Method for improving low-temperature and low-oxygen composite stress resistance of tobacco seedlings by using exogenous amino acid

Abstract

The invention relates to the technical field of plant cultivation, and discloses a method for improving low-temperature and low-oxygen composite stress resistance of tobacco seedlings by exogenous amino acids, which comprises the following steps: selecting tobacco seedlings in a two-leaf and one-core stage of floating seedling raising; preparing an L-pyroglutamic acid mother solution; performing exogenous treatment on the selected tobacco seedlings in the two-leaf and one-core stage by using the prepared L-pyroglutamic acid mother liquor; after treatment is completed under the low-temperature and low-oxygen condition for 3 days, taking a picture of a tobacco seedling, recording phenotype, sampling roots, and measuring root length and root activity; according to measured values under the treatment of adding different volumes of L-pyroglutamic acid mother liquor with the concentration of 1g / L, data is sorted by software, difference significance analysis is carried out by data processing software, and comprehensive evaluation is carried out by a multi-character fuzzy membership function method. The application comprehensively evaluates the effect of the L-pyroglutamic acid in improving the low-temperature and low-oxygen stress resistance of the tobacco seedlings, and provides important theoretical basis and practical guidance for improving the crop stress resistance in agricultural production.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, in particular to a method for improving the ability of tobacco seedlings to resist low temperature and hypoxia combined stress by using exogenous amino acids. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual herbaceous plant of the genus Nicotiana in the Solanaceae family native to South America. It is a thermophilic economic crop and is relatively sensitive to temperature. The optimum temperature for its growth is 25-28°C. When the ambient temperature is lower than 12°C, the tobacco germination rate is reduced and the seedlings grow slowly. Among them, seedling cultivation is an important link in tobacco leaf production. Temperature is an important factor affecting the strength of tobacco seedlings during the floating seedling cultivation process; during floating seedling cultivation, since the tobacco seedlings float on the nutrient solution, the heat capacity of the water is large and it is difficult to heat up. During the seedling cultivation period, it is easy to suffer from low temperature and chilling damage, resulting in weak and uneven growth of the seedlings. In addition, during the floating seedling cultivation process, the roots of tobacco are immersed in the nutrient solution for a long time, the seedling density is high, the roots require a lot of oxygen, and the contact area between the liquid surface and the air is small, which causes the oxygen content in the nutrient solution to drop rapidly, forming hypoxia stress; the growth of seedlings requires a lot of oxygen, and hypoxia stress will cause uneven emergence of seedlings, and the roots of seedlings are easy to rot, which will affect the absorption of water and nutrients by tobacco seedlings, thereby reducing the quality of seedling cultivation. In recent years, research on the response of plants to single stress of low temperature and low oxygen has made continuous progress, but the response to dual stress of low temperature and low oxygen needs further study. Amino acids play an important role in plant resistance to abiotic stresses, helping plants better cope with the challenges of abiotic stresses by enhancing stress resistance, promoting endogenous hormone synthesis, improving physiological metabolic processes, and balancing nutrient absorption.

[0003] Prior art 1, Chinese patent, application number: 202411099496.2 discloses an application of 3,5-dihydroxytoluene in improving the low temperature and hypoxia tolerance of tobacco seedlings, discovers and confirms the application of 3,5-dihydroxytoluene in improving the low temperature and hypoxia tolerance of tobacco seedlings, and can promote the growth of tobacco seedlings. Although the use of exogenous 3,5-dihydroxytoluene treatment 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 hypoxia stress in the production practice of tobacco floating seedlings, and has great application potential; however, as a chemical substance, 3,5-dihydroxytoluene may pose potential risks to the environment and human health through long-term use and residue; the mechanism of action may be more complicated, and there may be multiple side effects that affect the normal growth and development of tobacco seedlings; its performance may be more unstable under different environmental conditions; the production and use costs may be high, and it may not be easy to obtain in some areas.

[0004] Prior art 2, Chinese patent, application number: 202210077144.1 discloses an application of an exogenous selenium-molybdenum composition in alleviating chromium stress in tobacco. By conducting indoor hydroponic experiments, the effects of selenium-molybdenum combination on biomass, chlorophyll content, antioxidant enzyme activity, rhizome ultrastructure, leaf tissue structure, and Cr subcellular distribution in tobacco seedlings under Cr stress were analyzed. Although it has been confirmed that the selenium-molybdenum combination can improve the growth of tobacco under chromium stress, enhance the ability of tobacco to resist heavy metal stress, and provide an effective reference for exploring tobacco and other crops for safe production; however, the selenium-molybdenum composition is mainly aimed at chromium (Cr) stress, and its scope of action is relatively limited, and it can only alleviate specific heavy metal stress; the mechanism of action of the selenium-molybdenum composition may be more complicated, and there may be a variety of side effects, affecting the normal growth and development of tobacco seedlings; the effect of the selenium-molybdenum composition may be unstable, especially under different environmental conditions; the production and use costs may be high, and it may not be easy to obtain in some areas; although the selenium-molybdenum composition can alleviate chromium stress, its promoting effect on the overall growth of the plant is not significant.

[0005] Prior art three, Chinese patent, application number: 202111464074.7 discloses an application of chlorogenic acid in improving plant salt stress resistance. By measuring the growth of tobacco seedling root length and the changes in hydrogen peroxide and superoxide anion levels in tobacco seedlings, the role of chlorogenic acid in increasing plant salt stress tolerance is explored. It is found that chlorogenic acid can increase the growth rate of tobacco seedlings during salt stress, reduce the levels of hydrogen peroxide and superoxide anions, and reduce the toxic effects of oxidative stress (hydrogen peroxide and superoxide anions) on plant cells under salt stress. Although it enables tobacco to grow normally under high salt stress, it provides a new method for alleviating salt stress in saline-alkali plants; however, chlorogenic acid mainly targets salt stress, and its scope of action is relatively limited, and it can only alleviate specific salt stress.

[0006] At present, the existing technologies 1, 2 and 3 have the problem that the materials used are harmful to a certain extent, are easily restricted by the environment, and cannot effectively exert their performance; at the same time, the promoting effect on the overall growth of the plant is not significant. Therefore, the present invention provides a method for improving the ability of tobacco seedlings to resist low temperature and hypoxia combined stress by using exogenous amino acids. The tobacco seedling stage is selected, and the exogenous amino acid treatment test is carried out to achieve the purpose of improving the resistance to low temperature and hypoxia, promoting plant growth, and increasing the amount of material accumulation. Summary of the invention

[0007] The main purpose of the present invention is to provide a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids, so as to solve the problem that the materials used in the prior art have certain harmfulness, are easily subject to environmental constraints, and cannot effectively exert their performance; at the same time, the promoting effect on the overall growth of the plants is not significant.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids, the method comprising:

[0010] Select tobacco seedlings at the 2-leaf 1-heart stage grown in a light incubator and floating in nutrient solution;

[0011] Prepare L-pyroglutamic acid mother solution, weigh L-pyroglutamic acid, dissolve it in water, and prepare L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0012] The prepared L-pyroglutamic acid mother solution was used to treat the selected tobacco seedlings at the 2-leaf 1-heart stage exogenously; under low temperature and low oxygen conditions, an incubator was used, and anhydrous sodium sulfite was added to the hydroponic solution for low oxygen treatment; the L-pyroglutamic acid mother solution with a concentration of 1 g / L was added to the hydroponic solution;

[0013] Three days after the treatment under low temperature and low oxygen conditions, the tobacco seedlings were photographed and their phenotypes recorded. The roots were sampled to measure root length and root activity. New leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase, peroxidase and catalase activities, malondialdehyde content, soluble sugar content and soluble protein content.

[0014] According to the measured values ​​under the treatment of adding different volumes of L-pyroglutamic acid mother solution with a concentration of 1g / L, the data were sorted by software and the difference significance was analyzed by data processing software, and the multi-trait fuzzy membership function method was used for comprehensive evaluation.

[0015] As a further improvement of the present invention, tobacco seedlings at the 2-leaf 1-heart stage were selected and grown in a 25° C. light incubator with 16 h light / 8 h dark and floating in a 50 ppm nutrient solution.

[0016] As a further improvement of the present invention, the process of preparing a mother solution of L-pyroglutamic acid with a concentration of 1 g / L comprises:

[0017] Select L-pyroglutamic acid crystals and place them in a vacuum drying oven before use;

[0018] Weigh 1 g of L-pyroglutamic acid and transfer it to a pre-cooled dissolution container after each weighing; use water as the dissolution medium;

[0019] The weighed L-pyroglutamic acid was added to the pre-cooled aqueous solution, and a magnetic stirrer was used to stir at a low speed after each addition; after the last addition, an ultrasonic cleaner was used to perform ultrasonic treatment.

[0020] As a further improvement of the present invention, under low temperature and low oxygen conditions, in a 5°C incubator, anhydrous sodium sulfite is added to the hydroponic solution for low oxygen treatment; and L-pyroglutamic acid mother solution with a concentration of 1 g / L is added to the hydroponic solution.

[0021] As a further improvement of the present invention, the total volume of the solution in each treatment hydroponic box is 100 mL, and the final concentration of the L-pyroglutamic acid solution is 0 to 50 mg / L.

[0022] As a further improvement of the present invention, the control process of low-temperature and low-oxygen conditions utilizes a trained machine learning model to make real-time predictions on current environmental parameters and calculate the optimal control strategy; based on the prediction results, the temperature controller, gas mixing system and circulation pump are adjusted by the controller to achieve precise control of the low-temperature and low-oxygen environment.

[0023] As a further improvement of the present invention, the machine learning model is trained through multimodal data processing, time series modeling, multi-objective optimization, real-time updating and model fusion.

[0024] As a further improvement of the present invention, the final concentration of the L-pyroglutamic acid solution is 20 mg / L.

[0025] As a further improvement of the present invention, the process of measuring superoxide dismutase, peroxidase and catalase activities and malondialdehyde content, soluble sugar content and soluble protein content comprises the following steps:

[0026] Design equations to measure superoxide dismutase, peroxidase, and catalase activities, as well as malondialdehyde content, soluble sugar content, and soluble protein content;

[0027] The determination equations of the above indicators were integrated into a comprehensive equation system to evaluate the physiological responses of tobacco seedlings under low temperature and hypoxia stress.

[0028] As a further improvement of the present invention, the membership function calculation formula of the index positively correlated with the low temperature and hypoxia resistance is:

[0029] U ij =(X ij -X ijmin ) / (X ijmax -X ijmin );

[0030] The calculation formula of the index negatively correlated with the resistance to low temperature and hypoxia is:

[0031] U ij =1-(X ij -X ijmin ) / (X ijmax -X ijmin );

[0032] Among them, U ij represents the membership value of the jth index under the i-th treatment; X ij represents the measured value of the jth index under the i-th treatment, X ijmin With X ijmax Represents the minimum and maximum values ​​of the jth index under all treatments.

[0033] The present invention analyzes data such as plant growth, antioxidant enzyme activity, and osmotic substance content through exogenous treatment, and provides technical support for the large-scale construction and application of tobacco seedlings in floating seedling production. The method is simple, efficient, and low-cost, and can achieve normal growth of tobacco seedlings under low temperature and hypoxia stress during floating seedling cultivation, ensuring tobacco leaf production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic flow chart of the steps of one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress by exogenous amino acids of the present invention;

[0035] Figure 2 A schematic flow chart of the steps of preparing a mother solution of L-pyroglutamic acid with a concentration of 1 g / L in one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress with exogenous amino acids of the present invention;

[0036] Figure 3 A schematic flow chart of the steps of controlling low-temperature and low-oxygen conditions in one embodiment of the method for improving the low-temperature and low-oxygen combined stress tolerance of tobacco seedlings with exogenous amino acids of the present invention;

[0037] Figure 4 A schematic flow chart of the steps of measuring superoxide dismutase, peroxidase and catalase activities and malondialdehyde content, soluble sugar content and soluble protein content in one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress with exogenous amino acids of the present invention;

[0038] Figure 5 This is a schematic diagram of the effect of exogenous L-pyroglutamic acid treatment on the growth of tobacco seedlings in one embodiment of the method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids of the present invention;

[0039] Figure 6 This is a schematic diagram showing the effect of exogenous L-pyroglutamic acid treatment on the root length of tobacco seedlings in one embodiment of the method for improving the ability of tobacco seedlings to withstand combined stress of low temperature and hypoxia with exogenous amino acids of the present invention;

[0040] Figure 7 This is a schematic diagram of the effect of exogenous L-pyroglutamate treatment on SOD enzyme activity of tobacco seedlings in one embodiment of the method for improving the ability of tobacco seedlings to withstand combined stress of low temperature and hypoxia using exogenous amino acids of the present invention;

[0041] Figure 8 This is a schematic diagram showing the effect of exogenous L-pyroglutamate treatment on the POD enzyme activity of tobacco seedlings in one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress by exogenous amino acids of the present invention;

[0042] Fig. 9 This is a schematic diagram showing the effect of exogenous L-pyroglutamate treatment on CAT enzyme activity of tobacco seedlings in one embodiment of the method for improving the ability of tobacco seedlings to withstand combined stress of low temperature and hypoxia using exogenous amino acids of the present invention;

[0043] Fig.10 This is a schematic diagram showing the effect of exogenous L-pyroglutamic acid treatment on the MDA content of tobacco seedlings in one embodiment of the method for improving the ability of tobacco seedlings to withstand combined stress of low temperature and hypoxia with exogenous amino acids of the present invention;

[0044] Fig.11 A schematic diagram showing the effect of exogenous L-pyroglutamic acid treatment on the soluble sugar content of tobacco seedlings in one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress by exogenous amino acids of the present invention;

[0045] Fig.12 A schematic diagram showing the effect of exogenous L-pyroglutamic acid treatment on the soluble protein content of tobacco seedlings in one embodiment of the method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress by exogenous amino acids of the present invention;

[0046] Fig.13 The invention discloses an embodiment of a method for improving the tolerance of tobacco seedlings to low temperature and hypoxia combined stress by exogenous amino acids of the present invention, and shows an effect of exogenous L-pyroglutamic acid treatment on the root activity of tobacco seedlings. DETAILED DESCRIPTION

[0047] 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.

[0048] The terms "first", "second" and "third" in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined as "first", "second" and "third" can explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications (such as up, down, left, right, front, back...) in the embodiments of the present invention are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units that are not listed, or optionally also includes other steps or units inherent to these processes, methods, products or devices.

[0049] Reference to an "embodiment" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiment may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0050] like Figure 1 As shown, this embodiment provides an embodiment of a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids. In this embodiment, the raw materials and equipment used are all known products, which are obtained by purchasing commercially available products. Tobacco seeds K326 and tobacco floating seedling substrates are provided by Yunnan Yuxi Zhongyan Seed Co., Ltd., and L-pyroglutamic acid is purchased from MacLean Biotechnology Co., Ltd.; the method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids specifically includes the following steps:

[0051] Step S1: selecting tobacco seedlings at the 2-leaf 1-heart stage grown in a 25°C light incubator with 16 h light / 8 h dark and floating in a 50 ppm nutrient solution;

[0052] Step S2: preparing an L-pyroglutamic acid mother solution, weighing L-pyroglutamic acid, dissolving it in water, and preparing an L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0053] Step S3: exogenously treating the selected tobacco seedlings at the 2-leaf 1-heart stage with the prepared L-pyroglutamic acid mother solution; in a 5°C incubator under low temperature and low oxygen conditions, adding anhydrous sodium sulfite to the hydroponic solution for low oxygen treatment; adding different volumes of L-pyroglutamic acid mother solution with a concentration of 1 g / L to the hydroponic solution, the total volume of the solution in each treatment hydroponic box is 100 mL, and the final concentration of the L-pyroglutamic acid solution is 0 to 50 mg / L;

[0054] Step S4: After the treatment under low temperature and low oxygen conditions for 3 days, the tobacco seedlings were photographed and the phenotypes were recorded. The root length and root activity were measured after sampling. Fresh leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, malondialdehyde (MDA) content, soluble sugar content and soluble protein content;

[0055] Step S5: According to the measured values ​​under the treatment of adding different volumes of L-pyroglutamic acid mother solution with a concentration of 1 g / L, the data are sorted using software and the difference significance analysis (P<0.05) is performed using data processing software, and a comprehensive evaluation is performed using the multi-trait fuzzy membership function method.

[0056] The membership function calculation formula of the indicators positively correlated with low temperature and hypoxia resistance (root length, SOD, POD, CAT, soluble sugar content, soluble protein content, root activity) is as follows:

[0057] U ij =(X ij -X ijmin ) / (X ijmax -X ijmin );

[0058] The calculation formula of the negatively correlated index (malondialdehyde content) with the resistance to low temperature and hypoxia is:

[0059] U ij =1-(X ij -X ijmin ) / (X ijmax -X ijmin );

[0060] Among them, U ij represents the membership value of the jth index under the i-th treatment; X ij represents the measured value of the jth index under the i-th treatment, X ijmin With X ijmax Represents the minimum and maximum values ​​of the jth index under all treatments.

[0061] Preferably, step S1 of this embodiment selects tobacco seedlings, and selects tobacco seedlings in the 2-leaf 1-heart stage. This stage is a critical period for seedling growth, is sensitive to changes in the external environment, and is suitable for stress experiments. Significance: Ensure the consistency of the experimental subjects and the reliability of the experimental results, and provide a basis for stress treatment. Step S2 prepares L-pyroglutamic acid mother solution, and prepares L-pyroglutamic acid mother solution with a concentration of 1g / L to ensure that the concentration of amino acids used in the experiment is consistent, which is convenient for processing at different concentrations. Significance: Provide standardized amino acid solutions for the experiment to ensure the repeatability and scientificity of the experiment. Step S3 exogenously treats tobacco seedlings, and simulates a low temperature and low oxygen environment by adding different concentrations of L-pyroglutamic acid to the hydroponic solution to observe the effects of different concentrations of amino acids on the stress tolerance of tobacco seedlings. Significance: Explore the role of L-pyroglutamic acid in improving the tolerance of tobacco seedlings to low temperature and low oxygen stress, and provide a theoretical basis for improving crop resistance in agricultural production. Step S4: Sampling and analysis after treatment, recording phenotypes by taking photos, measuring root length and root activity, and analyzing SOD, POD, CAT activity, MDA content, soluble sugar and soluble protein content in leaves, to comprehensively evaluate the physiological response of tobacco seedlings under stress conditions. Significance: Through comprehensive analysis of multiple indicators, we can fully understand the physiological regulation mechanism of L-pyroglutamic acid on tobacco seedlings under low temperature and hypoxia stress, and provide data support for the experimental results. Step S5: Data analysis and comprehensive evaluation, using significant difference analysis (P<0.05) and multi-trait fuzzy membership function method, to comprehensively evaluate the data under different treatments to ensure the scientificity and reliability of the results. Significance: Through data analysis, the effect of L-pyroglutamic acid on the resistance of tobacco seedlings to low temperature and hypoxia stress at different concentrations is obtained, which provides a scientific basis for practical application and guides how to improve the stress resistance of crops through exogenous amino acids in agricultural production.

[0062] In summary, this embodiment comprehensively evaluates the role of L-pyroglutamic acid in improving the resistance of tobacco seedlings to low temperature and hypoxia stress through systematic steps, and provides important theoretical basis and practical guidance for improving crop stress resistance in agricultural production. This embodiment is completed by the steps of preparing exogenous substances, sampling and detecting resistance to low temperature and hypoxia, and by analyzing the phenotype of tobacco seedlings, antioxidant enzyme activity (including POD, SOD, CAT), soluble sugar content, soluble protein content, root activity and other data; exogenous substances and treatment methods that can improve the ability of tobacco seedlings to resist low temperature and hypoxia combined stress are obtained, and technical support for the large-scale construction and application of tobacco seedlings in floating seedling production is provided. This embodiment is simple, efficient, and low-cost, and can achieve the normal growth of tobacco seedlings under low temperature and hypoxia stress during floating seedling cultivation, ensuring tobacco leaf production. This example shows, through exogenous experiments, that the exogenous L-pyroglutamic acid reagent can significantly promote the growth of tobacco seedlings under low temperature and low oxygen conditions, enhance the antioxidant capacity and osmotic regulation capacity, thereby increasing the biomass of the population; assist tobacco in passing through the low temperature and low oxygen sensitive period during floating seedling cultivation, thereby promoting successful planting; and has the characteristics of simple operation, easy to master, extremely short cycle, quick effect, low cost, and convenient promotion and application.

[0063] Furthermore, if Figure 2 As shown, the process of preparing the L-pyroglutamic acid mother solution with a concentration of 1 g / L in step S2 specifically includes the following steps:

[0064] Step S21: selecting high-purity (≥99.9%) L-pyroglutamic acid crystals, and placing them in a vacuum drying oven before use, drying them at 40° C. for 2 hours to remove moisture adsorbed on the surface; preparing them in a sterile operating room, with the temperature in the operating room controlled at 22±1° C. and the relative humidity ≤40%;

[0065] Step S22: using a high-precision electronic balance (accuracy 0.0001 g), 1 g of L-pyroglutamic acid is weighed four times, and after each weighing, the mixture is transferred to a pre-cooled dissolution container; water is used as the dissolution medium;

[0066] Step S23: adding the weighed L-pyroglutamic acid to the precooled aqueous solution three times, stirring at a low speed (200 rpm) for 1 minute using a magnetic stirrer after each addition; after the last addition, using an ultrasonic cleaner with a frequency of 40 kHz for 15 minutes, and controlling the ultrasonic power at 50 W to further promote the rapid dissolution of L-pyroglutamic acid.

[0067] Among them, the magnetic stirrer control equation is:

[0068]

[0069] Ultrasonic cleaning machine control equation:

[0070]

[0071] Where, T(t) represents the temperature change of the magnetic stirrer at time t; T 0 represents the initial temperature (22°C); P s Indicates the power of the magnetic stirrer (assumed to be a constant, unit: W); C m represents the stirring efficiency coefficient of the magnetic stirrer; V represents the volume of the solution in the dissolution container (unit: L); k s represents the stirring speed coefficient of the magnetic stirrer (related to the rotation speed (200 rpm)); t represents time (unit: minute); E(t) represents the energy change of the ultrasonic cleaner at time t; E 0 represents the initial energy of the ultrasonic cleaning machine (assumed to be a constant, unit: J); k u Indicates the power coefficient of the ultrasonic cleaning machine (related to the ultrasonic power (50W)); P u Indicates the power of the ultrasonic cleaning machine (50W); f u Indicates ultrasonic frequency (40kHz); f u0 represents the base frequency of the ultrasonic cleaner (assumed to be a constant, unit: kHz); the magnetic stirrer equation takes into account the change of temperature over time during the stirring process, as well as the influence of stirring power, stirring efficiency and solution volume on the temperature change; the ultrasonic cleaner equation takes into account the change of ultrasonic energy over time, as well as the influence of ultrasonic frequency and power on energy change; these two equations can be used to accurately control the operation of the magnetic stirrer and ultrasonic cleaner to ensure rapid dissolution and uniform distribution of L-pyroglutamic acid.

[0072] Preferably, step S21 of this embodiment removes moisture from the surface of L-pyroglutamic acid crystals by vacuum drying to ensure the purity and stability of the crystals and avoid the influence of moisture on the subsequent dissolution process. Significance: Ensure that the concentration of the mother liquor is accurate and avoid concentration errors caused by residual moisture, thereby ensuring the reliability and repeatability of the experimental results. Step S22 uses a high-precision electronic balance to weigh L-pyroglutamic acid four times to ensure the accuracy and precision of the weighing; precooling the dissolution container and aqueous solution helps to slow down the dissolution rate of L-pyroglutamic acid and avoid local overheating. Significance: Improve the accuracy of weighing and ensure that the final prepared mother liquor concentration is 1g / L. At the same time, precooling treatment helps to maintain the stability of the solution and avoid solubility changes caused by temperature fluctuations. Step S23 gradually promotes the dissolution of L-pyroglutamic acid by adding in batches and stirring at a low speed to avoid uneven dissolution caused by one-time addition; ultrasonic treatment further accelerates the dissolution process to ensure that L-pyroglutamic acid is completely dissolved. Significance: Ensure that L-pyroglutamic acid is evenly dissolved in water and avoid the presence of undissolved particles, thereby ensuring the uniformity and stability of the mother solution and providing a high-quality solution basis for the experiment.

[0073] In summary, this embodiment ensures the high quality and stability of the L-pyroglutamic acid mother solution through accurate weighing, appropriate temperature control and effective dissolution method, and provides a reliable solution basis for experiments and applications.

[0074] Furthermore, if Figure 3 As shown, the control process of the low temperature and low oxygen conditions in step S3 specifically includes the following steps:

[0075] Step S31: Install high-precision temperature sensors at different positions in the incubator to collect temperature data in real time; install oxygen sensors in the incubator to monitor oxygen concentration in real time; install pH sensors and conductivity sensors in the hydroponic solution to monitor the pH and conductivity of the hydroponic solution in real time;

[0076] Step S32: collect data such as temperature, oxygen concentration, pH value and conductivity in real time through the sensor network; filter, denoise and standardize the collected data; collect a large amount of historical data to build a data set containing variables such as temperature, oxygen concentration, pH value, conductivity, etc.; select a deep neural network, use historical data to train the selected machine learning model, and optimize the model parameters;

[0077] Step S33: Use the trained machine learning model to make real-time predictions on the current environmental parameters and calculate the optimal control strategy; according to the prediction results, adjust the temperature controller, gas mixing system and circulation pump through the controller to achieve precise control of the low-temperature and low-oxygen environment; feed back the real-time control results to the machine learning model for online learning and optimization; set the alarm threshold, and when the environmental parameters exceed the set range, trigger the alarm system to notify the operator to handle it.

[0078] Preferably, the deployment of the sensor network in step S31 of this embodiment collects temperature data at different positions in the incubator in real time and accurately to ensure uniform temperature distribution; monitors oxygen concentration in real time to ensure that the oxygen content is within the set range; monitors the pH and conductivity of the hydroponic solution in real time to ensure the suitability of the hydroponic solution. Significance: Provide accurate and real-time environmental parameter data for data analysis and control; ensure that the experimental environment meets the requirements of low temperature and low oxygen conditions, and provide reliable basic conditions for the experiment. Step S32 data acquisition and preprocessing, collect temperature, oxygen concentration, pH value, conductivity and other data in real time through the sensor network to ensure the real-time and integrity of the data; filter, denoise and standardize the collected data to improve the quality and consistency of the data; collect a large amount of historical data, build a data set containing multiple variables, and provide rich training samples for the machine learning model; use machine learning models such as deep neural networks to train historical data, optimize model parameters, and improve the prediction accuracy of the model. Significance: Improve data quality through preprocessing to ensure the accuracy of model training; train the model with a large amount of historical data so that it can accurately predict and control environmental parameters and improve the accuracy and stability of control. Step S33 is real-time control and optimization. It uses the trained machine learning model to predict the current environmental parameters in real time and calculate the optimal control strategy. According to the prediction results, the temperature controller, gas mixing system and circulation pump are adjusted by the controller to achieve precise control of the low-temperature and low-oxygen environment. The real-time control results are fed back to the machine learning model for online learning and optimization to continuously improve the control accuracy and stability. The alarm threshold is set. When the environmental parameters exceed the set range, the alarm system is triggered to notify the operator to handle it. Significance: Through real-time prediction and control, the experimental environment is always in the set low-temperature and low-oxygen conditions, which improves the controllability and repeatability of the experiment. Through online learning and optimization, the control system can adapt to environmental changes and continuously improve the control effect. Through the abnormal detection and alarm system, abnormal situations are detected and handled in time to ensure the safety and reliability of the experiment.

[0079] In summary, this embodiment uses artificial intelligence technology to achieve precise control of low temperature and low oxygen environment, which not only improves the stability and repeatability of experimental conditions, but also ensures the safety and reliability of the experiment through adaptive optimization and anomaly detection. It provides reliable technical support for improving the ability of tobacco seedlings to withstand low temperature and low oxygen combined stress, and promotes the in-depth development of related research.

[0080] Furthermore, the process of using historical data to train the selected machine learning model in step S32 specifically includes the following steps:

[0081] Step S321: align the data of different sensors according to the timestamps, and use a multimodal autoencoder to perform joint feature extraction on the temperature, oxygen concentration, pH value and conductivity data; dynamically adjust the weight of feature extraction according to the volatility of each variable in the historical data;

[0082] Input layer: input temperature, oxygen concentration, pH value and conductivity data respectively;

[0083] Encoding layer: extract common features of multimodal data through a shared encoder;

[0084] Decoding layer: reconstruct each modality data separately to ensure the effectiveness of feature extraction;

[0085] Step S322: Use a long short-term memory network to model the time series data of temperature, oxygen concentration, pH value and conductivity; fuse the extracted time series features with the joint features extracted by the multimodal autoencoder to form the final input features;

[0086] Step S323: loss function design:

[0087] L=α·L 温度 +β·L 氧气浓度 +γ·L pH值 +δ·L 电导率

[0088] Where, L 温度 represents the mean square error (MSE) of temperature prediction, L 氧气浓度 represents the cross entropy loss of oxygen concentration prediction, L pH值 represents the Huber loss of pH value prediction (combining the advantages of MSE and MAE), L 电导率 represents the MSE of conductivity prediction; α, β, γ, and δ represent weights that are dynamically adjusted according to experimental requirements;

[0089] Adaptive learning rate strategy: initial learning rate: 0.001; weight decay: 0.01 to prevent model overfitting; dynamic adjustment of learning rate: according to the loss change of the validation set, the learning rate is adjusted every 10 epochs, and the cosine annealing strategy is adopted to ensure that the model can be finely optimized in the later stage of training;

[0090] Training in stages:

[0091] Phase 1: In the first 50 epochs, a larger learning rate (0.001) was used to achieve rapid convergence.

[0092] Phase 2: 50-100 epochs, the learning rate is gradually reduced (0.0005), and the model parameters are finely optimized;

[0093] Phase 3: After 100 epochs, the cosine annealing strategy is used to further optimize the model;

[0094] Step S324: collect real-time data every 5 minutes and add new data to the training set; use small batch gradient descent, and only use the most recent 100 data each time the model parameters are updated; update the model parameters every 10 minutes;

[0095] Data update strategy: Ratio of new data to historical data: 1:4; exclude abnormal data (such as data with temperature or oxygen concentration outside the set range); use incremental learning algorithms (such as EWC, Elastic Weight Consolidation) to retain the model's knowledge of historical data while adapting to new data; perform a complete incremental training every 24 hours to ensure that the model can adapt to environmental changes in the long term;

[0096] Step S325: Use a multi-objective optimization algorithm (such as NSGA-II) to find the optimal solution between temperature, oxygen concentration, pH value and conductivity; dynamically adjust the weight of each objective according to the needs of the experimental stage. For example, in the early stage of the experiment, prioritize the optimization of temperature and oxygen concentration; in the later stage of the experiment, prioritize the optimization of pH value and conductivity; the adversarial training frequency is once every 10 epochs; use isolation forest to detect anomalies in real-time data, and trigger the alarm system when abnormal data is detected; automatically adjust the control strategy, or notify the operator to intervene manually;

[0097] Step S326: Use the weighted average method to dynamically adjust the weights according to the validation set performance of each model; train each model separately, and finally perform weighted fusion on the output results; dynamically adjust the weights of each model according to the loss value of the validation set; use the pre-trained model to extract features from the source domain data; and migrate the parameters of the pre-trained model to a low-temperature, low-oxygen environment.

[0098] Preferably, step S321 of this embodiment is the joint feature extraction of multimodal data. Through the multimodal autoencoder, it is possible to simultaneously process and integrate data from different sensors (temperature, oxygen concentration, pH value and conductivity), extract the common features of these data, and dynamically adjust the weight of feature extraction according to the volatility of historical data. Significance: It enables the model to better understand the intrinsic connection between different data, improve the accuracy and efficiency of feature extraction, and provide high-quality input features for time series modeling. Step S322 is time series data modeling and feature fusion. It uses a long short-term memory network (LSTM) to model time series data, which can capture long-term dependencies in the data; at the same time, the time series features extracted by the LSTM are fused with the joint features extracted by the multimodal autoencoder to form the final input features. Significance: It can effectively process complex patterns in time series data and enhance the predictive ability of the model, especially in scenarios where time dependencies need to be considered. Step S323 Loss function design and adaptive learning rate strategy, by designing a multi-objective loss function, it can optimize multiple variables (temperature, oxygen concentration, pH value and conductivity) at the same time, and dynamically adjust the weight of each objective according to the experimental requirements; the adaptive learning rate strategy ensures that the model can converge quickly during the training process and perform fine optimization in the later stage. Significance: This strategy enables the model to flexibly adjust the optimization focus in different experimental stages, improve the adaptability and robustness of the model, and avoid overfitting. Step S324 Real-time data update and incremental learning, collect real-time data every 5 minutes and add it to the training set, use small batch gradient descent and incremental learning algorithms (such as EWC) to ensure that the model can adapt to new data while retaining knowledge of historical data. Significance: It enables the model to be updated in real time and adapt to changes in the environment, while keeping the memory of historical data, ensuring that the model remains efficient and accurate in long-term operation. Step S325 Multi-objective optimization and anomaly detection, use a multi-objective optimization algorithm (such as NSGA-II) to find the optimal solution among multiple objectives, and dynamically adjust the weight of each objective according to the needs of the experimental stage. At the same time, isolation forests are used for anomaly detection to ensure that timely alarms and corresponding measures can be taken when abnormal data is detected. Significance: It is able to find the optimal control strategy in a complex experimental environment to ensure the smooth progress of the experiment, and respond in time when anomalies occur to ensure the safety and reliability of the experiment. Step S326 Model fusion and transfer learning dynamically adjusts the weights of each model through the weighted average method to ensure the optimal performance of the model on the validation set; at the same time, the pre-trained model is used to extract features from the source domain data, and these parameters are migrated to the target environment. Significance: It can make full use of the advantages of different models to improve the performance of the overall model, and reduce the training cost in the new environment through transfer learning, thereby accelerating the model's adaptation process.

[0099] In summary, this embodiment ensures that the machine learning model can run efficiently and accurately in a complex environment through technical means such as multimodal data processing, time series modeling, multi-objective optimization, real-time updating and model fusion. It not only improves the prediction ability and adaptability of the model, but also ensures the safety and reliability of the experiment, providing strong support for practical applications.

[0100] Furthermore, if Figure 4 As shown, the process of measuring superoxide dismutase, peroxidase and catalase activities and malondialdehyde content, soluble sugar content and soluble protein content in step S4 specifically includes the following steps:

[0101] Step S41: designing equations for measuring superoxide dismutase, peroxidase and catalase activities and malondialdehyde content, soluble sugar content and soluble protein content;

[0102] Superoxide dismutase activity determination equation:

[0103]

[0104] In the formula, F 0 represents the initial fluorescence intensity (when no sample is added); F t represents the fluorescence intensity at time t (after adding the sample); V 总 V represents the total volume of the reaction system (mL); 样品· Indicates the sample volume (mL); k 猝灭 represents the fluorescence quenching rate constant (s-1);

[0105] Peroxidase (POD) activity determination equation:

[0106]

[0107] In the formula, I 氧化 Indicates the oxidation state current intensity (μA); I 还原 represents the reduction current intensity (μA); t represents the reaction time (s); V 总 V represents the total volume of the reaction system (mL); 样品· Indicates the sample volume (mL); k 电极 represents the electrode response coefficient (μA / μmol);

[0108] Catalase (CAT) activity determination equation:

[0109]

[0110] In the formula, Indicates the hydrogen peroxide absorption peak area (cm 2 ); Indicates water absorption peak area (cm 2 );k 吸收 The conversion coefficient between absorption peak area and enzyme activity (μmol / cm 2 );

[0111] Malondialdehyde (MDA) content determination equation:

[0112]

[0113] Among them, A 532 Indicates the absorbance value at 532nm; A 600 Indicates the absorbance value at 600nm; k MDA represents the molar extinction coefficient of MDA (L / μmol / cm);

[0114] Soluble sugar content determination equation:

[0115]

[0116] In the formula, A 340 Indicates the absorbance value at 340nm; k 糖 It represents the molar extinction coefficient of soluble sugar (L / μmol / cm);

[0117] Soluble protein content determination equation:

[0118]

[0119] In the formula, A 280 Indicates the absorbance value at 280nm; k 蛋白 It represents the molar extinction coefficient of soluble protein (L / μmol / cm);

[0120] Step S42: Integrate the determination equations of the above indicators into a comprehensive equation system for evaluating the physiological response of tobacco seedlings under low temperature and hypoxia stress:

[0121]

[0122] Preferably, the step S41 of this embodiment designs a measurement equation; by measuring the change in fluorescence intensity, the activity of SOD is calculated; the fluorescence quenching rate constant is used to correct the change in fluorescence intensity to ensure the accuracy of the measurement. Significance: SOD is an important antioxidant enzyme in plants, which can scavenge superoxide free radicals and protect cells from oxidative damage. By measuring the activity of SOD, the antioxidant capacity of plants under stress conditions can be evaluated. By measuring the changes in the current intensity of the oxidized and reduced states, the activity of POD is calculated. The electrode response coefficient is used to correct the change in current intensity to ensure the accuracy of the measurement. Significance: POD can catalyze the decomposition of hydrogen peroxide and reduce its toxic effects on cells; by measuring the activity of POD, the hydrogen peroxide scavenging ability of plants under stress conditions can be evaluated. By measuring the changes in the absorption peak areas of hydrogen peroxide and water, the activity of CAT is calculated; the absorption peak area and enzyme activity conversion coefficient is used to correct the changes in the absorption peak area to ensure the accuracy of the measurement. Significance: CAT is another important antioxidant enzyme in plants, which can catalyze the decomposition of hydrogen peroxide; by measuring the activity of CAT, the hydrogen peroxide scavenging ability of plants under stress conditions can be evaluated. The content of MDA is calculated by measuring the absorbance values ​​at 532nm and 600nm. The molar extinction coefficient of MDA is used to correct the change of absorbance value to ensure the accuracy of measurement. Significance: MDA is a product of lipid peroxidation, and its content can reflect the degree of oxidative damage to plant cell membranes; by measuring the content of MDA, the degree of oxidative damage to plants under stress conditions can be evaluated. The content of soluble sugar is calculated by measuring the absorbance value at 340nm; the molar extinction coefficient of soluble sugar is used to correct the change of absorbance value to ensure the accuracy of measurement. Significance: Soluble sugar is the main osmotic regulating substance in plants, which can maintain the osmotic balance of cells under stress conditions; by measuring the content of soluble sugar, the osmotic regulating ability of plants under stress conditions can be evaluated. The content of soluble protein is calculated by measuring the absorbance value at 280nm. The molar extinction coefficient of soluble protein is used to correct the change of absorbance value to ensure the accuracy of measurement. Significance: Soluble protein is the main antioxidant substance in plants, which can scavenge free radicals under stress conditions and protect cells from oxidative damage. By measuring the soluble protein content, the antioxidant capacity of plants under stress conditions can be evaluated. Step S42 integrates the comprehensive equation system, integrates the determination equations of various indicators into a comprehensive equation system, and comprehensively evaluates the physiological response of plants under stress conditions by calculating comprehensive physiological indicators. Significance: Comprehensive physiological indicators can reflect the overall physiological state of plants under stress conditions, including antioxidant capacity, osmotic regulation ability and degree of oxidative damage; through this indicator, we can have a more comprehensive understanding of the adaptation mechanism of plants under low temperature and hypoxia stress, and provide an important reference for plant stress resistance research.

[0123] In summary, this embodiment can systematically evaluate the physiological response of plants under stress conditions, revealing their specific performance in terms of anti-oxidation, osmotic regulation and oxidative damage. This not only helps to deeply understand the stress resistance mechanism of plants, but also provides a scientific basis for plant breeding and cultivation management, thereby improving the stress resistance and productivity of plants. Through the above-mentioned complex equation system, the physiological response of tobacco seedlings under low temperature and hypoxia combined stress can be comprehensively and accurately evaluated, providing a scientific basis for the study of exogenous amino acids (such as L-pyroglutamic acid) to improve stress resistance.

[0124] Furthermore, this embodiment provides a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids, which specifically includes the following steps:

[0125] a. Select tobacco seedlings at the 2-leaf 1-heart stage grown in a 25°C light incubator with 16h light / 8h dark and 50ppm nutrient solution;

[0126] b. Prepare L-pyroglutamic acid mother solution: weigh a certain amount of L-pyroglutamic acid, dissolve it in water, and prepare an L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0127] c. The tobacco seedlings at the 2-leaf 1-heart stage selected in step a are treated exogenously with the L-pyroglutamic acid mother solution prepared in step b. The treatment method is as follows: under low temperature and low oxygen conditions, 0 mL of L-pyroglutamic acid mother solution with a concentration of 1 g / L is added to the hydroponic solution. The total volume of the solution in each hydroponic box is 100 mL; the final concentration of the L-pyroglutamic acid solution is 0-50 mg / L; the low temperature and low oxygen treatment method is as follows: in a 5°C incubator, an appropriate amount of anhydrous sodium sulfite (Na2SO4) is added to the hydroponic solution. 2 SO 3 ) for hypoxia treatment;

[0128] d. Three days after the treatment in step c was completed, the tobacco seedlings were photographed and the phenotypes were recorded. The root length and root activity were measured after sampling. Fresh leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, malondialdehyde (MDA) content, soluble sugar content and soluble protein content;

[0129] e. Result analysis: According to the measured values ​​under each treatment, the data were sorted using Excel software and the Duncan method of SPSS software was used for significant difference analysis (P<0.05), and the membership function method was used for comprehensive evaluation.

[0130] Furthermore, this embodiment provides a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids, which specifically includes the following steps:

[0131] a. Select tobacco seedlings at the 2-leaf 1-heart stage grown in a 25°C light incubator with 16h light / 8h dark and 50ppm nutrient solution;

[0132] b. Prepare L-pyroglutamic acid mother solution: weigh a certain amount of L-pyroglutamic acid, dissolve it in water, and prepare an L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0133] c. The tobacco seedlings at the 2-leaf 1-heart stage selected in step a were treated with the L-pyroglutamic acid mother solution prepared in step b. The treatment method was as follows: under low temperature and low oxygen conditions, 1 mL of L-pyroglutamic acid mother solution with a concentration of 1 g / L was added to the hydroponic solution. The total volume of the solution in each hydroponic box was 100 mL. The final concentration of the L-pyroglutamic acid solution was 0-50 mg / L. The low temperature and low oxygen treatment method was as follows: in a 5°C incubator, an appropriate amount of anhydrous sodium sulfite (Na2SO4) was added to the hydroponic solution. 2 SO 3 ) for hypoxia treatment;

[0134] d. Three days after the treatment in step c was completed, the tobacco seedlings were photographed and the phenotypes were recorded. The root length and root activity were measured after sampling. Fresh leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, malondialdehyde (MDA) content, soluble sugar content and soluble protein content;

[0135] e. Result analysis: According to the measured values ​​under each treatment, the data were sorted using Excel software and the Duncan method of SPSS software was used for significant difference analysis (P<0.05), and the membership function method was used for comprehensive evaluation.

[0136] Furthermore, this embodiment provides a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids, which specifically includes the following steps:

[0137] a. Select tobacco seedlings at the 2-leaf 1-heart stage grown in a 25°C light incubator with 16h light / 8h dark and 50ppm nutrient solution;

[0138] b. Prepare L-pyroglutamic acid mother solution: weigh a certain amount of L-pyroglutamic acid, dissolve it in water, and prepare an L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0139] c. The tobacco seedlings at the 2-leaf 1-heart stage selected in step a were treated with the L-pyroglutamic acid mother solution prepared in step b. The treatment method was as follows: under low temperature and low oxygen conditions, 2 mL of L-pyroglutamic acid mother solution with a concentration of 1 g / L was added to the hydroponic solution. The total volume of the solution in each hydroponic box was 100 mL. The final concentration of the L-pyroglutamic acid solution was 0-50 mg / L. The low temperature and low oxygen treatment method was as follows: in a 5°C incubator, an appropriate amount of anhydrous sodium sulfite (Na2SO4) was added to the hydroponic solution. 2 SO 3 ) for hypoxia treatment;

[0140] d. Three days after the treatment in step c was completed, the tobacco seedlings were photographed and the phenotypes were recorded. The root length and root activity were measured after sampling. Fresh leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, malondialdehyde (MDA) content, soluble sugar content and soluble protein content;

[0141] e. Result analysis: According to the measured values ​​under each treatment, the data were sorted using Excel software and the Duncan method of SPSS software was used for significant difference analysis (P<0.05), and the membership function method was used for comprehensive evaluation.

[0142] Furthermore, this embodiment provides a method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress with exogenous amino acids, which specifically includes the following steps:

[0143] a. Select tobacco seedlings at the 2-leaf 1-heart stage grown in a 25°C light incubator with 16h light / 8h dark and 50ppm nutrient solution;

[0144] b. Prepare L-pyroglutamic acid mother solution: weigh a certain amount of L-pyroglutamic acid, dissolve it in water, and prepare an L-pyroglutamic acid mother solution with a concentration of 1 g / L;

[0145] c. The tobacco seedlings at the 2-leaf 1-heart stage selected in step a were treated with the L-pyroglutamic acid mother solution prepared in step b. The treatment method was as follows: under low temperature and low oxygen conditions, 5 mL of L-pyroglutamic acid mother solution with a concentration of 1 g / L was added to the hydroponic solution. The total volume of the solution in each hydroponic box was 100 mL. The final concentration of the L-pyroglutamic acid solution was 0 to 50 mg / L. The low temperature and low oxygen treatment method was as follows: in a 5°C incubator, an appropriate amount of anhydrous sodium sulfite (Na2SO4) was added to the hydroponic solution. 2 SO 3 ) for hypoxia treatment;

[0146] d. Three days after the treatment in step c was completed, the tobacco seedlings were photographed and the phenotypes were recorded. The root length and root activity were measured after sampling. Fresh leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, malondialdehyde (MDA) content, soluble sugar content and soluble protein content;

[0147] e. Result analysis: According to the measured values ​​under each treatment, the data were sorted using Excel software and the Duncan method of SPSS software was used for significant difference analysis (P<0.05), and the membership function method was used for comprehensive evaluation.

[0148] The embodiment detects the resistance to low temperature and hypoxia: Figure 5 As shown in the results, different dosages of L-pyroglutamic acid have a certain promoting effect on the growth of tobacco seedlings under low temperature and low oxygen. Figure 6 As shown, compared with CK, after treatment with exogenous L-pyroglutamic acid at concentrations of 10, 20, and 50 mg / L, the root length of seedlings increased by 41.22%, 29.39%, and 32.06%, respectively, and the effect of 10 mg / L L-pyroglutamic acid was the most significant. The content of soluble sugar, soluble protein, malondialdehyde content, root vitality and the activity of antioxidant enzymes such as SOD, POD, and CAT can all be used as important indicators for studying plant stress resistance. Under abiotic stress, a large number of free radicals will be produced in the plant body, and too many free radicals will cause peroxidation damage. Therefore, under abiotic stress, the activity of SOD, POD, and CAT enzymes in the plant body will increase significantly, thereby counteracting and blocking the damage to cells caused by free radicals and repairing damaged cells in a timely manner. Figure 7 As shown, compared with CK, the SOD enzyme activity increased by 56.08%, 50.67%, and 49.50% after treatment with 10, 20, and 50 mg / L exogenous L-pyroglutamate, respectively; Figure 8 As shown, compared with CK, the POD enzyme activity increased by 115.74%, 119.12%, and 129.15% after treatment with 10, 20, and 50 mg / L exogenous L-pyroglutamate, respectively; Fig. 9 As shown in the figure, compared with CK, CAT enzyme activity increased by 32.28%, 32.65%, and 43.81% after treatment with 10, 20, and 50 mg / L of exogenous L-pyroglutamate, respectively. When plant tissues are damaged by adversity, membrane lipid peroxidation intensifies. MDA is one of the products of membrane lipid peroxidation, and the accumulation of MDA can also be toxic to the body's cells, causing damage to the membrane structure and function. The intracellular MDA content and plasma membrane permeability can reflect the degree of membrane lipid peroxidation damage and the integrity of the membrane system. Fig.10As shown in the figure, compared with CK, after exogenous 10, 20, and 50 mg / L of L-pyroglutamic acid, the MDA content decreased by 10.92%, 15.03%, and 8.67%, respectively. The increase of osmotic regulating substances can reduce the osmotic potential of cells, enhance the water absorption capacity of plant cells, and alleviate the damage of salt stress to plants. Fig.11 As shown in Figure 2, after exogenous 10, 20, and 50 mg / L of L-pyroglutamic acid, the soluble sugar content increased by 12.40%, 46.25%, and 51.58%, respectively, compared with CK; Fig.12 As shown in the figure, after adding 10, 20, and 50 mg / L of exogenous L-pyroglutamic acid, the content of soluble protein increased by 161.88%, 141.01%, and 220.00%, respectively; Fig.13 As shown in the results, after exogenous L-pyroglutamic acid of 10, 20, and 50 mg / L, the root activity increased by 44.37%, 90.60%, and 43.31%, respectively. After exogenous L-pyroglutamic acid of 10, 20, and 50 mg / L, the MDA content of tobacco seedlings decreased, which alleviated the membrane lipid peroxidation caused by low temperature and hypoxia combined stress to a certain extent, and significantly increased the content of soluble sugar and soluble protein in tobacco seedlings, enhanced the root activity, and promoted the cells to resist the damage of adversity to seedlings. The membership function values ​​of each index under the four treatments were comprehensively evaluated by the multi-trait fuzzy membership function method (Table 1). The four treatments were arranged according to the D value, which was specifically L_20>L_50>L_10>CK, which showed that under the combined stress of low temperature and hypoxia, 10-50 mg / L L-pyroglutamic acid had a mitigating effect on tobacco seedlings, among which 20 mg / L L-pyroglutamic acid had the best mitigating effect.

[0149] Table 1 Membership function values ​​and comprehensive evaluation of each index under different concentrations of L-pyroglutamic acid treatment

[0150]

[0151] Comprehensively considering the changes in tobacco seedling biomass and stress resistance substance content, exogenous L-pyroglutamic acid at concentrations of 10, 20, and 50 mg / L can improve the tobacco seedlings' tolerance to low temperature and hypoxia to a certain extent, and 20 mg / L of L-pyroglutamic acid has the best effect. This result provides data support for tobacco floating seedling cultivation in production.

[0152] The specific implementation methods of the invention are described in detail above, but they are only examples, and the invention is not limited to the specific implementation methods described above. For those skilled in the art, any equivalent modification or substitution of the invention is also within the scope of the invention, therefore, the equalization, modification, improvement, etc. made without departing from the spirit and principle of the invention should be included in the scope of the invention.

Claims

1. A method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids, characterized in that: The method comprises: Select tobacco seedlings at the 2-leaf 1-heart stage grown in a light incubator and floating in nutrient solution; Prepare L-pyroglutamic acid mother solution, weigh L-pyroglutamic acid, dissolve it in water, and prepare L-pyroglutamic acid mother solution with a concentration of 1 g / L; The prepared L-pyroglutamic acid mother solution was used to treat the selected tobacco seedlings at the 2-leaf 1-heart stage exogenously; under low temperature and low oxygen conditions, an incubator was used, and anhydrous sodium sulfite was added to the hydroponic solution for low oxygen treatment; the L-pyroglutamic acid mother solution with a concentration of 1 g / L was added to the hydroponic solution; Three days after the treatment under low temperature and low oxygen conditions, the tobacco seedlings were photographed and their phenotypes recorded. The roots were sampled to measure root length and root activity. New leaves were collected and stored in liquid nitrogen for measuring superoxide dismutase, peroxidase and catalase activities, malondialdehyde content, soluble sugar content and soluble protein content. According to the measured values ​​under the treatment of adding different volumes of L-pyroglutamic acid mother solution with a concentration of 1g / L, the data were sorted by software and the difference significance was analyzed by data processing software, and the multi-trait fuzzy membership function method was used for comprehensive evaluation.

2. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 1, characterized in that: Tobacco seedlings at the 2-leaf 1-heart stage were selected and grown in a 25°C light incubator with 16 h light / 8 h dark and floating in a 50 ppm nutrient solution.

3. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 1, characterized in that: The process of preparing a mother solution of L-pyroglutamic acid with a concentration of 1 g / L includes: Select L-pyroglutamic acid crystals and place them in a vacuum drying oven before use; Weigh 1 g of L-pyroglutamic acid and transfer it to a pre-cooled dissolution container after each weighing; use water as the dissolution medium; The weighed L-pyroglutamic acid was added to the pre-cooled aqueous solution, and a magnetic stirrer was used to stir at a low speed after each addition; after the last addition, an ultrasonic cleaner was used to perform ultrasonic treatment.

4. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 1, characterized in that: Under low temperature and low oxygen conditions, in a 5°C incubator, anhydrous sodium sulfite is added to the hydroponic solution for low oxygen treatment; and L-pyroglutamic acid mother solution with a concentration of 1 g / L is added to the hydroponic solution.

5. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids according to claim 4, characterized in that: The total volume of the solution in each treatment hydroponic box was 100 mL, and the final concentration of the L-pyroglutamic acid solution was 0 to 50 mg / L.

6. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 4, characterized in that: The control process of low-temperature and low-oxygen conditions uses a trained machine learning model to make real-time predictions of current environmental parameters and calculate the optimal control strategy. Based on the prediction results, the temperature controller, gas mixing system and circulation pump are adjusted through the controller to achieve precise control of the low-temperature and low-oxygen environment.

7. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 6, characterized in that: Train machine learning models through multimodal data processing, time series modeling, multi-objective optimization, real-time updates, and model fusion.

8. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids according to claim 4, characterized in that: The final concentration of the L-pyroglutamic acid solution was 20 mg / L.

9. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by exogenous amino acids according to claim 1, characterized in that: The process of measuring superoxide dismutase, peroxidase and catalase activities and malondialdehyde content, soluble sugar content and soluble protein content comprises the following steps: Design equations to measure superoxide dismutase, peroxidase, and catalase activities, as well as malondialdehyde content, soluble sugar content, and soluble protein content; The determination equations of various indicators were integrated into a comprehensive equation system to evaluate the physiological responses of tobacco seedlings under low temperature and hypoxia stress.

10. The method for improving the ability of tobacco seedlings to withstand low temperature and hypoxia combined stress by using exogenous amino acids according to claim 1, characterized in that: The membership function calculation formula of the index that is positively correlated with the resistance to low temperature and hypoxia is: U ij =(X ij -X ijmin ) / (X ijmax -X ijmin ); The calculation formula of the index negatively correlated with the resistance to low temperature and hypoxia is: U ij =1-(X ij -X ijmin ) / (X ijmax -X ijmin ); Among them, U ij represents the membership value of the jth index under the i-th treatment; X ij represents the measured value of the jth index under the i-th treatment, X ijmin With X ijmax Represents the minimum and maximum values ​​of the jth index under all treatments.

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