2,4-epibrassinolid in regulating seed germination of chenopodium quinoa under saline stress

Abstract

The application discloses application of 2,4-epibrassinolide in regulating seed germination of quinoa under saline-alkali stress, and relates to the technical field of quinoa planting. ‑1 Before quinoa seeds are planted in saline-alkali soil, the quinoa seeds are soaked by using 2,4-epibrassinolide solution with a concentration of 0-2 mg / L. The 2,4-epibrassinolide solution with the above concentration has a promoting effect on the growth of quinoa in the germination stage and the seedling stage under saline-alkali stress. When the single salt concentration is 0.4-0.8 and the 2,4-epibrassinolide concentration is 0.2, the promoting effect is optimal, the osmotic regulation capacity of quinoa can be effectively improved, the oxidative damage of saline-alkali stress can be reduced, and the saline-alkali resistance of quinoa is improved. The research result can provide a theoretical reference for improving the tolerance of quinoa to saline-alkali stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the quinoa planting technical field, and particularly relates to application of 2,4-epibrassinolide in regulating seed germination of quinoa under saline-alkali stress BACKGROUND

[0002] Brassinolide (BR) is a kind of natural plant growth hormone, which is first extracted and separated from rape pollen, and is well known for its high efficiency, broad spectrum and non-toxic physiological characteristics. It has an important influence on the growth and development of plants and the response to abiotic stress. In addition, 2,4-epibrassinolide (EBR) is a kind of artificially synthesized high-activity brassinosteroid derivative. Under stress, EBR can act together with other plant hormones to regulate various physiological processes of plants, such as promoting seed germination, seedling growth, improving fruit quality and enhancing plant stress resistance, etc. Studies have shown that EBR can significantly improve photosynthetic efficiency by affecting the chlorophyll content, photosynthetic parameters and stomatal opening of cherry tomato leaves, affecting the photosynthesis and transpiration of tomato, and promoting the growth and development of cherry tomato to ensure its quality. Spraying EBR at an appropriate concentration can make the antioxidant enzyme activity and osmotic regulation ability of wheat under low temperature stress show an increasing trend, and the content of malondialdehyde shows a decreasing trend. Liu Zilu et al. explored the influence of EBR on the seed germination characteristics of rice, and found that EBR can alleviate the high-temperature damage suffered by seeds by regulating the growth of root sprouts and increasing the activity of antioxidant enzymes and reducing the content of malondialdehyde. In their research, Jin Yanan found that exogenous brassinolide (BR) can regulate the growth characteristics of barley seeds during the germination and seedling stages, which helps to enhance the tolerance of barley to alkaline environment, promote the germination and growth of seeds, and specifically, the germination potential, germination rate and germination index of seeds treated with exogenous BR are more than doubled compared with the control group. In summary, exogenous epibrassinolide can enhance the salt and alkali tolerance of plants to a certain extent.

[0003] EBR seed soaking or external application to alleviate the toxic effects of salt-alkali stress on crops has become one of the hotspots of plant stress resistance. Existing studies have shown that brassinolide can improve the stress resistance of wheat, rice and other crops, but as of now, there are few reports on the use of brassinosteroids to regulate the growth of quinoa during the germination period. Therefore, this paper takes the salt-sensitive quinoa “Longqu 1” as the test material to explore the effects of different concentrations of EBR on the seed germination and seedling growth of quinoa under salt-alkali stress, in order to provide technical methods for improving the seed viability of field quinoa in agricultural production, and to provide a theoretical basis for clarifying the salt-alkali tolerance mechanism of quinoa seeds during the germination period. SUMMARY

[0004] In order to solve the above technical problems, the application designs the application of 2,4-epibrassinolide in regulating the seed germination of quinoa under saline-alkali stress.

[0005] In order to achieve the above technical effects, the application is achieved by the following technical scheme: the application of 2,4-epibrassinolide in regulating the seed germination of quinoa under saline-alkali stress, characterized in that, before the quinoa seeds are planted in saline-alkali soil, the quinoa seeds are soaked with 2,4-epibrassinolide solution with a concentration of 0-2 mg . L -1 .

[0006] Further, the 2,4-epibrassinolide solution is an aqueous solution prepared by mixing 2,4-epibrassinolide with water.

[0007] Further, the water is pure water or distilled water.

[0008] Further, the soaking time of the seeds is 23-25 h.

[0009] Further, the concentration of the 2,4-epibrassinolide solution is 0.2-0.8 mg . L -1 .

[0010] Further, the PH of the saline-alkali soil is 9-11.

[0011] Further, the 2,4-epibrassinolide can improve the osmotic regulation ability of the quinoa seeds, reduce the oxidative damage of saline-alkali stress, and improve the saline-alkali resistance of the quinoa.

[0012] Further, the variety of the quinoa seeds is Longqu 1.

[0013] The application has the following beneficial effects:

[0014] The application provides a new application of 2,4-epibrassinolide, i.e., before the quinoa seeds are planted in saline-alkali soil, the quinoa seeds are soaked with 2,4-epibrassinolide solution with a concentration of 0-2 mg -1 L, and the 2,4-epibrassinolide solution with the above concentration can promote the growth of quinoa in the germination and seedling stages under saline-alkali stress, wherein, when the single salt concentration is 0.4-0.8 and the 2,4-epibrassinolide concentration is 0.2, the promotion effect is best, the osmotic regulation ability can be effectively improved, the oxidative damage of saline-alkali stress can be reduced, and the saline-alkali resistance of the quinoa can be improved; the research result can provide a theoretical reference for improving the tolerance of quinoa to saline-alkali stress. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the following description of the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and all other drawings obtained by those skilled in the art without creative labor on the basis of these drawings also belong to the protection scope of the present application.

[0016] Figure 1 is a line graph of the influence of 24-epibrassinolid on the seed germination process of different saline-alkali stresses of the present application;

[0017] Figure 2 is a schematic diagram of the influence of 24-epibrassinolid on the hypocotyl elongation of different saline-alkali stresses of the present application;

[0018] Figure 3 is a schematic diagram of the influence of 24-epibrassinolid on the fresh weight of the seedling of different saline-alkali stresses of the present application;

[0019] Figure 4 is a schematic diagram of the influence of 24-epibrassinolid on the malondialdehyde of the seedling of different saline-alkali stresses of the present application;

[0020] Figure 5 is a schematic diagram of the influence of 24-epibrassinolid on the proline content of the seedling of different saline-alkali stresses of the present application;

[0021] Figure 6 is a schematic diagram of the influence of 24-epibrassinolid on the antioxidant enzyme activity of the seedling of different saline-alkali stresses of the present application. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor also belong to the protection scope of the present application.

[0023] Embodiment 1

[0024] The steps are as follows to explore the role of 2,4-epibrassinolid (EBR) in regulating the seed germination of chenopodium under saline-alkali stress.

[0025] Step 1, variety selection: the test material is chenopodium variety "Longwei No. 1" provided by Gansu Academy of Agricultural Sciences. In the previous stage, the laboratory evaluated the salt tolerance of Longwei No. 1, Longwei No. 3 and NX-1 varieties, and the salt-alkali sensitivity was: Longwei No. 1 > Longwei No. 3 > NX-1, so Longwei No. 1 was selected as the test material. 2,4-EBR was purchased from Hefei Bomei Technology Co., Ltd.

[0026] Step 2, preparation of saline-alkali soil: saline-alkali treatment components, proportions and concentrations are shown in Table 1.

[0027]

[0028] Table 1

[0029] Step 3, 7 groups of 2,4-epibrassinolide (EBR) solution groups with the same concentration gradient were prepared according to the above prepared saline-alkali soil serial numbers, as follows:

[0030] CK+ (control group) (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0031] A+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0032] B+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0033] C+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0034] D+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0035] E+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0036] F+ (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg . L -1 EBR);

[0037] The experiment had a total of 56 treatments, each treatment was repeated 3 times.

[0038] Step 4, selection of seed: choose mature, full, uniform size, no disease and insect pests of seed, in 2% mercuric chloride for 15 min, then completely washed with distilled water.

[0039] Step 5, Specific Experiment: Selected quinoa seeds were placed in a 4℃ refrigerator at eight concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2 mg). . L -1 Seeds were soaked in a 24-epibrassinolide solution for 24 hours (meaning that each of the seven 24-epibrassinolide solution groups was soaked independently). After soaking, seeds from each group that showed uniform white sprouting were placed in a petri dish lined with two layers of filter paper, with 30 seeds per dish. 2 mL of the corresponding salt solution and 2 mL of EBR solution were added to each dish, while 4 mL of H2O was added to the control group. Each treatment was repeated three times. The plants were incubated in a light incubator with 4000 lx illumination, 25℃ temperature, and a photoperiod of 16 h / 8 h (day / night). The treatment solution was changed every 36 hours to maintain a constant concentration.

[0040] Step 6, Measurement Indicators and Methods

[0041] 6.1 Determination of germination rate

[0042] Seed germination is measured by the radicle reaching half the seed length, calculated using the following formula:

[0043] Germination rate = (Number of normally germinated seeds / Total number of seeds) × 100%;

[0044] Observe and measure the germination rate on the same day, and maintain this for 7 consecutive days.

[0045] 6.2 Determination of hypocotyl length and biomass in seedlings

[0046] Hypocotyl length: Ten seedlings were randomly selected from the culture dish of each treatment, arranged neatly with a ruler, photographed, and the hypocotyl length was measured using ImageJ software.

[0047] Biomass: Fresh weight of 30 seedlings was determined by weighing.

[0048] 6.3 Determination of seedling physiological indicators

[0049] Malondialdehyde (MDA) content was determined using the thiobarbituric acid method; proline (Pro) content was determined using the acidic ninhydrin method; superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium method; peroxidase (POD) activity was determined using the guaiacol method; and catalase (CAT) activity was determined using ultraviolet absorption spectrometry.

[0050] 6.4 Overall Evaluation

[0051] Salt and alkali tolerance was evaluated using the membership function method. The comprehensive membership function is U(Xi)=(Xi–XMIN) / (XMAX–XMIN), i=1,2,3,4....n. In the formula, Xi represents the i-th test index; XMIN represents the minimum value of the i-th test index; and XMAX represents the maximum value of the i-th test index.

[0052] 6.5 Data Analysis

[0053] The experimental data were organized and graphically presented using Microsoft Office Excel. One-way ANOVA and membership function analysis were performed using IBM SPSS 26.0 software. The criterion for statistical significance was set at P < 0.05. All data points in the experiment are presented as the average of three independent replicates. + It is expressed as standard error.

[0054] Step 7: The effect of exogenous 2,4-epibrassinolide (EBR) on quinoa seed germination under salt-alkali stress was examined, as follows:

[0055] 7.1, such as Figure 1 As shown, compared with the control (CK), both single salt stress and mixed salt-alkali stress led to a decrease in the germination rate of quinoa seeds. Specifically, under single salt stress treatments (NaCl, Na₂SO₄, NaHCO₃, and Na₂CO₃), the germination rate decreased by 40%, 35%, 15%, and 75%, respectively. Under mixed salt (1:1) stress, the germination rate decreased by 30%, and under mixed salt-alkali (1:1:1:1) stress, the germination rate decreased by 35%. This indicates that all salt-alkali stress treatments inhibited seed germination, with the Na₂CO₃ treatment showing the most significant effect. Soaking seeds in exogenous EBR solution before salt-alkali stress revealed that different concentrations of EBR increased the germination rate of quinoa seeds to varying degrees, and the germination rate initially increased and then decreased with increasing EBR concentration. Importantly, applying appropriate concentrations of EBR under NaCl, NaHCO3, mixed salt (1:1), and mixed salt-alkali (1:1:1:1) stress all resulted in 100% germination rate, increasing by 60.67%, 17.65%, 42.86%, and 53.85% respectively compared to the no-EBR stress. The optimal alleviating hormone concentrations were 0.05–0.4 mg. . L -1 0.2mg . L -1 0.2mg . L -1 0.4mg . L -1Applying EBR under Na₂SO₄ and Na₂CO₃ stress alone can increase the germination rate to as high as 90%, which is 0.38 and 2.8 times higher than that under salt stress, respectively. The optimal concentration for both is 0.4 mg. . L -1 This also indicates that 2,4-epibrassinolactone has the best mitigating effect on seed germination under Na2CO3 stress; Figure 1 In this study, all differences were expressed as P < 0.05, and all data were the average of three replicates. + Standard error representation.

[0056] 7.2 Effects of exogenous EBR on the growth of quinoa seedlings under salt-alkali stress

[0057] Depend on Figure 2 It was found that, compared with the control group (CK), various salt stresses significantly reduced the hypocotyl elongation ability of quinoa seeds. The inhibitory effects were most pronounced under single-salt stresses of NaHCO3 and Na2CO3, while the effects were relatively weaker under single-salt stresses of NaCl, Na2SO4, and mixed salt-alkali stresses (1:1 and 1:1:1:1). Under normal conditions, after EBR soaking, the hypocotyl elongation of quinoa seeds increased with increasing EBR concentration. EBR soaking also promoted hypocotyl elongation under various salt-alkali stresses. Under single-salt stress of NaCl, the increase in hypocotyl elongation was most significant when applying an appropriate concentration of EBR, with the optimal EBR concentration being 0.4 mg. . L -1 Applying 0.8 mg of [agent] to single salt Na₂SO₄, Na₂CO₃, and mixed salt (1:1) stress... . L -1 The EBR relief effect was most significant, with hypocotyl length increasing by 48.21%, 74.84%, and 107.57% compared to salt stress. Under NaHCO3 stress, 0.2 mg... . L -1 This is the optimal concentration. We discovered an interesting phenomenon: when the salt solution contains Na₂SO₄, applying 0.4 mg... . L -1 EBR not only fails to alleviate the damage caused by saline-alkali conditions, but its inhibitory effect is also more severe than that caused by salt stress alone. Figure 2 In this study, significant differences were expressed as P < 0.05, and all data were expressed as the mean plus standard error of three replicates.

[0058] Depend on Figure 3 The seedling fresh weight graph shows that, under normal conditions, the fresh weight of quinoa soaked in EBR increases with increasing EBR concentration, reaching a maximum at 0.8 mg / L. . L -1The fresh weight of seedlings reached the maximum. Compared with CK, various salt stresses significantly reduced the fresh weight of the germination stage of Chenopodium quinoa, and different degrees of mitigation were obtained by applying EBR, indicating that different concentrations had different effects. Compared with other salt stresses, the effect of EBR on the resistance of Chenopodium quinoa under NaCl stress was most obvious, and the fresh weight of seedlings under 0.4 mg . L -1 of EBR was restored to a level similar to that of the control group without EBR. Under mixed salt stress at a ratio of 1:1 and 1:1:1:1, 0.2 mg . L -1 of EBR had the most obvious effect on the increase of fresh weight, increasing by 28.79% and 41.59%, respectively. Under Na2CO3 stress, 0.8 mg . L -1 of EBR increased the fresh weight by 15.39%. Only under NaHCO3 stress, EBR had no significant effect on fresh weight. The above results show that salt stress not only affects the growth of hypocotyls, but also affects the growth of embryos. And it shows that appropriate concentration of EBR treatment can greatly promote the seed viability of Chenopodium quinoa under salt stress. Figure 3 The P<0.05 indicates the significant difference between the two groups, and the index data is the average of three replicates + The standard error is indicated.

[0059] 7.3 Effect of exogenous EBR on MDA and Pro content of Chenopodium quinoa under salt stress

[0060] From Figure 4 we found that the MDA content of Chenopodium quinoa seedlings was very low under normal conditions after EBR soaking, and the MDA content of Chenopodium quinoa seedlings increased significantly under salt stress. After applying appropriate concentration of EBR, the MDA content of each treatment decreased, and the content decreased to the lowest when the concentration was 0.2-0.8 mg . L -1 The application of 0.4 mg . L -1 of EBR under single salt NaCl stress significantly reduced the MDA content by 34.70%; under single salt Na2SO4 and Na2CO3 stress, 0.8 mg . L -1 of EBR had the best effect on salt stress, and the MDA content decreased by 19.04% and 28.19%, respectively; under single salt NaHCO3 and mixed salt 1:1:1:1 stress, 0.2 mg . L -1MDA content of EBR-treated seedlings was significantly decreased by 27.87% and 18.60% compared with the corresponding salt-alkaline stress. In summary, we found that salt-alkaline stress caused different degrees of damage to the seedlings of Chenopodium quinoa. Compared with neutral salt stress, the degree of membrane lipid peroxidation was higher under alkaline salt component stress, and the MDA content was larger, which more inhibited the growth and development and physiological activity of seedlings. The above results showed that exogenous EBR could alleviate the membrane lipid damage of Chenopodium quinoa seedlings under salt-alkaline stress to a certain extent, but it could not restore it to normal. Figure 4 The significant difference was represented by P<0.05, and the index data was the average value of three replicates + The standard error was represented.

[0061] As Figure 5 The change of proline content under normal conditions and under salt-alkaline stress after applying different concentrations of EBR. Overall, salt-alkaline stress may cause an increase in the content of proline in plants. Under normal conditions, EBR seed soaking had a slight effect on proline content, but after EBR seed soaking, the free proline content of Chenopodium quinoa seedlings under salt-alkaline stress was significantly increased. It was found that low-concentration EBR promoted proline content, and high-concentration inhibited the trend. Among them, under single salt NaCl, Na2SO4, Na2CO3 and mixed salt (1:1) treatment, 0.4 mg . L -1 Concentration of EBR solution can significantly improve the content of proline, and the effect is best under NaCl stress, which is increased by 35.50%. However, when the concentration exceeds 0.8 mg . L -1 EBR, it shows inhibitory effect. Under single salt NaHCO3 and mixed salt alkaline (1:1:1:1) treatment, 0.2 mg . L -1 EBR has the most effective increase in proline content, which is increased by 66.93% and 32.14% compared with no EBR. The test results show that the free proline content of Chenopodium quinoa seedlings under salt-alkaline stress increases, and exogenous application of different concentrations of EBR can effectively alleviate the damage of salt-alkaline stress, and significantly improve the content of free proline in plant leaves to maintain cell homeostasis and reduce oxidative damage. Figure 5 The significant difference was represented by P<0.05, and the index data was the average value of three replicates + The standard error was represented.

[0062] 7.4 Effect of exogenous EBR on antioxidant enzyme activity of Chenopodium quinoa under salt-alkaline stress

[0063] From Figure 6The activities of antioxidant enzymes POD, SOD and CAT in the seedlings under normal conditions and saline-alkali stress after EBR seed soaking can be seen. Compared with CK, due to the accumulation of active oxygen free radicals in the seedlings of quinoa under saline-alkali stress, the activities of CAT and POD enzymes increase sharply to eliminate the accumulation of hydrogen peroxide, hydroxyl radicals and other substances caused by saline-alkali to resist the oxidative damage caused by salt. Under the stress of NaCl, Na2SO4 and NaHCO3, the CAT activity increases by more than 4 times, and the POD activity increases by 1-2 times. Under the stress of mixed saline-alkali (1:1:1:1), the activities of CAT and POD do not increase significantly or even decrease. It is worth noting that under the stress of single salt (NaCl, Na2SO4, Na2CO3, NaHCO3), the activity of SOD decreases by 50-70%, and only under the stress of mixed salt (1:1 and 1:1:1:1), the activity increases by 5%-20%. After applying EBR with different concentrations, the activities of CAT and POD under different salt treatments reach the best at 0.2-0.8 mg . L -1 The activity of SOD decreases to the lowest at 0.2, 0.4 and 0.8 mg . L -1 The results show that various salt stresses force the activities of POD and CAT in seedlings to increase, and the application of EBR promotes the activities of antioxidant enzymes in seedlings, thereby improving the salt and alkali tolerance of quinoa. It can be known that each antioxidant enzyme responds differently to plant salt and alkali stress, and the ability to scavenge free radicals is also different. Figure 6 The significant difference in the mean values of the indicators is represented by P<0.05 + The standard error is represented.

[0064] 7.5 The membership function analysis of quinoa under different salt treatments is shown in Table 2.

[0065]

[0066] Table 2

[0067] Note: The comprehensive membership function U(Xi) = (Xi-XMIN).(XMAX-XMIN). The membership functions of each indicator are omitted in the table, and only the comprehensive evaluation value is shown.

[0068] Comprehensive evaluation value = U1 . W1+U2 . W2+……Ui . Wi (i value is determined by cumulative variation).

[0069] In summary, L. sativa 'Longqili No.1' has certain physiological and biochemical defense system under salt stress, but the ability of resistance is limited. Under salt stress, the seed germination rate, hypocotyl elongation and fresh weight were significantly lower than the control group. With the increase of EBR concentration, the seed germination rate, hypocotyl elongation and fresh weight were promoted. Salt stress affected the accumulation of osmotic adjustment substances in the seedling, the proline content and malondialdehyde content increased under stress, which would directly damage the membrane structure and function. Exogenous EBR could promote the increase of proline content to regulate the osmotic balance, and effectively reduce the content of malondialdehyde. Under partial salt stress, the ability of antioxidant enzymes was enhanced, and under EBR relief, the activity of antioxidant enzymes was induced to rapidly enhance to respond to the imbalance of oxidation and reduction. In summary, exogenous 2,4-epibrassinolide has a positive effect on the seed germination and seedling growth of L. sativa under salt stress, and the EBR solution of 0.2-0.8 mg / L has the most obvious effect on the seed germination and seedling growth of L. sativa under salt stress. . L -1 ​

Claims

1. Use of 2,4-epibrassinolid in regulating seed germination of chenopodium quinoa under saline stress, characterized in that, Before sowing quinoa seeds into saline-alkali soil, the quinoa seeds are soaked in a 2,4-epibrassinolide solution with a concentration of 0.2-0.8 mg / L.

2. Use of 2,4-epibrassinolide according to claim 1 for regulating seed germination of quinoa under saline stress, characterized in that, The 2,4-epibrassinolide solution is an aqueous solution prepared by mixing 2,4-epibrassinolide with water.

3. The use of 2,4-epibrassinolid according to claim 2 for the regulation of the seed germination of quinoa under saline stress, characterized in that, The water is pure water or distilled water.

4. The use of 2,4-epibrassinolide according to claim 1 for the regulation of seed germination of quinoa under saline stress, characterized by, The quinoa seeds are soaked for 23-25 hours.

5. The use of 2,4-epibrassinolide according to claim 1 for the regulation of seed germination of quinoa under saline stress, characterized in that, The saline-alkali soil has a pH of 9-11.

6. The use of 2,4-epibrassinolide according to claim 1 for the regulation of seed germination of quinoa under saline stress, characterized by the fact that, The 2,4-epibrassinolide improves the osmotic regulation ability of the quinoa seeds, reduces the oxidative damage caused by saline-alkali stress, and improves the saline-alkali resistance of the quinoa.

7. Use of 2,4-epibrassinolid according to any one of claims 1-6 for the regulation of the germination of seeds of quinoa under saline stress, characterized in that, The variety of the quinoa seeds is Longqu 1.

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