Application of exogenous trehalose in enhancing low-temperature tolerance of tobacco

Abstract

The invention discloses application of exogenous trehalose in enhancing low-temperature tolerance of tobacco, and belongs to the technical field of biology. According to the application of the exogenous trehalose in enhancing the low-temperature tolerance of the tobaccos, membrane lipid peroxidation caused by low-temperature stress can be slowed down by externally applying the trehalose, ROS accumulation can be inhibited, the activity of antioxidant enzymes and the content of osmotic regulation substances can be improved, and the low-temperature tolerance of the tobaccos can be enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to the application of exogenous trehalose in enhancing the low temperature tolerance of tobacco. Background Art

[0002] Low temperature is a common abiotic stress that affects plant growth and has an important impact on the distribution and yield of crops. Under low temperature stress, the types of reactive oxygen species (ROS) in plant cells increase, causing damage to mitochondrial structure and cellular energy supply. In this process, the activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in the antioxidant enzyme system increase, which can effectively remove the accumulation of ROS in plants, alleviate the peroxidative damage caused by ROS to cells, and maintain normal growth and development of plants. Studies have shown that the interaction between ROS and antioxidants constitutes a metabolic interface between metabolism and environmental signals. Through this interface, plants can regulate the initiation of adaptive processes or activate cell death programs. Low temperature treatment significantly increases the SP content in plants. As one of the osmotic regulators, soluble sugars can enhance intracellular osmotic force to alleviate cell volume shrinkage caused by low temperature stress, and reduce damage caused by chilling and dehydration by improving cell membrane stability. The increase in cell membrane permeability leads to the loss of unsaturated fatty acids, promotes the formation of membrane lipid peroxides, and thus increases the content of malondialdehyde (MDA).

[0003] The application of exogenous substances to regulate plant stress resistance is one of the most efficient and feasible methods. Studies have found that the effects of low temperature stress on crops can be alleviated by applying exogenous substances such as salicylic acid, spermidine, and brassinolide. The maximum photochemical efficiency, total chlorophyll and proline content of pepper seedlings were significantly increased by spraying spermidine at low temperatures, thereby effectively reducing the chilling injury index and mortality. Exogenous salicylic acid treatment can improve the plant's ability to defend against adversity by increasing the parameters related to photosynthetic electron transfer activity and the activity of antioxidant enzymes in wheat seedlings under low temperature stress. In addition, salicylic acid treatment can increase the activity of cell membrane protective enzymes in rice, tobacco, and watermelon, effectively remove the accumulation of reactive oxygen in plants, reduce the degree of plant membrane lipid peroxidation, and thus improve the cold tolerance of crops.

[0004] Trehalose (abbreviated as Tre) is a non-chemically reducing disucrose formed by two hydroxy glucoses connected by a, a1, 1 glycosidic bond. It is widely found in native bacteria, true insects and various invertebrates. During abiotic stress, trehalose has the functions of protecting intracellular active substances, regulating cell osmotic pressure, scavenging free radicals, and maintaining organelle integrity. Studies have shown that Tre mainly enhances the abiotic stress tolerance of crops by regulating metabolic processes such as antioxidant systems, photosynthesis and ion balance. Trehalose can enhance the resistance of plants to abiotic stress by activating antioxidant enzymes and reducing plasma membrane damage. Exogenous trehalose can maintain plant respiration by increasing plant accumulation of sugars, reduce the accumulation of starch content in nutritional organs, slow down the negative feedback mechanism of photosynthesis, and facilitate the transportation and distribution of starch to source leaves and sinks to improve photosynthesis, thereby increasing the accumulation of photosynthetic assimilates.

[0005] Tobacco (Nicotiana tabacum L.) originated in warm South America. It is a leaf-growing economic crop that likes warmth but is not cold-tolerant. During its growth and development, the optimum growth temperature for tobacco is generally 25℃ to 28℃. Low temperature stress is a common climate disaster during the tobacco transplanting period. In recent years, studies have found that when tobacco is subjected to low temperature stress during the seedling stage, the leaf thickness decreases, the chloroplast ultrastructure is damaged, and the light energy conversion efficiency and net photosynthetic rate decrease. At the same time, low temperature significantly inhibits the increase in leaf length, width and leaf area, which is not conducive to photosynthesis and the accumulation of organic matter, causing plant growth retardation and causing serious damage to the plant. At the same time, continuous low temperature will cause it to flower early, and it will not reach the expected plant height, number of leaves and leaf size, which will seriously affect the yield and quality of tobacco. Studies have found that during the cold weather in late spring, low temperature stress not only harms the roots of flue-cured tobacco seedlings, but also harms the aboveground parts of flue-cured tobacco seedlings; low temperature and rain have a certain relationship with some diseases and insect pests in the growth process of tobacco, such as affecting the occurrence of tobacco brown star pathogen, mosaic disease, and parasitic wasps. Low temperature and chilling damage can cause the accumulation of dry matter in tobacco to decrease, the leaves to become thinner after baking, and the oil content to decrease, thus affecting both yield and quality.

[0006] Therefore, providing the application of exogenous trehalose in enhancing the low temperature tolerance of tobacco is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides the use of exogenous trehalose in enhancing the low temperature tolerance of tobacco.

[0008] The present invention uses flue-cured tobacco seedlings as materials, and through exogenous application of trehalose solution of appropriate concentration, explores its effects on the growth, membrane lipid stability, ROS accumulation and antioxidant enzyme system of flue-cured tobacco seedlings under low temperature stress, in order to reveal the regulatory mechanism of trehalose on tobacco cold tolerance, which has theoretical and practical significance for the application of exogenous regulatory substances in agricultural production to improve the cold tolerance of flue-cured tobacco seedlings.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] Application of exogenous trehalose in enhancing low temperature tolerance of tobacco.

[0011] Furthermore, exogenous trehalose was used to improve the activities of catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) and reduced glutathione (GSH) in tobacco under low temperature stress.

[0012] Furthermore, exogenous trehalose was used to increase the content of proline (PRO) and soluble protein (SP) in tobacco under low temperature stress.

[0013] Furthermore, exogenous trehalose was used to reduce the contents of malondialdehyde (MDA), superoxide anion (OFR) and hydrogen peroxide (H2O2) in tobacco under low temperature stress.

[0014] Furthermore, the application of exogenous trehalose in reducing the relative electrical conductivity (EL) level of tobacco under low temperature stress.

[0015] Furthermore, the exogenous trehalose concentration is 15 mM.

[0016] Furthermore, the low temperature stress treatment temperature is 4°C.

[0017] When plants face adversity such as low temperature stress, a large amount of ROS will be produced. Excessive accumulation of ROS will cause disorders in plant physiological activities. Reactive oxygen species (ROS) are byproducts of metabolic processes in mitochondria, chloroplasts and peroxisomes. They have dual functions in adversity and have a dose effect. Under normal growth conditions, the production and removal of ROS in plants are in a state of dynamic equilibrium. In adversity, adverse environmental factors will induce the production of excessive hydroxyl free radicals (OH - ), superoxide anion radical (O 2- ) and hydrogen peroxide (H2O2) and other ROS toxic substances. The ROS staining results of the present invention show that the staining area of ​​the leaves of the seedlings treated with trehalose and then subjected to low temperature stress is smaller and the staining degree is lower; the biochemical results also show that 15mmol / L trehalose treatment can significantly reduce the OFR content and H2O2 content of tobacco leaves under low temperature stress, reduce the enrichment of ROS, and thus alleviate the peroxidation damage of tobacco induced by low temperature.

[0018] Excessive accumulation of ROS causes peroxidation of plant membrane lipids, produces a large amount of MDA, and causes serious damage to cells. At the same time, low temperature stress destroys the cell membrane structure of plants, increases cell membrane permeability, and causes electrolyte exosmosis. The exosmotic electrolytes can cause an increase in the conductivity of the solution. The results of the present invention show that the application of 15mmol / L trehalose can significantly reduce the MDA content and EL level of tobacco seedlings under low temperature stress, indicating that trehalose alleviates tobacco membrane lipid peroxidation caused by low temperature stress, improves the stability and fluidity of membrane lipids, and enhances tobacco low temperature tolerance.

[0019] In order to protect their own systems from damage by ROS, plants have formed a protection system, namely the antioxidant system, which includes enzymes and non-enzyme antioxidants. Antioxidant enzymes include SOD, POD, CAT, APX, etc., and GSH is an important non-enzyme antioxidant. When plants are subjected to low temperature stress, plants increase the activity of peroxidase to remove the reactive oxygen accumulated in the cells and alleviate the degree of cell membrane lipid peroxidation, thereby enhancing the adaptability of plants to adverse stress. The results of the present invention show that the application of 15mmol / L trehalose significantly increased the activity of CAT, APX, and GSH in tobacco seedlings under low temperature stress, which indicates that exogenous trehalose increases the activity of the antioxidant system, helps to enhance the removal of ROS by plants, and alleviates the peroxidation damage of low temperature stress to tobacco seedlings.

[0020] Under low temperature stress, osmotic regulating substances such as proline in plants will increase rapidly, improving the cold resistance of plants. As important osmotic regulating substances in plants, soluble protein and proline can effectively remove reactive oxygen free radicals in plants, improve the antioxidant capacity of plants, and thus improve the stress resistance of plants. Under adverse stress, osmotic regulating substances maintain cell turgor pressure, prevent excessive water loss of protoplasm, stabilize organelle structure, regulate certain physiological functions, and alleviate the damage of adverse stress to plants. The results of the present invention show that the exogenous application of trehalose significantly increases the content of proline and soluble protein in tobacco seedlings under low temperature stress, and improves the performance of the osmotic regulation system of tobacco under low temperature stress.

[0021] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses the application of exogenous trehalose in enhancing the low temperature tolerance of tobacco. The seedlings of the 6-8 leaf stage flue-cured tobacco (Nicotiana tabacum L.) variety 'Xiangyan No. 7' were used as test materials, and 200 mL of 15 mM trehalose solution was applied exogenously for 24 hours, and then transferred to a light incubator for 4°C low temperature stress treatment for 2 days, and the phenotype was observed and the physiological and biochemical indicators were measured. The results of phenotypic analysis showed that the exogenous application of trehalose significantly alleviated the inhibitory effect of low temperature stress on the growth of tobacco seedlings. The results of physiological and biochemical tests showed that exogenous trehalose significantly reduced the malondialdehyde (MDA) content, relative conductivity (EL) level, superoxide anion (OFR) and hydrogen peroxide (H2O2) content of tobacco under low temperature stress, and increased the activities of catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), reduced glutathione (GSH) and the content of proline (PRO) and soluble protein (SP). In summary, the application of exogenous trehalose improved the stability of tobacco cell plasma membrane under low temperature stress, increased its antioxidant enzyme activity and osmotic regulation substance content, reduced the accumulation of reactive oxygen, and thus enhanced the tolerance of tobacco to low temperature stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The accompanying drawings show the cold tolerance phenotype analysis of tobacco after trehalose was applied externally in the present invention; A: top view; B: side view;

[0024] Figure 2 The accompanying drawings show the changes in the ROS staining degree of tobacco seedlings under low temperature stress after trehalose is applied externally according to the present invention; A: NBT staining; B: DAB staining;

[0025] Figure 3 The accompanying drawings show the changes in OFR (A) and H2O2 (B) content of tobacco seedlings under low temperature stress after exogenous application of trehalose according to the present invention;

[0026] Figure 4 The accompanying drawings show the changes in MDA content (A) and EL value (B) of tobacco seedlings under low temperature stress after exogenous application of trehalose according to the present invention;

[0027] Figure 5The accompanying drawings show the changes in antioxidant enzyme activities of tobacco seedlings under low temperature stress after trehalose was applied externally according to the present invention; A: CAT; B: APX; C: POD; D: GSH;

[0028] Figure 6 The accompanying drawings show the changes in PRO (A) and SP (B) contents of tobacco seedlings under low temperature stress after exogenous application of trehalose according to the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] To ensure that the research results have wide applicability, the present invention uses the low-temperature resistant variety 'Xiangyan No. 7' as the experimental material. As a representative of cold-resistant varieties, 'Xiangyan No. 7' shows more robust physiological and biochemical responses under cold conditions. The tobacco variety 'Xiangyan No. 7' was provided by the China Tobacco Central and Southern Agricultural Experiment Station. Trehalose was purchased from Beijing Solebow Technology Co., Ltd.

[0031] Example 1

[0032] This experiment was conducted in the laboratory of Hunan Tobacco Science Research Institute (Central South Station). First, the surface of the seeds was disinfected with 75% alcohol for 30 seconds, and then disinfected with seed disinfectant (69.90% ddH2O: 30% sodium hypochlorite: 0.1% Tween-20) for five minutes. After being rinsed with distilled water, they were placed at 4℃ low-temperature vernalization for 48 hours, and then placed in an artificial climate chamber for germination in a seedling tray. The light intensity was set to 4000Lx, the day / night temperature was (25±2)℃, the humidity was 75%, and the photoperiod was 16h / 8h. When the tobacco seedlings grew to the four-leaf stage, they were uniformly fertilized, 200mL per plant (the fertilizer formula was: 150g tobacco-specific base fertilizer (N: P2O5: K2O = 7%: 14%: 8%), 75g potassium nitrate, and 75g potassium sulfate dissolved in 180L water). When the tobacco grew to the six-leaf and one-heart stage, tobacco seedlings with consistent growth were selected for experimental treatment.

[0033] There were 4 treatments in the experiment: CK-H2O (root irrigation with distilled water + normal temperature treatment), CK-Tre (root irrigation with trehalose + normal temperature treatment), LT-H2O (root irrigation with distilled water + low temperature treatment), and LT-Tre (root irrigation with trehalose + low temperature treatment). The optimal concentration of exogenous trehalose was 15mM in the preliminary experiment. 200ml of trehalose solution or distilled water was applied exogenously (root irrigation) to each tobacco seedling. After 24h of treatment, the CK-H2O and CK-Tre groups were placed in a 25℃ constant temperature and humidity incubator, and the LT-H2O and LT-Tre groups were placed in a pre-set 4℃ constant temperature and humidity incubator for 2 days (2d). Other culture conditions were 75% humidity, 16h of light, and 8h of darkness. Two days after treatment, the second to third true leaves of the flue-cured tobacco seedlings were taken from top to bottom for ROS tissue staining, and the fourth true leaf of the flue-cured tobacco seedlings was taken from top to bottom for determination of physiological and biochemical indices and relative conductivity content, with 5 biological replicates in each group.

[0034] Physiological and biochemical indexes:

[0035] (1) ROS tissue staining

[0036] DAB staining: Take the second true leaf of the seedling (counting from the top leaf) and soak it in DAB staining solution with a pH of 5.0, and treat it in the dark at room temperature for 12 hours. Remove the staining solution, add anhydrous ethanol, and boil it in a boiling water bath for 10 minutes to remove the chlorophyll in the leaves. Finally, transfer the leaves to 75% alcohol, take pictures, observe and preserve.

[0037] NBT staining: Take the third true leaf of the seedling (counting from the top leaf downwards) and immerse it in NBT staining solution. Stain it under light for 1-2 hours. When a color phenotype appears (blue leaves), remove the staining solution, add anhydrous ethanol and boil it in a boiling water bath for 10 minutes. When the chlorophyll on the leaves is removed, transfer the leaves to 75% alcohol, take pictures, observe and preserve them.

[0038] (2) Determination of biochemical indicators

[0039] 0.2 g of fresh leaves were weighed and the superoxide anion (OFR) content (Jiangsu Addison ADS-W-YH008), malondialdehyde (MDA) content (Nanjing Jiancheng A003-3-1), hydrogen peroxide (H2O2) content (Nanjing Jiancheng A064-1-1), catalase (CAT) activity (Nanjing Jiancheng A007-1-1), ascorbate peroxidase (APX) activity (Nanjing Jiancheng A123-1-1), trace reduced glutathione (GSH) content (Nanjing Jiancheng A006-2-1), peroxidase (POD) activity (A084-3-1), soluble protein (SP) content (A045-2) and proline (PRO) content (Suzhou Grace G0111WPRO) were determined using kits.

[0040] (3) Relative conductivity measurement

[0041] Take 0.2g of fresh tissue leaves from the fourth true leaf of the seedling (counting from the top leaf downward) with a circular puncher, put them into a centrifuge tube containing 25ml ddH2O, shake them at 37℃ for 24h, measure the first conductivity value of the exudate L1, then put the EP tube into an autoclave at 120℃ for 15 minutes, and measure the second conductivity value of the exudate L2 after the temperature cools to room temperature. Calculate the relative conductivity content (EL=L1 / L2*100%).

[0042] (4) Data Statistical Analysis

[0043] Microsoft Excel 2016 was used for data processing, analysis and drawing, and IBM SPSS 27.0 software was used for univariate analysis.

[0044] result:

[0045] 1) Effect of exogenous trehalose on the cold-tolerance phenotype of tobacco

[0046] In the cold tolerance phenotype analysis of tobacco seedlings, the physiological characteristics of tobacco variety 'Xiangyan No. 7' under low temperature stress were mainly observed. Figure 1 As shown in the figure, under normal temperature conditions, there was no significant difference between the CK-H2O and CK-Tre groups. However, after 24 hours of low temperature treatment, the leaves of the seedlings in the LT-H2O group wilted significantly, while there was no significant change in the LT-Tre group. This result shows that exogenous application of trehalose alleviated the effects of low temperature stress and enhanced the cold tolerance of tobacco seedlings ( Figure 1 ).

[0047] 2) Effect of exogenous trehalose on ROS accumulation in tobacco under low temperature stress

[0048] Due to the adverse stress caused by changes in the external environment or plant diseases or external biological damage, a large amount of ROS accumulates in the plant body. Excessive ROS usually causes serious oxidative damage to the plant. The amount of ROS accumulated in the plant body is one of the important indicators to measure the degree of damage to the plant after suffering from adverse stress. Under the reduction of superoxide anions, green nitro blue tetrazolium (NBT) can produce blue compounds; in the presence of POD, 3,3' diaminobenzidine (DAB) can be oxidized by hydrogen peroxide to produce a brown-red precipitate. Figure 2 As shown, there was no significant difference between CK-H2O and CK-Tre after NBT and DAB staining; low temperature stress caused the two staining levels to deepen, but compared with LT-H2O, the staining area of ​​LT-Tre leaves was smaller and the staining level was lighter, indicating that LT-Tre plants accumulated less OFR and H2O2.

[0049] Furthermore, the contents of ORF and H2O2 were determined. Figure 3 As shown in the figure, under normal temperature, there was no significant difference in the ORF content between CK-H2O and CK-Tre; low temperature stress led to the accumulation of OFR in the leaves of plants, and the OFR content in LT-Tre plants was significantly lower than that in LT-H2O ( Figure 3 A), which is consistent with Figure 2 The results of A were consistent. Similar to the superoxide anion content, there was no significant difference in the H2O2 content accumulated in the leaves of the seedlings of the CK-H2O and CK-Tre groups; low temperature stress led to the accumulation of H2O2 in tobacco seedlings, but compared with LT-H2O, the H2O2 content accumulated in the leaves of the seedlings of the LT-Tre group was significantly reduced ( Figure 3 B), which is consistent with Figure 2 The staining results were consistent with those of B. The above results indicate that exogenous application of trehalose can alleviate the accumulation of ROS induced by low temperature stress.

[0050] 3) Effect of exogenous trehalose on the stability of tobacco plasma membrane under low temperature stress

[0051] Plant cell membranes are the most sensitive organelles to low temperatures. MDA is a decomposition product of membrane lipid peroxidation in plants under adverse stress. The MDA content and relative conductivity (EL) can effectively reflect the degree of cell damage. Figure 4 As shown in the figure, under normal temperature, there was no significant difference between CK-H2O and CK-Tre; low temperature stress led to an increase in MDA content, but compared with LT-H2O, the MDA content in LT-Tre plants was significantly reduced ( Figure 4 A). Similarly, there was no significant difference in the EL values ​​between the CK-H2O and CK-Tre groups; low temperature treatment led to an increase in the EL level in the plant body, while the EL level of the LT-Tre group was significantly lower than that of the LT-H2O group under low temperature treatment ( Figure 4 B). The above results show that exogenous application of trehalose can significantly reduce the MDA content and EL of tobacco leaves under low temperature stress, which helps to enhance the stability of the plasma membrane.

[0052] 4) Effect of exogenous trehalose on antioxidant enzyme activities of tobacco under low temperature stress

[0053] like Figure 5 As shown in the figure, under normal temperature, the CAT activity in CK-Tre plants was significantly reduced compared with CK-H2O; low temperature stress led to an increase in CAT activity in plants, but compared with LT-H2O, the CAT activity in LT-Tre plants was significantly increased ( Figure 5A). Under normal temperature, the APX activity in CK-Tre plants was significantly higher than that in CK-H2O; low temperature treatment led to an increase in APX activity in plants, and the APX activity in LT-Tre plants was significantly higher than that in LT-H2O ( Figure 5 B). Under normal temperature, the POD activity in CK-Tre plants was significantly higher than that in CK-H2O plants; low temperature stress led to an increase in POD activity in plants, but compared with LT-H2O, there was no significant difference in POD activity in LT-Tre plants ( Figure 5 C). There was no significant difference in GSH activity between the CK-H2O and CK-Tre groups; low temperature treatment led to a decrease in GSH activity in plants, and the GSH activity in LT-Tre plants was significantly higher than that in LT-H2O ( Figure 5 D). The above results showed that exogenous application of trehalose significantly increased the antioxidant enzyme activities of tobacco seedlings under low temperature stress.

[0054] 5) Effect of exogenous trehalose on the content of osmotic regulating substances in tobacco under low temperature stress

[0055] like Figure 6 As shown in the figure, under normal temperature, the PRO content in CK-Tre plants was significantly higher than that in CK-H2O; low temperature stress led to an increase in PRO content, and the PRO content in LT-Tre plants was significantly higher than that in LT-H2O ( Figure 6 A). Under normal temperature, there was no significant difference in the SP content between the CK-H2O and CK-Tre groups; low temperature treatment led to an increase in the SP content in the plants, and the SP content in the LT-Tre plants was higher than that in the LT-H2O ( Figure 6 B). The above results show that exogenous application of trehalose significantly increased the content of osmotic regulating substances in tobacco seedlings under low temperature stress.

[0056] Conclusion: The results of the present invention show that low temperature stress leads to aggravated lipid peroxidation of tobacco cells, increased ROS content, changes in antioxidant enzyme activity and osmotic regulation substance content; while exogenous trehalose can slow down lipid peroxidation caused by low temperature stress, inhibit the accumulation of ROS, increase antioxidant enzyme activity and osmotic regulation substance content, and enhance tobacco low temperature tolerance.

[0057] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of exogenous trehalose in enhancing low temperature tolerance of tobacco.

2. Application of exogenous trehalose in improving the activities of catalase, ascorbate peroxidase, peroxidase and reduced glutathione in tobacco under low temperature stress.

3. Application of exogenous trehalose in increasing the proline and soluble protein content of tobacco under low temperature stress.

4. Application of exogenous trehalose in reducing the content of malondialdehyde, superoxide anion and hydrogen peroxide in tobacco under low temperature stress.

5. Application of exogenous trehalose in reducing the relative conductivity level of tobacco under low temperature stress.

6. The use according to any one of claims 1 to 5, characterized in that: The exogenous trehalose concentration was 15 mM.

7. The use according to any one of claims 1 to 5, characterized in that: The low temperature stress treatment temperature is 4°C.

Images