Compound seed soaking agent and application thereof in promoting lettuce seed germination and seedling growth under lead stress

Abstract

The invention discloses a compound seed soaking agent and application thereof in promoting lettuce seed germination and seedling growth under lead stress, the compound seed soaking agent is composed of a component I and a component II in a volume ratio of 1: 1-100, the component I takes water as a solvent and comprises 1-200mM of spermine and 0-150mM of gibberellin; and the component II is a festuca arundinacea extracting solution. According to the invention, spermine and gibberellin are taken as effective components of the seed soaking agent, and are combined with extract components of lead ion enrichment plants, so that a remarkable synergistic effect is generated, accumulation of lead content in lettuce seedlings can be effectively reduced through a seed soaking method, and the germination rate of lettuce seeds and the dry weight of the seedlings under lead stress are improved. The compound seed soaking agent takes water as a substrate, is simple in preparation process and convenient to use, does not produce product and environmental pollution, is not easy to produce phytotoxicity, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of agrochemistry, and particularly to a composite seed soaking agent and its application in promoting the germination of lettuce seeds and the growth of seedlings under lead stress. Background Art

[0002] In recent years, worldwide, with the advancement of urbanization and industrialization, the large-scale discharge of industrial waste water, waste gas and waste residue, the pollution of water sources for farmland irrigation by toxic and harmful components, the extensive use of highly toxic pesticides, harmful herbicides and traditional chemical fertilizers, and the continuous emergence of heavy metal pollution, environmental problems have become the main factors restricting crop production and quality improvement.

[0003] Lead (Pb) is a non-essential element for plants and has strong toxicity. It cannot be degraded by microorganisms in the soil and can only transform between different occurrence forms due to different conditions. The specific manifestations of the physiological toxicity of plants caused by Pb stress include reduced photosynthetic rate, impaired respiration, reduced protein content, generation of oxidative stress, and nutrient and hormone imbalance.

[0004] Lettuce (Lactuca sativa var. ramosa Hort.) is an important vegetable crop. According to the statistical data of the Food and Agriculture Organization of the United Nations (FAO), the sown area of lettuce and chicory vegetables in China reached 606,430 hectares in 2020, with a yield of approximately 14.3231 million tons, and it has become one of the most important vegetable varieties in the Chinese consumer market. However, heavy metal stress will significantly inhibit the germination of lettuce seeds and the growth of seedlings, and at the same time reduce the yield and quality of lettuce.

[0005] Spermine (Spm), with the molecular formula C 10 H 26 N4, is a common polyamine that participates in various plant growth and development processes and can improve the resistance of plants to various stresses such as high salt, low temperature, ozone and potassium deficiency. Spermine is an endogenous metabolite that can be biodegradable, non-toxic to humans, safe and residue-free, and is very suitable for the production of field and protected agriculture. Gibberellin is an important plant hormone that regulates plant seed dormancy and germination. It can induce the decomposition of storage substances, activate the key pathways of seed germination metabolism, break the dormancy of plant seeds and stimulate seed germination and seedling establishment.

[0006] Phytoremediation technology is an environmental purification technology developed in the 1990s to remove harmful metals from soil or water bodies through metal-accumulating plants. At present, many lead ion-accumulating plants have been discovered, such as alfalfa, Armeria maritima, tall fescue, etc., which have strong tolerance to lead stress. Summary of the Invention

[0007] The present invention provides a composite seed soaking agent, which can not only effectively promote the germination of lettuce seeds and the growth of seedlings under lead stress, but also has a simple preparation process and is safe and pollution-free.

[0008] The present invention also provides an application of the composite seed soaking agent in promoting the germination of lettuce seeds and the growth of seedlings under lead stress.

[0009] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0010] A composite seed soaking agent is composed of component I and component II with a volume ratio of 1:1 to 100; component I uses water as a solvent and contains: spermine 1 to 200 mM, gibberellin 0 to 150 mM; component II is a tall fescue extract. In the above composite seed soaking agent, gibberellin is a plant endogenous hormone and has the ability to break the dormancy of plant seeds and induce seed germination. Tall fescue (Festuca elata Keng ex E.B.Alexeev) is a perennial tufted herb of the genus Festuca in the family Poaceae. Tall fescue is a kind of plant that can enrich lead ions. In theory, the extracts of plants that can enrich lead ions can all be considered as components of the composite seed soaking agent of the present invention. However, experiments have found that the extracts of many lead ion-enriching plants do not achieve the expected effects. For example, pokeweed, alfalfa, etc. Only tall fescue has the effect of promoting the germination of lettuce seeds and the growth of seedlings under lead stress.

[0011] Preferably, the volume ratio of component I to component II is 1:1 to 60. More preferably, the volume ratio of component I to component II is 1:60. This optimal ratio can make the composite seed soaking agent achieve the best effect in promoting the germination rate of lettuce seeds and the growth of seedlings under lead stress.

[0012] Preferably, using water as a solvent, the composition of component I is: spermine 100 mM, gibberellin 120 mM. This concentration combination shows good balance in promoting seed germination and inhibiting lead ion absorption.

[0013] Preferably, the preparation method of the tall fescue extract includes: placing the powder obtained by air-drying and pulverizing tall fescue plants in water for extraction, removing insoluble substances, and obtaining the tall fescue extract. This preparation method can effectively extract the active ingredients in tall fescue that can relieve lead stress.

[0014] Preferably, the ratio of the powder to water is 1 g to 10:150 mL; the extraction temperature is 20 to 40 °C, and the time is 2 to 5 days. This parameter combination can ensure the maximum extraction rate of the active ingredients in the tall fescue extract.

[0015] The present invention also provides an application of the composite seed soaking agent in promoting the germination of lettuce seeds and the growth of seedlings under lead stress.

[0016] Preferably, the application is as follows: Lettuce seeds are placed on germination paper that has been moistened with the aforementioned compound soaking agent for slow water absorption treatment. The moistening treatment means that the germination paper has absorbed the compound soaking agent to keep it in a moist state. This treatment method can enable the seeds to uniformly absorb the active ingredients in the soaking agent and improve the lead stress resistance.

[0017] Preferably, for the moistening treatment, double-layer filter paper is laid in a germination box, and then the compound soaking agent is poured onto the double-layer filter paper. The dosage of the soaking agent is 5 - 10 mL, preferably 10 mL. This dosage ensures that the germination paper maintains an appropriate humidity and provides an ideal initial germination environment for the seeds.

[0018] Preferably, the temperature of the water absorption treatment is 5°C - 15°C, the time is 1 - 5 days, and it is carried out under dark conditions. The temperature of the water absorption treatment is preferably 10°C; the time of the water absorption treatment is preferably 2 days. This combination of conditions can promote the seeds to slowly absorb water, fully absorb the active ingredients of the soaking agent, and at the same time inhibit the absorption and accumulation of lead ions.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention uses spermine and gibberellin as the active ingredients of the soaking agent, combines with the extract components of lead ion-enriched plants, and produces a significant synergistic effect. Through soaking with an appropriate concentration, it effectively promotes the germination and seedling growth of lettuce seeds under lead stress.

[0021] (2) The compound soaking agent of the present invention uses water as the matrix, has a simple preparation process, is easy to use, does not cause product and environmental pollution, is not prone to phytotoxicity, and has broad application prospects. Description of the Drawings

[0022] Figure 1 It shows the effects of different treatments on the chlorophyll content of lettuce seedlings under lead stress. Detailed Embodiments

[0023] The following are specific examples to further illustrate the technical solutions of the present invention. It should be understood that the implementation of the present invention is not limited to the following examples, and any formal modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0024] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

[0025] The reagents used in the following examples can be obtained from conventional biochemical reagent stores unless otherwise specified.

[0026] In the following examples, spermine and gibberellin were purchased from Aladdin Reagent Co., Ltd.; the magnetic stirrer model was ZNCL-BS and was purchased from Shanghai Yuezhong Instrument and Equipment Co., Ltd.

[0027] Example 1

[0028] A composite seed soaking agent and its application in promoting the germination of lettuce seeds and the growth of seedlings under lead stress. The following is the specific experimental and verification process.

[0029] (1) Preparation of the composite seed soaking agent

[0030] Preparation of Component I: 2.022 g of spermine and 4.156 g of gibberellin were added to 100 ml of water and stirred well until dissolved to obtain Component I.

[0031] Preparation of Component II: The branches and stems of tall fescue were air-dried and ground into powder with a particle size of 0.10 - 0.15 mm. 100 g of the powder was immersed in 4 L of water at room temperature for 3 days, and then the insoluble substances were filtered off to obtain Component II.

[0032] Preparation of Component III: The branches and stems of Lushang were air-dried and ground into powder with a particle size of 0.10 - 0.15 mm. 100 g of the powder was immersed in 4 L of water at room temperature for 3 days, and then the insoluble substances were filtered off to obtain Component III.

[0033] Preparation of Component IV: The branches and stems of alfalfa were air-dried and ground into powder with a particle size of 0.10 - 0.15 mm. 100 g of the powder was immersed in 4 L of water at room temperature for 3 days, and then the insoluble substances were filtered off to obtain Component IV.

[0034] Composite seed soaking agent: Component I and Component II were mixed evenly at a ratio of 1:60 to obtain the composite seed soaking agent.

[0035] 2. Seed treatment

[0036] Nine seed soaking and germination treatments were set up, namely control CK (0 mg / L Pb), Pb (100 mg / L), Pb (100 mg / L) / (I + II), Pb (100 mg / L) / (I + III), Pb (100 mg / L) / (I + IV), Pb (100 mg / L) / I (diluted 60 times with water), Pb (100 mg / L) / II, Pb (100 mg / L) / III, Pb (100 mg / L) / IV.

[0037] Lay double-layer filter paper in a germination box (12 cm in length * 12 cm in width), pour 10 ml of seed soaking agent onto the double-layer filter paper, evenly spread lettuce seeds on the filter paper of each germination box at a rate of 0.5 g / germination box, soak them in the dark at 10 °C for 2 days, then take out the seeds, rinse them with deionized water for 1 minute and set aside.

[0038] 3. Seed germination test

[0039] After the lettuce seeds are soaked, germination is carried out on paper. Put 100 lettuce seeds in each germination box, with 4 replicates. Add pure water and 100 mg / L Pb(NO3)2 solution to the germination boxes with different treatments respectively. Place the germination boxes in a constant temperature incubator at 20 °C for germination, and set the light to 12 hours of light / 12 hours of darkness. During the test, change the treatment solution every 48 hours to ensure the consistency of Pb 2+ concentration among treatments.

[0040] Taking the radicle breaking through the seed coat as the germination standard, record the number of germinated seeds every day, and calculate the germination energy (GE) and germination percentage (GP) on the 4th and 7th days of germination respectively. After 7 days of germination, randomly select 10 representative seedlings and measure their dry weights of the seedlings.

[0041] 4. Determination of lead content

[0042] When sampling, first immerse the lettuce seedling samples in 20 mmol·L -1 Na2EDTA solution for 15 - 20 minutes, then rinse them with distilled water 3 times, and finally rinse them with deionized water once to remove the Pb 2+ attached to the surface of the samples. Dry the samples at 105 °C for 30 minutes, and then dry them at 70 °C for 72 hours. Grind the dried tissue and pass it through a 0.1 mm nylon sieve, take 0.5 g of dry sample for digestion with concentrated HNO3 - HClO3, and determine the Pb 2+ content using an atomic spectrophotometer (ASE, model SP9 - 400).

[0043] 5. Determination of chlorophyll content

[0044] Weigh about 0.1 g of fresh lettuce seedlings (germinated for 14 days) and put them into a 2 mL centrifuge tube, add 1.5 mL of a mixed solution of acetone, ethanol and water (v:v:v = 4.5:4.5:1). After mixing the extract, pipette 200 μL of each sample into a 96-well plate, and measure the absorbance at 645 nm and 663 nm using a microplate reader (TECAN / SPARK, TECAN Company, Austria). Obtain the chlorophyll content (mg / g) of the lettuce seedlings according to the standard curve.

[0045] 6. Effects of Different Treatments on Lead Content Accumulation in Lettuce Seedlings

[0046] As shown in Table 1, under Pb stress treatment, the Pb contents in lettuce seeds on the 4th and 7th days of germination were 6.33 μg / g and 7.14 μg / g respectively, which were 2.52 times and 2.21 times that of the control. Compared with the single Pb treatment, both Treatment I and Treatment II significantly reduced the lead content in seedlings on the 4th and 7th days of germination, and the lead content in seedlings after Treatment I+II was significantly lower than that of Treatment I and Treatment II alone, indicating that Component I and Component II can synergistically alleviate the accumulation of lead ions in lettuce seedlings during germination. The plant extract components III and IV did not achieve the corresponding effects.

[0047] Table 1 Effects of Compound Seed Soaking Agents on Lead Content Accumulation during Lettuce Seed Germination under Lead Stress

[0048]

[0049] *Lowercase letters in the same column indicate significant differences between treatments at the α = 0.05 (LSD test) level.

[0050] 7. Effects of Different Treatments on Lettuce Seed Germination under Lead Stress

[0051] As shown in Table 2, under Pb stress treatment, the germination potential, germination rate and dry weight of lettuce seeds were significantly reduced. Compared with the Pb treatment, both Treatment I and Treatment II alone significantly increased the germination potential, germination rate and dry weight of lettuce seeds under Pb stress.

[0052] After Treatment I+II, the germination potential, germination rate and dry weight of lettuce seeds were further higher than those of Treatment I and Treatment II alone, indicating that both Component I and Component II can promote the germination of lettuce seeds and the growth of seedlings under lead stress, and there is a mutually promoting effect between Component I and Component II. The plant extract components III and IV did not achieve the corresponding effects. Thus, the compound seed soaking agent (I+II) has the effect of improving the resistance of lettuce seeds to lead stress during germination.

[0053] Table 2 Effects of Different Treatments on Lettuce Seed Germination and Seedling Dry Weight under Lead Stress

[0054]

[0055] *Lowercase letters in the same column indicate significant differences between treatments at the α = 0.05 (LSD test) level.

[0056] 8. Effects of Different Treatments on Chlorophyll Content in Lettuce Seedlings under Lead Stress

[0057] The results are as Figure 1As shown, under Pb stress treatment, the chlorophyll content of lettuce seedlings germinated for 14 days decreased significantly. Compared with the Pb treatment, the I alone treatment significantly increased the chlorophyll content of lettuce seedlings under Pb stress. However, the II alone treatment had no significant effect on the chlorophyll content. However, the chlorophyll content of lettuce seedlings after the I+II treatment was further significantly higher than that of the I alone treatment, indicating that the I component can promote the photomorphogenesis of lettuce seedlings under lead stress. Although the II component had no significant effect on the chlorophyll content when acting alone, it can cooperate with the I component to further improve the lead stress resistance of lettuce seedlings.

[0058] Therefore, the composite soaking agent (I+II) has the effect of improving the establishment of lettuce seedlings after germination under lead stress.

[0059] The present invention uses spermine and gibberellin as the active ingredients of the soaking agent, combined with the extract components of lead ion-enriched plants, resulting in a significant synergistic effect. By the method of soaking seeds, it can effectively reduce the accumulation of lead content in lettuce seedlings and improve the germination rate and seedling dry weight of lettuce seeds under lead stress.

Claims

1. A composite seed soaking agent, characterized in that, It is composed of component I and component II with a volume ratio of 1:1 to 100; Component I uses water as a solvent and contains: 1 to 200 mM of spermine, and 0 to 150 mM of gibberellin; Component II is an extract of tall fescue.

2. The composite seed soaking agent according to claim 1, characterized in that, The volume ratio of component I to component II is 1:1 to 60.

3. The composite seed soaking agent according to claim 1, characterized in that The volume ratio of component I to component II is 1:

60.

4. The composite seed soaking agent according to claim 1, characterized in that, Using water as a solvent, the composition of component I is: 100 mM of spermine and 120 mM of gibberellin.

5. The composite seed soaking agent according to claim 1, wherein, The preparation method of the tall fescue extract includes: placing the powder obtained by air-drying and crushing tall fescue plants in water for leaching, removing insoluble substances, and obtaining the tall fescue extract.

6. The composite seed soaking agent according to claim 5, wherein, The ratio of the powder to water is 1 g to 10:150 mL; the temperature of the leaching is 20 to 40 °C, and the time is 2 to 5 days.

7. Use of the compound seed soaking agent according to any one of claims 1 to 6 in promoting the germination of lettuce seeds and the growth of seedlings under lead stress.

8. The application according to claim 7, wherein Place lettuce seeds on germination paper moistened with the described compound seed soaking agent for slow water absorption treatment. The moistening treatment means that the germination paper absorbs the compound seed soaking agent to keep it in a moist state.

9. The application according to claim 8, wherein This use is: in the germination box, lay double-layer filter paper, then pour the compound seed soaking agent on the double-layer filter paper, and the dosage of the seed soaking agent is 5 to 10 mL.

10. The application according to claim 9, characterized in that, The temperature of the water absorption treatment is 5 °C to 15 °C, the time is 1 to 5 days, and it is carried out under dark conditions.

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