Application of 3,5-dihydroxytoluene in improving low temperature and low oxygen tolerance of tobacco seedlings

Abstract

The application relates to the technical field of agricultural application, in particular to application of 3,5-dihydroxytoluene in improving low-temperature and low-oxygen tolerance of tobacco seedlings. The application finds and proves that the 3,5-dihydroxytoluene can be applied to improve the low-temperature and low-oxygen tolerance of the tobacco seedlings and promote the growth of the tobacco seedlings. Meanwhile, the exogenous 3,5-dihydroxytoluene treatment is easy to operate in practice, has effects, and is expected to become a good method for improving the low-temperature and low-oxygen stress resistance of tobacco floating seedling production practice, and has great application potential.

Description

Technical Field

[0001] This invention relates to the field of agricultural application technology, specifically to the application of 3,5-dihydroxytoluene in improving the low-temperature and low-oxygen tolerance of tobacco seedlings. Background Technology

[0002] As a widely cultivated leafy crop, low-temperature stress is one of the common abiotic stresses in tobacco production. It easily inhibits photosynthesis and organic matter accumulation in leaves, increases the total nitrogen content of leaves, and decreases the content of total phosphorus, total potassium, reducing sugar, and nicotine, thus affecting the quality of tobacco leaves. At the same time, in the process of tobacco floating seedling production, the coexistence of low temperature and low oxygen stress can easily cause seedling growth to be hindered or even lead to death, and reduce the vitality of mature plants, thereby seriously affecting tobacco production.

[0003] Phenolic acids are a class of organic acids containing a phenolic ring, formed from carbohydrate metabolism, and widely present as secondary metabolites in many plants. Currently, it is clear that phenolic acids play multiple important roles in plant resistance to abiotic stress. They help plants adapt to various adverse environments through mechanisms such as regulating plant growth, scavenging reactive oxygen species, antioxidation, influencing gene expression, and metabolic accumulation.

[0004] Exogenous treatment is an effective means of helping plants resist abiotic stress and can enhance their resistance. In agricultural production, studies have shown that exogenous application of phenolic acids can help plants resist abiotic stress. 3,5-Dihydroxytoluene, as a phenolic acid, has been widely used in the chemical and pharmaceutical fields as a raw material for organic synthesis, an analytical reagent, and a pharmaceutical intermediate. These applications demonstrate its good chemical stability and biological activity, providing a foundation for its potential applications in plant stress resistance. Summary of the Invention

[0005] This invention provides an application of 3,5-dihydroxytoluene in improving the low-temperature and low-oxygen tolerance of tobacco seedlings.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides an application of 3,5-dihydroxytoluene in improving the low-temperature and low-oxygen tolerance of tobacco seedlings.

[0008] This application investigated the treatment of tobacco seedlings under low-temperature and low-oxygen stress with 3,5-dihydroxytoluene (3,5-dihydroxytoluene). The results showed that this phenolic acid 3,5-dihydroxytoluene could induce tolerance to combined low-temperature and low-oxygen stress in tobacco seedlings and alleviate the damage caused by this stress. 3,5-dihydroxytoluene treatment significantly promoted the growth of tobacco seedlings under low-temperature and low-oxygen conditions. Furthermore, it significantly increased peroxidase activity, superoxide dismutase activity, catalase activity, ascorbate peroxidase activity, soluble sugar content, and soluble protein content compared to the control group without 3,5-dihydroxytoluene treatment, while significantly decreasing malondialdehyde content.

[0009] Furthermore, tobacco seedlings treated with 3,5-dihydroxytoluene exhibited significantly increased root length and relative chlorophyll content. Increased root length enhances the plant's efficiency in absorbing water and nutrients from the soil, which is crucial for maintaining normal growth under adverse conditions. Increased chlorophyll content helps maintain or improve photosynthetic levels, ensuring the plant obtains sufficient energy under adverse conditions. Therefore, it can be deduced that 3,5-dihydroxytoluene can serve as a potential regulator, improving the nutrient absorption capacity and photosynthetic efficiency of tobacco seedlings, thereby enhancing their survival and growth performance under low-oxygen and low-temperature conditions.

[0010] Here, 3,5-dihydroxytoluene is not limited to being added in the form of a prepared solution / formulation or a solid powder.

[0011] In summary, the applicant has discovered that 3,5-dihydroxytoluene promotes the growth of tobacco seedlings under low temperature and low oxygen stress, providing a new use for 3,5-dihydroxytoluene.

[0012] Preferably, the concentration of the 3,5-dihydroxytoluene is (0,8 mmol / L).

[0013] When 3,5-dihydroxytoluene is added in the form of a prepared solution / formulation, the concentration of 3,5-dihydroxytoluene in the culture medium after addition can be controlled to be (0-8 mmol / L). When 3,5-dihydroxytoluene is added in the form of a solid powder, the concentration of the culture medium can also be controlled by controlling the amount of solid powder added.

[0014] More preferably, the concentration of the 3,5-dihydroxytoluene is [3 mmol / L, 8 mmol / L].

[0015] More preferably, the concentration of the 3,5-dihydroxytoluene is [3 mmol / L, 5 mmol / L].

[0016] More preferably, the concentration of the 3,5-dihydroxytoluene is 5 mmol / L.

[0017] The present invention also provides a method for improving the low-temperature and low-oxygen tolerance of tobacco seedlings with 3,5-dihydroxytoluene, comprising: applying the 3,5-dihydroxytoluene at the 2-leaf-1-heart stage of tobacco seedlings.

[0018] Preferably, the application method is as follows: 3,5-dihydroxytoluene is added to the hydroponic solution of tobacco seedlings, ensuring that the roots of the tobacco seedlings are completely submerged in the hydroponic solution.

[0019] Preferably, the cultivation method of the tobacco seedlings is as follows: tobacco seeds are disinfected, rinsed with distilled water and drained, germinated at 25°C, and then cultivated until the seedlings reach the 2-leaf-1-heart stage.

[0020] Preferably, the germination method is as follows: 16 hours of light treatment followed by 8 hours of darkness treatment within one day, repeated for 7 to 8 days.

[0021] Preferably, the number of repeating days is 7 days.

[0022] To ensure the nutritional needs of the seedlings when they reach the 2-leaf-1-heart stage, seedlings that have been germinated for 7 days can be transferred to a seedling substrate (containing all the nutrients required for growth) for the next stage of cultivation.

[0023] Therefore, the present invention has the following beneficial effects:

[0024] (1) This invention discovers and confirms the application of 3,5-dihydroxytoluene in improving the low temperature and low oxygen tolerance of tobacco seedlings, while also promoting the growth of tobacco seedlings.

[0025] (2) The method of exogenous treatment of tobacco seedlings with 3,5-dihydroxytoluene provided by the present invention can improve the quality of tobacco and increase the content of soluble sugar and soluble protein in tobacco seedlings.

[0026] (3) The exogenous 3,5-dihydroxytoluene treatment of this invention is easy to operate and effective in practice. It is expected to become a good method to improve the ability to resist low temperature and low oxygen stress in tobacco floating seedling production practice, and has great application potential. Attached Figure Description

[0027] Figure 1 The effect of 3,5-dihydroxytoluene treatment on the growth phenotype of tobacco seedlings under low temperature and low oxygen conditions;

[0028] Figure 2 The effect of 3,5-dihydroxytoluene treatment on root length of tobacco seedlings under low temperature and low oxygen conditions;

[0029] Figure 3 The effect of 3,5-dihydroxytoluene treatment on the relative chlorophyll content of tobacco seedlings under low temperature and low oxygen conditions;

[0030] Figure 4The effect of 3,5-dihydroxytoluene treatment on POD enzyme activity in tobacco seedlings under low temperature and low oxygen conditions;

[0031] Figure 5 The effect of 3,5-dihydroxytoluene treatment on SOD enzyme activity in tobacco seedlings under low temperature and low oxygen conditions;

[0032] Figure 6 The effect of 3,5-dihydroxytoluene treatment on CAT enzyme activity in tobacco seedlings under low temperature and low oxygen conditions;

[0033] Figure 7 The effect of 3,5-dihydroxytoluene treatment on APX enzyme activity in tobacco seedlings under low temperature and low oxygen conditions;

[0034] Figure 8 The effect of 3,5-dihydroxytoluene treatment on MDA content in tobacco seedlings under low temperature and low oxygen conditions;

[0035] Figure 9 The effect of 3,5-dihydroxytoluene treatment on the soluble sugar content of tobacco seedlings under low temperature and low oxygen conditions;

[0036] Figure 10 The effect of 3,5-dihydroxytoluene treatment on the soluble protein content of tobacco seedlings under low temperature and low oxygen conditions;

[0037] Figure 11 The effect of 3,5-dihydroxytoluene treatment on root vigor of tobacco seedlings under low temperature and low oxygen conditions. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0039]

Example

[0040] Example 1

[0041] It is designated as group 3_3.

[0042] 1. Material preparation: tobacco 'K326' seeds, floating seedling substrate and 3,5-dihydroxytoluene solution.

[0043] 2. Seed treatment: Select tobacco seeds with normal color, plump kernels, and uniform size. Disinfect the seeds with 0.1% sodium hypochlorite for 15 minutes, rinse them 4 times with distilled water, and blot dry the water with sterile filter paper to obtain disinfected tobacco seeds.

[0044] 3. Seedling cultivation: The treated tobacco seeds were placed in a standard glass petri dish lined with three layers of filter paper and placed in a 25℃ incubator for germination (16h light / 8h darkness). After 7 days, seedlings with uniform growth were selected and transferred to hydroponic boxes containing floating seedling nutrient solution for floating seedling cultivation. When the seedlings reached the 2-leaf-1-heart stage, the tobacco seedlings were subjected to compound stress treatment.

[0045] 4. Test treatment:

[0046] 4.1 First, the preparation of the floating seedling nutrient solution: The tobacco floating seedling substrate was provided by Yunnan Yuxi China Tobacco Seed Co., Ltd., and a 50ppm nutrient solution was prepared: 1L of pure water was added for every 0.2275g of substrate (prepared fresh for use).

[0047] 4.2 Low temperature and low oxygen treatment method: When the tobacco plants are at the 2-leaf and 1-heart stage, the hydroponic box is moved to a 5℃ incubator for low temperature treatment, and an appropriate amount of anhydrous sodium sulfite (Na2SO3) is added to the nutrient solution for low oxygen treatment.

[0048] 4.3 Using low temperature and low oxygen as stress conditions, the concentration of 3,5-dihydroxytoluene solution was set at 3 mmol / L: Under low temperature and low oxygen conditions, the prepared 3,5-dihydroxytoluene stock solution was added to the hydroponic solution until the final concentration of 3,5-dihydroxytoluene in the hydroponic solution was 3 mmol / L, and the tobacco roots were completely immersed in the hydroponic solution.

[0049] Example 2

[0050] This embodiment is basically the same as Example 1, except that the final concentration of 3,5-dihydroxytoluene in "4.3" is 5 mmol / L, which is denoted as group 3_5.

[0051] Example 3

[0052] This embodiment is basically the same as Example 1, except that the final concentration of 3,5-dihydroxytoluene in "4.3" is 8 mmol / L, which is denoted as group 3-8.

[0053] Comparative Example 1

[0054] This comparative example is basically the same as Example 1, except that the application of 3,5-dihydroxytoluene solution in "4.3" is omitted, and it is designated as the CK group.

[0055] [Performance Testing]

[0056] Determination of various indicators: After 3 days of treatment in Examples 1-3 and Comparative Example 1, tobacco seedlings were sampled, root length was measured with a ruler, relative chlorophyll content of leaves was measured with a portable SPAD instrument, root activity was measured after root sampling and weighing, and fresh leaves were collected and stored in liquid nitrogen at -80℃. Then, the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), malondialdehyde (MDA) content, soluble sugar content, and soluble protein content were determined.

[0057] 1. Tobacco seedling growth

[0058] Figures 1-3 The effects of 3,5-dihydroxytoluene treatment on the growth phenotype, root length, and relative chlorophyll content of tobacco seedlings under low temperature and low oxygen conditions were investigated. Figures 2-3 The corresponding data results are shown in Table 1. Observation Figure 1 It can be seen that different concentrations of 3,5-dihydroxytoluene have a certain promoting effect on the growth of tobacco seedlings under low temperature and low oxygen conditions. Figure 2 It can be seen that, compared with the control (CK), exogenous treatment with 3,5-dihydroxytoluene solutions at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L increased the root length of seedlings by 31.68%, 29.77%, and 32.82%, respectively. Figure 3 It can be seen that, compared with the control (CK), the relative chlorophyll content of leaves increased by 14.73%, 18.09%, and 24.47% respectively after exogenous treatment with 3,5-dihydroxytoluene solutions at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L.

[0059] In summary, 3,5-dihydroxytoluene treatment significantly promoted the growth of tobacco seedlings under low temperature and low oxygen conditions. Specifically, increased root length improved the plant's efficiency in absorbing water and nutrients from the soil, which is crucial for maintaining normal growth under adverse conditions. Increased chlorophyll content helped maintain or improve photosynthetic levels, ensuring the plant could obtain sufficient energy under these conditions. Therefore, it can be preliminarily concluded that 3,5-dihydroxytoluene may serve as a potential regulator, improving the nutrient absorption capacity and photosynthetic efficiency of tobacco seedlings, thereby enhancing their survival ability and growth performance in low-oxygen and low-temperature environments.

[0060] Table 1. Effects of different treatments on root length and relative chlorophyll content of tobacco seedlings

[0061]

[0062] Note: Different lowercase letters indicate significant differences between different treatments at the 0.05 level.

[0063] 2. Antioxidant property test

[0064] Furthermore, the antioxidant index of tobacco seedlings treated with 3,5-dihydroxytoluene was determined, and the results are as follows: Figures 4-8 As shown, Table 2 is Figures 4-8 The corresponding data results.

[0065] Depend on Figure 4 As shown in Table 2, under low temperature and low oxygen conditions, the POD activity of each treatment group (Examples 1-3) and the control group (Comparative Example 1) changed significantly throughout the treatment period (P<0.05). The POD activity of tobacco seedlings treated with exogenous 3,5-dihydroxytoluene solutions at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L was significantly higher than that of the control group, exceeding the CK treatment by 107.28%, 144.58%, and 130.85%, respectively. Figure 5 As shown in Table 2, the SOD activity changes were significant in each treatment group (P<0.05). The SOD activity of tobacco seedlings treated with exogenous 3,5-dihydroxytoluene solution at a concentration of 3 mmol / L was 22.86% higher than the control group; the SOD activity of tobacco seedlings treated with exogenous 3,5-dihydroxytoluene solution at a concentration of 5 mmol / L was 74.86% higher than the control group; and the SOD activity of tobacco seedlings treated with exogenous 3,5-dihydroxytoluene solution at a concentration of 8 mmol / L was 54.48% higher than the control group. Figure 6 As shown in Table 2, the CAT enzyme activity in tobacco seedlings increased by 25.47%, 58.96%, and 50.81%, respectively, after exogenous treatment with 3,5-dihydroxytoluene solutions at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L, compared to the control (CK). Figure 7 As can be seen from the APX data in Table 2, compared with the control (CK), the APX enzyme activity of tobacco seedlings increased by 5.90%, 22.87%, and 14.42% respectively after exogenous treatment with 3,5-dihydroxytoluene solution at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L.

[0066] The results indicate that exogenous 3,5-dihydroxytoluene solution can regulate the increase of antioxidant enzyme activity in plants, significantly enhancing the antioxidant capacity and stress tolerance of tobacco seedlings. Among these, a 5 mmol / L 3,5-dihydroxytoluene solution showed the best effect in mitigating oxidative damage caused by the dual stresses of low temperature and low oxygen.

[0067] In addition, by Figure 8As shown in Table 2, the MDA content of tobacco seedlings not treated with 3,5-dihydroxytoluene was significantly higher than that of those treated with 3,5-dihydroxytoluene. The MDA content of tobacco seedlings treated with exogenous 3,5-dihydroxytoluene solutions at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L was reduced by 11.00%, 23.00%, and 17.84% respectively compared with the control (CK) treatment. This indicates that exogenous 3,5-dihydroxytoluene solution treatment can alleviate the damage to the endometrial system of tobacco seedlings caused by low temperature and low oxygen stress.

[0068] Table 2 Effects of different treatments on antioxidant indices of tobacco seedlings

[0069]

[0070] Note: Different lowercase letters indicate significant differences between different treatments at the 0.05 level.

[0071] 3. Nutrient content determination and root vitality

[0072] 20. Further, the soluble sugar content and soluble sugar content of tobacco seedlings treated with 3,5-dihydroxytoluene were...

[0073] The protein content was determined, and the results are as follows: Figures 9-10 As shown in Table 3.

[0074] like Figure 9 As shown, treatment with exogenous 3,5-dihydroxytoluene at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L increased the soluble sugar content by 54.65%, 19.19%, and 8.02%, respectively, compared to the control (CK). Figure 10 As shown, treatment with exogenous 3,5-dihydroxytoluene at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L increased the content of soluble protein by 162.37%, 236.86%, and 167.51%, respectively. Therefore, it can be inferred that 3,5-dihydroxytoluene can be used to improve the quality of tobacco.

[0075] Figure 11 To investigate the effect of 3,5-dihydroxytoluene treatment on root vigor of tobacco seedlings under low temperature and low oxygen conditions, this study observed... Figure 11 It was found that treatment with exogenous 3,5-dihydroxytoluene at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L increased root activity by 18.61%, 22.81%, and 63.11%, respectively. Treatment with exogenous 3,5-dihydroxytoluene at concentrations of 3 mmol / L, 5 mmol / L, and 8 mmol / L decreased the MDA content in tobacco seedlings, thus alleviating membrane lipid peroxidation caused by low temperature and hypoxia stress to some extent. Furthermore, it significantly increased the content of soluble sugars and soluble proteins in tobacco seedlings, enhanced root activity, and consequently promoted cell resistance to adverse conditions affecting seedlings.

[0076] Table 3 Effects of different treatments on nutrient composition and root activity of tobacco seedlings

[0077]

[0078] Note: Different lowercase letters indicate significant differences between different treatments at the 0.05 level.

[0079] 4. Optimal concentration screening

[0080] Results Analysis and Screening of Optimal 3,5-Dihydroxytoluene Concentration: Data processing was performed using Microsoft Excel 2019 software, and statistical analysis of experimental data was conducted using SPSS 26.0 software. Duncan's new multiple range test was used for multiple comparisons, and significance analysis was performed on each treatment. Different lowercase letters represent significant differences between different treatments at the 0.05 level. The case-ranked summation method was used to process the exogenous effects of different concentrations of 3,5-dihydroxytoluene and calculate the total score. The total scores were sorted in ascending order, and negatively correlated indicators were first positively oriented. The results are recorded in Table 4.

[0081] As shown in Table 4, under combined low temperature and low oxygen stress, 3–8 mmol / L of 3,5-dihydroxytoluene had a mitigating effect on tobacco seedlings, with 5 mmol / L showing the best mitigating effect.

[0082] Table 4. Case ranking values ​​and comprehensive evaluation of each indicator under different concentrations of 3,5-dihydroxytoluene treatment.

[0083]

[0084]

[0085] Based on comprehensive analysis of various indicators in tobacco seedlings, exogenous 3,5-dihydroxytoluene (3,5-dihydroxytoluene) can effectively increase the activity of antioxidant enzymes in tobacco leaves under the dual stress of low temperature and low oxygen, and reduce the content of reactive oxygen species, thereby mitigating the damage to tobacco plants caused by low temperature and low oxygen. Specifically, the exogenous 3,5-dihydroxytoluene treatment showed the following effects: 5 mmol / L > 8 mmol / L > 3 mmol / L. Treatment with exogenous 3,5-dihydroxytoluene is easy to implement in practice, has proven effective, and shows great promise as a method to improve the resistance to low temperature and low oxygen stress in tobacco floating seedling production, with significant application potential.

Claims

1. The application of 3,5-dihydroxytoluene in improving the low-temperature and low-oxygen tolerance of tobacco seedlings, characterized in that, The concentration of the 3,5-dihydroxytoluene is [3 mmol / L, 8 mmol / L]; The method of applying the 3,5-dihydroxytoluene is as follows: add the 3,5-dihydroxytoluene to the hydroponic solution of tobacco seedlings at the 2-leaf-1-heart stage, and ensure that the roots of the tobacco seedlings are completely submerged in the hydroponic solution.

2. The application as described in claim 1, characterized in that, The cultivation method for tobacco seedlings is as follows: tobacco seeds are disinfected, rinsed with distilled water and drained, germinated at 25℃, and then cultivated until the seedlings reach the 2-leaf-1-heart stage.

3. The application as described in claim 2, characterized in that, The germination method is as follows: 16 hours of light treatment followed by 8 hours of darkness treatment within one day, repeated for 7-8 days.

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