A forage growth promoter and application of daidzein, genistein or palmitic acid in promoting forage growth

Abstract

The present invention discloses a forage growth promoter and the application of daidzein, genistein or palmitic acid in promoting forage growth. The active ingredient of the forage growth promoter is a combination of one or more of daidzein, genistein or palmitic acid. The three allelopathic substances extracted and separated from alfalfa, daidzein, genistein or palmitic acid, have a growth-promoting effect on alfalfa or ryegrass within a certain concentration range. The three components can be used as the effective ingredients of the forage growth promoter to promote the planting of high-quality forage such as alfalfa or ryegrass. In addition, at high concentrations, daidzein, genistein or palmitic acid, four natural substances present in plants, can be used as ecological herbicides to inhibit forage growth, which has the advantages of environmental protection and pollution-free.

Description

Technical Field

[0001] The invention relates to the technical field of animal husbandry forage cultivation, and in particular to a forage growth promoter and application of daidzein, genistein or palmitic acid in promoting forage growth. Background Art

[0002] Alfalfa is a high-quality legume forage grass with high nutritional value and good palatability. It is known as the "king of forage grasses". Two problems are often encountered in alfalfa cultivation: weed control and continuous cropping self-toxicity. Both problems will affect the quality and yield of alfalfa. In addition, ryegrass is also a common high-quality forage grass.

[0003] my country is a large agricultural country, and animal husbandry occupies an important position in our economy. Alfalfa, as a high-quality legume forage, plays an important role in animal husbandry. As a high-protein and high-yield forage crop, alfalfa is widely planted throughout the world. In the United States, it is the third largest economic crop after corn and soybeans. Planting alfalfa not only has economic benefits but also ecological benefits. As a legume forage, alfalfa has a significant effect in biological nitrogen fixation, which can increase the content of soil organic matter and improve soil structure. Not only that, the developed root system of alfalfa can also reduce soil erosion. However, two problems are often encountered in alfalfa cultivation: weed control and continuous cropping self-toxicity effect. Both of these problems will affect the quality and yield of alfalfa.

[0004] Therefore, under the above development background, how to promote the growth of alfalfa and ryegrass is an important issue, and the market has an urgent need for eco-friendly growth promoters for these types of forage.

[0005] On the other hand, weed control has not been effectively solved in agricultural production. Today, the use of herbicides is the fastest and most effective method of weed control. However, the extensive use of chemical herbicides has a negative impact on the environment, and large-scale exposure to chemical herbicides may lead to many health problems, such as reproductive problems, nerve damage, pancreatic cancer, immune dysfunction, and silicosis. In addition, chemical herbicides are not suitable for organic farming. Therefore, the development of ecological herbicides is imperative. Summary of the invention

[0006] In view of the lack of environmentally friendly grass growth promoters and ecological herbicides, the present application successfully extracted three active ingredients, namely, daidzein, genistein, and tryptamine or palmitic acid, from alfalfa. It was found that these four ingredients have a growth-promoting effect on alfalfa or ryegrass within a certain concentration range and can be used as active ingredients of grass growth promoters. In addition, at high concentrations, daidzein, genistein or palmitic acid can be used as ecological herbicides to inhibit grass growth, which has the advantages of being environmentally friendly and pollution-free.

[0007] To solve the above problems, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides a forage growth promoter, the active ingredient of which is a combination of one or more of daidzein, genistein or palmitic acid.

[0009] In a second aspect, the present application provides the use of daidzein, genistein, and palmitic acid in promoting forage growth, wherein daidzein, genistein, or palmitic acid is used as an active ingredient of the forage promoter.

[0010] Preferably, the forage grass is alfalfa or ryegrass. The test results show that daidzein, genistein or palmitic acid has the effect of promoting germination, growth or increasing fresh weight of alfalfa or ryegrass within a certain concentration range.

[0011] Preferably, the three substances, daidzein, genistein and palmitic acid, are used to promote the growth of forage, wherein the concentration of daidzein applied to ryegrass is 200-400 μg / ml; the concentration of genistein is 50-100 μg / ml; and the concentration of palmitic acid is 25-100 μg / ml.

[0012] The test results prove that:

[0013] (1) Soybean aglycone at a concentration of about 25 μg / ml can promote the length of the radicle of alfalfa. The results showed that the length of the radicle of alfalfa increased by 11% under the treatment of 25 μg / ml concentration.

[0014] At a concentration of 50-100 μg / ml, daidzein promoted the growth of ryegrass radicles, and at a concentration of 50 and 100 μg / ml, the root length of ryegrass was extended by 9.3% and 8.9%, respectively. At a concentration of 200-400 μg / ml, daidzein increased the fresh weight of ryegrass, and at a concentration of 200 and 400 μg / ml, the fresh weight of ryegrass increased by 16.7% and 26.5%, respectively.

[0015] (2) Genistein promoted the growth of alfalfa embryos in the range of 50-100 μg / ml.

[0016] On the one hand, genistein promoted the elongation of the radicle of alfalfa in the concentration range of 50-100 μg / ml. The experimental results showed that the radicle of alfalfa elongated by 5.2% at a concentration of 50 μg / ml and by 27% at a concentration of 100 μg / ml. On the other hand, the hypocotyl of alfalfa elongated by 23.4% and 36.5% respectively under the treatment of genistein at concentrations of 25, 50, and 100 μg / ml.

[0017] Genistein significantly promoted the growth of ryegrass seedlings within the concentration range of 25~50μg / ml. Genistein at concentrations of 25 and 50μg / ml increased the length of ryegrass seedlings by 4.9% and 7.7%.

[0018] (3) Palmitic acid significantly promoted the elongation of alfalfa hypocotyls at a concentration of 100 μg / ml (P<0.05). This concentration caused the elongation of alfalfa hypocotyls to be 14.8%.

[0019] Palmitic acid significantly promoted the growth of ryegrass seedlings within the concentration range of 25-50 μg / ml. Specifically, the growth of ryegrass seedlings was extended by 9.6% and 6.6% at concentrations of 25 and 50 μg / ml.

[0020] Palmitic acid significantly increases the fresh weight of ryegrass within the concentration range of 100~400μg / ml. Experiments have shown that palmitic acid at concentrations of 100 and 200 increases the fresh weight of ryegrass by 20.5% and 17.2%, respectively.

[0021] From the above content, it can be seen that preferably, palmitic acid can significantly promote the growth of ryegrass in a concentration range of 25-100 μg / ml.

[0022] Preferably, in the application, the method for using the daidzein, genistein, tryptamine or palmitic acid is: soaking the forage seeds with a solution of a certain concentration of daidzein, genistein or palmitic acid or spraying the solution on the forage seedlings.

[0023] In a third aspect, the present application provides a herbicide, the active ingredient of which is a combination of one or more of daidzein, genistein, tryptamine or palmitic acid.

[0024] In a fourth aspect, the present application provides the use of daidzein, genistein, tryptamine or palmitic acid as active ingredients of herbicides in inhibiting weed growth. Experiments have found that at high concentrations, these four allelopathic substances derived from plants have the effect of inhibiting the germination and growth of alfalfa and ryegrass.

[0025] Preferably, in the application, the weeds are alfalfa or ryegrass.

[0026] Preferably, daidzein, genistein, tryptamine or palmitic acid significantly inhibits germination, radicle growth and fresh weight of alfalfa at 200-400 μg / ml, and has a good control effect on alfalfa, especially seeds and seedlings.

[0027] The experimental results show that within a certain concentration range (under higher concentration conditions), daidzein, genistein, tryptamine or palmitic acid inhibit the germination, radicle and hypocotyl of alfalfa and ryegrass, and reduce the fresh weight of these two weeds, as follows:

[0028] A. Daidzein

[0029] When the concentration of daidzein reaches 400μg / ml, daidzein will significantly inhibit the germination of alfalfa, reducing the germination rate of alfalfa by 14%. At a concentration of 100-400μg / ml, daidzein inhibits the radicle length of alfalfa. 100, 200, and 400μg / ml daidzein shorten the radicle length of alfalfa by 10.6%, 22.3%, and 30%, respectively. At a concentration of 25-400μg / ml, daidzein inhibits the growth of the hypocotyl of alfalfa; under the treatment of daidzein at a concentration of 25, 50, 100, 200, and 400μg / ml, the hypocotyl of alfalfa shortens by 7.6%, 5.7%, 14.6%, 39%, and 23.9%, respectively. When the concentration of daidzein reached 400 μg / ml, it had a significant inhibitory effect on the fresh weight of alfalfa. After treatment with this concentration, the fresh weight of alfalfa decreased by 20.2%.

[0030] Preferably, when the concentration of daidzein reaches or exceeds 400 μg / ml, it effectively inhibits the germination, radicle and hypocotyl growth of alfalfa, and inhibits the fresh weight of alfalfa to the greatest extent. Therefore, at this concentration, daidzein has a better control effect on alfalfa.

[0031] B. Genistein

[0032] Genistein inhibited the germination of alfalfa in the concentration range of 100~400μg / ml. The germination rate of alfalfa was reduced by 16%, 13% and 16% at concentrations of 100, 200 and 400μg / ml, respectively. Genistein inhibited the radicle length of alfalfa at a concentration of 200μg / ml~400μg / ml. Genistein at 200 and 400μg / ml inhibited the elongation of the radicle of alfalfa, shortening the radicle by 6.2% and 21%. When the concentration of genistein reached 400μg / ml, it had a significant inhibitory effect on the fresh weight of alfalfa. The fresh weight of alfalfa treated with this concentration was reduced by 14.6%.

[0033] Preferably, genistein significantly inhibits germination, radicle growth and fresh weight of alfalfa at a concentration of 200-400 μg / ml, and has a good control effect on alfalfa, especially seeds and seedlings.

[0034] Genistein had no effect on the germination rate and fresh weight of ryegrass seedlings.

[0035] C. Tryptamine

[0036] Tryptamine inhibited the germination of alfalfa and ryegrass in the range of 200-400 μg / ml. Tryptamine at concentrations of 200 and 400 μg / ml reduced the germination rate of alfalfa by 11.5% and 14%, respectively. Tryptamine at concentrations of 200 and 400 μg / ml reduced the germination rate of ryegrass by 15.6% and 32.5%, respectively.

[0037] Tryptamine inhibited the growth of hypocotyl and radicle of alfalfa at concentrations of 25-400 μg / ml; the hypocotyl of alfalfa shortened by 10.8%, 20%, 45.1%, 60.9% and 92% respectively under the treatment of tryptamine at concentrations of 25, 50, 100, 200 and 400 μg / ml. After the alfalfa was treated with tryptamine at concentrations of 25, 50, 100, 200 and 400 μg / ml, its radicle shortened by 22%, 43%, 57%, 68% and 93% respectively.

[0038] Tryptamine at 50-400 μg / ml had a significant inhibitory effect on the seedling and root length of ryegrass. At 50, 100, 200, and 400 μg / ml concentrations, the seedling length of ryegrass was shortened by 8.6%, 18.5%, 24.8%, and 33.3%, respectively. Tryptamine at 25, 50, 100, 200, and 400 μg / ml shortened the root length of ryegrass by 18.4%, 22.3%, 30.3%, 52.2%, and 87.2%, respectively.

[0039] The concentrations of 200 and 400 μg / ml of tryptamine reduced the fresh weight of alfalfa by 20.6% and 17.8% respectively (P<0.05).

[0040] It can be seen that tryptamine significantly inhibited the germination, radicle growth and fresh weight of alfalfa and ryegrass under the condition of 200-400 μg / ml, and had a good control effect on alfalfa, especially seeds and seedlings.

[0041] D. Palmitic acid

[0042] Palmitic acid inhibited the germination of alfalfa at 200-400 μg / ml. The germination rate of alfalfa decreased by 14% and 13% at 200 and 400 μg / ml respectively. Palmitic acid significantly inhibited the germination of ryegrass seeds at a concentration of 400 μg / ml. P <0.05), which reduced the germination rate of ryegrass by 6.5%.

[0043] Palmitic acid inhibited the growth of radicle of alfalfa at 25-400 μg / ml. After alfalfa was treated with palmitic acid at concentrations of 25, 50, 100, 200, and 400 μg / ml, its radicle shortened by 14.3%, 11.5%, 30.7%, 30.5%, and 32%, respectively. Palmitic acid began to significantly inhibit the root length of ryegrass at a concentration of 100-400 μg / ml (P<0.05), and the root length of ryegrass shortened by 6.7%, 9%, and 7.6% at concentrations of 100, 200, and 400 μg / ml.

[0044] Preferably, palmitic acid at 200-400 μg / ml can effectively inhibit the germination and radicle growth of alfalfa and ryegrass, and has a good control effect on alfalfa, especially seeds and seedlings.

[0045] Preferably, in the application, the method for using the daidzein, genistein, tryptamine or palmitic acid is: soaking the forage seeds with a solution of daidzein, genistein, tryptamine or palmitic acid or spraying the solution on the forage seedlings.

[0046] The present invention has the following beneficial effects:

[0047] 1. The present invention extracts four allelopathic substances, namely daidzein, genistein, tryptamine or palmitic acid, from alfalfa for the first time, and discovers for the first time that daidzein, genistein and palmitic acid have new functions and new uses in promoting the growth of two high-quality forages, alfalfa and ryegrass, within a certain concentration range, which is mainly reflected in significantly promoting the seed germination, radicle and hypocotyl growth of these two forages and increasing their fresh weight. These three substances can be used as forage growth promoters, which is helpful to the development of animal husbandry.

[0048] 2. The present application also found that the four allelopathic substances, namely, daidzein, genistein, tryptamine or palmitic acid, have inhibitory effects on alfalfa and ryegrass at high concentrations, and can be used as active ingredients of herbicides to control alfalfa and ryegrass at higher concentrations.

[0049] 3. Although the above four components, daidzein, genistein, tryptamine or palmitic acid, have long existed, they all play completely different roles in other technical fields. This application first discovered their dual performance as forage growth promoters or herbicides under different conditions. This discovery shows that daidzein, genistein, tryptamine or palmitic acid have multiple application prospects and high utilization rates. Moreover, these four components come from the plant alfalfa, making them safer to use; as ecological forage growth promoters or ecological herbicides, they have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the multi-peak diagram of MRM metabolite detection;

[0051] Figure 2 The effects of four substances on the germination rate of alfalfa and ryegrass; Daidzein: soybean aglycone, Genistein:: genistein, Tryptamine: tryptamine, Hexadecanoic acid: palmitic acid; Figure A: germination rate of alfalfa. Figure B: germination rate of ryegrass; lowercase letters indicate significant differences between treatments (P<0.05);

[0052] Figure 3 The effects of four substances on the length of radicle and hypocotyl of alfalfa; Daidzein: soybean aglycone, Genistein:: genistein, Tryptamine: tryptamine, Hexadecanoic acid: palmitic acid; Figure A: radicle length of alfalfa, Figure B: hypocotyl length of alfalfa. Lowercase letters indicate significant differences among treatments (P<0.05);

[0053] Figure 4 The effects of four substances on the growth of alfalfa seedlings; D: daidzein, T: tryptamine, G: genistein, Ha: palmitic acid; 0: 0μg / ml, 1: 25μg / ml, 2: 50μg / ml, 3: 100μg / ml, 4: 200μg / ml, 5: 400μg / ml;

[0054] Figure 5 The effects of four substances on the root length and seedling length of ryegrass; Daidzein: soybean aglycone, Genistein: genistein, Tryptamine: tryptamine, Hexadecanoic acid: palmitic acid; Figure A: ryegrass root length, Figure B: ryegrass seedling length; lowercase letters indicate significant differences among treatments (P<0.05);

[0055] Figure 6 The effects of four substances on the growth of ryegrass seedlings; D: daidzein, T: tryptamine, G: genistein, Ha: palmitic acid; 0: 0μg / ml, 1: 25μg / ml, 2: 50μg / ml, 3: 100μg / ml, 4: 200μg / ml, 5: 400μg / ml;

[0056] Figure 7 The figure shows the effects of four substances on the fresh weight of alfalfa and ryegrass; Daidzein, Genistein, Tryptamine and Hexadecanoic acid; Figure A: fresh weight of alfalfa, Figure B: fresh weight of ryegrass; lowercase letters indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0058] Example 1 Extraction of the active ingredients of alfalfa

[0059] 1.1 Test methods

[0060] 1.1.1 Collection of test materials

[0061] Alfalfa materials were collected in the Modern Science and Technology Demonstration Garden of Qingdao Agricultural University. The aboveground part and the root of about 20 cm underground of 3-year-old Suntory alfalfa were taken. Random sampling was adopted and the sampling was repeated 3 times. The alfalfa seeds (Suntory) and ryegrass seeds used for bioactivity determination were purchased from Best Seed Company.

[0062] 1.1.2 Test material processing

[0063] Cut the alfalfa roots into about 1 cm lengths and dry them together with the stems and leaves at 37°C. After drying, use a grinder to crush the samples, and pass them through a 40-mesh sieve. Weigh 2 kg of the crushed and sieved samples into a 10L wide-mouth bottle, and use distilled water to extract the samples. The samples were extracted in the wide-mouth bottle for 24 hours. The solution obtained after extraction was first filtered with 4 layers of gauze, and then filtered with quantitative filter paper to collect the extract. Repeat the extraction 4 times, mix the 4 extracts, and remove the solvent by vacuum rotary evaporation at 40°C to obtain an extract.

[0064] 1.1.3 Detection and separation of extracts

[0065] In this embodiment, the identification of the components in the above extracts relies on the Maiwei metabolome testing technology and the plant secondary metabolite database and public database independently constructed by Maiwei for material qualitative analysis. The instrument systems for collecting and analyzing material information are divided into two, one is Ultra High Performance Liquid Chromatography (UPLC), and the other is Tandem mass spectrometry (MS / MS), and the conditions are as follows:

[0066] (1) The conditions for ultra-high performance liquid chromatography are:

[0067] Chromatographic column: Waters ACQUITY UPLC HST3C18 1.8µm, 2.1mm*100mm

[0068] Mobile phase: The aqueous phase is ultrapure water with acetic acid added, and the proportion of acetic acid is 0.04%; the organic phase is acetonitrile with acetic acid added, and the proportion of acetic acid in acetonitrile is also 0.04%.

[0069] Flow rate: 0.4 ml / min, column temperature: 40 °C, injection volume: 5 µl.

[0070] Elution gradient: as shown in Table 1.

[0071] Table 1 Gradient elution method

[0072]

[0073] (2) The conditions for tandem mass spectrometry are shown in Table 2

[0074] Table 2 Mass spectrometry conditions

[0075]

[0076] 1.1.4 Relative quantification and qualitative analysis of metabolites

[0077] The qualitative analysis of the substances in this invention is based on the self-built MWDB (metwaredatabase) of Maiwei Metabolism and the public metabolite database, and the primary and secondary spectrum data detected by mass spectrometry are analyzed and qualitatively analyzed. The isotope signals of some substances are removed during qualitative analysis, including K + 、Na + NH4 + The metabolite structures were analyzed with reference to MassBank ( http: / / www.massbank.jp / )、KNAPSAcK( http: / / kanaya.naist.jp / KNApSAcK / )、HMDB( http: / / www.hmdb.ca / ) (Wishartetal.2013), MoToDB ( http: / / www.ab.wur.nl / moto / )、METLIN( http: / / metlin.scripps.edu / index.php ) and other existing public mass spectrometry databases.

[0078] Relative quantification of metabolites is accomplished using the multiple reaction monitoring (MRM) analysis of a triple quadrupole mass spectrometer. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, excluding ions corresponding to other molecular weight substances to preliminarily eliminate interference. The precursor ions are induced to ionize in the collision chamber and then break to form many fragment ions. The fragment ions are then filtered through the triple quadrupole to select the required characteristic fragment ions, eliminating interference from the target ions, making quantification more accurate and more repeatable. After obtaining the metabolite mass spectrometry analysis data of different samples, the peak areas of all mass spectrometry peaks are integrated, and the mass spectrometry peaks of the same metabolite in different samples are integrated and corrected.

[0079] 1.2 Experimental results and analysis

[0080] (1) Qualitative test results of alfalfa extract

[0081] For details on the multi-peak diagram of metabolite detection, see Figure 1 After identification and analysis, a total of 273 substances were detected from the aforementioned alfalfa extract, including flavonoids, phenolic acids, alkaloids, terpenes, lignin and coumarins, quinones, fatty acids and other types.

[0082] The proportions of various substances in these substances are flavonoids 48%, phenolic acids 27.5%, alkaloids 11%, terpenes 2.6%, lignin and coumarins 2.2%, quinones 0.73%, fatty acids 0.37%, and other substances 7.6%. Among them, 81 substances have reached the A level in qualitative level.

[0083] According to the qualitative level and relative content analysis of substances, four new components that have not been reported in alfalfa were found: daidzein, genistein, tryptamine and palmitic acid. The information of the four components is shown in Table 3.

[0084] Table 3 Information of four substances

[0085]

[0086] Example 2 Activity test of four potential allelopathic substances

[0087] The purpose of this example is to study the growth-promoting effects of four allelochemicals isolated from alfalfa, namely daidzein, genistein, tryptamine and palmitic acid, on alfalfa and ryegrass, respectively.

[0088] 2.1 Experimental Materials

[0089] The four active substances used in this example, daidzein, genistein, tryptamine and palmitic acid, were purchased from Shanghai Yuanye Biological Company.

[0090] 2.2 Experimental methods

[0091] (1) Soak the alfalfa and ryegrass seeds required for the experiment in 70% ethanol for 3 minutes for disinfection. Then wash the seeds 5 times with distilled water and then wash the seeds 5 times with ultrapure water. After washing the seeds, dry them in a fume hood.

[0092] (2) The above four substances were dissolved in dimethyl sulfoxide to prepare a 100 mg / ml stock solution, and then the stock solution was diluted to prepare a gradient concentration of 25, 50, 100, 200, and 400 μg / ml. Alfalfa and ryegrass were used as receptor plants for subsequent seed germination and seedling growth research experiments.

[0093] (3) Place two layers of filter paper in a culture dish (9 cm in size), add 5 ml of the four substances of different concentrations to each culture dish, and add the same volume of dimethyl sulfoxide as a blank control. Place all culture dishes in a fume hood to dry until the dimethyl sulfoxide is completely evaporated. Then add 5 ml of distilled water to each culture dish, soak the filter paper completely, and plant 20 seeds disinfected as described above in each culture dish. Repeat each concentration and blank setting 4 times. To prevent the water in the culture dish from evaporating, seal the culture dish with plastic wrap.

[0094] (4) All culture dishes containing seeds were placed in an artificial incubator. The seeds were germinated in the dark at 23°C for 3 days. The germination rate was measured on the 3rd day. The seeds were then cultured in the light for 4 days at a light / darkness ratio of 25°C / 23°C. The radicle length and hypocotyl length of alfalfa, and the root length and seedling length of ryegrass were measured on the 7th day.

[0095] 2.3 Measurement items and methods

[0096] Seed germination rate: When the seeds are white and the sprout length exceeds 1 / 2 of the seed itself, they are considered to have germinated. The radicle length, hypocotyl length, root length and seedling length of alfalfa, ryegrass are all measured with a ruler.

[0097] The data were statistically collated and analyzed using office365 and SPSS22.0 software, and graphs were drawn using Origin2017.

[0098] 2.4 Experimental results and analysis

[0099] 2.4.1 Effects of the four substances on the germination rate of alfalfa and ryegrass

[0100] (1) From Figure 2 A It can be seen that the four substances, daidzein, genistein, tryptamine and palmitic acid, will inhibit the germination of alfalfa, and the higher the concentration, the stronger the inhibition.

[0101] ① When the concentration of daidzein reaches 400μg / ml, daidzein will significantly inhibit the germination of alfalfa ( P <0.05), which reduced the germination rate of alfalfa by 14%.

[0102] ②When the concentration of genistein was 100, 200 and 400 μg / ml, the germination rate of alfalfa decreased by 16%, 13% and 16% respectively.

[0103] ③ Tryptamine at concentrations of 200 and 400 μg / ml reduced the germination rate of alfalfa by 11.5% and 14%, respectively.

[0104] ④ Palmitic acid at concentrations of 200 and 400 μg / ml reduced the germination rate of alfalfa by 14% and 13%, respectively.

[0105] (2) By Figure 2 B shows that the four substances, daidzein, genistein, tryptamine and palmitic acid, have different effects on the germination rate of ryegrass.

[0106] ① High concentrations of tryptamine have a significant inhibitory effect on the germination rate of ryegrass ( P <0.05), and the influence pattern was that the inhibition became stronger with the increase of concentration. 200 and 400 μg / ml tryptamine reduced the germination rate of ryegrass by 15.6% and 32.5%, respectively.

[0107] ② Palmitic acid significantly inhibited the germination of ryegrass seeds when the concentration reached 400 μg / ml ( P <0.05), which reduced the germination rate of ryegrass by 6.5%.

[0108] ③Daidzein and genistein had no significant effect on the germination rate of ryegrass in the experiment ( P >0.05).

[0109] 2.4.2 Effects of four substances on the growth of alfalfa seedlings

[0110] 2.4.2.1 Effects of four substances on the growth of alfalfa radicle

[0111] from Figure 3 A and Figure 4 It can be seen that all four substances have an effect on the growth of alfalfa radicle, and the specific effects are as follows:

[0112] (1) The effects of daidzein and genistein on the radicle growth of alfalfa first promoted and then inhibited with the increase of concentration.

[0113] ① Daidzein

[0114] Promoting effect: 25 μg / ml daidzein significantly promoted the radicle length of alfalfa (P<0.05). Under this concentration, the radicle length of alfalfa increased by 11%.

[0115] Inhibitory effect: 100μg / ml daidzein inhibited the radicle length of alfalfa. 100, 200, and 400μg / ml daidzein shortened the radicle length of alfalfa by 10.6%, 22.3%, and 30%, respectively.

[0116] ②Genistein

[0117] Promoting effect: 50 and 100 μg / ml genistein can promote the elongation of the radicle of alfalfa, making the radicle elongate by 5.2% and 27%;

[0118] Inhibitory effect: 200 and 400 μg / ml genistein will inhibit the elongation of the radicle of alfalfa, shortening the radicle by 6.2% and 21% respectively.

[0119] (2) The effect of tryptamine and palmitic acid on the radicle length of alfalfa showed that the inhibitory effect gradually increased with the increase of concentration. When the concentration was 25 μg / ml, it had a significant inhibitory effect on the radicle of alfalfa (P<0.05).

[0120] ① According to the analysis of tryptamine data, the radicle of alfalfa was shortened by 22%, 43%, 57%, 68% and 93% respectively under the treatment of 25, 50, 100, 200 and 400 μg / ml concentrations;

[0121] ②According to the data analysis of palmitic acid, under the treatments of 25, 50, 100, 200 and 400 μg / ml concentrations, the radicle of alfalfa was shortened by 14.3%, 11.5%, 30.7%, 30.5% and 32%, respectively.

[0122] 2.4.2.2 Effects of four substances on the growth of hypocotyls of alfalfa

[0123] from Figure 3 B and Figure 4 It can be seen that all four substances have an effect on the growth of alfalfa radicle, and the specific effects are as follows:

[0124] (1) The effects of genistein and palmitic acid on the hypocotyls of alfalfa are promoting.

[0125] ① The data of genistein showed that all concentrations of genistein significantly promoted the elongation of alfalfa hypocotyls (P<0.05). Under the treatments of 25, 50, 100, 200 and 400 μg / ml concentrations, the hypocotyls of alfalfa elongated by 23.4%, 36.5%, 24.2%, 20.9% and 19.2%, respectively.

[0126] ②The data of palmitic acid showed that the substance significantly promoted the elongation of alfalfa hypocotyls at a concentration of 100 μg / ml (P<0.05). This concentration elongated the alfalfa hypocotyls by 14.8%, while other concentrations had no significant effect (P>0.05).

[0127] (2) The effects of daidzein and tryptamine on alfalfa hypocotyls showed that the inhibition increased with the increase of concentration;

[0128] ① Daidzein: When the concentration is 25μg / ml, daidzein will have a significant inhibitory effect on the hypocotyls of alfalfa (P<0.05), and the inhibition increases with the increase of concentration. Under the treatment of daidzein concentrations of 25, 50, 100, 200, and 400μg / ml, the hypocotyls of alfalfa were shortened by 7.6%, 5.7%, 14.6%, 39%, and 23.9%, respectively.

[0129] ② Tryptamine: Tryptamine has the most obvious inhibitory effect on the hypocotyls of alfalfa. With the increase of concentration, the degree of inhibition increases. Under the treatment of tryptamine concentrations of 25, 50, 100, 200, and 400 μg / ml, the hypocotyls of alfalfa were shortened by 10.8%, 20%, 45.1%, 60.9%, and 92%, respectively.

[0130] 2.4.3 Effects of four substances on the growth of ryegrass seedlings

[0131] 2.4.3.1 Effects of four substances on ryegrass root growth

[0132] from Figure 5 A and Figure 6 It can be seen that the effects of daidzein, genistein, tryptamine and palmitic acid on the root growth of ryegrass are different.

[0133] (1) The effect of daidzein on ryegrass root growth is that it first promotes and then inhibits as the concentration increases.

[0134] 50 and 100 μg / ml of daidzein extended the root length of ryegrass by 9.3% and 8.9%, respectively. 400 μg / ml of daidzein significantly inhibited the root length of ryegrass (P<0.05), shortening it by 9.6% compared with the blank.

[0135] (2) The effect of genistein and tryptamine on ryegrass root growth is that the inhibition gradually increases with the increase of concentration.

[0136] ① When the concentration was 25 μg / ml, genistein had a significant inhibitory effect on the root length of ryegrass (P<0.05). Under the treatments of 25, 50, 100, 200, and 400 μg / ml concentrations, the root length of ryegrass was shortened by 10.9%, 14.4%, 16%, 24.3%, and 29.1%, respectively.

[0137] ②The inhibitory effect gradually increased with the increase of concentration. When the concentration was 25 μg / ml, tryptamine had a significant inhibitory effect on the root length of ryegrass (P<0.05). Each concentration (25, 50, 100, 200, 400 μg / ml) shortened the root length of ryegrass by 18.4%, 22.3%, 30.3%, 52.2%, and 87.2%, respectively.

[0138] (3) Palmitic acid data showed that at low concentrations (25 and 50 μg / ml), this substance had no significant effect on the root length of ryegrass (P>0.05). When the concentration reached 100 μg / ml, this substance began to significantly inhibit the root length of ryegrass (P<0.05). The root length of ryegrass was shortened by 6.7%, 9% and 7.6% at 100, 200 and 400 μg / ml concentrations.

[0139] 2.4.3.2 Effects of four substances on ryegrass seedling growth

[0140] from Figure 5 B and Figure 6 It can be seen that the effects of daidzein, genistein, tryptamine and palmitic acid on the growth of ryegrass seedlings are different:

[0141] (1) The effect of genistein and palmitic acid in this experiment is that low concentrations promote the growth of ryegrass seedlings.

[0142] ① Genistein at low concentrations (25, 50 μg / ml) significantly promotes the growth of ryegrass seedlings. Genistein at concentrations of 25 and 50 μg / ml increases the length of ryegrass seedlings by 4.9% and 7.7%.

[0143] ② Palmitic acid at low concentrations (25, 50 μg / ml) significantly promoted the growth of ryegrass seedlings (P<0.05), extending the length of ryegrass seedlings by 9.6% and 6.6%.

[0144] (2) The effects of daidzein and tryptamine on ryegrass seedling growth showed that the inhibitory effect increased with increasing concentration.

[0145] ① Daidzein had a significant inhibitory effect on the seedling length of ryegrass at a low concentration (25μg / ml) (P<0.05), and the higher the concentration of the substance, the stronger the inhibitory effect. Under the treatments of 25, 50, 100, 200, and 400μg / ml concentrations, the length of ryegrass seedlings was shortened by 8.2%, 5%, 8.2%, 10.5%, and 12.4%, respectively.

[0146] ②When the concentration of tryptamine was 50μg / ml, it had a significant inhibitory effect on the seedling length of ryegrass (P<0.05). Under the treatments of 50, 100, 200 and 400μg / ml concentrations, the seedling length of ryegrass was shortened by 8.6%, 18.5%, 24.8% and 33.3%, respectively.

[0147] From the above, we can see that genistein has no effect on the germination rate and fresh weight of ryegrass seedlings, but only inhibits the root growth of ryegrass in the range of 25μg / ml~400μg / ml.

[0148] 2.4.4 Effects of four substances on fresh weight of alfalfa and ryegrass seedlings

[0149] (1) From Figure 7 A showed that high concentrations of daidzein, genistein and tryptamine had a significant inhibitory effect on the fresh weight of alfalfa (P<0.05).

[0150] ① When the concentration of daidzein reached 400μg / ml, it had a significant inhibitory effect on the fresh weight of alfalfa (P<0.05). The fresh weight of alfalfa treated with this concentration decreased by 20.2%.

[0151] ② When the concentration of genistein reached 400μg / ml, it had a significant inhibitory effect on the fresh weight of alfalfa (P<0.05). After treatment with this concentration, the fresh weight of alfalfa decreased by 14.6%.

[0152] ③200 and 400 μg / ml tryptamine reduced the fresh weight of alfalfa by 20.6% and 17.8% respectively (P<0.05).

[0153] ④ Palmitic acid had no significant effect on the fresh weight of alfalfa in this experiment (P>0.05).

[0154] (2) From Figure 7 B shows that daidzein and palmitic acid significantly increased the fresh weight of ryegrass under the concentration treatment set in this experiment ( P <0.05).

[0155] ① The concentrations of 200 and 400 μg / ml of daidzein increased the fresh weight of ryegrass by 16.7% and 26.5%, respectively.

[0156] ② Palmitic acid at concentrations of 100, 200, and 400 μg / ml significantly increased the fresh weight of ryegrass ( P <0.05), increased by 20.5%, 17.2%, and 16.7%, respectively.

[0157] In addition, genistein and tryptamine had no significant effect on the fresh weight of ryegrass at the concentrations set in this experiment.

[0158] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. The application of genistein in promoting the growth of forage grass, characterized in that: Genistein is used as an active ingredient of a forage promoter, the forage is alfalfa or ryegrass, and the specific application is: genistein in the concentration range of 50-100 μg / ml can promote the elongation of the radicle and hypocotyl of alfalfa; genistein in the concentration range of 25-50 μg / ml can significantly promote the elongation of the seedling length of ryegrass.

2. The use according to claim 1, characterized in that: The method for using the genistein is as follows: soaking the forage grass seeds with the genistein solution or spraying the forage grass seedlings.

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