A method for rapidly improving the salt tolerance of salt-sensitive rice

Abstract

A method for rapidly improving the salt tolerance of salt-sensitive rice belongs to the field of rice planting technology. The steps are as follows: Step 1. Dissolve DL-piperidinic acid with a 3-10 wt.‰ NaCl solution to prepare a DL-piperidinic acid solution with a concentration of 50-400 mg / L, and then disinfect the surface of the rice seeds; Step 2. Soak the disinfected rice seeds in the DL-piperidinic acid solution at a temperature of 20-30°C for 16-24 h to obtain treated rice seeds, so that they germinate and grow normally under a salt stress environment. The present invention utilizes exogenous DL-piperidinic acid to soak rice seeds and irrigate them, which can improve the germination rate, root length and growth rate of salt-sensitive rice seeds under salt stress conditions, improve the salt tolerance of salt-sensitive rice, meet the urgent demand of my country's coastal mudflats for high-yield, high-quality, disease-resistant, salt-tolerant, direct-seeding rice varieties, and improve saline-alkali land, with high application potential and value.

Description

Technical Field

[0001] The invention relates to the technical field of rice planting, and in particular to a method for rapidly improving the salt tolerance of salt-sensitive rice. Background Art

[0002] Soil salinization affects seed germination and restricts crop growth and development, making it the second-leading abiotic stress contributing to yield reductions. Given the pressures of a growing population, shrinking arable land, and severe freshwater shortages, the rational development and utilization of saline-alkali land is a critical issue worldwide.

[0003] Planting rice in saline-alkali soils not only expands the area of grain crops but also improves saline-alkali land. Rice is a moderately salt-sensitive crop, and its seed germination and seedling stages are important stages in its life cycle. It is highly sensitive to salt stress. Excessive salt in the soil can cause salt damage symptoms such as low germination rate and uneven seedling emergence during seed germination. Previous research has focused on improving rice salt tolerance, and nearly a thousand QTLs associated with salt tolerance have been reported for different growth stages of rice. However, only the KCL1 and HST1 genes have been positionally cloned, and Saltol from Pokkali has been precisely mapped. Furthermore, only the Saltol salt-tolerance gene has been successfully used for molecular improvement of rice salt tolerance. Therefore, using molecular marker-assisted selection to introduce salt-tolerance genes into salt-sensitive rice to improve its salt tolerance has the disadvantages of a long breeding cycle and difficulty in application.

[0004] Under salt stress, rice undergoes complex physiological changes to combat salt damage, accumulating numerous metabolites. For example, proline levels are increased to regulate osmotic balance during salt stress. The application of metabolomics to studying plant stress has provided new insights into the physiological mechanisms of plant salt tolerance. However, current research has largely focused on alterations in metabolites or metabolic pathways in response to stress. Studies on the metabolomics of rice salt tolerance are limited, particularly those examining how to leverage rice's own metabolites under salt stress to enhance salt tolerance. Summary of the Invention

[0005] Technical problems to be solved: In response to the above technical problems in the prior art, the present invention provides a method for rapidly improving the salt tolerance of salt-sensitive rice. The method is low in cost and can effectively improve the germination rate, root length, and growth rate of salt-sensitive rice under salt stress.

[0006] Technical solution: A method for rapidly improving the salt tolerance of salt-sensitive rice, comprising the following steps:

[0007] Step 1. Dissolve DL-2-piperidinic acid in 3-10 wt.‰ NaCl solution to prepare a DL-2-piperidinic acid solution with a concentration of 50-400 mg / L, and then disinfect the surface of the rice seeds;

[0008] Step 2. Soak the sterilized rice seeds in a DL-2-piperidinic acid solution at 20-30° C. for 16-24 hours to obtain treated rice seeds, which are then allowed to germinate and grow normally under a salt stress environment.

[0009] Preferably, the method for rapidly improving the salt tolerance of salt-sensitive rice is specifically a method for rapidly improving the germination rate, root length and growth rate of salt-sensitive rice seeds under salt stress conditions.

[0010] Preferably, the surface disinfection of rice seeds in step 1 is specifically performed as follows: selecting mature rice seeds with plump grains and no mold spots, quickly rinsing them with 70-75 wt.% ethanol solution 2-3 times for 20-40 s each time, rinsing them with sterile water 2-3 times, then sterilizing them with 5-10 wt.% NaClO solution for 5-15 min, rinsing them with sterile water 3-5 times, and finally absorbing water attached to the seed surface with sterile filter paper.

[0011] Preferably, in the step 1, 5 wt.‰ NaCl solution is used to dissolve DL-2-piperidinic acid to prepare a DL-2-piperidinic acid solution with a concentration of 100 mg / L.

[0012] Preferably, in step 2, the salt stress environment is the irrigation water salt stress solution is a mixed solution of DL-2-piperidinic acid and NaCl, wherein the concentration of DL-2-piperidinic acid is 0-400 mg / L and the concentration of NaCl is 3-10 wt.‰.

[0013] Preferably, the method for testing the germination rate, root length and growth rate of rice seeds in step 2 comprises the following steps: placing the rice seeds treated in step 2 in a germination box for a germination test, padding the germination box with two layers of sterile filter paper, placing 100 seeds in each germination box, adding a mixed solution of DL-2-piperidinic acid and NaCl of equal concentration as in the step to keep the filter paper moist, and covering the germination box with a lid, placing the germination box in a dark culture at 20-30° C. for 2-3 days, and then performing light culture at a light intensity of 4000-10000 lx. After 2-4 days of light culture, the germination rate is counted, and 30 seedlings with consistent growth are selected for a seedling growth test, with 3 replicates of the test; transferring the germinated seedlings into a 96-well PCR plate with the bottom cut off, placing one seedling in each well, and culturing in an artificial climate incubator at 25-30° C. and a light intensity of 4000-10000 lx. lx, humidity 60-80%, light culture: dark culture for 16h:8h, then use the same concentration of DL-2-piperidinic acid and NaCl mixed solution for hydroculture, change the hydroponic solution every 3 days, and measure the seedling height, root length and dry weight after 14 days.

[0014] Preferably, when calculating the germination rate, a sum of the root length and the seedling height greater than 0.5 cm is considered to be germination.

[0015] Beneficial Effects: 1) The present invention provides a method for rapidly increasing the germination rate, root length, and growth rate of salt-sensitive rice seeds under salt stress conditions, effectively improving the salt tolerance of salt-sensitive rice. Under no salt stress, the germination rate of Yanshendao 83006 seeds was approximately 100%, indicating that the selected seeds had a high and uniform germination rate, with an average plant height of 6.49 cm and a root length of 9.89 cm. Under 5‰ salt stress, the germination rate dropped sharply to 72.33%, with a plant height of 4.06 cm and a root length of 7.67 cm. Seed soaking treatment with exogenous DL-2-piperidinic acid (100-400 mg / L) significantly improved the germination rate of seeds under 5‰ salt stress. Treatment with 100 mg / L resulted in a germination rate of 87.33%, a plant height of 5.42 cm, and a root length of 12.25 cm.

[0016] 2) The DL-2-piperidinic acid used in this invention is a salt-tolerance-related metabolite discovered in previous research by the inventors. Studies have reported that it is crucial for plant immunity, but there are no reports of its application in plant salt tolerance. This invention utilizes exogenous DL-2-piperidinic acid to enhance the salt tolerance of salt-sensitive rice varieties, potentially addressing the urgent need for high-yield, high-quality, disease-resistant, salt-tolerant, direct-seeded rice varieties on my country's coastal mudflats.

[0017] 3) Compared with the inventor's prior patent application (publication number CN112020930 B), the 5-hydroxy-L-norvaline used in the prior application is difficult to purchase and is expensive, costing approximately RMB 2,000 per 100 mg, making it difficult to apply in large quantities to salt-tolerant rice production. The DL-2-piperidinic acid proposed in the present invention, on the other hand, is inexpensive, costing approximately RMB 200 per 100 grams. Furthermore, DL-2-piperidinic acid has a significant effect on promoting root growth. Under 5‰ salt stress, the root lengths of plants treated with 100-400 mg / L of DL-2-piperidinic acid were significantly longer than those under no salt stress. However, 5-hydroxynorvaline had no significant effect on promoting root growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison of the germination rate of seeds after soaking in 100 mg / L DL-2-piperidinic acid and the untreated control grown under 5‰ salt stress for 7 days. Figure a is the germination of seeds of the untreated control grown under 5‰ salt stress for 7 days, and b is the germination of seeds after soaking in 100 mg / L DL-2-piperidinic acid solution and grown under 5‰ salt stress for 7 days.

[0019] Figure 2This is a comparison of the growth of plants containing 100 mg / L DL-2-piperidinic acid and the control under 5‰ salt stress for 14 days. Figure a shows the phenotype of the untreated control grown under 5‰ salt stress for 14 days, and b shows the phenotype of plants containing 100 mg / L DL-2-piperidinic acid grown under 5‰ salt stress for 14 days. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] In the examples herein, rice (Oryza sativa L.) was selected from Yanshen rice 83006. DL-2-piperidinic acid (CAS No. 4043-87-2) was provided by Sangon Biotech (Shanghai) Co., Ltd., with a content of >97.5%. NaCl was a conventional chemical reagent.

[0022] Example 1

[0023] A method for rapidly improving salt tolerance of salt-sensitive rice, comprising the following steps:

[0024] Step 1. Dissolve DL-2-piperidinic acid in 3 wt.‰ NaCl solution to prepare a 50 mg / L DL-2-piperidinic acid solution. Disinfect the surface of rice seeds as follows: Select mature rice seeds with plump grains and no mold spots. Quickly rinse them twice with 70 wt.% ethanol solution for 20 seconds each, rinse twice with sterile water, sterilize them with 5 wt.% NaClO solution for 5 minutes, rinse three times with sterile water, and finally remove any surface water with sterile filter paper.

[0025] Step 2. Soak rice seeds in the DL-2-piperidinic acid solution prepared in Step 1 at 20°C for 16 hours to obtain treated rice seeds, which are then allowed to germinate and grow normally under salt stress. The salt stress environment is a mixture of DL-2-piperidinic acid and NaCl in the irrigation water, wherein the concentration of DL-2-piperidinic acid is 50 mg / L and the concentration of NaCl is 3 wt.‰.

[0026] The method for testing the germination rate, root length and growth rate of the rice seeds treated in step 2 is as follows: the rice seeds treated in step 2 are placed in a germination box for germination test, two layers of sterile filter paper are padded in the germination box, 100 seeds are placed in each germination box, a mixed solution of DL-2-piperidinic acid and NaCl of the same concentration as in the step is added to keep the filter paper moist, and the germination box is covered with a lid, and the germination box is placed at 20°C for dark culture for 2 days, and then light culture is carried out with a light intensity of 4000 lx, and after 2 days of light culture, the germination rate was counted, and 30 seedlings with consistent growth were selected for a seedling growth test, with 3 replicates. When counting the germination rate, a sum of the root length and the seedling height greater than 0.5 cm was considered to have germinated; the germinated seedlings were transferred to a 96-well PCR plate with the bottom cut off, with one seedling placed in each well, and cultured in an artificial climate incubator under the conditions of 25°C, a light intensity of 4000 lx, and a humidity of 60-80%, with a light culture:dark culture ratio of 16 h:8 h, and then hydroponically cultured with a mixed solution of DL-2-piperidinic acid and NaCl of equal concentrations as in the same step, with the hydroponic solution changed every 3 days. After 14 days, the seedling height, root length, and dry weight were measured.

[0027] Example 2

[0028] A method for rapidly improving salt tolerance of salt-sensitive rice, comprising the following steps:

[0029] Step 1. Dissolve DL-2-piperidinic acid in 10 wt.‰ NaCl solution to prepare a 400 mg / L DL-2-piperidinic acid solution. Disinfect the surface of rice seeds as follows: Select mature rice seeds with plump grains and no mold spots. Rinse them quickly with 75 wt.% ethanol solution three times for 40 seconds each, rinse three times with sterile water, sterilize them with 10 wt.% NaClO solution for 15 minutes, rinse five times with sterile water, and finally remove any surface water with sterile filter paper.

[0030] Step 2. Soak rice seeds in the DL-2-piperidinic acid solution prepared in Step 1 at 30°C for 24 hours to obtain treated rice seeds, which are then allowed to germinate and grow normally under salt stress. The salt stress environment is the irrigation water. The salt stress solution is a mixture of DL-2-piperidinic acid and NaCl, wherein the concentration of DL-2-piperidinic acid is 400 mg / L and the concentration of NaCl is 10 wt.‰.

[0031] The method for testing the germination rate, root length and growth rate of the rice seeds treated in step 2 is as follows: the rice seeds treated in step 2 are placed in a germination box for germination test, two layers of sterile filter paper are padded in the germination box, 100 seeds are placed in each germination box, a mixed solution of DL-2-piperidinic acid and NaCl of the same concentration as in the step is added to keep the filter paper moist, and the germination box is covered with a lid, and the germination box is placed at 30°C for dark culture for 3 days, and then light culture is carried out with a light intensity of 10,000 lx, and after 4 days of light culture, the germination rate was counted, and 30 seedlings with consistent growth were selected for a seedling growth test. The test was repeated 3 times. When counting the germination rate, the sum of the root length and the seedling height was greater than 0.5 cm and was considered to be germinated; the germinated seedlings were transferred to a 96-well PCR plate with the bottom cut off, with one seedling placed in each well, and cultured in an artificial climate incubator under the culture conditions of 30°C, a light intensity of 10,000 lx, and a humidity of 60-80%, with a light culture:dark culture ratio of 16 hours:8 hours, and then hydroponically cultured with a mixed solution of DL-2-piperidinic acid and NaCl of equal concentrations as in the same step, and the hydroponic solution was changed every 3 days. After 14 days, the seedling height, root length, and dry weight were measured.

[0032] Example 3

[0033] A method for rapidly improving salt tolerance of salt-sensitive rice, comprising the following steps:

[0034] Step 1. Dissolve DL-2-piperidinic acid in 5 wt.‰ NaCl solution to prepare a 100 mg / L DL-2-piperidinic acid solution. Disinfect the surface of rice seeds as follows: Select mature rice seeds with plump grains and no mold spots. Quickly rinse them twice with 70 wt.% ethanol solution for 30 seconds each, rinse twice with sterile water, sterilize them with 5 wt.% NaClO solution for 5 minutes, rinse five times with sterile water, and finally remove any surface water with sterile filter paper.

[0035] Step 2. Soak the rice seeds in the DL-2-piperidinic acid solution prepared in step 1 at 25°C for 24 hours to obtain the treated rice seeds, and allow them to germinate and grow normally under salt stress. The salt stress environment is the irrigation water, and the salt stress solution is a mixed solution of DL-2-piperidinic acid and NaCl with the same concentration as in the same step.

[0036] The method for testing the germination rate, root length and growth rate of the rice seeds treated in step 2 is as follows: the rice seeds treated in step 2 are placed in a germination box (19 cm x 14 cm x 12 cm) for germination test, two layers of sterile filter paper are padded in the germination box, 100 seeds are placed in each germination box, a mixed solution of DL-2-piperidinic acid and NaCl of the same concentration as in the step is added to keep the filter paper moist, and the germination box is covered with a lid. The germination box is placed at 28°C for dark culture for 3 days, and then light culture is carried out with a light intensity of 4000 lx, after 3 days of light culture, the germination rate was counted, and 30 seedlings with consistent growth were selected for seedling growth test, the test was repeated 3 times, and when counting the germination rate, the sum of the root length and the seedling height was greater than 0.5 cm, which was considered to be germinated; the germinated seedlings were transferred to a 96-well PCR plate with the bottom cut off, with one seedling placed in each well, and cultured in an artificial climate incubator under the culture conditions of 25°C, a light intensity of 4000 lx, a humidity of 60-80%, and a light culture: dark culture ratio of 16 hours:8 hours. Then, a mixed solution of DL-2-piperidinic acid and NaCl with equal concentrations as in the same step was used for hydroponics, and the hydroponic solution was changed every 3 days. After 14 days, the seedling height, root length, and dry weight were measured.

[0037] Example 4

[0038] Same as Example 3, except that in step 1, 5 wt.‰ NaCl solution is used to dissolve DL-2-piperidinic acid to prepare a DL-2-piperidinic acid solution with a concentration of 200 mg / L.

[0039] Example 5

[0040] Same as Example 3, except that in step 1, 5 wt.‰ NaCl solution is used to dissolve DL-2-piperidinic acid to prepare a DL-2-piperidinic acid solution with a concentration of 400 mg / L.

[0041] Comparative Example 1

[0042] Same as Example 3, except that in step 1, the 200 mg / L DL-2-piperidinic acid solution was replaced with an equal volume of sterile water.

[0043] Comparative Example 2

[0044] Same as Example 3, except that in step 1, the 200 mg / L DL-2-piperidinic acid solution was replaced with an equal volume of 5 wt.‰ NaCl solution.

[0045] The germination rates and seedling growth conditions of rice seeds after treatment in Examples 3-5 and Comparative Examples 1 and 2 are shown in the following table:

[0046] Effects of different concentrations of DL-2-piperidinic acid on seed germination rate and seedling growth under 5‰ salt stress

[0047]

[0048] Figure 1 This is a comparison of the germination rate of seeds treated with 100 mg / L DL-2-piperidinic acid and the untreated control grown under 5‰ salt stress for 7 days. Figure 2 The comparison chart of the growth of 100 mg / L DL-2-piperidinic acid and the control under 5‰ salt stress for 14 days is shown in the table and Figure 1 and Figure 2 As can be seen from the figure, under no salt stress conditions, the germination rate of Yanshen Rice 83006 seeds was approximately 100%, indicating that the selected seeds had a high and uniform germination rate. Under 5‰ salt stress, the germination rate dropped sharply to 72.33%. Exogenous DL-2-piperidinic acid 100-400 mg / L seed soaking treatment significantly increased the germination rate under 5‰ salt stress to 86.67-88.67%. Compared with NaCl treatment alone, 100-400 mg / L DL-2-piperidinic acid treatment increased the biomass of seedlings under salt stress, with the 100 mg / L treatment having the best effect (see photos of seedlings after 14 days of growth). Figure 2 b). Therefore, 100 mg / L of DL-2-piperidinic acid was selected as the optimal concentration.

[0049] Due to laboratory limitations, the salt stress environment used in the examples herein was determined to be a mixture of DL-2-piperidinic acid and NaCl in the irrigation water. The concentration of DL-2-piperidinic acid was 0-400 mg / L, and the concentration of NaCl was 3-10 wt.‰. In actual planting, the treated rice seeds were planted in either saline soil or a salt stress environment.

[0050] The above embodiments do not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for rapidly improving salt tolerance of salt-sensitive rice, characterized in that: The method for rapidly improving the salt tolerance of salt-sensitive rice is specifically a method for rapidly improving the germination rate, root length and growth rate of salt-sensitive rice seeds under salt stress conditions, and the steps are as follows: Step 1. Dissolve DL-2-piperidinic acid in 3-10 wt.‰ NaCl solution to prepare a DL-2-piperidinic acid solution with a concentration of 50-400 mg / L, and then disinfect the surface of the rice seeds; Step 2. Soak the sterilized rice seeds in a DL-2-piperidinic acid solution at 20-30°C for 16-24 hours to obtain treated rice seeds, so that they germinate and grow normally under a salt stress environment. The salt stress environment is a mixed solution of DL-2-piperidinic acid and NaCl in irrigation water, wherein the concentration of DL-2-piperidinic acid is 0-400 mg / L and the concentration of NaCl is 3-10 wt.‰.

2. The method for rapidly improving salt tolerance of salt-sensitive rice according to claim 1, characterized in that: The specific steps for surface disinfection of rice seeds in step 1 are as follows: select mature rice seeds with plump grains and no mold spots, quickly rinse them with 70-75 wt.% ethanol solution 2-3 times for 20-40 seconds each time, rinse them with sterile water 2-3 times, then sterilize them with 5-10 wt.% NaClO solution for 5-15 minutes, rinse them with sterile water 3-5 times, and finally absorb the water attached to the seed surface with sterile filter paper.

3. The method for rapidly improving salt tolerance of salt-sensitive rice according to claim 1, characterized in that: In the step 1, 5 wt.‰ NaCl solution is used to dissolve DL-2-piperidinic acid to prepare a DL-2-piperidinic acid solution with a concentration of 100 mg / L.

4. The method for rapidly improving salt tolerance of salt-sensitive rice according to claim 1, characterized in that: The method for testing the germination rate, root length and growth rate of rice seeds in step 2 comprises the following steps: placing the rice seeds treated in step 2 in a germination box for a germination test, padding the germination box with two layers of sterile filter paper, placing 100 seeds in each germination box, adding a mixed solution of DL-2-piperidinic acid and NaCl of the same concentration as in the step to keep the filter paper moist, and covering the germination box with a lid; placing the germination box at 20-30° C. for dark culture for 2-3 days, and then performing light culture with a light intensity of 4000-10000 lx; counting the germination rate after 2-4 days of light culture, and selecting 30 seedlings with consistent growth for a seedling growth test, with three replicates; transferring the germinated seedlings into a 96-well PCR plate with the bottom cut off, placing one seedling in each well, and culturing in an artificial climate incubator with culture conditions of 25-30° C. and a light intensity of 4000-10000 lx. lx, humidity 60-80%, light culture: dark culture for 16h:8h, then use the same concentration of DL-2-piperidinic acid and NaCl mixed solution for hydroculture, change the hydroponic solution every 3 days, and measure the seedling height, root length and dry weight after 14 days.

5. The method for rapidly improving salt tolerance of salt-sensitive rice according to claim 4, characterized in that: When calculating the germination rate, the sum of the root length and the seedling height greater than 0.5 cm was considered to be germinated.

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