CN107114386A - The application of Sitosterolum and preparation - Google Patents

Abstract

The invention relates to the technical field of agricultural production, in particular to the application of β-sitosterol and its preparation, especially to the application of β-sitosterol in improving the drought resistance of plants. Experiments have shown that β‑sitosterol can effectively delay the wilting of leaves of white clover under drought stress, alleviate the growth inhibition caused by drought, reduce the degradation of chlorophyll and peroxidation damage, etc., and significantly improve the drought resistance of white clover. Moreover, the application effect is not limited by the application time, and has a good drought-resistant effect on both drought-sensitive white clover varieties and drought-tolerant white clover varieties. In addition, compared with other exogenous additives such as ABA, SA, and brassinosteroids, β-sitosterol is cheap and low in production cost, and is suitable for large-scale promotion and application.

Description

Application and preparation of β-sitosterol

technical field

The invention relates to the technical field of agricultural production, in particular to the application of β-sitosterol and its preparation.

Background technique

With the frequent occurrence of extreme climates in the world, the uneven distribution of water resources and the reduction of available irrigation water resources, the impact of drought on crop yields around the world continues to expand, becoming one of the most important factors limiting plant growth and development. Since the middle of last century, the area affected by crop drought in my country has increased year by year.

White clover (Trifolium repens L.) is a perennial high-quality forage grass belonging to the leguminosae genus Trifolium. One of the main leguminous pastures used is widely used in the establishment of artificial mixed-seeding grasslands and improving natural grasslands. At the same time, due to the characteristics of strong creeping growth characteristics, fast regeneration speed and beautiful leaf shape, white clover is also used in ornamental lawns, playing a huge role in urban greening, soil and water conservation and ecological construction. In recent years, the cultivation area of white clover has been continuously expanding in various places. However, white clover likes cold and humid climate, belongs to cold-season shallow-rooted grass species, has poor drought and heat tolerance, and needs a lot of water for plant growth, and its anti-transpiration ability is weak. In subtropical and temperate regions, drought has become a The main adversity stress factors that limit its production and utilization. Therefore, in the cultivation of white clover, it is urgent to explore cultivation measures and methods to improve the drought resistance of white clover.

In agricultural production, the application of exogenous plant growth regulators to improve crop stress resistance has the characteristics of simple operation, quick results and high practical value. It has been practically applied to crops such as vegetables, rice and forage. There are few reports on the improvement of the drought resistance of white clover. Research reports have shown that exogenous application of plant growth regulator substances abscisic acid (ABA), salicylic acid (SA), brassinolide (BR), trinexapac-ethyl (TE), paclobutrazol (PP33 3), chlormequat (CCC ) can improve plant drought resistance. However, the price of these hormones or growth regulators is relatively high, and the use effect varies with different plant species. If they are used in a large area in production, they will face problems such as high production costs. Therefore, it is of great practical value to find a new, efficient and low-cost exogenous additive.

β-sitosterol (β-sitosterol) is one of the components of phytosterols, which belongs to tetracyclic and tricyclic compounds, and is widely found in various vegetable oils, nuts, seeds, etc. in nature. At present, β-sitosterol is widely used in the pharmaceutical industry due to its unique biological characteristics and physical and chemical properties. Studies have shown that the intake of β-sitosterol is related to the incidence of human chronic diseases. Studies have shown that it can significantly reduce serum The effect of cholesterol can replace cholesterol as liposome membrane material. However, there is no research on the role of β-sitosterol in plant drought resistance.

Contents of the invention

The invention provides the application of β-sitosterol in improving the drought resistance of plants.

Drought stress, also known as water deficit stress or water stress, usually refers to the disturbance of normal physiological functions of plants caused by drought and water shortage. Studies have shown that drought stress can inhibit plant growth and cause plant leaves to wilt, accompanied by a series of physiological and biochemical changes in plants, such as electrolyte permeability, malondialdehyde content, SOD activity, POD activity, CAT activity and APX activity. Reduce leaf green content. The present invention finds that root application or foliage spraying of β-sitosterol can effectively delay the wilting of leaves of white clover under drought stress, alleviate the growth inhibition and degradation of chlorophyll caused by drought; and significantly reduce the electrolyte permeability and the accumulation of malondialdehyde, increase the activity of enzymes such as SOD, POD, CAT and APX, thereby reducing peroxidative damage, and finally significantly improving the drought resistance of white clover. Therefore, the present invention provides the application of β-sitosterol in plant drought resistance.

In some specific embodiments provided by the present invention, the plant is trifolium trifoliate.

As a preference, the method of applying the β-sitosterol is foliar spray or root application; preferably, the root application concentration of β-sitosterol is 25-100 mg/L; preferably, the foliar spray of β-sitosterol The concentration of application is 25 ~ 50mg/L.

In some embodiments provided by the present invention, when foliar spraying or root spraying is performed, it is preferred to dissolve β-sitosterol in 100% hot ethanol to prepare a mother liquor, and then mix it with a solvent before applying. In a specific embodiment provided by the present invention, β-sitosterol is dissolved in 100% hot ethanol and dissolved to make a mother liquor of 0.2g/mL, and then the mother liquor is mixed with Hoagland nutrient solution to prepare a treatment solution for β-sitosterol. Leaves were treated with root application. In another specific embodiment provided by the present invention, β-sitosterol is dissolved in 100% hot ethanol to make a 0.2 g/mL mother liquor, and 1% Tween-80 is used as a solvent to prepare a β-sitosterol treatment solution Apply foliar sprays to white clover.

The invention also provides a drought-resistant drug preparation, which includes β-sitosterol and pesticide acceptable auxiliary materials. Preferably, the weight ratio of the β-sitosterol to the pesticide acceptable excipient is 0.01-0.1: 1-20.

As a preference, the pesticide-acceptable auxiliary material is selected from Tween-80, Tween-20, sodium dodecylbenzenesulfonate, sodium octadecylsulfosuccinate, polyoxyethylene alkylphenol and polyoxyethylene One or a mixture of two or more of oxyethylene fatty alcohol, phenethylphenol polyoxyethylene ether, alkylsulfonate, and alkylnaphthalenesulfonate. However, the pesticide acceptable excipients recognized by those skilled in the art are all within the protection scope of the present invention, and the present invention is not limited here.

In some embodiments provided by the present invention, the agrochemically acceptable excipient is Tween-80.

The present invention is aimed at exogenous application of abscisic acid (ABA), salicylic acid (SA), brassinolide (BR), trinexapac-ethyl (TE), paclobutrazol (PP333), chlormequat (CCC) in large areas in the prior art. ) and other exogenous additives are relatively expensive, and the effect varies with different plant species. A new type of exogenous additive, β-sitosterol, has been developed to improve plant drought resistance. The present invention takes white clover as the research object, and finds that exogenous application of β-sitosterol can effectively delay the wilting of white clover leaves under drought stress, relieve growth inhibition caused by drought, chlorophyll degradation and peroxidation damage, etc., and significantly improve Drought resistance of white clover. In addition, compared with other exogenous additives such as ABA, SA, brassinosteroids, etc., the price is cheap, the production cost is low, and it is suitable for large-scale popularization and application.

detailed description

The invention discloses the application of β-sitosterol and its preparation, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

Below in conjunction with embodiment, further set forth the present invention:

The screening of embodiment 1β-sitosterol root application concentration

Test material: widely used 'Latinuo' white clover as the test material

Material cultivation: first soak the seeds of white clover with 0.1% potassium permanganate solution for 10 minutes to sterilize, wash them with deionized water for ³ times, and sow the seeds at a rate of 1g/m2 at a length of 20cm, a width of 15cm, and a height of 5cm. Germinate in plastic pots filled with quartz sand in a light incubator (temperature day/night: 23/19°C; duration: 12 hours; relative humidity: 70%; light intensity: 700μmol m ^-2 s ^-1 ) . After the seeds germinated for 7 days, the seedlings were continued to be cultivated with Hoagland nutrient solution until 30 days, and the materials with consistent growth were selected for subsequent experiments.

Experimental design: A total of 7 β-sitosterol concentration gradients were set up, which were: 0mg/L (control, direct drought stress), 5mg/L, 25mg/L, 50mg/L, 100mg/L, 250mg/L, 500mg/L L, a total of 7 treatments, with 4 independent repetitions for each treatment. Drought stress treatment was performed with 18% polyethylene glycol 6000 (PEG-6000).

Exogenous addition and drought stress treatment: β-sitosterol was dissolved in 100% hot ethanol (0.2 g/mL), cooled naturally to room temperature and used as mother liquor for later use. The nutrient solution was used as a solvent, and β-sitosterol treatment solutions of different concentrations were prepared with β-sitosterol mother liquor, and the material was pretreated with β-sitosterol treatment solution for 6 days, and then replaced with 18% (W/V) PEG-6000 The Hoagland complete nutrient solution was used for drought stress treatment. During the treatment, the treatment solution was replaced every 3 days, 300 mL each time. All materials are placed in an intelligent light incubator, and the conditions are the same as that of the materials. Start timing when changing to the treatment solution containing 18% (W/V) PEG-6000, investigate the time when the wilting symptoms and severe wilting of the white clover leaves appear, and the results are shown in the table.

Table 1 Effects of root application of different concentrations of β-sitosterol on improving drought resistance of white clover

It can be seen from Table 1 that when 25-100 mg/L of β-sitosterol is applied to the root, it can significantly improve the drought resistance of white clover, and the effect of improving the cold resistance of white clover is better. Under the intensity of drought stress, the leaf wilting of white clover can be delayed for 3-5 days.

Example 2 The screening of foliar spraying β-sitosterol concentration

Test material: Drought-sensitive 'Latinuo' white clover was used as the test material.

Material cultivation: Mix garden soil and nutrient soil at a ratio of 1:1, add insecticides and fungicides and mix evenly, and store it outdoors for 3 days before use. Adopting a pot with a diameter of 20 cm and a depth of 25 cm to plant white clover, fill the pot with an equal amount of mixed soil, scatter the white clover seeds at a seeding rate of 1 g/m ² , cover with shallow soil and then water. The materials were placed in the greenhouse and cultivated for 3 months, and 250mL Hoagland full nutrient solution was poured into each pot every week, and the routine management of fertilization, weeding, watering and pests and diseases was uniformly carried out. The average temperature of the greenhouse is 23°C, the relative humidity is 65%, the light time is 12 hours, and the light intensity is 500 μmol m ^-2 s ^-1 . After the seeds germinated for 7 days, the seedlings were continued to be cultivated with Hoagland nutrient solution until 30 days, and the materials with consistent growth were selected for subsequent experiments.

Experimental design: A total of 7 β-sitosterol concentration gradients were set up, which were: 0mg/L (control, direct drought stress), 5mg/L, 10mg/L, 15mg/L, 25mg/L, 50mg/L, 100mg/L L, 250mg/L, a total of 8 treatments, each set 4 independent repetitions.

Exogenous spraying and drought stress treatment: β-sitosterol was fully dissolved in 100% hot ethanol (0.2g/mL) as a mother liquor for later use, and 1% Tween-80 was used as a solvent to dilute the mother liquor to prepare different concentrations of β-sitosterol solutions , the control sprayed the corresponding solvent without β-sitosterol. Carry out foliar spraying at 9:00 in the morning each time, the spraying amount is 150mL, spray 3 times continuously, each interval is 1 day. During the spraying of exogenous additives, normal watering was performed. After the treatment, all materials were stopped from watering and placed in the greenhouse for natural drought stress. Start timing from the natural drought stress (that is, stop watering), and investigate the time when the wilting symptoms and severe wilting of the white clover leaves appear. The results are shown in Table 2.

Table 2 Effect of foliar spraying different concentrations of β-sitosterol on improving drought resistance of white clover

As can be seen from Table 2, when the β-sitosterol of 10-25mg/L is sprayed on the leaves, the drought resistance of the white clover can be significantly improved. Compared with the control group, the severe wilting of the white clover under natural drought stress can Delayed by 5-7 days.

Example 3 Effect of foliar spraying of β-sitosterol on physiological indexes of drought resistance of white clover

Test material: Drought-sensitive 'Latinuo' white clover was used as the test material

Material cultivation: with embodiment 2

Design of the test: The optimal leaf spraying concentration 15mg/L screened out according to Experimental Example 2 is the spraying concentration of this experiment. There are 4 treatments in the test, and 4 independent repetitions are set for each treatment. They are respectively: 1. normal condition control (CK ): 18 days of normal culture in the greenhouse after spraying foliage without β-sitosterol as a control; ② CK+β-sitosterol: normal culture in the greenhouse after foliar spraying of 15 mg/L β-sitosterol 18 days; ③Direct drought stress: 18 days of natural drought in the greenhouse after spraying a solvent without β-sitosterol on the leaves; ④Drought + β-sitosterol: After spraying 15mg/L β-sitosterol on the leaves Naturally dry in the greenhouse for 18 days.

Drought stress treatment: Spray foliage at 9:00 a.m. for each spraying, spray 100mL equivalent liquid evenly on the leaves of each treatment until all leaves are covered with water droplets, spray three times in a row, each time 1 day interval. After the treatment, the control group (CK and CK+β-sitosterol) were watered normally, and the drought stress group (drought and drought+β-sitosterol) stopped watering and placed in the greenhouse for natural drought stress. Greenhouse condition is with embodiment 2.

Observation indicators: After 18 days of drought stress, measure the dry weight and relative growth of a single plant reflecting the growth status of white clover; the relative water content reflecting the water status of leaves; and the chlorophyll content, electrolyte permeability, malondialdehyde content, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) activities. The results are shown in Table 3.

Table 3 Changes of Physiological and Biochemical Indexes Related to Drought Resistance of White Clover by Foliar Spraying β-sitosterol (15mg/L)

It can be seen from Table 3 that under normal conditions, spraying exogenous β-sitosterol pretreatment can promote the growth and chlorophyll synthesis of white clover plants; under drought stress, spraying exogenous β-sitosterol pretreatment can significantly Relieve the growth inhibition caused by drought, reduce the degradation of chlorophyll, and slow down the peroxidative damage caused by drought by increasing the activity of various antioxidant enzymes, and significantly reduce the electrolyte permeability and the accumulation of membrane lipid peroxidation product malondialdehyde. These growth and physiological and biochemical indicators further proved that foliar spraying of β-sitosterol pretreatment can effectively improve the drought resistance of white clover.

Embodiment 4: The impact of foliar spraying β-sitosterol application time on application effect

(1) Test material: the drought-sensitive 'Latinuo' white clover was used as the test material.

(2) material cultivation: with embodiment 2

(3) Experimental design and treatment: The optimal leaf spraying concentration of 15 mg/L screened out according to Experimental Example 2 is the spraying concentration of this experiment. Foliar spraying was carried out at 9:00 in the morning, 11:00 in the morning, 14:00 in the afternoon, 17:00 in the evening and 20:00 in the evening, respectively, and each treatment was repeated 4 times independently. Material drought treatment and greenhouse conditions are the same as in Example 2. Start timing from natural drought stress (that is, stop watering), and investigate the time when the leaf wilting symptoms and severe wilting of the white clover appear. The results are shown in Table 4.

Table 4 Effect of foliar spraying β-sitosterol (15mg/L) on improving drought resistance of white clover at different application times

It can be seen from Table 4 that different foliar spraying times had no significant effect on the effect of β-sitosterol on improving the drought resistance of white clover.

Embodiment 5: The application effect of foliar spraying β-sitosterol on different white clover varieties

(1) Test materials: two white clover varieties 'Latinuo' (drought sensitive type) and 'Haifa' (drought tolerant type).

(2) material cultivation: with embodiment 2.

(3) Experimental design and treatment: The optimal leaf spraying concentration of 15 mg/L screened out according to Experimental Example 2 was the spraying concentration of this experiment. Foliar spray treatments were performed at 9:00 AM, and each treatment was set up with 4 independent replicates. Material drought treatment and greenhouse conditions are the same as in Example 2. Start timing from natural drought stress (that is, stop watering), and investigate the time when the leaf wilting symptoms and severe wilting of the white clover appear.

Table 5 Effect of foliar spraying β-sitosterol (15mg/L) on improving drought resistance of different white clover varieties

It can be seen from Table 5 that under the same cultivation and drought stress conditions, without pretreatment, the drought resistance of 'Haifa' was stronger than that of 'Latinuo'; The drought resistance of all varieties was significantly improved compared with the control treatment. Comparing the two varieties, the effect of exogenous spraying of β-sitosterol on drought-sensitive 'Latinuo' was more significant than that of 'Haifa'.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (9)

1. application of the cupreol in plant drought resistance is improved.

2. application according to claim 1, it is characterised in that the plant is Trifolium repense.

3. application according to claim 1, it is characterised in that the method for application of the cupreol is foliage-spray or root Apply.

4. application according to claim 3, it is characterised in that the root of the cupreol applies concentration for 25~100mg/L.

5. application according to claim 3, it is characterised in that the concentration of the foliage-spray of the cupreol is 10~ 25mg/L。

6. a kind of drought resisting pharmaceutical preparation, it is characterised in that including acceptable auxiliary material on cupreol and Pesticide Science.

7. drought resisting pharmaceutical preparation according to claim 6, it is characterised in that can in the cupreol and the Pesticide Science The weight ratio of the auxiliary material of receiving is 0.01~0.1：1~20.

8. drought resisting pharmaceutical preparation according to claim 6, it is characterised in that acceptable auxiliary material is selected from the Pesticide Science Tween-20, Tween-80, neopelex, octadecyl sulphur sodium succinate, polyoxyethylene alkylphenol and polyoxyethylene Mixture more than one or both of fatty alcohol, phenethyl phenol polyethenoxy ether, alkylsulfonate, alkylnaphthalene sulfonate.

9. drought resisting pharmaceutical preparation according to claim 8, it is characterised in that acceptable auxiliary material is to tell in the Pesticide Science Temperature -80.