Application of rice leaf extract in inhibiting plant growth

Abstract

The present invention discloses the application of rice leaf extract in inhibiting plant growth. The present invention provides the application of rice leaf extract in any one of the following: inhibiting plant growth; preparing a product for inhibiting plant growth; dwarfing plants; preparing a product for dwarfing plants; inhibiting the elongation of plant hypocotyls; preparing a product for inhibiting the elongation of plant hypocotyls. The rice leaf extract provided by the present invention has the effect of inhibiting plant growth and can inhibit the germination of rapeseed seeds and / or the elongation of hypocotyls. The present invention is of great significance for controlling plant excessive growth and dwarfing plants.

Description

Technical Field

[0001] The present invention relates to the field of agriculture, and specifically to the application of rice leaf extract in inhibiting plant growth. Background Art

[0002] Flavonoids are widely present in plants in nature and belong to plant secondary metabolites. Flavonoids are a class of yellow pigments derived from flavone (2-phenylchromone) as the parent nucleus, including isomers of flavone and their hydrogenated and reduced products, that is, a series of compounds with a basic carbon skeleton of C6-C3-C6. Flavonoids are widely distributed in the plant kingdom and mostly exist in the form of glycosides or carbon glycosides bound to sugars in plants, and some exist in free form. According to the degree of oxidation of the central three-carbon chain, whether it forms a ring, and the position of the B-ring connection (2- or 3-position), etc., the main natural flavonoids can be divided into flavonoids, flavonols, dihydroflavonoids, dihydroflavonols, anthocyanins, isoflavonoids, etc.

[0003] There are many compounds with medicinal value among flavonoids. These compounds are used to prevent and treat cardiovascular and cerebrovascular diseases, such as reducing the brittleness of blood vessels, improving blood vessel permeability, reducing blood lipids and cholesterol, preventing and treating senile hypertension, cerebral hemorrhage, coronary heart disease, angina pectoris, dilating coronary blood vessels, and increasing coronary blood flow. Many flavonoid components have activities of relieving cough, reducing phlegm, relieving asthma and antibacterial, and at the same time have functions of protecting the liver, detoxifying the liver, antifungal, treating acute and chronic hepatitis, liver cirrhosis, and anti-free radical and antioxidant effects. In addition, flavonoids also have the same effects as phytoestrogens. In the production of livestock animals, the application of flavonoids can significantly improve animal production performance, improve the disease resistance of animal bodies, and improve the immune function of animal bodies.

[0004] Rice leaves contain a large number of secondary metabolites, including flavonoids, which play an important role in rice against ultraviolet rays and diseases (Peng et al., 2017; Zhan et al., 2020). However, it is not very clear at present whether rice leaf metabolites can inhibit plant growth and other issues. Summary of the Invention

[0005] The purpose of the present invention is to provide a rice leaf extract and its application in inhibiting plant growth.

[0006] In the first aspect, the present invention claims to protect the application of rice leaf extract in any one of the following:

[0007] P1, inhibiting plant growth;

[0008] P2, preparing a product for inhibiting plant growth;

[0009] P3, dwarfing plants;

[0010] P4. Preparation of products for dwarfing plants;

[0011] P5. Inhibiting plant seed germination and / or hypocotyl elongation;

[0012] P6. Preparation of products for inhibiting plant seed germination and / or hypocotyl elongation.

[0013] The inhibition of plant growth is the inhibition of plant etiolation.

[0014] Wherein, the rice leaf extract is an ethanol extract of rice leaves.

[0015] Furthermore, the rice leaf extract is an extract obtained by extracting rice leaves with 75% (volume percentage) ethanol.

[0016] Even further, the rice leaf extract can be prepared by a method including the following steps: adding rice leaves in a ratio of 1 Kg (fresh weight) to 1 L to 75% (volume percentage) ethanol, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid.

[0017] Furthermore, after soaking, there is also a step of rotary evaporation and concentration.

[0018] Furthermore, the mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract can be (1 - 1.5):1. Such as 1.35:1.

[0019] In a specific embodiment of the present invention, the total concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the stock solution of the rice leaf extract obtained after rotary evaporation and concentration reaches about 2000 mg / L. Such as 2040 mg / L (the concentration of tricin-5-O-glucoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L).

[0020] In the second aspect, the present invention claims any of the following methods:

[0021] Method I: A method for inhibiting plant growth (a method for inhibiting plant etiolation), which may include the following steps: applying the rice leaf extract described in the second aspect above to plant seeds or hypocotyls or seedlings.

[0022] Method II: A method for dwarfing plants, which may include the following steps: applying the rice leaf extract described in the second aspect above to plant seeds or hypocotyls or seedlings.

[0023] Method III: A method for inhibiting plant seed germination and / or hypocotyl elongation, comprising the following steps: applying the rice leaf extract described in the second aspect above to plant seeds and / or hypocotyls.

[0024] Specifically, it can be: treating plant seeds, hypocotyls or seedlings with the solution of the rice leaf extract described in the second aspect above (the total working concentration of the target compounds, vitexin-5-O-glucoside and isoorientin, is 100 mg / L). For example, placing plant seeds on filter paper moistened with the solution of the rice leaf extract described in the second aspect above (adding 2 mL of the solution to a filter paper with a diameter of Φ6 cm). The treatment time can be 72 h or more.

[0025] In the first aspect to the second aspect, the rice is indica rice.

[0026] Furthermore, the indica rice can be any variety of indica rice such as Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and / or YOU-I B, etc.

[0027] In a specific embodiment of the present invention, the rice leaves are a quality mixture of the leaves of Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B.

[0028] In a specific embodiment of the present invention, the plant growth is manifested as seed germination and / or hypocotyl elongation.

[0029] In a specific embodiment of the present invention, the plant is wheat.

[0030] In a third aspect, the present invention claims the use of vitexin-5-O-glucoside and / or isoorientin in any of the following:

[0031] P1. Inhibiting plant growth;

[0032] P2. Preparing a product for inhibiting plant growth;

[0033] P3. Dwarfing plants;

[0034] P4. Preparing a product for dwarfing plants;

[0035] P5. Inhibiting plant seed germination and / or hypocotyl elongation;

[0036] P6. Preparing a product for inhibiting plant seed germination and / or hypocotyl elongation.

[0037] The inhibition of plant growth is the inhibition of plant excessive growth.

[0038] Among them, in the said mixture, the mass ratio of tricin-5-O-glucoside to isoorientin can be (1 - 1.5):1, such as 1.35:1.

[0039] In the said application, dissolve the mixture composed of tricin-5-O-glucoside and isoorientin in ethanol (such as 75% ethanol) so that the total working concentration of tricin-5-O-glucoside and isoorientin is 100 mg / L.

[0040] In the specific embodiments of the present invention, the plant growth is embodied as seed germination and / or hypocotyl elongation. The dwarfing of the plant is achieved by inhibiting the elongation of the plant hypocotyl.

[0041] In each of the above aspects, the plant can be a dicotyledon or a monocotyledon.

[0042] Furthermore, the dicotyledon can be a cruciferous plant; the monocotyledon can be a gramineous plant.

[0043] Even further, the cruciferous plant can be a Brassica plant; the gramineous plant can be an Oryza plant.

[0044] More specifically, the Brassica plant can be rapeseed; the Oryza plant can be rice.

[0045] Experiments have proved that the rice leaf extract provided by the present invention has the effect of inhibiting plant growth and can inhibit the seed germination and / or hypocotyl elongation of rapeseed. The present invention is of great significance for controlling plant excessive growth. For example, it can be used for fruit trees to control the excessive growth of new shoots of fruit trees and promote the ripening of autumn shoots; it can also be used for crops such as wheat, soybean, rapeseed, and peanut, having the effects of controlling vegetative growth, shortening internodes, dwarfing plants (strengthening seedlings, resisting lodging), and breaking apical dominance. Specific Embodiments

[0046] The present invention will be further described in detail below in combination with specific embodiments. The examples given are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0047] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0048] Example 1. Preparation of Rice Leaf Extract

[0049] (1)Collect fresh leaves of rice (indica rice variety) about 60 days after sowing (the fresh leaves of indica rice varieties Montakcl, J.P.5, PD46, PATNAI 6, Shui Ya Jien, and YOU-I B are mixed in equal mass. These indica rice varieties are all recorded in "Supplemental Table 1" of "Wang et al., The power of inbreeding: NGS based GWAS of rice reveals convergent evolution during rice domestication. 2016 Molecular Plant 9:975", which is publicly available from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes). Then, soak the rice leaves in 75% ethanol (volume percentage, with the other 25% being water) at a ratio of 1 Kg of fresh leaves to 1 L of 75% ethanol for 30 min. After 30 min, collect the rice leaf extract obtained from the soaking solution, and add an appropriate amount of 75% ethanol to the leaves again for extraction once more.

[0050] (2) Collect all the obtained leaf extracts (the liquid after soaking), and concentrate them using a rotary evaporator. During this process, continuously detect the concentration of the concentrate until the total concentration of the target substances (tricin-5-O-glucopyranoside and isoorientin) in the rice leaf extract reaches approximately 2000 mg / L. The detailed detection method is as follows: Pipette 200 μL of the concentrate of the rice leaf extract into a 1.5 mL Eppendorf centrifuge tube, centrifuge at 4 °C and 12,000 rpm for 15 min. Pipette 100 μL of the supernatant into an Agilent injection vial containing a liner with a volume of 200 μL. Then, detect it using UPLC-MS. Mobile phase A: 0.1% formic acid aqueous solution (% represents volume percentage); B phase: acetonitrile. Elution gradient: 0 - 2 min: 5% B - 10% B, 2 - 12 min: 10% B - 25% B, 12 - 18 min: 25% B - 70% B, 18 - 23 min: 70% B - 90% B, 23 - 25 min: 90% B - 100% B, 25 - 30 min: 100% B, post-run for 5 min, and % all represent volume percentage. Flow rate is 0.3 mL / min, column temperature: 40 °C, injection volume: 5 μL. Use an electrospray ionization source (ESI), detect in positive ion mode, carrier gas is high-purity nitrogen, pressure is 40 psi, temperature is 325 °C. Finally, using the standard curve of the target substances (tricin-5-O-glucopyranoside and isoorientin) drawn, convert the peak area of the target substances in the obtained mass spectrum into the absolute content of the target substances in the concentrate. After detection, the total concentration of the target substances tricin-5-O-glucopyranoside and isoorientin in the concentrate is 2040 mg / L (the concentration of tricin-5-O-glucopyranoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L). Dilute the obtained rice leaf extract with water to make the total amount of the active substances (the total amount of the target substances tricin-5-O-glucopyranoside and isoorientin) 1440 mg / L. Use the obtained rice leaf extract as the stock solution of the biological pesticide (WSE) for the subsequent experiments.

[0051] The structural formula of tricin-5-O-glucopyranoside is as follows:

[0052]

[0053] The structural formula of isoorientin is as follows:

[0054]

[0055] Example 2: Performance test of the plant regulator of the biological pesticide WSE

[0056] I. Experimental materials

[0057] Test targets: rapeseed hypocotyl, wheat sprout sheath, cucumber cotyledon expansion, cucumber cotyledon rooting.

[0058] Experimental crops: rapeseed, wheat, cucumber.

[0059] Instruments and equipment: electronic analytical balance, mortar, pressure steam sterilizer, pipette, etc.

[0060] Test reagents: different multiple dilutions of the biological pesticide WSE prepared in Example 1 (the diluent is water).

[0061] 2. Experimental Methods

[0062] 1. Rapeseed hypocotyl elongation inhibition assay

[0063] The rapeseed variety for the test is Mayvine. After soaking the seeds, select uniform seeds for use. The sample preparation adopts the filter paper method in the determination of plant hormone active substances, and the final concentration of the sample test is 100, 10, 1, and 0.1 mg / L. According to the initial concentration of WSE, it is diluted to 1000, 100, 10, and 1 mg / L, and then 0.2 mL of the diluted solution is added with 1.8 mL of sterile water and evenly dripped on a Φ6 cm filter paper, that is, the sample treatment with a final concentration of 100, 10, 1, and 0.1 mg / L. After the solvent is air-dried, a filter paper containing the sample is placed in a Φ6 cm culture dish, and 2 mL of distilled water is added. 10 seeds are evenly placed on the filter paper containing the sample. Distilled water is used as the control. Cultured in a dark environment at 25°C, the length of the hypocotyl is measured after 72 hours. Each treatment is repeated 3 times and the results are recorded. The results are averaged.

[0064] 2. Determination of wheat bud sheath elongation

[0065] The wheat variety tested was Lunkang No. 6. After soaking, the seeds were sown in a covered enamel dish containing 0.7% agar and cultured in a dark environment at 25°C for 72 hours. When the seedlings were 2.5-3.0 cm tall, the top 3 mm of the sheath was removed, and 5 mm long sheath segments were cut and placed in distilled water for 1 hour to remove endogenous hormones. The sample preparation adopted the filter paper method in the determination of plant hormone active substances, and the final concentrations of the sample tests were 100, 10, 1, and 0.1 mg / L. According to the initial concentration of WSE, it was diluted to 1000, 100, 10, and 1 mg / L, and then 0.3 mL of the diluted solution was added with 2.7 mL of sterile water and evenly dropped on a Φ6 cm filter paper, which was a sample treatment with a final concentration of 100, 10, 1, and 0.1 mg / L. After the solvent was air-dried, a filter paper containing the sample was placed in a Φ6 cm culture dish, and 0.01 mol·L containing 2% sucrose was added. -13 mL of phosphate-citrate buffer (pH = 5) was evenly placed into 10 segments. Phosphate-citrate buffer was used as the control. They were cultured in the dark at 25 °C. After 18 - 20 h, the total length of every 10 segments was measured. Each treatment was repeated 3 times and the results were recorded. The results were averaged.

[0066] 3. Cucumber cotyledon expansion determination

[0067] The cucumber variety used was Jinyan No. 4. After soaking the seeds, they were sown in a covered enamel tray containing 0.7% agar and cultured in the dark at 26 °C for 72 h. Cotyledons with uniform size were selected for use. The filter paper method in the determination of plant hormone active substances was used for sample preparation. The final concentrations of the samples for testing were 100, 10, 1, and 0.1 mg / L. According to the initial concentration of WSE, they were diluted to 1000, 100, 10, and 1 mg / L. Then, 0.3 mL of the diluted medicinal liquid was added to 2.7 mL of sterile water and evenly dropped onto filter papers with a diameter of Φ6 cm, which was the sample treatment with final concentrations of 100, 10, 1, and 0.1 mg / L. After the solvent was air-dried, 1 filter paper containing the sample was placed in a petri dish with a diameter of Φ6 cm, 3 mL of distilled water was added, and 10 cotyledons were added. Distilled water was used as the control. They were cultured at 26 °C in an environment of 3000 Lux. After 72 h, the fresh weight of every 10 cotyledons was measured. Each treatment was repeated 3 times and the results were recorded. The results were averaged.

[0068] 4. Cucumber cotyledon rooting determination

[0069] The cucumber variety used was Jinyan No. 4. After soaking the seeds, they were sown in a covered enamel tray containing 0.7% agar and cultured in the dark at 26 °C for 72 h. Cotyledons with uniform size were selected for use. The filter paper method in the determination of plant hormone active substances was used for sample preparation. The final concentrations of the samples for testing were 100, 10, 1, and 0.1 mg / L. According to the initial concentration of WSE, they were diluted to 1000, 100, 10, and 1 mg / L. Then, 0.3 mL of the diluted medicinal liquid was added to 2.7 mL of sterile water and evenly dropped onto filter papers with a diameter of Φ6 cm, which was the sample treatment with final concentrations of 100, 10, 1, and 0.1 mg / L. After the solvent was air-dried, 1 filter paper containing the sample was placed in a petri dish with a diameter of Φ6 cm, 3 mL of distilled water was added, and 10 cotyledons were added. Distilled water was used as the control. They were cultured in the dark at 26 °C. After 5 d, the number of roots at the base of the petiole of every 10 cotyledons was measured. Each treatment was repeated 3 times and the results were recorded. The results were averaged.

[0070] 5. Evaluation criteria

[0071] Calculation method:

[0072] Wheat coleoptile:

[0073] Cotyledon expansion:

[0074] Cotyledon rooting:

[0075] Rapeseed hypocotyl:

[0076]

[0077] Plant hormone activity grading:

[0078] Wheat coleoptile: +++ ≥ 40%; ++ < 40% and ≥ 25%; + < 25% and ≥ 10%; - < 10%.

[0079] Cotyledon expansion: +++ ≥ 40%; ++ < 40% and ≥ 25%; + < 25% and ≥ 10%; - < 10%.

[0080] Cotyledon rooting: +++ ≥ 150%; ++ < 150% and ≥ 100%; + < 100% and ≥ 50%; - < 50%.

[0081] Rapeseed hypocotyl: +++ ≥ 70%; ++ < 70% and ≥ 50%; + < 50% and ≥ 30%; - < 30%.

[0082] The more "+", the better the corresponding effect; "-" means no corresponding effect.

[0083] III. Results and Analysis

[0084] The results are shown in Table 1.

[0085] Table 1. Evaluation of the phytoregulatory function of biological pesticide WSE

[0086]

[0087] Note: Treatment with 100 mg / L rapeseed severely inhibited seed germination, and the elongation of hypocotyls of individual germinations was also severely inhibited, reaching the level of "+++".

[0088] The investigation results show that the biological pesticide WSE has a certain effect on inhibiting rapeseed hypocotyls, reaching the level of "+++" at a concentration of 100 mg / L. Through this experiment, it is determined that the biological substance WSE has a certain effect on inhibiting plant growth and has certain application value in inhibiting crop overgrowth and dwarfing crops.

[0089] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. Use of rice leaf extract in any of the following: P1. Inhibiting the elongation of plant hypocotyls; P2. Preparing a product for inhibiting the elongation of plant hypocotyls; The rice leaf extract is prepared by a method comprising the following steps: adding rice leaves in a ratio of 1 kg fresh weight to 1 L to 75% (v / v) ethanol, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid; after soaking, a step of rotary evaporation and concentration is further included; The rice is indica rice; the indica rice is a mass mixture of Montakcl, J.P. 5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B, etc.; The plant is rapeseed.

2. A method for inhibiting the elongation of plant hypocotyls, comprising the following steps: applying the rice leaf extract described in claim 1 to plant seeds and / or hypocotyls; The plant is rapeseed; wherein, The total working concentration of the target compounds, tricin-5-O-glucoside and isoorientin, in the rice leaf extract is 100 mg / L.

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