A method for promoting germination and growth of alfalfa seeds under zinc stress by synergistically using 24-epibrassinolide and gibberellin

Abstract

The invention discloses a method and application of enhancing zinc tolerance of alfalfa seeds by using 24-epibrassinolide and gibberellin. A method for promoting germination and growth of alfalfa seeds under zinc stress by applying 24-epibrassinolide and gibberellin exogenously is provided. Taking alfalfa seeds as test materials, the positive effects of different concentration ratios and implementation methods of exogenous 24-epibrassinolide and gibberellin on germination characteristics, root growth and antioxidant enzyme system of alfalfa seeds under different zinc concentration stress are obtained. The different concentration ratios of exogenous 24-epibrassinolide and gibberellin and the specific method proposed can significantly improve the zinc tolerance of alfalfa seeds and promote root growth, providing technical guarantee for the promotion and planting of alfalfa in zinc-contaminated soil and the repair of zinc-contaminated soil.

Description

Technical Field

[0001] The invention relates to the technical field of alfalfa cultivation under zinc stress, and in particular to a method for promoting seed germination and root growth of alfalfa under zinc stress by synergistically utilizing 24-epibrassinolide and gibberellin. Background Art

[0002] Phytoremediation technology has the advantages of being green, environmentally friendly, low cost, and having little disturbance to the soil. By taking simple auxiliary management measures, such as the application of plant hormones, the tolerance of plants to heavy metal-contaminated soil can be improved, thereby improving the efficiency of phytoremediation of zinc-contaminated soil.

[0003] Alfalfa (Medicago sativa L.) is a perennial leguminous forage plant with wide adaptability, large biomass, good palatability and high quality. At the same time, its strong stress resistance and nitrogen fixation and soil improvement characteristics make it a pioneer plant for repairing soil contaminated by heavy metal tailings and has been widely planted in my country.

[0004] Zinc is one of the trace elements necessary for plant growth and development, and is involved in the synthesis of various enzyme proteins, auxin and chlorophyll and other biological processes. However, excessive zinc can poison plants, inhibit plant seed germination and root elongation, damage photosynthesis and respiration, destroy plant cell membranes, proteins and genetic materials, etc., and zinc enriched in plants will eventually be absorbed by the human body through the food chain, endangering human health.

[0005] Using extremely low concentrations of exogenous plant hormones to treat plants will trigger multiple signal transduction pathways to alleviate the stress of heavy metals on plants. Brassinosteroids are known as the "sixth plant hormone" and are a class of polyhydroxy steroid plant hormones that can regulate a variety of physiological functions of plants, such as embryogenesis and seed germination, cell division and elongation, differentiation and growth of pollen tubes, and aging and death of leaves. Gibberellic acid is a natural plant growth regulator that plays a very important regulatory role in plant growth and development, including cell elongation and division, breaking dormancy and reproductive development. For example, in the invention patent CN115152360A, a method for breaking the dormancy of wild eggplant seeds and promoting germination is provided, which sequentially soaks wild eggplant seeds in sodium chloride solution and gibberellin solution, which can effectively break the dormancy of wild eggplant seeds and promote the germination of wild eggplant seeds. For example, in the invention patent CN115067159A, a method for efficient and rapid germination and breeding of Polygonatum sibiricum seeds is provided, which uses a mixed solution of cytokinin, salicylic acid and brassinolide to soak the seeds, and then applies ultrasonic treatment, thereby shortening the germination period of the Polygonatum sibiricum seeds and improving the germination rate of the Polygonatum sibiricum seeds.

[0006] In summary, although there are technical solutions or studies showing that brassinosteroids or gibberellins promote the germination or root growth of some seeds, there is no technology or research showing the positive effects of brassinosteroids or gibberellins on plant seed germination under zinc stress. In particular, there is no report in the prior art on how to promote the germination and root growth of alfalfa in zinc-contaminated soil. Therefore, it is very important to develop a simple, environmentally friendly and efficient new technology method to promote the germination and growth of alfalfa seeds in zinc-contaminated soil, so as to utilize the growth characteristics of alfalfa plants to repair heavily zinc-contaminated soil. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provides a method for utilizing 24-epibrassinolide and gibberellin to synergistically promote the germination of alfalfa seeds and the growth of the root system under zinc stress.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A method for promoting alfalfa seed germination and root growth under zinc stress by synergistically using 24-epibrassinolide and gibberellin, comprising the following steps:

[0010] (1) Soaking alfalfa seeds in disinfected water containing 1% to 5% effective chlorine for 10 to 30 minutes, then taking out the alfalfa seeds and rinsing them with sterile water for 2 to 7 times;

[0011] (2) The alfalfa seeds treated in step (1) are cultured in a 0.3% to 1.5% water agar medium for 6 to 10 days, wherein the water agar medium contains Zn 2+ Concentration 150 g L -1 ~450g L -1 , hormone concentration is 0.1mmol L -1 The hormone types are 24-epibrassinolide and gibberellin, and the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = (0.5-1.3): (0.8-12), and the concentration of 24-epibrassinolide is 8-12 mmol L -1 The concentration of gibberellin is 8-120 mmol L -1 .

[0012] As a further improvement to the above scheme, the seeds are evenly spaced and spread on a water agar medium for culture, and the culture dish is placed in an artificial climate box.

[0013] As a further improvement to the above scheme, when the seeds are evenly spaced and spread on a water agar medium for cultivation, the interval between adjacent seeds is 2.5 to 3 cm.

[0014] As a further improvement of the above scheme, an artificial climate chamber is used to culture alfalfa seeds under a day-night photoperiod cycle, specifically: first, 16 hours of light culture is carried out, and the temperature of the light culture is maintained at 23°C to 25°C, and then 8 hours of light-free culture is carried out, and the temperature of the light-free culture is maintained at 21 to 23°C.

[0015] As a further improvement to the above scheme, the disinfectant water in step (1) is a chemically pure sodium hypochlorite solution with an effective chlorine content of 10%, which is diluted 2-5 times before use.

[0016] As a further improvement of the above scheme, when the Zn2+ treatment concentration in the water agar medium is 300 g L-1, the root length of alfalfa seeds cultured with the addition of 24-epibrassinolide and gibberellin can be increased by up to 3.22 times compared with the root length of alfalfa seeds cultured without the addition of 24-epibrassinolide and gibberellin.

[0017] As a further improvement of the above scheme, when the Zn2+ treatment concentration in water agar medium is 450 g L-1, the biomass of alfalfa seeds cultured with the addition of 24-epibrassinolide and gibberellin can be increased by up to 2.42 times compared with the biomass of alfalfa seeds cultured without the addition of 24-epibrassinolide and gibberellin.

[0018] As a further improvement to the above scheme, the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = 1:10.

[0019] As a further improvement to the above scheme, the gibberellin is specifically at least one of gibberellic acid, gibberellin A1, gibberellin A4, and gibberellin A7.

[0020] The present application also protects a method for using 24-epibrassinolide and gibberellin to synergistically promote alfalfa seed germination and root growth under zinc stress, and its application in using alfalfa to repair zinc-contaminated soil.

[0021] Beneficial technical effects

[0022] The present invention is the first to apply 24-brassinolide and gibberellin exogenously during the germination process of alfalfa seeds in a water agar medium. Through the synergistic promotion and stimulation of the two hormones, in particular, brassinolide is used to regulate the proliferation, differentiation and elongation of root cells, and the processes such as lateral root differentiation, root hair formation and vascular tissue differentiation in the growth and development of plant roots are affected. At the same time, gibberellin breaks seed dormancy and promotes germination by increasing the growth potential of the embryo and overcoming the mechanical obstacles of the embryo surrounding layer. At the same time, it also regulates the cell replication and elongation process of the root tip apical tissue. The synergistic effect of the two well promotes and increases the germination rate, germination potential, root length and biomass of alfalfa seeds in a living environment with excessive zinc metal, that is, under zinc stress. According to the method of the present invention, under the stress of different amounts of metallic zinc, the germination rate, germination potential, root length and biomass of alfalfa seeds are all improved and improved to a certain extent under the synergistic effect of 24-brassinolide and gibberellin compared with no addition of any hormones. For example, in a water agar medium with Zn 2+ The treatment concentration is 450gL -1 When Zn was added to the water agar medium, the biomass of alfalfa seeds cultured with 24-epibrassinolide and gibberellin increased by 2.42 times at most compared with that of alfalfa seeds cultured without 24-epibrassinolide and gibberellin. 2+ The treatment concentration was 300 g L -1 When the root length of alfalfa seeds cultured with 24-epibrassinolide and gibberellin was increased by 3.22 times, the root length of alfalfa seeds cultured without 24-epibrassinolide and gibberellin was increased by 3.22 times. 2+ The treatment concentration was 150 g L -1 , 300g L -1 , 450g L -1When only one of 24-epibrassinolide or gibberellin is added, the root length of alfalfa seeds is shorter than that of the latter, and the biomass is also significantly less than that of the latter, and the biomass of alfalfa seeds cultured with gibberellin is increased by 2.42 times at most compared with the biomass of alfalfa seeds cultured without adding 24-epibrassinolide and gibberellin. It can be seen that the synergistic effect of the above two hormones is very obvious. In addition, the synergistic application of brassinolide and gibberellin significantly enhances the stress resistance of plants, and promotes them to grow normally in adverse natural environments, thereby providing a good technical reference for using the growth of alfalfa to improve the nitrogen fixation and soil improvement of areas where zinc is seriously exceeded. At the same time, the synergistic effect of the above two hormones is obvious, and the method is simple, easy, economical, environmentally friendly, and efficient. It can effectively improve the germination of alfalfa under zinc stress and promote the healthy growth of the root system, providing important technical support for the promotion of alfalfa cultivation in heavy metal contaminated soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are actual photographs of the effects of the two hormones (brassinolide and gibberellin) of the present invention on the growth of alfalfa under different concentrations of zinc stress. DETAILED DESCRIPTION

[0024] The technical solutions of the specific embodiments of the present invention will be clearly and completely described below in combination with various specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained through ordinary commercial channels.

[0025] The present invention relates to a method for promoting the germination of alfalfa seeds and root growth under zinc stress by using 24-epibrassinolide and gibberellin in a coordinated manner, comprising the following steps:

[0026] (1) Soak the alfalfa seeds in disinfectant containing 1% to 5% effective chlorine for 10 to 30 minutes, then take out the alfalfa seeds and rinse them with sterile water for 2 to 7 times. The percentage of the effective chlorine in the disinfectant can be 1%, 2%, 3%, 4%, 5%, etc., the disinfection time can be 10, 15, 20, 25, 30 minutes, etc., and the number of rinses can be 2, 3, 4, 5, 6, or 7 times. Through the above soaking and rinsing, the influence of dust, mold and other impurities and bacteria on the seed surface on seed culture can be effectively reduced, and the contamination of the above impurities on the water agar medium in the next step of water agar culture can be reduced, which is conducive to the normal germination of seeds and reduces the exogenous error between different seeds.

[0027] (2) The alfalfa seeds treated in step (1) are cultured in a 0.3% to 1.5% water agar medium for 6 to 10 days, wherein the Zn2+ concentration in the water agar medium is 150 g L-1 to 450 g L-1, the hormone concentration is 0.1 mmol L-1, the hormone types are 24-epibrassinolide and gibberellin, and the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = (0.5 to 1.3): (0.8 to 12), the concentration of 24-epibrassinolide is 8 to 12 mmol L-1, and the concentration of gibberellin is 8 to 120 mmol L-1. Wherein, the water agar base can specifically be 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, the culture days can be 6 or 7 or 8 or 9 or 10 days, the Zn2+ concentration in the water agar medium is 150g L-1, 300g L-1, 450g L-1, and the water agar medium and the two synergistic hormones are preferably selected as mentioned above, and the addition ratio and concentration of the two are optimized, so as to minimize the adverse effects of zinc stress on seed germination and growth, and improve the normal germination and growth of alfalfa seeds under the adverse environmental factor of zinc stress, so as to provide reference and technical support for the improvement of soil in zinc-contaminated areas, such as nitrogen fixation and soil improvement.

[0028] Furthermore, the above-mentioned culture specifically spreads the seeds evenly at intervals on the water agar medium for culture, and the culture dish is placed in an artificial climate box. The evenly spaced arrangement and the growth environment setting of the artificial climate box are conducive to each alfalfa seed to evenly absorb the nutrients of the culture medium, reduce the occurrence of problems such as cross-growth and competition for nutrients, and provide convenience for the subsequent observation and measurement of growth conditions such as root length. At the same time, through the use of the artificial climate box, the real climate conditions are restored to the greatest extent, making the whole method closer to the natural environment, which is convenient for providing real and reliable method support for the actual planting of alfalfa and improving zinc-contaminated soil.

[0029] Furthermore, when the seeds are evenly spaced and spread on a water agar medium for culture, the spacing between adjacent seeds is 2.5 to 3 cm, specifically: a 10×10 cm culture dish, 10 seeds per culture dish, and the spacing between adjacent seeds is about 2.5 to 3 cm. Reasonable spacing parameters are conducive to the lateral growth of seed roots and reduce the cross-entanglement of roots between adjacent seeds, which is convenient for the determination of parameters such as root length and biomass, providing accurate and reliable data support for the evaluation of the entire method.

[0030] Furthermore, an artificial climate chamber was used to culture alfalfa seeds under a day-night cycle photoperiod, specifically: first, 16 hours of light culture was carried out, and the temperature of the light culture was maintained at 23℃~25℃, and then 8 hours of light-free culture was carried out, and the temperature of the light-free culture was maintained at 21℃~23℃. Among them, the light culture temperature can be maintained at 23℃, 24℃, 25℃, etc., and the light-free culture temperature can be maintained at 21℃, 22℃, 23℃, etc. Through experiments and explorations of day-night cycle photoperiod culture at reasonable temperatures, the optimal culture conditions were screened out, which was conducive to the acquisition of culture methods and parameters for alfalfa seeds under zinc stress conditions, and provided reliable and simple technical conditions for the application of alfalfa seeds in soil remediation in zinc-stressed soil environments.

[0031] Furthermore, the disinfectant in step (1) is a chemically pure sodium hypochlorite solution with an effective chlorine content of 10%, which is diluted 2-5 times before use. By selecting sodium hypochlorite solution as the disinfectant and selecting a suitable dilution multiple, it is possible to ensure thorough disinfection of the seed surface and reduce the side effects of chlorine in the disinfectant on seed activity, such as inhibition of seed germination rate.

[0032] Furthermore, when the Zn2+ treatment concentration in water agar medium was 300g L-1, the root length of alfalfa seeds cultured with 24-epibrassinolide and gibberellin increased by 3.22 times at most compared with the root length of alfalfa seeds cultured without 24-epibrassinolide and gibberellin. By investigating the root length growth of alfalfa seeds treated with two hormones compared with no hormones when the Zn2+ treatment concentration was 300g L-1, data support was provided for the cooperation of the two hormones in promoting the germination and growth of alfalfa seeds under certain zinc ion pollution conditions, which provided an important reference for the subsequent planting of alfalfa in zinc-contaminated soil.

[0033] Furthermore, when the Zn2+ treatment concentration in water agar medium was 450g L-1, the biomass of alfalfa seeds cultured with 24-epibrassinolide and gibberellin increased by 2.42 times at most compared with the biomass of alfalfa seeds cultured without 24-epibrassinolide and gibberellin. By examining the biomass of alfalfa seeds treated with two hormones and without any hormones at a Zn2+ treatment concentration of 450g L-1, it provides important comparative data support for the cooperation of the two hormones in promoting the germination and growth of alfalfa seeds under certain zinc ion pollution conditions.

[0034] Furthermore, the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = 1: 10. By optimizing the ratio of the two, the synergistic effect of the two is improved to the best, and the germination and growth of alfalfa seeds are promoted to the maximum extent.

[0035] Furthermore, the gibberellin used is at least one of gibberellic acid, gibberellin A1, gibberellin A4 and gibberellin A7.

[0036] Furthermore, under the premise that other culture conditions are the same, the ratio of the germination rate of alfalfa seeds when the Zn2+ treatment concentration in the water agar medium is 150g L-1 to 450g L-1 to the germination rate of alfalfa seeds when the Zn2+ treatment concentration in the water agar medium is 0g L-1 is ≥0.6045. The data support and reference of the germination rate are helpful for technicians to select and control the seeding amount when considering the use of alfalfa to repair zinc-contaminated soil, providing support for ensuring the stability of the germination rate and the effect of the repair.

[0037] Furthermore, the water agar medium corresponding to the treatment prepared in step (2) was added to each 10×10 cm culture dish with 40 ml.

[0038] Furthermore, in step (2), the number of seeds that germinate is counted every 24 hours during the culture process, and the germination standard is when the radicle or embryo breaks through the seed coat and extends 5 mm. On the last day of culture, plant samples are collected and fresh weight and root length are measured.

[0039] Further, in each embodiment, water agar medium was prepared: 4 different gradients of Zn 2+ Treatment concentration 0g L -1 , 150g L-1, 300g L-1, 450g L-1, 24-epibrassinolide and gibberellin treatment concentrations of 0.1mmol L-1, respectively, were prepared into the corresponding water agar medium. Zinc treatment, 24-epibrassinolide and gibberellin were randomized in block design, with a total of 16 treatment experimental combinations. Three groups of 100 seeds were set as three biological replicates for each treatment.

[0040] The parameters and calculation methods of alfalfa seed germination and root growth mentioned in the subsequent embodiments are as follows:

[0041] Germination rate (%) = (number of germinated seeds on the last day of the test / total number of tested seeds) / 100%;

[0042] Germination potential (%) = (number of germinated seeds on the middle day of the experiment / total number of seeds tested) / 100%; (the middle day means that if the total number of days of the experiment is N days and N is a cardinality, the middle day is the (N+1) / 2 day; if the total number of days of the experiment is N days and N is an even number, the middle day is the N / 2 day);

[0043] Germination index = Σ(Gt / Dt), Dt is the number of days of the experiment, Gt is the number of germinated seeds on that day;

[0044] Vitality index = germination index × average root length.

[0045] Biomass calculation method: Place the culture medium with seedlings in distilled water, gently wash out the complete seedlings, dry the surface water with a paper towel, put it on a scale, and weigh the fresh weight of the whole plant as the biomass value. The washing and water absorption process must be done quickly to avoid errors caused by the seedlings absorbing water and evaporating water.

[0046] The present application also protects a method for using 24-epibrassinolide and gibberellin to synergistically promote alfalfa seed germination and root growth under zinc stress, and its application in using alfalfa to repair zinc-contaminated soil.

[0047] Example 1

[0048] A method for using 24-epibrassinolide and gibberellin to synergistically promote alfalfa seed germination and root growth under zinc stress comprises:

[0049] (1) Soaking alfalfa seeds in disinfected water containing 3% available chlorine for 20 minutes, then taking out the alfalfa seeds and rinsing them with sterile water for 5 times;

[0050] (2) The alfalfa seeds treated in step (1) are cultured in a 0.8% water agar medium for 8 days, wherein the Zn2+ concentration in the water agar medium is 150 g L-1 to 450 g L-1, the hormone concentration is 0.1 mmol L-1, the hormone types are 24-epibrassinolide and gibberellin, and the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = 1:10, the concentration of 24-epibrassinolide is 8 to 12 mmol L-1, and the concentration of gibberellin is 8 to 120 mmol L-1.

[0051] The seeds were evenly spaced and plated on water agar medium for cultivation, and the culture dishes were placed in an artificial climate chamber.

[0052] When the seeds are evenly spaced and spread on water agar medium for cultivation, the interval between adjacent seeds is 2.5 to 3 cm.

[0053] The alfalfa seeds were cultured under a day-night photoperiod using an artificial climate chamber, specifically: firstly, they were cultured under light for 16 hours with the temperature kept at 25°C, and then they were cultured without light for 8 hours with the temperature kept at 22°C.

[0054] The disinfectant in step (1) is a chemically pure sodium hypochlorite solution having an effective chlorine content of 10%, which is diluted 3.33 times before use.

[0055] In step (2), 10 mmol L-1 mother solutions of 24-epibrassinolide and gibberellin are prepared respectively. 4.78 mg of 24-epibrassinolide powder is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml; 3.32 mg of gibberellin is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml. The prepared mother solutions of the two hormones are stored in a -20°C refrigerator for standby use. During the experimental treatment, 10 ul of the two hormone mother solutions are respectively taken and added to 1 L of water agar medium and mixed. At this time, the hormone concentration of the medium is the final treatment concentration of 0.1 mmol L-1.

[0056] Prepare the corresponding water agar medium in step (2) and add 40 ml to each 10×10 cm culture dish.

[0057] In step (3), the number of seeds that have germinated is counted every 24 hours, and the germination standard is the radicle or plumule breaking through the seed coat and extending 5 mm.

[0058] At the same time, parallel control experiments were carried out without adding any hormones, adding only 24-epibrassinolide, and adding only gibberellin.

[0059] At the end of the experiment on the 8th day, plant samples were collected to measure fresh weight and root length, and statistics and calculations were performed according to the previously recorded calculation methods for germination rate, germination potential, germination index, and vitality index.

[0060] Example 1 Experimental data statistics table

[0061] Table 1-1 Effects of brassinolide and gibberellin on germination rate of alfalfa under different concentrations of zinc stress (%)

[0062]

[0063] Table 1-2 Effects of brassinolide and gibberellin on germination potential of alfalfa under different concentrations of zinc stress (%)

[0064]

[0065] Table 1-3 Effects of brassinolide and gibberellin on germination index of alfalfa under different concentrations of zinc stress

[0066]

[0067] Table 1-4 Effects of brassinolide and gibberellin on the vitality index of alfalfa under different concentrations of zinc stress

[0068]

[0069] Table 1-5 Effects of brassinolide and gibberellin on root length (cm) of alfalfa under different concentrations of zinc stress

[0070]

[0071]

[0072] Table 1-6 Effects of brassinolide and gibberellin on alfalfa biomass (mg) under different concentrations of zinc stress

[0073]

[0074] The data results of germination indexes and growth indexes in Table 1-1 to Table 1-6 show that: with the increasing concentration of zinc stress, the inhibition of alfalfa germination and growth is stronger. Under 300g L-1 and 450g L-1 zinc stress, the germination and vitality of alfalfa seeds were significantly inhibited, and the root length and biomass accumulation were significantly reduced. The exogenous synergistic application of 24-epibrassinolide and gibberellin in a reasonable ratio significantly increased the germination rate, germination potential, germination index, vitality index and root length of alfalfa; and under the same concentration of hormone treatment, the promoting effect of brassinolide was slightly higher than that of gibberellin; when 24-epibrassinolide and gibberellin were applied at the same time, the germination promotion effect on alfalfa seeds was the best. 24-Epibrassinolide and gibberellin also significantly promoted the root length and biomass of alfalfa, and significantly promoted root elongation under 300g L-1 and 450g L-1 zinc stress. Figure 1 It can also be seen that the growth of alfalfa under the treatment of exogenous simultaneous application of 24-epibrassinolide and gibberellin is significantly better than that under zinc stress. This method shows that brassinolide and gibberellin can significantly improve the germination rate and germination potential of alfalfa, promote root growth, and enhance the zinc tolerance of alfalfa seeds and seedlings. It is conducive to expanding its application to the remediation of zinc-contaminated soil and plant cultivation.

[0075] Example 2

[0076] A method for using 24-epibrassinolide and gibberellin to synergistically promote alfalfa seed germination and root growth under zinc stress comprises:

[0077] (1) Soak alfalfa seeds in disinfected water containing 1% available chlorine for 30 minutes, then take out the alfalfa seeds and rinse them twice with sterile water;

[0078] (2) The alfalfa seeds treated in step (1) are cultured in a 0.3% water agar medium for 10 days, wherein the Zn2+ concentration in the water agar medium is 150 g L-1 to 450 g L-1, the hormone concentration is 0.1 mmol L-1, the hormone types are 24-epibrassinolide and gibberellin, and the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = 0.5:0.8, the concentration of 24-epibrassinolide is 8 to 12 mmol L-1, and the concentration of gibberellin is 8 to 120 mmol L-1.

[0079] The seeds were evenly spaced and plated on water agar medium for cultivation, and the culture dishes were placed in an artificial climate chamber.

[0080] When the seeds are evenly spaced and spread on water agar medium for cultivation, the interval between adjacent seeds is 2.5 to 3 cm.

[0081] The alfalfa seeds were cultured under a day-night photoperiod using an artificial climate chamber, specifically: firstly, they were cultured under light for 16 hours with the temperature kept at 24°C, and then they were cultured without light for 8 hours with the temperature kept at 23°C.

[0082] The disinfectant in step (1) is a chemically pure sodium hypochlorite solution having an effective chlorine content of 10%, which is diluted 3.33 times before use.

[0083] In step (2), 10 mmol L-1 mother solutions of 24-epibrassinolide and gibberellin are prepared respectively. 4.78 mg of 24-epibrassinolide powder is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml; 3.32 mg of gibberellin is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml. The prepared mother solutions of the two hormones are stored in a -20°C refrigerator for standby use. During the experimental treatment, 10 ul of the two hormone mother solutions are respectively taken and added to 1 L of water agar medium and mixed. At this time, the hormone concentration of the medium is the final treatment concentration of 0.1 mmol L-1.

[0084] Prepare the corresponding water agar medium in step (2) and add 40 ml to each 10×10 cm culture dish.

[0085] In step (3), the number of seeds that have germinated is counted every 24 hours, and the germination standard is the radicle or plumule breaking through the seed coat and extending 5 mm.

[0086] At the same time, parallel control experiments were conducted without adding any hormones, adding only 24-epibrassinolide, and adding only gibberellin. Plant samples were collected at the end of the experiment on the 10th day, and fresh weight and root length were measured. Statistics and calculations were performed according to the previously recorded calculation methods for germination rate, germination potential, germination index, and vitality index.

[0087] Example 2 Experimental data statistics table

[0088] Table 2-1 Effects of brassinolide and gibberellin on germination rate of alfalfa under different concentrations of zinc stress (%)

[0089]

[0090] Table 2-2 Effects of brassinolide and gibberellin on germination potential of alfalfa under different concentrations of zinc stress (%)

[0091]

[0092] Table 2-3 Effects of brassinolide and gibberellin on germination index of alfalfa under different concentrations of zinc stress

[0093]

[0094] Table 2-4 Effects of brassinolide and gibberellin on the vitality index of alfalfa under different concentrations of zinc stress

[0095]

[0096] Table 2-5 Effects of brassinolide and gibberellin on root length (cm) of alfalfa under different concentrations of zinc stress

[0097]

[0098] Table 2-6 Effects of brassinolide and gibberellin on alfalfa biomass (mg) under different concentrations of zinc stress

[0099]

[0100] As can be seen from the table above, the specific method adopted in Example 2 has a lower germination rate, germination potential, germination index and vitality index of alfalfa seeds than the specific method adopted in Example 1, indicating that the parameter setting of Example 1 is more reasonable. However, it can be seen from the comparison of the experimental results of Example 2 that the synergistic effect of the two hormones has a good promoting effect on the seed germination and root growth of alfalfa seeds under zinc stress, and the promoting effect is more obvious compared with the promotion of a single hormone or no hormone promotion at all.

[0101] Example 3

[0102] A method for using 24-epibrassinolide and gibberellin to synergistically promote alfalfa seed germination and root growth under zinc stress comprises:

[0103] (1) Soaking alfalfa seeds in disinfected water containing 5% available chlorine for 10 minutes, then taking out the alfalfa seeds and rinsing them with sterile water for 7 times;

[0104] (2) The alfalfa seeds treated in step (1) are cultured in a 1.5% water agar medium for 6 days, wherein the Zn2+ concentration in the water agar medium is 150 g L-1 to 450 g L-1, the hormone concentration is 0.1 mmol L-1, the hormone types are 24-epibrassinolide and gibberellin, and the mixed volume ratio of 24-epibrassinolide and gibberellin is 24-epibrassinolide: gibberellin = 1.3:12, the concentration of 24-epibrassinolide is 8 to 12 mmol L-1, and the concentration of gibberellin is 8 to 120 mmol L-1.

[0105] The seeds were evenly spaced and plated on water agar medium for cultivation, and the culture dishes were placed in an artificial climate chamber.

[0106] When the seeds are evenly spaced and spread on water agar medium for cultivation, the interval between adjacent seeds is 2.5 to 3 cm.

[0107] The alfalfa seeds were cultured under a day-night photoperiod using an artificial climate chamber, specifically: firstly, they were cultured under light for 16 hours with the temperature kept at 23°C, and then they were cultured without light for 8 hours with the temperature kept at 21°C.

[0108] The disinfectant in step (1) is a chemically pure sodium hypochlorite solution having an effective chlorine content of 10%, which is diluted 3.33 times before use.

[0109] In step (2), 10 mmol L-1 mother solutions of 24-epibrassinolide and gibberellin are prepared respectively. 4.78 mg of 24-epibrassinolide powder is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml; 3.32 mg of gibberellin is weighed and dissolved in 500 ul of anhydrous ethanol, and then distilled water is added to dilute to 1 ml. The prepared mother solutions of the two hormones are stored in a -20°C refrigerator for standby use. During the experimental treatment, 10 ul of the two hormone mother solutions are respectively taken and added to 1 L of water agar medium and mixed. At this time, the hormone concentration of the medium is the final treatment concentration of 0.1 mmol L-1.

[0110] Prepare the corresponding water agar medium in step (2) and add 40 ml to each 10×10 cm culture dish.

[0111] In step (3), the number of seeds that have germinated is counted every 24 hours, and the germination standard is the radicle or plumule breaking through the seed coat and extending 5 mm.

[0112] At the same time, parallel control experiments were carried out without adding any hormones, adding only 24-epibrassinolide, and adding only gibberellin.

[0113] At the end of the experiment on the 6th day, plant samples were collected to measure fresh weight and root length, and statistics and calculations were performed according to the previously recorded calculation methods for germination rate, germination potential, germination index, and vitality index.

[0114] Example 3 Experimental data statistics table

[0115] Table 3-1 Effects of brassinolide and gibberellin on germination rate of alfalfa under different concentrations of zinc stress (%)

[0116]

[0117] Table 3-2 Effects of brassinolide and gibberellin on germination potential of alfalfa under different concentrations of zinc stress (%)

[0118]

[0119] Table 3-3 Effects of brassinolide and gibberellin on germination index of alfalfa under different concentrations of zinc stress

[0120]

[0121] Table 3-4 Effects of brassinolide and gibberellin on the vitality index of alfalfa under different concentrations of zinc stress

[0122]

[0123] Table 3-5 Effects of brassinolide and gibberellin on root length (cm) of alfalfa under different concentrations of zinc stress

[0124]

[0125] Table 3-6 Effects of brassinolide and gibberellin on alfalfa biomass (mg) under different concentrations of zinc stress

[0126]

[0127] As can be seen from the above table, the specific method adopted in Example 3 has a slightly lower germination rate, germination potential, germination index, and vitality index of alfalfa seeds than the specific method adopted in Example 1, but is better than the effect shown in the method of Example 2, indicating that the parameter setting of Example 1 is more reasonable, followed by Example 3. However, even if the effect of the method of Example 3 is not as good as that of the method shown in Example 1, it can still be seen that the synergistic effect of the two hormones has a good promoting effect on the seed germination and root growth of alfalfa seeds under zinc stress, and the promoting effect is obvious compared with the promotion of a single hormone or no hormone promotion at all.

[0128] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin, characterized in that: The processing steps include: (1) Soak alfalfa seeds in disinfected water containing 3% available chlorine for 20 minutes, then take out the alfalfa seeds and rinse them with sterile water for 5 times; (2) Using 0.8% water agar medium to culture the alfalfa seeds treated in step (1) for 8 days, wherein the water agar medium contains Zn 2+ Concentration: 150 g·L -1 ~450g·L -1 The hormones are 24-epibrassinolide and gibberellin; the method of adding the hormones is: preparing 10 mmol·L of 24-epibrassinolide and gibberellin respectively. -1 Weigh 4.78 mg of 24-epibrassinolide powder and dissolve it in 500 ul of anhydrous ethanol, then add distilled water to dilute to 1 ml; weigh 3.32 mg of gibberellin and dissolve it in 500 ul of anhydrous ethanol, then add distilled water to dilute to 1 ml; store the prepared mother solutions of the two hormones in a -20 ℃ refrigerator for later use; take 10 ul of the two hormone mother solutions and add them to 1 L of water agar medium and mix well. At this time, the final treatment concentrations of the two hormones in the medium are 0.1 μmol·L -1 .

2. The method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin according to claim 1, characterized in that: The seeds were evenly spaced and plated on water agar medium for cultivation, and the culture dishes were placed in an artificial climate chamber.

3. The method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin according to claim 2, characterized in that: When the seeds are evenly spaced and spread on water agar medium for cultivation, the interval between adjacent seeds is 2.5 cm to 3 cm.

4. The method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin according to claim 2, characterized in that: The alfalfa seeds were cultured under a day-night photoperiod using an artificial climate chamber, specifically: firstly, they were cultured under light for 16 hours with the temperature kept at 25°C, and then they were cultured without light for 8 hours with the temperature kept at 22°C.

5. The method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin according to claim 1, characterized in that: The disinfectant in step (1) is a chemically pure sodium hypochlorite solution having an effective chlorine content of 10%, which is diluted 3.33 times before use.

6. The method for promoting alfalfa seed germination and root growth under zinc stress by using 24-epibrassinolide and gibberellin according to claim 1, characterized in that: The gibberellin is specifically at least one of gibberellin A1, gibberellin A4 and gibberellin A7.

7. Use of the method for promoting alfalfa seed germination and root growth under zinc stress using 24-epibrassinolide and gibberellin according to any one of claims 1 to 6 in remediating zinc-contaminated soil using alfalfa.

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