Method for testing influence of exogenous putrescine on physiological characteristics of alfalfa seeds and seedlings

Abstract

The invention discloses a method for testing the influence of exogenous putrescine on physiological characteristics of alfalfa seeds and seedlings, relates to the technical field of testing of physiological characteristics of alfalfa seeds, and solves the problems that the exogenous putrescine can improve the drought tolerance of plants and can be used as a ROS source and a potential ROS scavenger, and the influence of the exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings is poor. The invention aims to solve the problems that in the prior art, polyamines with different concentrations are used for relieving the germination concentration of alfalfa seeds in the germination period and play a role in redox steady-state regulation in plants, but at present, external putrescine does not have related research on the drought restoration capability of different drought-tolerant alfalfa in the germination period and the seedling period. The invention screens the concentrations of different concentrations of polyamines for relieving the germination of alfalfa seeds in the germination period; the selected putrescine is used for carrying out drought stress treatment on two stages of seed germination and seedling growth of the medicago sativa with different drought tolerance, so that the influence of the exogenous putrescine on the physiological characteristics of the seed germination and the seedling of the medicago sativa under the drought stress is explored, and the physiological adaptation mechanism of the exogenous putrescine for relieving the drought stress is analyzed; therefore, theoretical support is provided for drought-tolerant breeding of alfalfa.

Description

Technical Field

[0001] The invention relates to the technical field of testing the physiological characteristics of alfalfa seeds, in particular to a testing method for the influence of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings. Background Art

[0002] Drought is a worldwide problem that restricts agricultural production, ecological environment construction and plant distribution. Global climate change has increased the frequency, intensity and duration of droughts. Drought is one of the main factors affecting plant growth and yield. Studying the physiological mechanism of plant response to drought stress, discovering key metabolites involved in plant drought stress response and conducting functional studies on them are of great theoretical significance for improving plant drought resistance, breeding water-saving and drought-resistant varieties, and developing drought-resistant and high-yield agriculture. Alfalfa, as one of the most important forage grasses, has high yield, high nutritional value and wide adaptability. It is one of the most widely distributed and earliest cultivated high-quality legume forage grasses in the world.

[0003] The increasingly severe drought is one of the important factors limiting its large-scale and industrialized cultivation. In areas with water resource shortages, the only way to increase and stabilize the yield of dryland alfalfa is to cultivate and apply drought-resistant alfalfa varieties.

[0004] Comparative analysis of the morphological structure, physiological and biochemical and molecular response differences of alfalfa varieties with significant differences in drought resistance under drought stress conditions will help reveal the key mechanism of alfalfa drought resistance and provide a theoretical basis for in-depth research on the drought resistance mechanism of alfalfa.

[0005] Exogenous putrescine can improve the drought tolerance of plants. Putrescine can serve as a source of ROS and a potential ROS scavenger, and play a role in regulating redox homeostasis in plants. However, there is currently no research on the effect of exogenous putrescine on the drought repair ability of different drought-tolerant alfalfa during the germination and seedling stages; therefore, it does not meet the existing needs. In this regard, we proposed a test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings. Summary of the invention

[0006] The purpose of the present invention is to provide a testing method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings, so as to solve the problem raised in the above background technology that there is currently no research related to the drought repair ability of exogenous putrescine in the germination and seedling stages of different drought-tolerant alfalfa.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for testing the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings, comprising the following steps:

[0008] Step A: different concentrations of PEG-6000 were set to simulate drought stress, and alfalfa seeds with full grains and uniform size were selected from Dryland and WL354HQ, soaked and disinfected in 5% sodium hypochlorite solution for 15 minutes, rinsed with distilled water, and excess water was absorbed with filter paper. Thirty seeds were sown in each dish and placed in a constant temperature environment of 25°C, with 14 hours of light and 8 hours of dark culture. Based on the culture under different putrescine treatment conditions, a seed germination period test was carried out;

[0009] Step B: Evenly sowing alfalfa seeds in plastic pots containing nutrient soil and vermiculite of equal weight, with 10 to 15 seeds in each pot, and after germination, thinning the seedlings to 4 plants per pot, and growing the potted plants in a greenhouse at a room temperature of 25° C. and a relative humidity of 40% for 4 weeks, and making all the plants grow in soil containing 75% to 80% of the field water holding capacity by weighing method, and conducting a drought stress test at the seedling stage;

[0010] Step C: performing seed germination index determination, seedling growth index determination and physiological index determination, wherein the physiological index determination includes: performing peroxidase POD activity determination, superoxide dismutase SOD activity determination, free proline content determination, malondialdehyde MDA content determination and catalase CAT activity determination on germination stage materials and seedling stage tissue materials collected under different treatment conditions;

[0011] Step D: Performing statistical analysis on the measurement results to clarify the alleviating effect of exogenous putrescine on the drought stress of alfalfa seeds during the seed germination and seedling stages.

[0012] Preferably, the seed germination period test comprises the following steps:

[0013] S1: The effects of different exogenous polyamines on the germination period of alfalfa under drought stress were studied. Under the treatment conditions of distilled water CK, 10% PEG6000, 15% PEG6000 and 20% PEG6000, 0.1mmol / L putrescine, spermine and spermidine were added exogenously respectively;

[0014] S2: During the culture period, the filter paper and solution were replaced every two days, and the number of seed germination was recorded daily. The germination test was terminated after 14 days, and three biological replicates were performed;

[0015] S3: The effect of exogenous putrescine on the germination period of alfalfa under drought stress was studied. The seeds were cultured under different putrescine treatment conditions. During the culture period, the filter paper and solution were replaced every two days, and the number of seed germination was recorded daily. The germination test was terminated after 14 days, and three biological replicates were performed.

[0016] Preferably, the treatment conditions of different putrescine include distilled water CK, distilled water plus 0.1mmol / L putrescine, 5% PEG6000, 10% PEG6000, 15% PEG6000, 20% PEG6000, 5% PEG6000 plus 0.1mmol / L putrescine, 10% PEG6000 plus 0.1mmol / L putrescine, 15% PEG6000 plus 0.1mmol / L putrescine and 20% PEG6000 plus 0.1mmol / L.

[0017] Preferably, the seedling stage drought stress test comprises the following steps:

[0018] D1: The cultivated alfalfa plants were randomly divided into three groups, including:

[0019] Control group A: continuous watering and maintaining soil moisture content at 75%-80%;

[0020] Drought stress group A: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%;

[0021] Drought stress plus exogenous putrescine treatment group A: stop watering, and spray 3 mL of 0.1 mmol / L exogenous putrescine solution on the leaves for 3 consecutive days;

[0022] D2: Samples were taken from the control group A and the drought stress group A to observe morphological indicators and measure various physiological indicators. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed.

[0023] D3: The cultivated alfalfa plants were randomly divided into three groups, including:

[0024] Control group B: continuous watering and maintaining the soil moisture content at 75%-80%, and setting a control treated with exogenous putrescine under normal watering conditions;

[0025] Drought stress group B: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%;

[0026] Drought stress plus exogenous putrescine treatment group B: stop watering, and spray 3 mL of 0.1 mmol / L exogenous putrescine solution on the leaves for 3 consecutive days;

[0027] D4: Samples were taken from the control group B and the drought stress group B for morphological observation and determination of various physiological indices. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed.

[0028] Preferably, the exogenous putrescine solution is an aqueous solution containing putrescine.

[0029] Preferably, the seed germination index measurement starts from the day after the seeds are placed in the bed, and when the length of the embryo is half of the seed, it is determined to have germinated; after the test, the seed germination potential, germination rate, germination index and vitality index are calculated respectively.

[0030] Preferably, the calculation formulas for seed germination potential, germination rate, germination index and vitality index are:

[0031] Germination potential = number of germinated seeds counted on the third day / number of test seeds × 100%;

[0032] Germination rate = total number of germinated seeds counted on the 14th day / number of test seeds × 100%;

[0033] Germination index = total number of germinated seeds / total number of seeds × 100%;

[0034] Vitality index = seedling growth × germination index.

[0035] Preferably, the seedling growth index is determined by randomly selecting 6 seedlings in a group under different putrescine treatment conditions, and determining the growth height and fresh weight of the plants after each treatment.

[0036] Preferably, the intensity of illumination in step A is 3000 μ mol·m-2·s-1.

[0037] Application of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings: Exogenous putrescine is used to improve the seed germination rate of alfalfa under drought stress conditions, and exogenous putrescine alleviates the damage of drought stress to the germination of alfalfa seeds.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention screened different concentrations of polyamines to alleviate the germination of alfalfa seeds during the germination period, and then selected 0.1 mol / L putrescine to treat two alfalfa seeds with different drought tolerance at the germination and seedling growth stages with drought stress, so as to explore the effects of exogenous putrescine on the germination of alfalfa seeds and the physiological characteristics of seedlings under drought stress, analyze the physiological adaptation mechanism of exogenous putrescine to alleviate drought stress, and thus provide theoretical support for the drought-tolerant breeding of alfalfa;

[0040] 2. The present invention uses scientific and diversified testing methods based on cultivation under different putrescine treatment conditions to clarify that drought stress significantly inhibits the germination of alfalfa seeds and the growth of seedlings. Putrescine can effectively alleviate the inhibition of drought stress on the germination of alfalfa seeds and the growth of seedlings, which is specifically manifested in increasing the germination rate, plant height and fresh weight under drought stress, increasing peroxidase activity, reducing the degree of membrane lipid peroxidation, and ultimately enhancing the drought resistance of alfalfa, effectively obtaining the best form of exogenous putrescine addition. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a bar graph showing the effects of different exogenous polyamines on the germination of alfalfa seeds with different drought tolerances;

[0042] Figure 2 This is a bar graph showing the effect of exogenous putrescine on the germination of alfalfa seeds with different drought tolerances;

[0043] Figure 3 This is a bar graph showing the effect of exogenous putrescine on POD activity and MDA content of alfalfa seeds with different drought tolerances;

[0044] Figure 4 This is a bar graph showing the effect of exogenous putrescine on the phenotype, plant height and fresh weight of alfalfa seedlings with different drought tolerances;

[0045] Figure 5 This is a bar graph showing the effect of exogenous putrescine of the present invention on the physiological characteristics of alfalfa seedlings with different drought resistance. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be described clearly and completely 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 the embodiments.

[0047] See also Figures 1 to 5 The present invention provides an embodiment: a method for testing the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings, comprising the following steps:

[0048] Step A: different concentrations of PEG-6000 were set to simulate drought stress, and alfalfa seeds with full grains and uniform size were selected from Dryland and WL354HQ, soaked and disinfected in 5% sodium hypochlorite solution for 15 minutes, rinsed with distilled water, and excess water was absorbed with filter paper. 30 seeds were sown in each dish and placed in a constant temperature environment of 25°C, with 14 hours of light and 8 hours of dark culture, and the intensity of light was 3000 μ mol·m-2·s-1. On the basis of culture under different putrescine treatment conditions, a seed germination period test was carried out;

[0049] Step B: Evenly sowing alfalfa seeds in plastic flower pots containing nutrient soil and vermiculite (v / v, 1:1) of equal weight, the size of the plastic flower pots being 12 cm in height and 10 cm in diameter, with 10 to 15 seeds in each pot, thinning out the seedlings to 4 plants per pot after emergence, and growing the potted plants in a greenhouse at a room temperature of 25° C. and a relative humidity of 40% for 4 weeks, and making all the plants grow in soil containing 75% to 80% of the field water holding capacity by weighing method, and conducting a drought stress test at the seedling stage;

[0050] Step C: performing seed germination index determination, seedling growth index determination and physiological index determination, wherein the physiological index determination includes: performing peroxidase POD activity determination, superoxide dismutase SOD activity determination, free proline content determination, malondialdehyde MDA content determination and catalase CAT activity determination on germination stage materials and seedling stage tissue materials collected under different treatment conditions;

[0051] Seed germination index determination starts from the day after the seeds are placed in the bed. When the length of the embryo is half of the seed, it is judged to have germinated. After the test, the seed germination potential, germination rate, germination index and vitality index are calculated respectively.

[0052] The calculation formulas for seed germination potential, germination rate, germination index and vitality index are as follows:

[0053] Germination potential = number of germinated seeds counted on the third day / number of test seeds × 100%;

[0054] Germination rate = total number of germinated seeds counted on the 14th day / number of test seeds × 100%;

[0055] Germination index = total number of germinated seeds / total number of seeds × 100%;

[0056] Vitality index = seedling growth × germination index;

[0057] The seedling growth index was determined by randomly selecting 6 seedlings in the groups under different putrescine treatment conditions, and measuring the growth height and fresh weight of the plants after each treatment.

[0058] Step D: The measurement results were statistically analyzed using Excel 2023 software, IBM SPSS Statistics 27 was used for variance analysis and Duncan's significant difference test (α=0.05), and GraphPad Prism 8 software was used for plotting;

[0059] To clarify the alleviating effect of exogenous putrescine on drought stress in alfalfa seeds during the seed germination and seedling stages.

[0060] The seed germination test includes the following steps:

[0061] S1: The effects of different exogenous polyamines on the germination period of alfalfa under drought stress were studied. Under the treatment conditions of distilled water CK, 10% PEG6000, 15% PEG6000 and 20% PEG6000, 0.1mmol / L putrescine, spermine and spermidine were added exogenously respectively;

[0062] S2: During the culture period, the filter paper and solution were replaced every two days, and the number of seed germination was recorded every day. The germination test was terminated after 7 days, and three biological replicates were performed;

[0063] S3: The effect of exogenous putrescine on the germination period of alfalfa under drought stress was studied. The seeds were cultured under different putrescine treatment conditions, including distilled water CK, distilled water plus 0.1mmol / L putrescine, 5% PEG6000, 10% PEG6000, 15% PEG6000, 20% PEG6000, 5% PEG6000 plus 0.1mmol / L putrescine, 10% PEG6000 plus 0.1mmol / L putrescine, 15% PEG6000 plus 0.1mmol / L putrescine and 20% PEG6000 plus 0.1mmol / L. During the culture period, the filter paper and solution were replaced every two days, the number of seed germination was recorded daily, the germination test was ended after 14 days, and three biological replicates were performed.

[0064] The seedling drought stress test includes the following steps:

[0065] D1: The cultivated alfalfa plants were randomly divided into three groups, including:

[0066] Control group A: continuous watering and maintaining soil moisture content at 75%-80%;

[0067] Drought stress group A: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%;

[0068] Drought stress plus exogenous putrescine treatment group A: stop watering, and spray 3 mL of 0.1 mmol / L exogenous putrescine solution on the leaves for 3 consecutive days;

[0069] D2: Samples were taken from the control group A and the drought stress group A to observe morphological indicators and measure various physiological indicators. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed.

[0070] D3: The cultivated alfalfa plants were randomly divided into three groups, including:

[0071] Control group B: continuous watering and maintaining the soil moisture content at 75%-80%, and setting a control treated with exogenous putrescine under normal watering conditions;

[0072] Drought stress group B: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%;

[0073] Drought stress plus exogenous putrescine treatment group B: stop watering, and spray 3 mL of 0.1 mmol / L exogenous putrescine solution on the leaves for 3 consecutive days. The exogenous putrescine solution is an aqueous solution containing putrescine.

[0074] D4: Samples were taken from the control group B and the drought stress group B for morphological observation and determination of various physiological indices. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed.

[0075] Application of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings: Exogenous putrescine is used to improve the seed germination rate of alfalfa under drought stress conditions, and exogenous putrescine alleviates the damage of drought stress to the germination of alfalfa seeds.

[0076] The seeds tested were alfalfa Dryland and WL354HQ, and the purity of exogenous hormones Putrescine Put, spermine Spm and spermidine Spd was greater than 99.0%.

[0077] Results and Analysis:

[0078] Effects of different types of exogenous polyamines on the germination and seedling stages of alfalfa with different drought tolerance: Figure 1 As shown in the results, drought stress significantly inhibited the germination of alfalfa seeds. With the gradual increase of PEG6000 concentration, the inhibition was more significant, but the degree of inhibition on the drought-sensitive variety WL345HQ was more obvious. The effects of exogenous addition of 0.1mmol / L putrescine, spermine and spermidine on the germination of alfalfa seeds under different concentrations of PEG6000 stress were compared. It was found that exogenous putrescine had a significant alleviating effect on alfalfa varieties under drought stress. At the seedling stage of alfalfa, 0.1mmol / L putrescine, spermine and spermidine were sprayed exogenously in the early stage of drought stress. Under moderate and severe drought stress conditions, the seedlings of the drought-sensitive variety WL354HQ wilted severely, but after exogenous spraying of putrescine and spermidine, the seedlings could still grow normally. In summary, exogenous putrescine has a relatively good alleviating effect on the seed germination and seedling stages of alfalfa seeds under drought stress.

[0079] Effects of exogenous putrescine on germination of alfalfa seeds with different drought tolerance: Figure 2The different concentrations of PEG6000 treatment shown significantly inhibited the germination of two alfalfa varieties with different drought tolerance (P < 0.05). Under the treatment condition of 20% PEG6000, both varieties could not germinate normally. Compared with CK, the germination rate, germination potential and germination index of Dryland began to decrease significantly under the treatment condition of 15% PEG6000 (P < 0.05), decreasing by 16.8%, 21.9% and 23.2% respectively. However, the germination rate, germination potential and germination index of WL354HQ began to decline significantly (P < 0.05) under the treatment of 10% PEG6000, decreasing by 15.1%, 29.4% and 20.0% respectively; under the treatment of 15% PEG6000, the germination rate, germination potential and germination index decreased by 22.7%, 42.3% and 17.0% respectively, indicating that Dryland has better drought resistance than WL354HQ during the seed germination period.

[0080] Under normal conditions, exogenous addition of 0.1mmol / L putrescine had no significant effect on the germination of the two varieties (P>0.05). However, under PEG6000 stress, exogenous putrescine treatment significantly promoted the seed germination of the two alfalfa varieties (P<0.05). Compared with the 15% PEG6000 treatment, the germination rate, germination potential and germination index of Dryland increased by 12.7%, 22.4% and 22.5% respectively under exogenous putrescine treatment, and those of WL354HQ increased by 11.5%, 64.4% and 64.4% respectively. Under 20% PEG6000 treatment, the two germplasms almost did not germinate, but after exogenous putrescine treatment, the germination rate, germination potential and germination index of Dryland increased to 25.6%, 22.2% and 22.2% respectively, and those of WL354HQ increased to 24.4%, 22.2% and 23.2% respectively. The above results show that the exogenous addition of 0.1mmol / L putrescine can significantly alleviate the inhibitory effect of drought stress on alfalfa seed germination, and the alleviating effect is better for drought-sensitive varieties.

[0081] Effects of exogenous putrescine on the physiological characteristics of alfalfa seeds with different drought resistance during the germination period: PEG6000 treatment significantly inhibited the germination and root growth of both seeds. Consistent with the above results, the degree of inhibition was more significant under the treatment condition of 15% PEG6000. Exogenous putrescine can effectively alleviate the inhibition.

[0082] like Figure 3As shown in the results, the POD activity during seed germination increased first and then decreased with the increase of PEG6000 concentration. Under normal conditions, the POD activity of WL354HQ was 22.3% higher than that of Dryland. Under 5% PEG6000 stress, the POD activity of the two varieties reached the highest level. After drought stress treatment, exogenous putrescine could significantly increase the POD activity of the two varieties, and the POD activity of Dryland was significantly higher than that of WL354HQ, increasing by 0.89%, 71.5% and 194.0% under 5%, 10% and 15% PEG6000 stress conditions, respectively. Exogenous putrescine could significantly increase the POD activity of the two varieties (P < 0.05), especially under 15% PEG6000 stress, the POD activity of Dryland could increase by 6.89%, and the POD activity of WL354HQ could increase by 57.1%;

[0083] Drought stress promoted the accumulation of MDA in the two varieties. Under the treatment of 20% PEG6000, the MDA content of Dryland and WL354HQ reached the highest. Under the stress of 5% PEG6000, the MDA of Dryland decreased significantly (P < 0.05), indicating that low concentration of PEG6000 treatment can promote the germination of drought-tolerant varieties. Compared with CK, the increase of MDA content of WL354HQ after drought stress was greater, further indicating that Dryland was more drought-tolerant than WL354HQ. Exogenous putrescine treatment significantly reduced the MDA content of Dryland and WL354HQ after drought stress, which was more significant under 15% PEG6000 stress. Compared with CK, the MDA content decreased by 49.0% and 5.36%, respectively, further indicating that exogenous putrescine can effectively alleviate the damage of drought stress to the two varieties.

[0084] Effects of exogenous putrescine on plant height and fresh weight of alfalfa seedlings with different drought tolerance: Figure 4 As shown in the results, drought stress significantly reduced the plant height and fresh weight of the two varieties (P < 0.05). After drought stress, compared with CK, the plant height of Dryland and WL354HQ decreased by 21.0% and 25.4%, respectively. After exogenous putrescine, the plant height increased by 8.9% and 10.0%, respectively. After drought stress, the fresh weight of the two varieties decreased significantly (P < 0.05), and the fresh weight of Dryland and WL354HQ decreased by 110.0% and 132.7%, respectively. Exogenous putrescine can significantly increase the fresh weight of the two varieties, which increased by 37.4% and 65.3%, respectively, compared with the control. The above results show that exogenous putrescine can improve the resistance of alfalfa seedlings to drought stress, but the effect on drought-sensitive varieties is the best.

[0085] Effects of exogenous putrescine on physiological characteristics of alfalfa seedlings with different drought tolerance: Figure 5As shown in the results, under normal conditions, compared with CK, exogenous putrescine significantly increased the POD enzyme activity of Dryland and WL354HQ leaves by 57.9% and 51.9%, respectively. Under drought stress conditions, the POD activity of both varieties increased significantly (P < 0.05). After exogenous putrescine, the POD enzyme activity decreased significantly by 17.3% and 9.19%, respectively. Exogenous putrescine significantly increased the MDA content of Dryland (P < 0.05), while the increase in WL354HQ was not significant (P > 0.05). After drought stress, the MDA content of WL354HQ was higher than that of Dryland in fresh weight. Under exogenous putrescine, the MDA content of both varieties decreased significantly (P < 0.05). SOD activity decreased significantly after exogenous putrescine. After drought stress, the SOD activity of both varieties was the lowest under exogenous putrescine.

[0086] Compared with Dryland, WL354HQ had higher CAT activity. Both exogenous putrescine and drought reduced the CAT activity of Dryland. The CAT activity of WL354HQ increased significantly under drought stress and decreased significantly after exogenous putrescine (P<0.05).

[0087] Seed germination and seedling establishment are influenced by both internal physiological and biochemical regulation and the external environment. They are the most sensitive stages to external stimuli. Insufficient water supply will seriously affect seed germination and seedling growth and development, causing plant wilting or even death.

[0088] Studies have shown that exogenous hormones or growth regulators can change the growth process of plants by affecting their physiological characteristics, photosynthetic characteristics, and the synthesis of endogenous hormones, effectively alleviating the damage suffered by plants under adverse conditions and enhancing their tolerance to various abiotic stresses.

[0089] Polyamines PAs are a type of low molecular weight aliphatic nitrogenous base that exist in a free state in higher plants and are considered a new class of plant hormones. The most abundant polyamines in plants include putrescine Put, spermidine Spd, and spermine Spm.

[0090] Under drought stress, exogenous polyamines can improve plant photosynthesis and water utilization, promote the synthesis and accumulation of soluble sugars, proteins and low molecular weight amino acids, increase the activity of protective enzymes, and reduce the degree of cell membrane lipid peroxidation.

[0091] Before drought stress, tobacco leaves were treated with putrescine for 1 hour, which not only inhibited water loss, but also increased the intracellular putrescine concentration and restored photosynthetic parameters to normal values, confirming that putrescine and spermine play a key role in regulating plant tolerance to osmotic stress, and can also directly participate in the process of stress signal transduction as signal molecules.

[0092] Based on the above conclusions, exogenous putrescine can significantly improve the seed germination rate of alfalfa under drought stress conditions, especially under 15% and 20% PEG6000 conditions, the relief effect is more significant, and the relief effect is better for drought-sensitive varieties.

[0093] At the seedling stage, drought stress significantly reduced the plant height and fresh weight of the two varieties, which was consistent with the results at the germination stage. Exogenous putrescine could significantly improve the tolerance, plant height and fresh weight of the two varieties under drought stress, and the alleviating effect on drought-sensitive varieties was more significant.

[0094] The above results show that exogenous putrescine can improve the resistance of alfalfa to drought stress during the germination and seedling stages, and the effect is best on drought-sensitive varieties.

[0095] Under drought stress conditions, oxygen in chloroplasts will be activated into reactive oxygen species, such as superoxide anions, hydroxyl radicals, singlet oxygen, hydrogen peroxide and other reactive oxygen species, which will cause varying degrees of damage to the microstructure, submicrostructure and function of cells; the antioxidant defense system of plants plays a very important role in adverse stress. POD mainly eliminates reactive oxygen species in the body by catalyzing the decomposition of peroxides, and SOD catalyzes the dismutation reaction of superoxide. They can both reduce the toxic effects of reactive oxygen species on plant cells.

[0096] When the main active oxygen scavenging system in the chloroplast cannot eliminate these active oxygen in time, it will cause membrane lipid peroxidation and produce a large amount of MDA, thereby inhibiting protein synthesis, destroying the chloroplast cell membrane system, causing chlorophyll degradation, and further affecting leaf photosynthesis.

[0097] During the germination period, under drought stress, the POD activity of the drought-tolerant variety Dryland was higher than that of the sensitive variety WL354HQ. Exogenous putrescine could increase the POD activity of both varieties, but the effect on the sensitive variety WL354HQ was most obvious, indicating that Dryland had better osmotic regulation and antioxidant capacity, which to a certain extent explained why Dryland had higher drought resistance, and exogenous putrescine could increase the antioxidant capacity of alfalfa during the germination period under drought conditions.

[0098] Drought stress promoted the accumulation of MDA in the two varieties. Under the treatment of 20% PEG6000, the MDA content of Dryland and WL354HQ reached the highest, while exogenous putrescine treatment significantly reduced the MDA content of Dryland and WL354HQ after drought stress, which was more significant under 15% PEG6000 stress. This shows that exogenous putrescine can effectively alleviate the damage of drought stress to seed germination of the two varieties.

[0099] At the seedling stage, the changing trends of POD and SOD in the two varieties were consistent. Drought stress led to enhanced activity. Exogenous putrescine reduced the activity of POD and SOD enzymes in the two varieties. POD mainly catalyzed the decomposition of peroxides to eliminate reactive oxygen species in the body, and SOD catalyzed the dismutation reaction of superoxide. They can both reduce the toxic effects of reactive oxygen species on plant cells.

[0100] Exogenous putrescine reduced the activity of POD and SOD enzymes in the two varieties after drought stress. The effects of POD and SOD are dual. In the early stage of adverse stress, they are expressed to remove H202, showing a protective effect. In the later stage of adverse stress, they are expressed to participate in the generation of reactive oxygen species and chlorophyll degradation, thereby inducing membrane lipid peroxidation. Under normal conditions, exogenous putrescine significantly increased the MDA content in the leaves of the two varieties. Under drought stress, it significantly reduced the MDA content in the leaves of the two varieties. This shows that exogenous putrescine has no promoting effect on the growth of alfalfa seedlings under normal conditions, but under drought conditions, it can significantly alleviate the damage of drought stress to seedlings.

[0101] CAT is the key enzyme in plants that is responsible for breaking down H202 into water and oxygen. After drought stress, the CAT activity in the leaves of the two varieties increased significantly, and the activity in the leaves of Dyland was significantly higher than that in the leaves of WL354HQ. Exogenous putrescine reduced the activity of CAT after drought stress, further demonstrating that Dryland has stronger drought resistance than WL354HQ in the seedling stage, and exogenous putrescine can alleviate the damage caused by drought stress in the seedling stage of alfalfa.

[0102] In summary, drought stress significantly inhibited the germination of alfalfa seeds and the growth of seedlings, and the exogenous addition of 0.1 mmol / L putrescine can effectively alleviate the inhibition of drought stress on the germination of alfalfa seeds and the growth of seedlings, which is specifically manifested in increasing the germination rate, plant height and fresh weight under drought stress, increasing the peroxidase activity, reducing the degree of membrane lipid peroxidation, and ultimately enhancing the drought resistance of alfalfa.

[0103] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings, characterized in that: The steps include: Step A: different concentrations of PEG-6000 were set to simulate drought stress, and alfalfa seeds with full grains and uniform size were selected from Dryland and WL354HQ, soaked and disinfected in 5% sodium hypochlorite solution for 15 minutes, rinsed with distilled water, and excess water was absorbed with filter paper. Thirty seeds were sown in each dish and placed in a constant temperature environment of 25°C, with 14 hours of light and 8 hours of dark culture. Based on the culture under different putrescine treatment conditions, a seed germination period test was carried out; Step B: Evenly sowing alfalfa seeds in plastic pots containing nutrient soil and vermiculite of equal weight, with 10 to 15 seeds in each pot, and after germination, thinning the seedlings to 4 plants per pot, and growing the potted plants in a greenhouse at a room temperature of 25° C. and a relative humidity of 40% for 4 weeks, and making all the plants grow in soil containing 75% to 80% of the field water holding capacity by weighing method, and conducting a drought stress test at the seedling stage; Step C: performing seed germination index determination, seedling growth index determination and physiological index determination, wherein the physiological index determination includes: performing peroxidase POD activity determination, superoxide dismutase SOD activity determination, free proline content determination, malondialdehyde MDA content determination and catalase CAT activity determination on germination stage materials and seedling stage tissue materials collected under different treatment conditions; Step D: Performing statistical analysis on the measurement results to clarify the alleviating effect of exogenous putrescine on the drought stress of alfalfa seeds during the seed germination and seedling stages.

2. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 1, characterized in that: The seed germination period test comprises the following steps: S1: The effects of different exogenous polyamines on the germination period of alfalfa under drought stress were studied. Under the treatment conditions of distilled water CK, 10% PEG6000, 15% PEG6000 and 20% PEG6000, 0.1mmol / L putrescine, spermine and spermidine were added exogenously respectively; S2: During the culture period, the filter paper and solution were replaced every two days, and the number of seed germination was recorded daily. The germination test was terminated after 14 days, and three biological replicates were performed; S3: The effect of exogenous putrescine on the germination period of alfalfa under drought stress was studied. The seeds were cultured under different putrescine treatment conditions. During the culture period, the filter paper and solution were replaced every two days, and the number of seed germination was recorded daily. The germination test was terminated after 14 days, and three biological replicates were performed.

3. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 2, characterized in that: The treatment conditions of different putrescine included distilled water CK, distilled water plus 0.1mmol / L putrescine, 5% PEG6000, 10% PEG6000, 15% PEG6000, 20% PEG6000, 5% PEG6000 plus 0.1mmol / L putrescine, 10% PEG6000 plus 0.1mmol / L putrescine, 15% PEG6000 plus 0.1mmo1 / L putrescine and 20% PEG6000 plus 0.1mmol / L.

4. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 3, characterized in that: The seedling stage drought stress test comprises the following steps: D1: The cultivated alfalfa plants were randomly divided into three groups, including: Control group A: continuous watering and maintaining soil moisture content at 75%-80%; Drought stress group A: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%; Drought stress plus exogenous putrescine treatment group A: stop watering, and spray 3 mL of 0.1 mmol / L exogenous putrescine solution on the leaves for 3 consecutive days; D2: Samples were taken from the control group A and the drought stress group A to observe morphological indicators and measure various physiological indicators. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed. D3: The cultivated alfalfa plants were randomly divided into three groups, including: Control group B: continuous watering and maintaining the soil moisture content at 75%-80%, and setting a control treated with exogenous putrescine under normal watering conditions; Drought stress group B: stop watering, spray 3 mL of water on the leaves for 3 consecutive days, and maintain the soil moisture content at 40%-50%; Drought stress plus exogenous putrescine treatment group B: stop watering, and spray 3mL 0.1mmol / L exogenous putrescine solution on the leaves for 3 consecutive days; D4: Samples were taken from the control group B and the drought stress group B for morphological observation and determination of various physiological indices. At the same time, germination experiments were carried out using seed bags. After 14 days, samples were collected for phenotypic and physiological and biochemical index determination, and three biological replicates were performed.

5. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 4, characterized in that: The exogenous putrescine solution is an aqueous solution containing putrescine.

6. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 5, characterized in that: The determination of seed germination indicators begins from the day after the seeds are placed in the bed. When the length of the embryo is half of the seed, it is determined to have germinated. After the experiment, the seed germination potential, germination rate, germination index and vitality index are calculated respectively.

7. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 6, characterized in that: The calculation formulas for seed germination potential, germination rate, germination index and vitality index are as follows: Germination potential = number of germinated seeds counted on the third day / number of test seeds × 100%; Germination rate = total number of germinated seeds counted on the 14th day / number of test seeds × 100%; Germination index = total number of germinated seeds / total number of seeds × 100%; Vitality index = seedling growth × germination index.

8. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 7, characterized in that: The seedling growth index is determined by randomly selecting 6 seedlings in a group under different putrescine treatment conditions, and determining the growth height and fresh weight of the plants after each treatment.

9. The test method for the effect of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings according to claim 1, characterized in that: The intensity of light in step A is 3000 μmol·m-2·s-1.

10. Application of exogenous putrescine on the physiological characteristics of alfalfa seeds and seedlings, characterized in that: Exogenous putrescine was used to improve the seed germination rate of alfalfa under drought stress conditions, and exogenous putrescine alleviated the damage of drought stress to the germination of alfalfa seeds.

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