Seed priming method for improving wax content of sweet sorghum leaves under drought condition
Soaking sweet sorghum seeds through salicylic acid solution improves the wax content of its leaves, enhances drought resistance, solves the problem of insufficient wax of sweet sorghum under drought conditions, and is suitable for use in arid areas in agricultural production.
Patent Information
- Application Number
- CN202510610902.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-08-29
AI Technical Summary
Under drought conditions, the wax content of sweet sorghum leaves is low, resulting in insufficient drought resistance. In the prior art, seed initiation method is less used in improving drought resistance of sweet sorghum.
Sweet sorghum seeds were soaked in salicylic acid solution, with a concentration of 50-250mg·L-1, and the soaking time was 24 hours. Then potted planting experiments were conducted to observe the changes in wax content and moisture loss under drought conditions.
It significantly improves the wax content of sweet sorghum leaves, enhances its drought resistance, reduces the moisture loss rate of the stratum corneum, provides preventive protection, and is suitable for applications in arid areas in agricultural production.
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Figure CN120548815A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of forage cultivation under adverse conditions, and particularly relates to a seed priming method for increasing the wax content of sweet sorghum leaves under drought conditions. Background Art
[0002] Cuticular wax is an organic mixture composed of compounds such as primary alcohols, aldehydes, fatty acids, and alkanes. It covers the plant surface and forms a stress-resistant barrier, effectively limiting non-stomatal water loss. Studies have found that the content and chemical composition of plant cuticular waxes vary depending on the plant species, developmental stage, and organ location. For example, the main components of tomato leaf wax are alkanes and triterpenoids, the main components of wheat leaf wax are primary alcohols and alkanes, and the main components of sorghum leaf wax are alkanes and aldehydes. The function of cuticular wax is affected by its composition. For example, increased levels of C29 and C31 alkanes are more beneficial for preventing excessive water loss in plants. Therefore, the deposition of cuticular wax helps improve plant drought resistance.
[0003] Sweet sorghum (Sorghum dochna (Forssk.) Snowden) has significant application value. Its stems are high in sugar, making it an important dual-purpose crop for grain and feed. Currently, my country is optimizing its agricultural production structure by shifting from the traditional two-component cropping system (grain crops / cash crops) to a three-component cropping system (TCCS: grain crops / cash crops / forage grasses) to promote grain conversion and accelerate the development of herbivorous livestock farming. Therefore, increasing sweet sorghum's drought resistance is beneficial for its cultivation and application, and is of great value in alleviating my country's feed resource shortages and promoting economic development.
[0004] Seed priming can improve seedling resistance to abiotic stresses such as drought. Priming is widely used in a variety of horticultural crops, medicinal plants, food crops, and cash crops due to its ease of use and low production costs. However, research and application in improving sweet sorghum's drought tolerance is limited. Summary of the Invention
[0005] The purpose of the present invention is to provide a seed priming method for increasing the wax content of sweet sorghum leaves under drought conditions.
[0006] The purpose of the present invention can be achieved through the following technical solutions.
[0007] Application of salicylic acid in increasing wax content in plant leaves under drought conditions.
[0008] The above application is to soak seeds with salicylic acid solution as a seed initiator.
[0009] A seed priming method for increasing the wax content of sweet sorghum leaves under drought conditions comprises soaking the seeds in a salicylic acid solution.
[0010] The concentration of the salicylic acid solution is 50-250 mg·L -1 ; More preferably 150mg L -1 .
[0011] The plant leaves are leaves of sweet sorghum, but are not limited thereto.
[0012] The soaking was carried out at 22° C. for 24 hours.
[0013] The advantages of the present invention are: the present invention improves drought resistance: by increasing the wax content of the leaves, the adaptability of plants to drought environments can be enhanced, because the wax layer can effectively reduce the evaporation and loss of water through the cuticle, which helps plants survive under water-deficient conditions; preventive measures: as a pre-sowing treatment method, this method can provide a layer of protection for plants before they grow and develop, thereby preventing drought stress that plants may encounter during their growth process and reducing the damage caused thereby; simple application: if the treatment method involved in this invention is simple and easy to use, then it may be widely used in agricultural production, especially in areas prone to drought. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The effect of different concentrations of SA on the total amount of wax in sweet sorghum leaves under drought stress.
[0015] The horizontal axis numbers 0, 1, 2, 3, 4, and 5 in the figure correspond to the triggering concentrations of 0, 50, 100, 150, 200, and 250 mg / L, respectively. -1 Lowercase letters represent the results of significance analysis, and different letters indicate significant differences among treatments.
[0016] Figure 2 150mg L -1 Effects of SA induction on the content of wax components and total wax content in sweet sorghum leaves.
[0017] The SA triggering concentration in the figure is 150 mg L -1 Lowercase letters represent the results of significance analysis, and different letters indicate significant differences among treatments.
[0018] Figure 3 150mg L under drought stress -1 Effects of SA priming on water loss from the cuticle of sweet sorghum leaves. DETAILED DESCRIPTION
[0019] The following is a clear and complete description of the technical solutions in the experimental examples of the present invention. Obviously, the experimental examples described are only a part of the experimental examples of the present invention, not all of the experimental examples. Based on the experimental examples of the present invention, all other experimental examples obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.
[0020] Unless otherwise specified, the materials and reagents used in the following experimental examples can be obtained from commercial sources. The quantitative tests in the following experimental examples were repeated four times, and the results were averaged.
[0021] In the following experimental examples, the tested Hercules and Mule seeds were from commercial varieties, and the Pin 05206 seeds were from the Shanxi Academy of Agricultural Sciences.
[0022] The chemical initiator salicylic acid (SA) used in the following experimental examples was of analytical grade.
[0023] SPSS statistical analysis software was used to perform variance analysis on the data, and the LSD method was used for significance analysis.
[0024] Experimental Example 1 Effect of different concentrations of salicylic acid on the wax content of sweet sorghum
[0025] (1) Test groups and test methods
[0026] A total of 5 priming concentrations were set, with no priming as the control. The treatment details of each group are as follows:
[0027] SA treatment group: Different masses of SA were mixed with water to obtain five salicylic acid initiators with concentrations of 50 mg L -1、 100mg L -1 、150mg L -1 , 200mg L -1 , 250mg L -1To ensure experimental accuracy, the required salicylic acid concentration for each group must be precisely prepared. Here are the steps for preparing these solutions: Calculate the mass of salicylic acid required: For each liter of solution, calculate the mass of salicylic acid required to achieve the desired concentration (50 mg L⁻¹, 100 mg L⁻¹, 150 mg L⁻¹, 200 mg L⁻¹, and 250 mg L⁻¹). Prepare the solution: For a 50 mg L⁻¹ solution, weigh 50 mg of salicylic acid and dissolve it in 1 L of water. Similarly, for higher concentrations, weigh the corresponding amount of salicylic acid and dissolve it in 1 L of water. For the control group, use only 1 L of water without adding salicylic acid. Mix and dissolve: Accurately weigh the salicylic acid powder using an appropriate tool (such as a digital balance). Slowly add the weighed salicylic acid to 1 L of water and stir thoroughly until completely dissolved. Ensure that each step is performed in a sterile environment to avoid contamination. Calibration and storage: After preparation, the exact concentration of each solution needs to be calibrated to ensure the validity of the experiment. If the solution concentration deviates, the ratio needs to be readjusted. Once the concentration is confirmed to be correct, each solution should be stored in a suitable container until later use.
[0028] (2) Seed priming treatment
[0029] Healthy sweet sorghum seeds of uniform size were soaked in the aforementioned priming solution at 22°C in the dark for 24 hours. This ensured that each seed group was treated under identical conditions to eliminate environmental influences on the experimental results. During this period, the solution temperature was kept constant and exposure to light was avoided, as temperature and light can affect the effectiveness of salicylic acid. Primed seeds were obtained, along with unprimed seeds as a control. After priming, the seeds were removed and thoroughly rinsed with running water to remove any residual priming solution. The seeds were gently wiped dry with absorbent paper or a towel. A control group was prepared, in which seeds were not primed but simply soaked in water under the same conditions. The treated seeds were allowed to return to their original weight at room temperature. This ensured that the seeds were at the same starting point before potting. Once the seeds had returned to their original weight, potting experiments were performed. This effectively primed the sweet sorghum seeds, allowing for subsequent potting experiments under controlled conditions to evaluate the effects of salicylic acid priming on sweet sorghum growth and development.
[0030] (3) Potted plant experiment: the specific steps are as follows:
[0031] Prepare an appropriate amount of potting soil and divide the pots into the desired number of replicates (four in this experiment). Sow six primed or unprimed sorghum seeds evenly in each pot. Ensure all seeds are sown at a relatively consistent depth to ensure uniform growing conditions. When the seeds germinate and reach the three-leaf stage, begin watering regularly, keeping the soil moist but not overly saturated. When the sorghum seedlings reach a certain size, thin them out, leaving three plants per pot. This ensures that the remaining plants have sufficient resources for continued growth. After one week of water restriction, reduce watering to a soil moisture content of 35% to 40%. The purpose of the drought treatment is to simulate water-limited conditions and observe the physiological responses of sorghum plants to suboptimal conditions. Maintain controlled water conditions (35% to 40% soil moisture content) for two weeks. During this period, ensure that growing conditions (such as light and temperature) remain consistent across all pots. Sampling and Measurement: At the end of the drought treatment, collect samples from the sorghum plants. A range of physiological indicators, such as leaf relative water content, stomatal conductance, net photosynthesis rate, and chlorophyll content, can be measured to assess sorghum's response to drought stress. Data recording and analysis: Data from all observed indicators are recorded and statistically analyzed to reveal differences in sorghum seed drought stress before and after salicylic acid priming. These steps allow researchers to assess the potential impact of salicylic acid priming on sweet sorghum drought tolerance in a controlled potted environment and analyze the underlying physiological mechanisms.
[0032] (4) Leaf wax content extraction: Leaf selection and preparation: The second fully expanded healthy leaf from the top leaf on the sweet sorghum plant was selected as the sample. The leaf area was measured using the WinFOLIA professional leaf image analysis system and digital scanner to facilitate subsequent data analysis. The leaves were placed in a 50 mL test tube, ensuring that the leaves were not folded or damaged. About 3 mL of chloroform solution containing 1 μg μL-1 internal standard (hexadecane) was added to the test tube at room temperature, and the extraction was shaken for 30 seconds. This process was then repeated 3 times to ensure that the wax in the leaves was fully extracted. The extract was blown dry with nitrogen at 40°C to remove excess chloroform. 20 μL each of the derivatizing agents pyridine and BSTFA were added. The function of these two reagents is to convert the fatty acids in the wax into a form that is easy to detect. The reaction was carried out at 70°C for 45 minutes to allow the derivatization reaction to proceed fully. The solvent was blown dry again using a nitrogen blower and prepared for the next step of analysis. The wax extract was dissolved in 0.5 mL of chloroform to ensure that the sample had a suitable concentration and fluidity for gas chromatography and mass spectrometry analysis.
[0033] (5) Determination of leaf wax content: Gas chromatograph settings: A Fuli 9790Ⅱ series gas chromatograph was used for analysis. The specifications of the GC capillary column were 30 m long, 0.32 mm in diameter, and 0.25 μm in liquid film thickness. Nitrogen was used as the carrier gas. The injection volume was 2 μl. The column membrane and FID detector temperatures were 300°C and 320°C, respectively. The split ratio was set to 3:1. Temperature program: The initial temperature was 80°C, and the temperature was increased to 260°C at a rate of 15°C per minute and maintained for 10 min. Then the temperature was increased to 290°C at a rate of 2°C per minute and maintained for 1 min. Finally, the temperature was increased to 320°C at a rate of 5°C per minute and maintained for 10 min. Content determination by internal standard method: An internal standard (hexadecane in this case) was used to calculate the wax content per unit area of the plant. By comparing the peak area with that of the internal standard, the extraction efficiency and analysis results of different samples can be corrected. Gas chromatography-mass spectrometry analysis: To identify the unknown waxy components, GC-MS (model GCMS-QP2010Ultra) was used. The same temperature program was performed on the GC-MS to keep the separation conditions consistent with those of the GC analysis.
[0034] Effects of different concentrations of priming on the total amount of leaf wax can be found in Figure 1 With the increase of initiation concentration, the total amount of wax increases first and then decreases. -1 The wax content was highest during the priming treatment. Under drought conditions, the wax content increased by 13.24%, 94.35% and 32.85% compared with the drought control, respectively. Therefore, the concentration with the most significant effect on increasing wax content was 150 mg L -1 .
[0035] Experimental Example 2 Effect of salicylic acid priming on the content of wax components in sweet sorghum
[0036] (1) Seed priming treatment
[0037] Healthy sweet sorghum seeds of uniform size were soaked in the aforementioned priming solution at 22°C in the dark for 24 hours. This ensured that each seed group was treated under identical conditions to eliminate environmental influences on the experimental results. During this period, the solution temperature was kept constant and exposure to light was avoided, as temperature and light can affect the effectiveness of salicylic acid. Primed seeds were obtained, along with unprimed seeds as a control. After priming, the seeds were removed and thoroughly rinsed with running water to remove any residual priming solution. The seeds were gently wiped dry with absorbent paper or a towel. A control group was prepared; seeds in this group were not primed but simply soaked in water under the same conditions. The treated seeds were allowed to return to their original weight at room temperature. This ensured that the seeds were at the same starting point before potting. Once the seeds had returned to their original weight, potting experiments could be performed.
[0038] (2) Potted plant experiment: the specific steps are as follows:
[0039] Prepare an appropriate amount of potting soil and divide the pots into the desired number of replicates (four in this experiment). Sow six primed or unprimed sorghum seeds evenly in each pot. Ensure all seeds are sown at a relatively consistent depth to ensure uniform growing conditions. When the seeds germinate and reach the three-leaf stage, begin watering regularly, keeping the soil moist but not overly saturated. When the sorghum seedlings reach a certain size, thin them out, leaving three plants per pot. This ensures that the remaining plants have sufficient resources for continued growth. After one week of water restriction, reduce watering to a soil moisture content of 35% to 40%. The purpose of the drought treatment is to simulate water-limited conditions and observe the physiological responses of sorghum plants to suboptimal conditions. Maintain controlled water conditions (35% to 40% soil moisture content) for two weeks. During this period, ensure that growing conditions (such as light and temperature) remain consistent across all pots. Sampling and Measurement: At the end of the drought treatment, collect samples from the sorghum plants. A range of physiological indicators, such as leaf relative water content, stomatal conductance, net photosynthesis rate, and chlorophyll content, can be measured to assess sorghum's response to drought stress. Data recording and analysis: Data from all observed indicators are recorded and statistically analyzed to reveal differences in sorghum seed drought stress before and after salicylic acid priming. These steps allow researchers to assess the potential impact of salicylic acid priming on sweet sorghum drought tolerance in a controlled potted environment and analyze the underlying physiological mechanisms.
[0040] (3) Leaf wax content extraction: Leaf selection and preparation: The second fully expanded healthy leaf from the top leaf on the sweet sorghum plant was selected as the sample. The leaf area was measured using the WinFOLIA professional leaf image analysis system and digital scanner to facilitate subsequent data analysis. The leaves were placed in a 50 mL test tube, ensuring that the leaves were not folded or damaged. About 3 mL of chloroform solution containing 1 μg μL-1 internal standard (hexadecane) was added to the test tube at room temperature, and the extraction was shaken for 30 seconds. This process was then repeated 3 times to ensure that the wax in the leaves was fully extracted. The extract was blown dry with nitrogen at 40°C to remove excess chloroform. 20 μL each of the derivatizing agents pyridine and BSTFA were added. The function of these two reagents is to convert the fatty acids in the wax into a form that is easy to detect. The reaction was carried out at 70°C for 45 minutes to allow the derivatization reaction to proceed fully. The solvent was blown dry again using a nitrogen blower and prepared for the next step of analysis. The wax extract was dissolved in 0.5 mL of chloroform to ensure that the sample had a suitable concentration and fluidity for gas chromatography and mass spectrometry analysis.
[0041] (4) Gas chromatograph settings: A Fuli 9790Ⅱ series gas chromatograph was used for analysis. The specifications of the GC capillary column were 30 m long, 0.32 mm in diameter, and 0.25 μm in liquid film thickness. Nitrogen was used as the carrier gas. The injection volume was 2 μl. The column membrane and FID detector temperatures were 300°C and 320°C, respectively. The split ratio was set to 3:1. Temperature program: The initial temperature was 80°C, and the temperature was increased to 260°C at a rate of 15°C per minute and maintained for 10 min. Then the temperature was increased to 290°C at a rate of 2°C per minute and maintained for 1 min. Finally, the temperature was increased to 320°C at a rate of 5°C per minute and maintained for 10 min. Content determination by internal standard method: An internal standard (hexadecane in this case) was used to calculate the wax content per unit area of the plant. By comparing the peak area with that of the internal standard, the extraction efficiency and analysis results of different samples can be corrected. Gas chromatography-mass spectrometry (GC-MS) analysis: To identify the unknown waxy components, GC-MS (model GCMS-QP2010 Ultra) was used. The same temperature program was performed on the GC-MS to maintain the same separation conditions as those used in GC analysis.
[0042] (5) Determination of water loss rate of cuticle: Before collecting leaves, the plants need to adapt to the dark for 12 hours. This is to close the stomata so that the water loss measured later is mainly attributed to water loss from the cuticle rather than transpiration through the stomata. Leaf selection and pretreatment: The second fully expanded healthy leaf from the top of the plant was selected for the experiment. The fresh weight of the leaf was measured, which represents the weight of the leaf in its natural state. The leaves were immersed in distilled water for 3 hours and then their saturated weight was measured. This step is to allow the leaves to absorb enough water so that the total water content of the leaves can be calculated. Continuous dehydration and monitoring: The leaves were placed in the dark for continuous dehydration and weighed regularly (every 15 minutes) during the dehydration process to record the water loss of the leaves. The dehydration process lasted for 150 minutes to ensure that the dynamic changes in water loss from the cuticle could be captured during this period. Calculation of water loss rate: The water loss rate at each time point was calculated using the given formula: Water loss rate (%) = (saturated weight - weight at each time point) / total water content × 100%. The total water content is obtained by subtracting the dry weight from the saturated weight in step 2. By measuring the weight change over time, the rate of water loss from the leaves can be estimated. The results of this experiment can reflect the role of the cuticle structure in reducing non-transpiratory water loss.
[0043] Figure 2 It was shown that SA priming could significantly increase the total amount of wax under drought conditions ( Figure 2Under drought conditions, SA priming significantly increased the total wax content, primary alcohol, aldehyde, and amyl alcohol contents of Hercules sorghum by 13.2%, 57.0%, 38.9%, and 77.1%, respectively, compared to the drought control. SA priming also significantly increased the total wax content, alkane, primary alcohol, and amyl alcohol contents of Mule 8000 by 33.3%, 42.0%, 164.7%, and 257.9%, respectively. The wax content and primary alcohol contents of Pin 05206 also significantly increased by 12.3% and 50.2%, respectively.
[0044] Experimental Example 3 Effect of salicylic acid on the water loss rate of sweet sorghum cuticle
[0045] (1) Seed priming treatment
[0046] Healthy sweet sorghum seeds of uniform size were soaked in the aforementioned priming solution at 22°C in the dark for 24 hours. This ensured that each seed group was treated under identical conditions to eliminate environmental influences on the experimental results. During this period, the solution temperature was kept constant and exposure to light was avoided, as temperature and light can affect the effectiveness of salicylic acid. Primed seeds were obtained, along with unprimed seeds as a control. After priming, the seeds were removed and thoroughly rinsed with running water to remove any residual priming solution. The seeds were gently wiped dry with absorbent paper or a towel. A control group was prepared; seeds in this group were not primed but simply soaked in water under the same conditions. The treated seeds were allowed to return to their original weight at room temperature. This ensured that the seeds were at the same starting point before potting. Once the seeds had returned to their original weight, potting experiments could be performed.
[0047] (2) Potted plant experiment: the specific steps are as follows:
[0048] Prepare an appropriate amount of potting soil and divide the pots into the desired number of replicates (four in this experiment). Sow six primed or unprimed sorghum seeds evenly in each pot. Ensure all seeds are sown at a relatively consistent depth to ensure uniform growing conditions. When the seeds germinate and reach the three-leaf stage, begin watering regularly, keeping the soil moist but not overly saturated. When the sorghum seedlings reach a certain size, thin them out, leaving three plants per pot. This ensures that the remaining plants have sufficient resources for continued growth. After one week of water restriction, reduce watering to a soil moisture content of 35% to 40%. The purpose of the drought treatment is to simulate water-limited conditions and observe the physiological responses of sorghum plants to suboptimal conditions. Maintain controlled water conditions (35% to 40% soil moisture content) for two weeks. During this period, ensure that growing conditions (such as light and temperature) remain consistent across all pots. Sampling and Measurement: At the end of the drought treatment, collect samples from the sorghum plants. A range of physiological indicators, such as leaf relative water content, stomatal conductance, net photosynthesis rate, and chlorophyll content, can be measured to assess sorghum's response to drought stress. Data recording and analysis: Data from all observed indicators are recorded and statistically analyzed to reveal differences in sorghum seed drought stress before and after salicylic acid priming. These steps allow researchers to assess the potential impact of salicylic acid priming on sweet sorghum drought tolerance in a controlled potted environment and analyze the underlying physiological mechanisms.
[0049] (3) Determination of water loss rate through the cuticle: The plants were placed in the dark for 12 hours to close the stomata. This ensured that the measured water loss was mainly due to the cuticle rather than transpiration through open stomata. Leaf selection and processing: The second fully expanded healthy leaf from the top was selected. The fresh weight of the leaf was weighed. The leaf was immersed in distilled water for 3 hours and then weighed to its saturated weight. This step is to measure the total amount of water that the leaf can absorb. Dehydration process: The leaves were continuously dehydrated under dark conditions and weighed regularly during the dehydration process to monitor water loss. The dehydration process lasted for 150 minutes and was weighed every 15 minutes. Calculation of water loss rate: The water loss rate was calculated according to the formula: Water loss rate (%) = (saturated weight - weight at each time point) / total water content × 100%. The total water content was obtained by subtracting the dry weight from the saturated weight.
[0050] Figure 3 The results showed that SA priming could reduce the water loss rate of leaves of Dalishi, Mule 8000 and Pin 05206 under drought conditions ( Figure 3 ).
[0051] In summary, SA induction under drought stress can reduce the water loss rate of the cuticle by increasing the wax content in the cuticle of sweet sorghum leaves.
[0052] Although the present invention has been described in detail through the preferred experimental examples above, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention is defined by the appended claims.
Claims
1. Application of salicylic acid in increasing the wax content of sweet sorghum leaves under drought stress.
2. The use according to claim 1, characterized in that: Plant seeds were treated with salicylic acid as a seed priming agent.
3. The use according to claim 2, characterized in that: The concentration of the seed initiator salicylic acid is 50-250 mg·L -1 ; More preferably 150mg L -1 .
4. The use according to any one of claims 1 to 3, characterized in that: The sweet sorghum varieties are leaves of hybrid lines Dalishi and Mule 8000 and inbred line 05206.
5. A method for increasing the wax content of sweet sorghum leaves, characterized by: (1) Soak the seeds with salicylic acid as a seed priming agent, then rinse and dry the seeds.
6. The method for increasing the wax content of sweet sorghum leaves according to claim 5, wherein: The concentration of the seed initiator salicylic acid is 50-250 mg L -1 .
7. The method for increasing the wax content of sweet sorghum leaves according to claim 6, wherein: The concentration of the seed initiator salicylic acid is 150 mg L -1 .
8. The method for increasing the wax content of sweet sorghum leaves according to claim 7, wherein: The soaking is carried out at 22°C.
9. The method for increasing the wax content of sweet sorghum leaves according to claim 8, characterized in that: The soaking was carried out at 22° C. for 24 hours.
10. The method for increasing the wax content of sweet sorghum leaves according to claims 5 to 9, characterized in that: The sweet sorghum varieties are leaves of hybrid lines Dalishi and Mule 8000 and inbred line 05206.