Application of alpha-salicin or / and alpha-isosalicin in basil planting

Abstract

The invention relates to application of alpha-salicin or / and alpha-isosalicin in basil planting, belongs to the field of plant planting, and finds that the alpha-salicin or / and alpha-isosalicin can promote germination and growth of basil seeds, especially under drought stress, and can promote germination and growth of basil seeds. Alpha-salicin or / and alpha-isosalicin can improve the germination rate of basil seeds and improve the growth condition, and in the planting process, the content of nutrient substances in basil plants can be increased by irrigating the basil plants with an alpha-salicin or / and alpha-isosalicin solution.

Description

Technical Field

[0001] The present invention belongs to the field of plant cultivation, and particularly relates to the application of α - salicin or / and α - isosalicin in the cultivation of Ocimum basilicum L. Background Art

[0002] Glycoside compounds play an indispensable role in physiological processes such as plant growth and development, and defense responses. In recent years, their unique biological activities have attracted much attention in the scientific research community and shown great potential in plant stress resistance regulation. Many glycoside products are closely related to the physiological functions of salicylic acid, and they can be interconverted through specific metabolic pathways in plants. As a common glycoside compound, β - salicin has been proven to have biological activities such as anti - inflammation and antioxidant. In plants, β - salicin can be metabolically converted into salicylic acid to exert its physiological functions.

[0003] Recent studies have shown that the α / β - configuration of glycosides significantly affects their biological activities through steric hindrance effects. For example, β - salicin can be hydrolyzed into salicylic acid (SA) by β - glucosidase to indirectly activate the stress resistance signaling pathway, while the α - configuration is difficult to be recognized by conventional glycosidases due to the steric hindrance between the sugar group and the phenolic hydroxyl group, and may trigger downstream responses by directly binding to membrane receptors or regulating transcription factors (such as NAC, WRKY).

[0004] Ocimum basilicum L. is an annual herbaceous plant of the genus Ocimum in the Lamiaceae family, which is distributed in many countries around the world. It is usually cultivated as a medical supply and a spice, and is an aromatic plant with both culinary and medicinal values. The growing market demand has promoted the expansion of its cultivation scale. However, drought stress severely limits the cultivation and growth of Ocimum basilicum L. in arid regions, resulting in a decline in yield and quality, and hindering the development of the Ocimum basilicum L. industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of α - salicin or / and α - isosalicin in the cultivation of Ocimum basilicum L., and the α - salicin and α - isosalicin can promote the seed germination and growth of Ocimum basilicum L., and at the same time improve the drought resistance of Ocimum basilicum L.

[0006] The present invention is achieved by the following technical solutions:

[0007] The present invention provides one of the applications of α - salicin or / and α - isosalicin in the cultivation of Ocimum basilicum L., and the application is to promote the seed germination and growth of Ocimum basilicum L. by using α - salicin or / and α - isosalicin.

[0008] Furthermore, the application method is to soak Ocimum basilicum seeds with α - salicin and α - isosalicin solutions, and irrigate them with α - salicin or / and α - isosalicin solutions during the growth process.

[0009] As a preferred technical solution, when used alone, the concentrations of both α - salicin and α - isosalicin solutions are 0.100 mmol·L -1 , and when they are used in combination, the concentrations of both α - salicin and α - isosalicin solutions are 0.0500 mmol·L -1 .

[0010] The present invention provides a second application of α - salicin or / and α - isosalicin in the cultivation of Ocimum basilicum. The application is to promote the germination and growth of Ocimum basilicum seeds with α - salicin or / and α - isosalicin under drought stress.

[0011] Furthermore, the application method is to soak Ocimum basilicum seeds with α - salicin or / and α - isosalicin solutions, and irrigate them with α - salicin or / and α - isosalicin solutions during the growth process.

[0012] As a preferred technical solution, when used alone, the concentrations of both α - salicin and α - isosalicin solutions are 0.100 mmol·L -1 , and when they are used in combination, the concentrations of both α - salicin and α - isosalicin solutions are 0.0500 mmol·L -1 .

[0013] The present invention provides a third application of α - salicin or / and α - isosalicin in the cultivation of Ocimum basilicum. The application is to promote the increase and accumulation of nutrients in Ocimum basilicum plants with α - salicin or / and α - isosalicin solutions under drought conditions.

[0014] Furthermore, the application method is to irrigate Ocimum basilicum plants with α - salicin or / and α - isosalicin solutions.

[0015] As a preferred technical solution, when used alone, the concentrations of both α - salicin and α - isosalicin solutions are 0.100 mmol·L -1 , and when they are used in combination, the concentrations of both α - salicin and α - isosalicin solutions are 0.0500 mmol·L -1 .

[0016] Advantages of the present invention compared with the prior art: The present invention discovers that during the normal planting process, α-salicin and α-isosalicin can promote the germination and growth of Ocimum basilicum seeds and significantly promote the accumulation of nutrients. Under drought stress, α-salicin and α-isosalicin can also promote the germination and growth of Ocimum basilicum seeds, improving the germination rate and growth of seeds under drought stress. Under drought stress during the planting process, irrigating Ocimum basilicum plants with α-salicin or / and α-isosalicin solution can increase the nutrient content therein. Description of the Drawings

[0017] Figure 1 It is a reaction formula diagram of sucrose phosphorylase for the biosynthesis of α-salicin and α-isosalicin;

[0018] Figure 2 It is a diagram showing the effects of different concentrations of α-salicin, α-isosalicin, and salicyl alcohol on the soluble sugar content of Ocimum basilicum;

[0019] Figure 3 It is a diagram showing the effects of different concentrations of α-salicin, α-isosalicin, and salicyl alcohol on the soluble protein content of Ocimum basilicum;

[0020] Figure 4 It is a diagram showing the effects of different concentrations of α-salicin, α-isosalicin, and salicyl alcohol on the chlorophyll content of Ocimum basilicum;

[0021] Figure 5 It is a diagram showing the effects of applying different substances on the soluble sugar content of Ocimum basilicum seedling leaves under drought stress;

[0022] Figure 6 It is a diagram showing the effects of applying different substances on the soluble protein content of Ocimum basilicum seedling leaves under drought stress;

[0023] Figure 7 It is a diagram showing the effects of applying different substances on the chlorophyll content of Ocimum basilicum seedling leaves under drought stress;

[0024] Figure 8 It is a diagram showing the effects of applying different substances on the MDA content of Ocimum basilicum seedling leaves under drought stress;

[0025] Figure 9 It is a diagram showing the effects of applying different substances on the SOD content of Ocimum basilicum seedling leaves under drought stress;

[0026] Figure 10 It is a diagram showing the effects of applying different substances on the POD content of Ocimum basilicum seedling leaves under drought stress;

[0027] Figure 11 It is a diagram showing the effects of applying different substances on the CAT content of Ocimum basilicum seedling leaves under drought stress. Detailed Embodiments

[0028] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] Experimental materials: Ocimum basilicum seeds of consistent size and plump grains were selected. α - Salicin and α - isosalicin were catalytically synthesized by the sucrose phosphorylase Suc75290 in our laboratory (purity ≥ 98%) (the amino acid sequence number of sucrose phosphorylase Suc75290 is PP496818); PEG6000 is of analytical purity. Other reagents used in the experiment are all of analytical purity.

[0030] Enzymatic preparation of α - salicin and α - isosalicin:

[0031] 1. Preparation method:

[0032] The reaction system contains 50 mM MES - NaOH buffer (pH 7.5), 240 g / L sucrose, 12 g / L o - hydroxybenzyl alcohol, and purified enzyme Suc75290 (150 U·mL-1). The reaction is carried out at 45 °C for 20 h. The products are quantified by HPLC (C18 column, methanol: water = 35:65, flow rate 1.0 mL·min-1, detection wavelength 280 nm). The conversion rate calculation formula is: (product peak area / initial substrate peak area) × 100%. The reaction equation is as Figure 1 shown.

[0033] 2. Separate the samples using a semi - preparative column and purify the samples. After collecting the samples, perform rotary evaporation for loading. Check whether the samples are single by high - performance liquid chromatography and analyze the purity of the purified samples by TLC. High - performance liquid chromatography (HPLC) detection conditions: Shimadzu PDA detector; semi - preparative C18 column; mobile phase: water: methanol = 70:30; flow rate: 3 mL / min; column temperature 30 °C; detection wavelength 280 nm.

[0034] 3. Freeze - dry and collect the o - hydroxybenzyl alcohol glycosylation products

[0035] Carry out rotary evaporation for 30 min on the effluent containing the target products respectively. The water bath temperature is 40 °C, the pressure is 50 mbar, and the rotation speed is 100 r / min. Concentrate by rotary evaporation to about 2 ml, and collect the solid substances after freeze - drying, which are the glycosylation products.

[0036] Example 1. Effects of different concentrations of α - salicin and α - isosalicin on seed germination and growth

[0037] 1. Experimental design

[0038] The Ocimum basilicum seeds were disinfected by soaking in 0.05% sodium hypochlorite solution for 5 min, then rinsed with ultrapure water 5 - 6 times and soaked for another 24 h. Sixteen treatment groups were set up, including treatment groups with different concentrations (0.005 mmol·L-1, 0.01 mmol·L-1, 0.05 mmol·L-1, 0.100 mmol·L-1, 0.500 mmol·L-1) of α - salicin, α - isosalicin and salicyl alcohol (o - hydroxybenzyl alcohol) solutions, with distilled water as the control (CK). Fifty seeds were selected for each treatment group, with 3 replicates. The treated seeds were placed in a 10 - cm Petri dish, with three layers of filter paper laid at the bottom as the germination bed, and germinated in an incubator at 25℃. The filter paper and treatment solution were changed daily, and the seed germination situation was recorded.

[0039] Table 1 Different Treatments of Ocimum basilicum Seeds

[0040]

[0041] 2. Data Statistics Observation started from the day when the seeds were placed on the bed. Taking the radicle length being greater than or equal to 2 times the seed diameter as the germination standard, the number of germinated seeds was recorded daily. The day when the first seed germinated in one of the three replicates was taken as the start period of germination for this treatment, and the day when the seed germination rate of each treatment did not change significantly was taken as the end period of germination. The number of germinated seeds was counted daily. The germination energy (GE) was calculated at 3 d of germination, and the germination rate (GR) was calculated at 10 d.

[0042] Germination energy (GE) = (Number of germinated seeds on the 3rd day / Number of tested seeds) x 100%;

[0043] Germination rate (GP) = (Number of germinated seeds on the 10th day / Number of tested seeds) x 100%.

[0044] The data were expressed as mean ± standard deviation. One - way analysis of variance (ANOVA) was performed using SPSS 22.0. The differences between groups were tested by the LSD method (p < 0.05). In the charts, "*" indicates a significant difference from the control (p < 0.05), and "**" indicates a highly significant difference (p < 0.01).

[0045] 3. The experimental results showed that 0.100mmol·L-1 α-salicin and α-isosalicin treatment significantly increased the germination rate, germination potential, root length and fresh weight of basil seeds (Table 2, Table 3), indicating that these two compounds can effectively promote the germination of basil seeds and seedling growth. This promotion may be related to their regulation of plant hormone signaling pathways. As shown in Tables 2 and 3, the germination rates of the 0.100mmol·L-1 α-salicin (T4) and α-isosalicin (M4) treatment groups were 91.33% and 91.67%, respectively, which were significantly higher than the control (CK, 85.33%) (p<0.05). The root length increased by 23.8%, indicating that α-configuration glycosides can break through the metabolic limitations of β-configuration and directly promote seedling growth.

[0046] Table 2 Effects of different concentrations of α-salicin on the growth indexes of basil seeds

[0047]

[0048] Table 3 Effects of different concentrations of α-isosalicylic acid on the growth indexes of basil seeds

[0049]

[0050] Soluble sugar is an important source of energy in the growth and development of plants, and is also a key substance involved in osmotic regulation. As an important nutrient and osmotic regulator, the increase in soluble protein content helps maintain the stability of the intracellular environment, thereby effectively promoting the germination of plant seeds and the growth of seedlings. Figure 3 and Figure 4 As shown, the soluble sugar and soluble protein contents of the treatment groups with α-salicin and α-isosalicin were higher than those of the CK (clear water) group, and the most significant addition was 0.100mmol·L-1. At this concentration, the soluble sugar content of the α-salicin treatment group increased by 45.9% and the soluble protein content increased by 33.8% compared with the CK group; the soluble sugar content of the α-isosalicin treatment group increased by 46.2% and the soluble protein content increased by 42.2% compared with the CK group. This fully demonstrates that the addition of α-salicin and α-isosalicin effectively promotes the increase and accumulation of nutrients in plants, providing a more sufficient material and energy basis for seed germination and seedling growth.

[0051] The level of chlorophyll content directly reflects the level of plant photosynthesis and growth status. In this experiment, compared with the CK group, the chlorophyll content of plants treated with α-salicylin and α-isosalicylin was significantly increased ( Figure 5). Taking the 0.100mmol·L-1 treatment as an example, the chlorophyll content of the α-salicin treatment group increased by 29.1% compared with the CK group, and the α-isosalicin treatment group increased by 27.9%. The significant increase in chlorophyll content indicates that these two compounds can significantly promote the efficiency of photosynthesis, enabling plants to capture more light energy, convert carbon dioxide and water into more carbohydrates, provide more energy and material guarantees for plant growth, and further promote the healthy growth of plants.

[0052] Example 2 Drought stress experiment

[0053] A solution containing 15% PEG-6000 was selected for drought stress concentration configuration, as shown in Table 4, and PEG+0.100mmol·L-1 salicyl alcohol (S0+DR), α-salicylin (S1+DR), and α-isosalicylin (S2+DR) treatment groups were set, the positive control was salicylic acid (S3+DR) with distilled water (CK), and the negative control was PEG6000 (DR). According to the above seed pretreatment steps, each group of solutions was added to a culture dish, each group was set up with 3 replicates, and each group of solutions was applied 5ml per day, and there was no flowing solution in the culture dish. Other culture conditions were the same as in Example 1;

[0054] Table 4 Experimental design of different concentrations of substances on seed resistance to drought stress

[0055]

[0056] 2. Physiological index measurement

[0057] After the 10th day of seed growth, the physiological indicators of basil leaves were measured. The chlorophyll content was determined by ethanol extraction, and the concentrations of chlorophyll a and b and the total chlorophyll content were calculated by measuring the absorbance values ​​at 663nm and 645nm. The soluble sugar was determined by anthrone sulfate colorimetry, the soluble protein was determined by biuret method, the malondialdehyde (MDA) content was determined by thiobarbituric acid colorimetry, the peroxidase (POD) content was determined by guaiacol colorimetry, the superoxide dismutase (SOD) content was determined by nitroblue tetrazolium reduction method, and the catalase (CAT) content was determined by ultraviolet absorption colorimetry, all according to the instructions of the corresponding kits.

[0058] 3. Statistical Analysis

[0059] The data are expressed as mean ± standard deviation. SPSS22.0 was used for one-way analysis of variance (ANOVA). The differences between the groups were tested by the LSD method (p<0.05). In the charts, "*" indicates a significant difference from the control (p<0.05), and "**" indicates extremely significant (p<0.01).

[0060] 4. Results and Analysis

[0061] 4.1 Effects of Applying α - Salicin and α - Iso - salicin on Seed Germination and Growth under Drought Stress

[0062] Under drought stress conditions, the germination rate, germination potential, and physiological indices such as root length, leaf length, leaf width, and fresh weight of Ocimum basilicum seeds decreased significantly. After applying α - salicin and α - iso - salicin solutions containing 12% PEG - 6000 at different concentrations, as can be seen from the data in Table 5, the germination rate of the α - salicin (S1+DR) treatment group increased by 24.67% compared with the DR group (12% PEG - 6000 solution), and the root length increased by 1.29 cm; the germination rate of the α - iso - salicin (S2+DR) treatment group increased by 21.34% compared with the DR group, and the root length increased by 1.62 cm.

[0063] This indicates that under drought - stress environments, α - salicin and α - iso - salicin can enhance the tolerance of plants to drought stress by regulating the osmotic - regulation mechanism and antioxidant system of plants, relieve the inhibitory effect of drought on the germination of Ocimum basilicum seeds and the growth of seedlings to a certain extent, and maintain a relatively good growth state. However, compared with the normal CK group, there are still certain gaps in each growth index, indicating that the impact of drought stress is still significant, but it is alleviated after treatment with the compounds.

[0064] Table 5 Effects of Applying Different Substances on the Growth Indexes of Ocimum basilicum Seedlings under Drought Stress

[0065]

[0066] 4.3 Effects of Applying α - Salicin and α - Iso - salicin on the Physiological Indexes of Seeds under Drought Stress

[0067] Under drought stress, plants maintain the osmotic pressure inside cells by accumulating substances such as soluble sugars and soluble proteins, thereby relieving water stress. The contents of soluble sugars and soluble proteins in the PEG - 6000 treatment group (DR) are higher than those in the CK, which is a defense mechanism of plants to cope with drought by themselves. When α - salicin and α - iso - salicin are externally applied under PEG - 6000 treatment, the contents of soluble sugars and soluble proteins show an increasing trend. For example, the soluble sugar content in the α - salicin (S1+DR) treatment group increased by 124.9% compared with the DR group, and the soluble protein content increased by 22.4%; the soluble sugar content in the α - iso - salicin (S2+DR) treatment group increased by 124.3% compared with the DR group, and the soluble protein content increased by 32.5%. This shows that α - salicin and α - iso - salicin can enhance the ability of plants to resist drought stress by further increasing the content of osmotic - regulation substances in plants.

[0068] In the PEG-6000 treatment group (DR), the chlorophyll content of Ocimum basilicum significantly decreased, while the chlorophyll content of Ocimum basilicum with exogenous application of α - salicin and α - isosalicin under PEG-6000 treatment showed a significant increase. The chlorophyll content in the α - salicin (S1+DR) treatment group increased by 30.5% compared with the DR group, and that in the α - isosalicin (S2+DR) treatment group increased by 33.9%. This indicates that under drought stress conditions, exogenous application of α - salicin and α - isosalicin promotes the chlorophyll synthesis of Ocimum basilicum, can alleviate the photosynthetic inhibition caused by drought damage, enable the plant to maintain a certain photosynthesis level in the drought environment, and provide energy and material support for plant growth.

[0069] The content of malondialdehyde (MDA) is an important indicator for evaluating the degree of cell membrane damage. Under drought stress, the content of MDA in Ocimum basilicum leaves increased significantly, while the treatment with α - salicin and α - isosalicin can significantly reduce the MDA content. Among them, the MDA content in the α - salicin (S1+DR) treatment group decreased by 55.6% compared with the DR group, and that in the α - isosalicin (S2+DR) treatment group decreased by 58.6%. This shows that they can protect the integrity of the cell membrane and maintain the normal physiological functions of cells by reducing lipid peroxidation. At the same time, water deficiency and osmotic stress will cause oxidative stress in plant cells, and plants resist reactive oxygen species damage through the antioxidant enzyme system. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) play important roles in the defense system of plants against biotic and abiotic stresses. The activities of SOD, POD and CAT enzymes in the drought treatment group (DR) were higher than those in the CK group. When α - salicin and α - isosalicin were added exogenously under drought treatment, the contents of these three enzymes could be further increased. Taking the α - salicin (S1+DR) treatment group as an example, the SOD activity increased by 26.7% compared with the DR group, the POD activity increased by 42.8%, and the CAT activity increased by 35.5%; a similar trend was also observed in the α - isosalicin (S2+DR) treatment group. This shows that α - salicin and α - isosalicin can be used as exogenous elicitors to activate the antioxidant enzyme activity, enhance the ability of plants to scavenge reactive oxygen species, and effectively resist the damage of drought to plant cells.

[0070] Example 3

[0071] Based on the experimental results of Examples 1 and 2, this example uses a 1:1 mixture of α - salicin and α - isosalicin, so that the concentrations of α - salicin and α - isosalicin in the solution are both 0.05 mmol·L-1. This mixed solution has the same technical effect on the germination and growth of Ocimum basilicum seeds as that of using a single component. Even under drought stress, it can also reduce the damage of drought to seeds and plants, improve the germination and growth of seeds, and promote the accumulation of nutrients, just like using α - salicin and α - isosalicin alone.

Claims

1. The application of α-salicin and / or α-isosalicin in the cultivation of large-leaf basil, characterized in that: The application is to utilize α-salicin and / or α-isosalicin to promote the germination and growth of large-leaf basil seeds.

2. The use according to claim 1, characterized in that: The application is to soak the large-leaf basil seeds with α-salicin or / and α-isosalicin solution, and to water them with the α-salicin or / and α-isosalicin solution during the growth process.

3. The use according to claim 2, characterized in that: When used alone, the concentration of α-salicylin and α-isosalicylin solutions is 0.100 mmol·L -1 When the two are mixed, the concentrations of α-salicylin and α-isosalicylin solutions are both 0.0500mmol·L -1 .

4. The use according to claim 1, characterized in that: The α-salicin and α-isosalicin are prepared by the following method: the reaction system contains 50mM pH 7.5MES-NaOH buffer, 240g / L sucrose, 12g / L o-hydroxybenzyl alcohol and 150U·mL-1 purified enzyme Suc75290, reacts at 45°C for 20h, and then separates and purifies the fermentation product to obtain α-salicin and α-isosalicin.

5. The use of α-salicin or / and α-isosalicin in the cultivation of Ocimum basilicum according to claim 1, characterized in that: The application is to utilize α-salicin and / or α-isosalicin to promote the germination and growth of large-leaf basil seeds under drought stress.

6. The use of α-salicin or / and α-isosalicin in the cultivation of Ocimum basilicum according to claim 5, characterized in that: The application is to promote the increase and accumulation of nutrients in large-leaf basil plants by using α-salicin or / and α-isosalicin solution under drought stress.

7. The use according to claim 6, characterized in that: The application method is to use α-salicin or / and α-isosalicin solution to irrigate large-leaf basil plants.

8. The use according to claim 7, characterized in that: When used alone, the concentration of α-salicylin and α-isosalicylin solutions is 0.100 mmol·L -1 When the two are mixed, the concentrations of α-salicylin and α-isosalicylin solutions are both 0.0500mmol·L -1 .

9. The use according to claim 8, characterized in that: The α-salicin and α-isosalicin are prepared by the following method: the reaction system contains 50mM pH 7.5MES-NaOH buffer, 240g / L sucrose, 12g / L o-hydroxybenzyl alcohol and 150U·mL-1 purified enzyme Suc75290, reacts at 45°C for 20h, and then separates and purifies the fermentation product to obtain α-salicin and α-isosalicin.

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