P-13428) strain or a proteolytic enzyme derived from the strain to act on soy protein raw material has an action to promote adventitious root formation in plants. A plant adventitious root formation promoter characterized by containing. 2. The adventitious root formation promoter according to claim 1, wherein the soy protein raw material is soybean koji. 2. The adventitious root formation promoter according to claim 1, wherein the soybean protein degradation product having an adventitious root formation promoting action is a peptide having a molecular weight of 5000 or less. 2. The adventitious root formation promoter according to claim 1, wherein the plant is selected from the family of solanaceous, leguminous, asteraceae and dianthus. A method for promoting adventitious root formation of a plant, comprising contacting the adventitious root formation promoter according to claim 1 with a plant. 6. The method for promoting adventitious root formation according to claim 5, wherein the plant is selected from the family of solanaceous, legume, asteraceae and dianthus.

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