Use of spermine to promote germination and seedling growth under drought conditions and methods

Abstract

The application provides application and methods of spermine in promoting germination and seedling growth of plants under drought conditions, and relates to the technical field of plant cultivation. The application of spermine and the method thereof provided by the application are simple in operation, can quickly and effectively promote the germination of alfalfa seeds and the growth of seedlings under drought stress, and are beneficial to popularization in the planting and production of alfalfa in arid areas.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, and in particular relates to the application and method of spermine in promoting plant germination and seedling growth under drought conditions. Background Technology

[0002] Drought is one of the major abiotic stresses affecting plant growth and development, posing a severe challenge to agricultural development. Alfalfa (Medicago sativa L.) is one of the most widely cultivated forage grasses in the world, possessing advantages such as rich nutrition, strong adaptability, and high yield. However, increasingly severe drought conditions pose a serious threat to the planting area and yield of alfalfa, especially affecting seed germination.

[0003] Polyamines (PAs) are bioactive, low-molecular-weight aliphatic nitrogenous bases produced during biological metabolism. Common PAs in plants include putrescine (Put), spermidine (Spd), and spermine (Spm). The promoting effects of polyamines on seed germination have been widely reported. However, current technology still lacks more in-depth methods for promoting plant germination and seedling growth under drought conditions. Summary of the Invention

[0004] In view of this, the present invention provides the application and method of spermine in promoting plant germination and seedling growth under drought conditions, so as to improve the stress resistance of alfalfa seeds under drought stress.

[0005] This invention provides the application of spermine in promoting seed germination in plants under drought conditions.

[0006] The present invention also provides the application of spermine in promoting seedling growth of plants under drought conditions.

[0007] In this invention, the plant includes alfalfa.

[0008] The present invention provides a formulation for promoting seed germination and seedling growth of plants under drought conditions, the formulation comprising an aqueous solution of spermine.

[0009] In this invention, the concentration of the spermine aqueous solution is 0.1 mM to 1.0 mM.

[0010] The present invention also provides a method for promoting seed germination and seedling growth of plants under drought conditions, the method comprising the following steps:

[0011] (1) Disinfect the seeds of the plant to obtain disinfected plant seeds; the disinfection process in step (1) includes: soaking, rinsing and drying the seeds of the plant in a 75% ethanol aqueous solution in sequence.

[0012] Furthermore, the present invention preferably selects plump, uniformly sized alfalfa seeds free from disease and pest damage, disinfects them with a 75% ethanol aqueous solution for 10 minutes, rinses them with deionized water, and then uses filter paper to absorb the moisture from the seed surface.

[0013] (2) The sterilized plant seeds obtained in step (1) are soaked in a spermine aqueous solution to obtain plant seeds that have undergone soaking treatment; the concentration of the spermine aqueous solution in step (2) is 0.1 mM to 1.0 mM; the soaking time is 10 h; the soaking environment is a dark environment without light;

[0014] In this invention, the sterilized seeds are preferably placed in beakers containing aqueous solutions of spermine at concentrations of 0.1 mM, 0.5 mM, and 1.0 mM.

[0015] (3) Rinse and dry the soaked plant seeds obtained in step (2);

[0016] In this invention, rinsing is preferably done quickly with deionized water, and drying is preferably done by absorbing the moisture from the seed surface with filter paper.

[0017] Beneficial effects:

[0018] 1. Soaking seeds in a solution of spermine (Spm) can effectively improve the germination rate, germination potential, germination index, vitality index, radicle length, plumule length, root-to-bud ratio, and fresh weight of alfalfa seeds under drought stress.

[0019] 2. The seed treatment method for plants provided by this invention is simple to operate and has a straightforward procedure. It can quickly and effectively promote the germination of alfalfa seeds and the growth of seedlings under drought stress, especially for weakly drought-resistant alfalfa seeds. This method can be promoted and applied to improve the drought resistance of alfalfa seeds in actual planting and production. Detailed Implementation

[0020] 1. Materials and Methods

[0021] 1.1 Materials

[0022] 1.1.1 Alfalfa materials

[0023] The materials were two different drought-resistant alfalfa varieties: the highly drought-resistant Longzhong alfalfa (Medicago sativa L.cv. Longzhong, hereinafter referred to as Longzhong) and the weakly drought-resistant Gannong No.3 alfalfa (Medicago sativa L.cv. Gannong No.3, hereinafter referred to as Gannong No.3).

[0024] 1.1.2 Reagents

[0025] Polyethylene glycol (hereinafter referred to as PEG-6000) and Spm were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0026] 1.2 Methods

[0027] (1) Select plump, uniformly sized alfalfa seeds that are free from disease and pest damage. Disinfect them with a 70% ethanol aqueous solution for 10 minutes, then rinse them with deionized water, and then use filter paper to absorb the moisture from the seed surface.

[0028] (2) The present invention sets three Spm soaking concentrations of 0.1mM, 0.5mM and 1.0mM.

[0029] Seeds of two varieties of alfalfa were soaked in 100 mL each of distilled water, 0.1 mM, 0.5 mM and 1.0 mM SpM solutions, respectively. After being placed in the dark for 10 hours, the seeds were taken out, rinsed quickly with deionized water, and the moisture on the seed surface was absorbed with filter paper.

[0030] (3) Place the treated seeds in a 9cm diameter petri dish lined with two layers of filter paper, with 25 seeds per dish.

[0031] (4) Studies on the effects of exogenous substances on plant seed germination under drought stress are generally conducted using semi-lethal drought stress concentrations.

[0032] When the PEG-6000 gradient was -0.6 MPa, the germination rate of both varieties was approximately 50% of the control. Furthermore, a PEG-6000 stress of -1.2 MPa was a sensitive stress water potential for distinguishing the differences in growth and physiological responses of Gannong 3 and Longzhong alfalfa seedlings under drought stress. The above two representative PEG-6000 gradients were established.

[0033] Therefore, nine treatments were set up for each of the two varieties, as follows:

[0034] 1) CK: Alfalfa seeds soaked in distilled water are placed on a paper bed moistened with distilled water and marked as group 1;

[0035] 2) -0.6MPa: Alfalfa seeds soaked in distilled water were placed on a paper bed moistened with 7mL of -0.6MPa PEG-6000 solution and marked as 2 groups;

[0036] 3) -0.6MPa + 0.1mM Spm: Alfalfa seeds soaked in 0.1.0mM Spm were placed on a paper bed moistened with 7mL of -0.6MPa PEG-6000 solution and labeled as 3 groups;

[0037] 4) -0.6MPa + 0.5mM Spm: Alfalfa seeds soaked in 0.5mM Spm were placed on a paper bed moistened with 7mL of -0.6MPa PEG-6000 solution and labeled as 4 groups;

[0038] 5) -0.6MPa + 1.0mM Spm: Alfalfa seeds soaked in 0.5mM Spm were placed on a paper bed moistened with 7mL of -0.6MPa PEG-6000 solution and labeled as 5 groups;

[0039] 6) -1.2MPa: Alfalfa seeds soaked in distilled water were placed on a paper bed moistened with 7mL of -1.2MPa PEG-6000 solution and marked as 6 groups;

[0040] 7) -1.2MPa + 0.1mM Spm: Alfalfa seeds soaked in 0.1mM Spm were placed on a paper bed moistened with 7mL of -1.2MPa PEG-6000 solution and labeled as 7 groups;

[0041] 8) -1.2MPa + 0.5mM Spm: Alfalfa seeds soaked in 0.5mM Spm were placed on a paper bed moistened with 7mL of -1.2MPa PEG-6000 solution and labeled as 8 groups;

[0042] (9) -1.2MPa+1.0mM Spm: Alfalfa seeds soaked in 1.0mM Spm were placed on a paper bed moistened with 7mL of -1.2MPa PEG-6000 solution and labeled as 9 groups.

[0043] Each treatment was repeated in triplicate. The treatments were placed in a constant temperature incubator at 25°C with 12 hours of light and 12 hours of darkness, and the lost moisture was replenished daily by weighing.

[0044] 2 Results

[0045] 2.1 Effects of Spm soaking on germination potential, germination rate and germination index of alfalfa seeds under drought stress

[0046] Under drought stress, the germination potential, germination rate, and germination index of two different drought-resistant alfalfa varieties all decreased. Among them, the germination potential, germination rate, and germination index of Longzhong alfalfa seeds decreased less than those of Gannong No. 3 compared with the control group. Details are shown in Table 1.

[0047] At -0.6 MPa, Spm soaking treatment had little effect on the germination potential of the Longzhong variety. However, different concentrations of Spm soaking treatment significantly increased the germination potential of the Gannong 3 variety. The germination potential of Gannong 3 under 1.0 mM Spm soaking treatment increased by 779.96% compared with simple drought stress (p<0.05). At -1.2 MPa, the germination potential of both varieties was 0 regardless of whether they were treated with Spm.

[0048] Compared with drought stress alone, the Spm soaking treatment improved the germination rate of two different drought-resistant alfalfa seeds. At -0.6 MPa, the germination rate of the Longzhong variety only showed a significant increase of 28.21% (p<0.05) under the 1.0 mM Spm soaking treatment compared to drought stress alone, while the germination rate of the Gannong 3 variety significantly increased under Spm soaking treatment, with a 61.54% increase (p<0.05) under the 1 mM Spm soaking treatment compared to drought stress alone. At -1.2 MPa, the germination rate of the Longzhong variety only showed a significant increase of 90.02% (p<0.05) under the 0.5 mM Spm soaking treatment compared to drought stress alone, while the germination rate of the Gannong 3 variety significantly increased under Spm soaking treatment, with a 424.84% increase (p<0.05) under the 0.5 mM Spm soaking treatment compared to drought stress alone.

[0049] Compared with drought stress alone, the Spm soaking treatment improved the germination index of two different drought-resistant alfalfa seeds. At -0.6 MPa, the germination index of the Longzhong variety increased significantly by 25.54% under the 1.0 mM Spm soaking treatment compared with drought stress alone (p<0.05), while the germination index of the Gannong 3 variety increased significantly under the Spm soaking treatment, with an increase of 131.92% under the 1.0 mM Spm soaking treatment compared with drought stress alone (p<0.05). At -1.2 MPa, the germination index of the Longzhong variety increased significantly by 117.96% under the 0.5 mM Spm soaking treatment compared with drought stress alone (p<0.05), while the germination index of the Gannong 3 variety increased significantly under the Spm soaking treatment, with an increase of 899.74% under the 0.5 mM Spm soaking treatment compared with drought stress alone (p<0.05).

[0050] The above results indicate that Spm soaking treatment can improve the germination potential, germination rate, and germination index of alfalfa. At -0.6 MPa, the optimal Spm soaking concentration for improving the germination potential, germination rate, and germination index of alfalfa is 1.0 mM; at -1.2 MPa, the optimal concentration is 0.5 mM.

[0051] Table 1

[0052]

[0053]

[0054] 2.2 Effects of Spm seed soaking on alfalfa seed vigor index under drought stress

[0055] Under drought stress, the vigor index of both alfalfa seeds decreased significantly. Compared with Longzhong, drought stress had a greater inhibitory effect on the vigor index of Gannong 3 seeds, as shown in Table 2.

[0056] At -0.6 MPa, the vigor index of the Longzhong variety increased significantly by 58.94% (p<0.05) only under the 1.0 mM Spm seed soaking treatment compared to the drought stress treatment alone. The germination index of the Gannong 3 variety increased significantly under both Spm and 1.0 mM Spm treatments, with an increase of 321.16% (p<0.05) compared to the drought stress treatment alone. At -1.2 MPa, the vigor index of the Longzhong variety increased significantly by 468.72% (p<0.05) only under the 0.5 mM Spm seed soaking treatment compared to the drought stress treatment alone. The vigor index of the Gannong 3 variety increased significantly under both the 0.1 mM and 0.5 mM Spm treatments compared to the drought stress treatment alone (p<0.05), with increases of 4655.91% and 4459.75%, respectively.

[0057] These results indicate that at -0.6 MPa, the optimal concentration of Spm for improving the vigor index of alfalfa is 1.0 mM; and at -1.2 MPa, the optimal concentration is 0.5 mM.

[0058] Table 2

[0059] deal with Longzhong Vitality Index Gannong No. 3 Vitality Index Group 1 219.06±2.82a 253.1±20.91a 2 groups 35.17±2.05c 33.55±2.16d 3 groups 35.25±8.89c 125.04±19.57b 4 groups 23.58±4.04d 97.8±12.42c 5 groups 55.9±7.22b 141.26±12.51b 6 groups 2.21±0.24f 0.67±0.03e 7 groups 8.62±1.78ef 31.76±6.69d 8 groups 12.58±1.9e 30.45±3.37d 9 groups 5.15±0.75ef 6.42±2.96e

[0060] 2.3 Effects of Spm seed soaking on seed plumule length, radicle length, total seedling length, and root-to-bud ratio of alfalfa under drought stress

[0061] Drought stress inhibited the radicle length, plumule length, and total seedling length of two different drought-resistant alfalfa varieties. Compared with the drought-resistant Longzhong variety, drought stress had a stronger inhibitory effect on the radicle length, plumule length, and total seedling length of Gannong 3, as detailed in Table 3.

[0062] Compared with drought stress alone, Spm soaking treatment increased the radicle length of alfalfa seeds from two different drought-resistant varieties. At -0.6 MPa, the radicle length of the Longzhong variety increased significantly by 33.72% (p<0.05) and 28.97% (p<0.05) under 0.1 mM Spm and 1 mM Spm treatments compared to drought stress alone. Similarly, the radicle length of the Gannong 3 variety increased significantly by 82.06% (p<0.05) and 75.80% (p<0.05) under 0.1 mM Spm and 1 mM Spm treatments compared to drought stress alone. At -1.2 MPa, the radicle length of both varieties increased significantly under different concentrations of Spm soaking treatment compared to drought stress alone, with 0.1 mM Spm soaking treatment showing the best promoting effect on alfalfa seed radicle length. The radicle length of Longzhong and Gannong 3 alfalfa varieties was significantly increased by 231.38% (p<0.05) and 593.83% (p<0.05) respectively under the 0.1 mM Spm treatment compared to the drought stress treatment alone. Compared to the drought stress treatment alone, the Spm soaking treatment increased the plumule length of seeds from two different drought-resistant alfalfa varieties. At -0.6 MPa, the plumule length of the Longzhong variety was not significantly different under the Spm soaking treatment compared to the drought stress treatment alone, while the plumule length of the Gannong 3 variety was significantly increased by 91.31% (p<0.05) and 127.55% (p<0.05) respectively under the 0.5 mM Spm and 1.0 mM Spm soaking treatments compared to the drought stress treatment alone. At -1.2 MPa, the plumule length of *Alfalfa* var. *longensis* significantly increased by 64.29% (p<0.05) and 71.45% (p<0.05) under 0.1 mM Spm and 1.0 mM Spm soaking treatments compared to drought stress alone. The plumule length of *Alfalfa* var. *ganong* var. *longensis* significantly increased by 78.61% (p<0.05) and 107.16% (p<0.05) under 0.1 mM Spm and 0.5 mM Spm soaking treatments compared to drought stress alone. Compared to drought stress alone, Spm soaking treatment increased the total seedling length of two different drought-resistant alfalfa varieties.

[0063] At -0.6 MPa, the total seedling length of the Longzhong variety increased significantly by 30.14% (p<0.05) and 25.92% (p<0.05) under 0.1 mM Spm and 1.0 mM Spm soaking treatments, respectively, compared to the drought stress treatment alone. Similarly, the total seedling length of the Gannong 3 variety increased significantly (p<0.05) under 0.1 mM Spm and 1.0 mM Spm soaking treatments, respectively, compared to the drought stress treatment alone, with increases of 77.26% and 80.92%, respectively. At -1.2 MPa, the total seedling length of both Longzhong and Gannong 3 varieties increased significantly (p<0.05) under Spm soaking treatments, with the largest increases observed under 0.1 mM Spm soaking treatment, with increases of 207.17% and 455.09%, respectively.

[0064] The root-to-shoot ratio of alfalfa seedlings in both Longzhong and Gannong 3 varieties was significantly increased compared to the control (CK) under a drought stress treatment of -0.6 MPa (p<0.05), with increases of 52.48% and 51.13%, respectively. At -0.6 MPa, the root-to-shoot ratio of Longzhong seedlings was significantly increased under 0.1 mM Spm and 1.0 mM Spm treatments compared to the drought stress treatment alone (p<0.05), with increases of 42.01% and 34.96%, respectively. The root-to-shoot ratio of Gannong 3 seedlings was significantly increased by 36.65% under the 0.1 mM Spm treatment compared to the drought stress treatment alone (p<0.05). The root-to-shoot ratio of alfalfa seedlings in Longzhong showed no significant difference compared to the CK under a drought stress treatment of -1.2 MPa, while the root-to-shoot ratio of alfalfa seedlings in Gannong 3 was significantly decreased by 54.59% compared to the CK under the -1.2 MPa drought stress treatment (p<0.05). At -1.2 MPa, the root-to-shoot ratio of seedlings in Longzhong was significantly increased by 98.29% (p<0.05) and 88.06% (p<0.05) under 0.1 mM Spm and 0.5 mM Spm treatments, respectively, compared with the drought stress treatment alone. The root-to-shoot ratio of seedlings of Gannong No. 3 was significantly increased under different concentrations of Spm seed soaking treatments compared with the drought stress treatment alone (p<0.05), and increased by 285.57% under 0.1 mM Spm seed soaking treatment compared with the drought stress treatment alone.

[0065] The above results indicate that Spm soaking can increase the radicle length, plumule length, total seedling length and root-to-bud ratio of alfalfa under drought stress, with 0.1 mM Spm soaking treatment being more beneficial to the growth of alfalfa radicle under drought stress.

[0066] Table 3

[0067]

[0068]

[0069] 2.4 Effects of Spm seed soaking on the fresh weight of alfalfa seedlings under drought stress

[0070] Drought stress led to a decrease in the fresh weight of individual alfalfa seedlings of two different drought-resistant varieties. The decrease in the fresh weight of individual seedlings of Gannong 3 under drought stress was greater than that of the Longzhong variety, as detailed in Table 4.

[0071] Spm soaking treatment can increase the fresh weight of alfalfa seedlings. At -0.6 MPa, the fresh weight of seedlings of Longzhong variety and Gannong 3 was significantly higher under 1.0 mM Spm treatment than under drought stress alone (p<0.05), with increases of 15.24% and 38.32%, respectively. At -1.2 MPa, the fresh weight of seedlings of both varieties was significantly increased under different concentrations of Spm soaking treatment compared to drought stress alone (p<0.05). Under 0.1 mM Spm soaking treatment, the fresh weight of seedlings of Longzhong and Gannong 3 increased significantly by 179.37% and 183.67%, respectively, compared to drought stress alone.

[0072] The results above indicate that at -0.6 MPa, a seed soaking concentration of 1.0 mM Spm is more beneficial for increasing the fresh weight of individual seedlings of both varieties; and at -1.2 MPa, a seed soaking concentration of 0.1 mM Spm is more beneficial for increasing the fresh weight of individual seedlings of both varieties.

[0073] Table 4

[0074] deal with Fresh weight of single seedling in Longzhong Fresh weight of single seedling of Gannong No. 3 Group 1 0.03±0a 0.03±0a 2 groups 0.01±0cd 0.02±0c 3 groups 0.02±0bc 0.02±0c 4 groups 0.01±0de 0.02±0c 5 groups 0.02±0b 0.02±0b 6 groups 0.01±0g 0.01±0f 7 groups 0.01±0cd 0.02±0c 8 groups 0.01±0ef 0.02±0c 9 groups 0.01±0f 0.01±0d

[0075] A comprehensive evaluation of the effects of 2.5 Spm seed soaking on alfalfa seed germination and seedling growth under drought stress.

[0076] Because the Spm soaking concentrations required to improve various indicators differed, the membership function values ​​of germination rate, germination potential, germination index, radicle length, plumule length, root-to-shoot ratio, seedling fresh weight, and vigor index of Longzhong and Gannong No. 3 alfalfa seeds under different treatments were calculated. The seeds were then ranked according to the average value of the membership function to analyze the strength of the effect of different Spm soaking concentrations on promoting alfalfa germination and seedling growth under drought stress (see Tables 5 and 6). The results showed that at -0.6 MPa, the Spm soaking concentration ranking for both alfalfa seeds was: 1.0 mM > 0.1 mM > 0.5 mM; while at -1.2 MPa, the ranking was: 0.1 mM > 0.5 mM > 1 mM. Under drought stress, the membership function values ​​of all Spm soaking treatments were greater than those of the simple drought stress treatment. Furthermore, the membership function values ​​of Gannong 3 under all Spm soaking treatments were greater than those of Longzhong under the corresponding Spm soaking treatments. These results indicate that Spm soaking can improve the drought resistance of alfalfa during germination, and the improvement is more significant for the weakly drought-resistant Gannong 3 alfalfa seeds. Meanwhile, the optimal Spm soaking concentration varies under different drought stress levels. To screen the most suitable Spm soaking concentration for improving the drought resistance of alfalfa seeds during germination, the average membership function values ​​of various alfalfa indicators under the same Spm soaking concentration at different drought stress levels were calculated. The results showed that the membership function values ​​of both alfalfa varieties were highest under the 0.1 mM Spm soaking treatment. This indicates that a 0.1 mM Spm soaking concentration is the optimal concentration for promoting alfalfa seed germination and seedling growth.

[0077] Table 5. Membership function values ​​of different concentrations of Spm seed soaking treatments to alleviate different drought stresses in alfalfa seeds in Longzhong.

[0078]

[0079] Table 6. Membership function values ​​of different concentrations of Spm seed soaking treatments to alleviate different drought stresses for Gannong No. 3 alfalfa seeds and seedling growth.

[0080]

[0081]

[0082] In summary, Spm soaking can improve the germination rate, germination potential, germination index, and vigor index of alfalfa seeds under drought stress, and promote the accumulation of radicle length, plumule length, root-to-bud ratio, and fresh weight per plant in seedlings, thereby alleviating the inhibitory effect of drought stress on alfalfa seed germination and seedling growth. Compared with drought-resistant varieties, Spm soaking has a better alleviating effect on drought-sensitive varieties. Furthermore, the optimal Spm soaking concentration varies under different drought stress gradients, but overall, a 0.1 mM Spm soaking concentration is the optimal concentration for improving drought resistance during the germination period of alfalfa seeds. Therefore, it is recommended to use 0.1 mM Spm for seed soaking before sowing alfalfa seeds in arid areas, which can effectively promote seed germination and seedling growth.

[0083] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. Any variations and improvements made to the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of spermine in promoting seed germination and / or seedling growth in plants under drought conditions; Its features are, The plant in question is alfalfa; In the aforementioned application, the preparation of spermine is an aqueous solution of spermine, and the concentration of the aqueous solution of spermine is 0.1 mM. The method of application is as follows: (1) Disinfect the seeds of plants to obtain disinfected plant seeds; (2) Soak the sterilized plant seeds obtained in step (1) in an aqueous solution of spermine to obtain plant seeds that have undergone soaking treatment. (3) Rinse and dry the plant seeds obtained in step (2) after soaking.

2. The application according to claim 1, characterized in that, The disinfection process in step (1) includes soaking, rinsing and drying the plant seeds in a 75% ethanol aqueous solution.

3. The application according to claim 1, characterized in that, The concentration of the spermine aqueous solution in step (2) is 0.1 mM; The soaking time is 10 hours; The immersion environment is a dark environment without light.

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