A method for screening of apium nigrum stress-resistant mutants

Abstract

The application belongs to the technical field of plant breeding, and relates to a screening method of a celery stress-resistant mutant, comprising the following steps: step 1, obtaining celery aseptic seedlings; step 2, inducing celery callus; step 3, inducing loose embryogenic callus of celery; step 4, determining EMS mutagenesis and PEG-6000 screening stress; and step 5, obtaining the celery stress-resistant mutant. The application significantly improves the efficiency of obtaining the target stress-resistant mutant by optimizing the loose embryogenic callus induction system and adopting the strategy of mild mutagenesis and strict screening.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology and relates to a method for screening stress-resistant mutants of celery. Background Technology

[0002] Celery (Apium graveolens L.) is an important vegetable crop widely cultivated in my country, possessing both high nutritional and economic value. However, in actual production, celery growth and development are easily affected by abiotic stresses such as low temperatures and drought, leading to decreased yield and deterioration in quality, severely hindering the stable development of the celery industry.

[0003] Traditional celery breeding suffers from problems such as long cycles, low efficiency, and narrow genetic backgrounds, making it difficult to meet the needs of rapid breeding for stress resistance. Plant mutation breeding is an effective means of creating new germplasm and broadening genetic variation. Ethyl methanesulfonate (EMS), as a highly efficient chemical mutagen, mainly induces point mutations and has advantages such as simple operation, low cost, and a broad mutation spectrum, making it widely used in crop mutation breeding. In the targeted screening of stress-resistant mutants, polyethylene glycol (PEG) cannot permeate cell membranes and can create a stable low water potential environment, making it an ideal screening agent for simulating drought stress.

[0004] In vitro cultured callus cells are uniform in size and have sufficient contact with mutagens, which can effectively avoid the formation of chimeras, making them excellent mutagenic materials. Among them, loose embryogenic callus cells have a loose structure, a high proportion of embryogenic cells, and strong regeneration ability, making them ideal starting materials for mutagenesis breeding.

[0005] Currently, there are some studies on celery tissue culture and mutation breeding, such as hypocotyl-induced callus, but existing techniques still have significant shortcomings:

[0006] 1. Callus induction often uses hypocotyls as explants, resulting in low induction rates and embryogenic quality. There is a lack of systematically optimized induction conditions for high-quality, loosely embryogenic callus.

[0007] 2. Chemical mutagenesis typically uses a median lethal dose (LD50). 50 Determining the EMS concentration, while ensuring a certain survival rate, is problematic because high concentrations of EMS severely impair cell regeneration capacity, resulting in a small number of regenerable mutants and limiting the size of the mutant library.

[0008] 3. A complete system for creating stress-resistant mutants of celery, from explant preparation and efficient mutagenesis to targeted screening, has not yet been established. The efficiency of obtaining mutants is low and the stress resistance traits are not prominent, which cannot meet the actual needs of stress-resistant breeding of celery.

[0009] Therefore, developing an efficient, systematic, and stable screening method for obtaining highly stress-resistant celery mutants is of great significance for celery stress-resistant breeding and industrial development. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for screening celery stress-resistant mutants.

[0011] The technical problem solved by this invention is achieved through the following technical solution:

[0012] A method for screening stress-resistant mutants in celery, characterized by the following steps:

[0013] Step 1: Obtain sterile celery seedlings;

[0014] Step 2, induction of celery callus:

[0015] Cotyledonary nodes of sterile celery seedlings were used as explants and inoculated onto callus induction medium. The explants were cultured at a temperature of 25±2℃, a light intensity of 1000~1500lx, and a 24h light condition to induce callus production.

[0016] Step 3, induction of loose embryogenic callus from celery:

[0017] The induced callus was transferred to embryogenic callus induction medium for subculture (culture conditions were the same as for callus) to obtain loose celery embryogenic callus.

[0018] Step 4, Determination of EMS mutagenesis and PEG-6000 screening pressure:

[0019] The loose embryogenic callus of celery obtained in step 3 was placed on regeneration medium containing different concentrations of EMS and different concentrations of PEG-6000 and cultured. The appropriate concentrations for mutagenesis and screening were determined according to the principle of semi-regeneration inhibition.

[0020] Step 5, Obtaining the celery stress-resistant mutant:

[0021] The loose embryogenic callus of celery obtained in step 3 was first placed in a regeneration medium containing 0.04% EMS for mutagenesis treatment; then the mutagenized callus was transferred to a regeneration medium containing 4% PEG-6000 for directional screening culture. The regenerated shoots that survived the culture were the celery stress-resistant mutants.

[0022] Furthermore, it also includes step 6, verification of the celery stress-resistant mutant:

[0023] The celery mutants obtained in step 5 were hardened off and transplanted. Physiological and biochemical indicators of the mutants and control plants were measured in terms of leaf anatomy, stomatal morphology, antioxidant enzyme activity, osmotic regulation substance content, membrane lipid peroxidation degree and cold resistance. New celery germplasm with significantly enhanced stress resistance was screened out.

[0024] Furthermore, the method for obtaining sterile celery seedlings in step 1 is as follows:

[0025] Sterile seedlings were obtained by disinfecting celery seeds. The disinfection method was to treat the seedlings with 75% ethanol for 30 seconds and then soak them in 4% sodium hypochlorite solution for 10 minutes.

[0026] Furthermore, the callus induction medium is MS medium supplemented with plant growth regulators, with the following formula: MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D; the embryogenic callus induction medium is MS medium supplemented with plant growth regulators, with the following formula: MS + 0.1 mg / L KT + 0.5 mg / L 2,4-D.

[0027] Furthermore, in step 4, the loose celery embryogenic callus obtained in step 3 is cultured on regeneration media containing different concentrations of EMS and PEG-6000. The appropriate concentrations for mutagenesis and screening are determined according to the principle of semi-regeneration inhibition. The appropriate treatment concentration of EMS is 0.04%, and the appropriate screening concentration of PEG-6000 is 4%. The regeneration media is MS medium with added plant growth regulators. The formula is: MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D (culture conditions are the same as for callus).

[0028] Furthermore, in step 6, the method for hardening off and transplanting the celery stress-resistant mutant obtained in step 5 is as follows: when the regenerated shoots grow to 3-4 cm, first loosen the cap of the tissue culture bottle and culture it under natural light in the culture room for 2-3 days, then fully open the cap and continue to culture for 2-3 days; then take out the plant, wash off the culture medium from the roots, and transplant it into a mixed substrate with a peat moss:vermiculite:perlite mass ratio of 3:1:1, cover it with plastic film to keep it moist, and gradually uncover the film to ventilate after 7 days.

[0029] Furthermore, the physiological and biochemical indicators in step 6 include: stomatal density, guard cell length, superoxide dismutase activity, catalase activity, peroxidase activity, soluble sugar content, malondialdehyde content, and relative conductivity and degree of damage under low temperature stress.

[0030] Furthermore, the superoxide dismutase (SOD) activity comparison was determined using the nitroblue tetrazolium (NBT) photoreduction method; the peroxidase (POD) activity comparison was determined using the guaiacol method; the catalase (CAT) activity comparison was determined using ultraviolet spectrophotometry (H2O2 decomposition rate at 240 nm); the malondialdehyde (MDA) content comparison was determined using the thiobarbituric acid (TBA) colorimetric method; the soluble sugar content comparison was determined using the anthrone colorimetric method; and the cold resistance comparison was determined using the conductivity method.

[0031] The advantages and positive effects of this invention are:

[0032] 1. This invention is the first to determine that cotyledonary nodes of sterile celery seedlings are the explants, and combined with an optimized hormone combination (MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D), the callus induction rate reached as high as 98.3%, significantly higher than that using traditional explants such as hypocotyls. Simultaneously, through subsequent subculture in embryogenic callus induction medium (MS + 0.1 mg / L KT + 0.5 mg / L 2,4-D), loosely structured embryogenic callus with a high proportion of embryogenic cells and typical cytological characteristics (small cells, large nuclei, and high nucleocytoplasmic ratio) was successfully obtained, providing high-quality starting material for subsequent mutagenesis and screening.

[0033] 2. This invention abandons the traditional "half-lethal dose" mutagenesis principle and innovatively adopts a two-factor strategy of "mild mutagenesis + rigorous screening." First, a low concentration (0.04%) of EMS is used for treatment. While ensuring a high survival rate (95.67%), the regeneration capacity is suppressed to approximately 50% (45.67%), effectively inducing mutations while avoiding severe damage to cell regeneration. Subsequently, a high concentration (4%) of PEG-6000 is used for rigorous screening. At this concentration, the callus survival rate remains high (93.33%), but the regeneration capacity is almost completely suppressed (regeneration rate only 1.26%), thus efficiently eliminating non-mutated cells. This results in a callus screening success rate (mutation frequency) of up to 10.4% after EMS treatment, far exceeding the control group. This strategy significantly improves the efficiency of obtaining the target anti-mutant mutant.

[0034] 3. The celery mutants obtained through the method of this invention exhibit excellent stress resistance characteristics at multiple levels. For example... Figure 4 As shown, at the physiological and biochemical level: the mutant's antioxidant enzyme system was significantly activated, with superoxide dismutase (SOD) and catalase (CAT) activities increasing by approximately 86% and 27%, respectively, compared to the control; simultaneously, the content of soluble sugars, an osmotic regulator, increased by approximately 34.6%; while the content of malondialdehyde (MDA), a membrane lipid peroxidation product, decreased. At the morphological level: the mutant's leaf stomatal density was significantly reduced (from 2.20 stomata / 10000 μm). 2 Reduced to 1.72 per 10000 μm 2The length of guard cells increased significantly (from 21.12 μm to 24.63 μm), a structural change that helps reduce water transpiration and improve water use efficiency. Regarding cold resistance: under low-temperature stress, the relative conductivity (28.56%) and damage level (15.87%) of the mutant were significantly lower than those of the control strain (88.89% and 84.96%, respectively), indicating better cell membrane system stability and significantly enhanced cold resistance. In summary, the mutant obtained in this invention possesses comprehensive stress resistance advantages including strong antioxidant capacity, good osmotic regulation capacity, high membrane structure stability, and optimized stomatal structure.

[0035] 4. This invention establishes for the first time a complete technical system in celery that integrates "efficient induction of loose embryogenic callus - mild EMS mutagenesis - rigorous PEG screening - physiological, biochemical and molecular verification", filling a technical gap in related fields.

[0036] 5. The technical solution of this invention is highly operable, reproducible, and efficient in obtaining mutants. It not only provides important technical support and germplasm resources for the targeted improvement of stress-resistant celery varieties, but also provides important technical reference and guidance for mutation breeding research of Apiaceae and other vegetable crops. Attached Figure Description

[0037] Figure 1 This is a cytological observation image (400×) of loose embryogenic callus tissue of celery in this invention (the image shows that the embryogenic cells are small, round, with large and centrally located nuclei and a high nucleocytoplasmic ratio, while the non-embryonic cells are large, irregular in shape, and have obvious vacuolization).

[0038] Figure 2 The image shows a comparison of the stomatal morphology of the leaves of the celery stress-resistant mutant and the control plant (400×) ((a) is the control plant, with a high stomatal density; (b) is the mutant plant, with a low stomatal density and longer guard cells).

[0039] Figure 3 Images of the mature, induced, loose embryogenic callus tissue of this invention;

[0040] Figure 4 This is a comparative table of physiological and chemical indicators of celery for the present invention. Detailed Implementation

[0041] The embodiments of the present invention will be further described in detail below with reference to the accompanying drawings:

[0042] Example 1: Induction of loose embryogenic callus from celery

[0043] Select plump, disease-free "Shiqin" seeds. Treat them with 75% ethanol for 30 seconds in a clean bench, then soak them in 4% sodium hypochlorite solution for 10 minutes (with constant shaking). Rinse three times with sterile water before inoculating onto MS medium. Culture for 30 days at (25±2)℃, light intensity 1000-1500 lx, and 24h light to obtain sterile seedlings with a seed germination rate of 90.3% and a contamination rate of only 1.7%. Cut cotyledonary nodes (approximately 0.5 cm long) from the sterile seedlings and inoculate them onto callus induction medium (MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D). Culture for 30 days at (25±2)℃, light intensity 1000-1500 lx, and 24h light. After 30 days of culture, the callus induction rate reached 98.3%. Figure 3 As shown, the callus tissue is yellowish-green, loosely structured, soft in texture, and grows rapidly.

[0044] The callus was transferred to embryogenic callus induction medium (MS + 0.1 mg / L KT + 0.5 mg / L 2,4-D) and subcultured every 15 days for three consecutive times to obtain loose celery embryogenic callus. After acetic carmine staining, microscopic observation showed that the embryogenic callus cells were small, round, with large, centrally located nuclei and a high nucleocytoplasmic ratio, and mitotic cells were visible.

[0045] Example 2: EMS mutagenesis, determination of PEG-6000 screening concentration, and acquisition of stress-resistant mutants

[0046] The loose embryogenic callus of celery obtained in Example 1 was inoculated onto regeneration medium (MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D) containing different concentrations of EMS (0.02%, 0.04%, 0.06%, 0.08%) and different concentrations of PEG-6000 (3%, 4%, 5%, 6%), as shown in the table below. After 20 days of culture, the survival rate and regeneration rate were counted.

[0047] The results showed that when the EMS concentration was 0.04%, the callus survival rate was 95.67% and the regeneration rate was 45.67%; when the PEG-6000 concentration was 4%, the callus survival rate was 93.33% and the regeneration rate was only 1.26%. Based on this, the EMS mutagenic concentration was determined to be 0.04%, and the PEG-6000 screening concentration was determined to be 4%.

[0048] Screening criteria:

[0049] Selection criteria for EMS mutagenesis pressure: In chemical mutagenesis breeding research, the dosage of mutagen is generally selected by referring to the median lethal dose (LD50). 50This dosage corresponds to a survival rate of approximately 50% for the target material after treatment. Chen Zhihua et al. (2016) conducted EMS mutagenesis experiments on yam callus, setting various combinations of concentrations and treatment times, and using a survival rate of 50% as the criterion to determine the optimal mutagenesis conditions. Similar to the study by Yan Feng et al. (2024), they also used the same method to determine the appropriate mutagenesis dose. All of the above studies indicate that the median lethal dose (LD50) is a key indicator in research on quinoa seeds, crape myrtle seeds, etc. The purpose of this method is to efficiently complete gene mutations while ensuring good survival of the tested samples, laying a good foundation for subsequent selection.

[0050] The mutagenesis strategy used in this study differs fundamentally from traditional mutagenesis methods. While the regeneration rate of loose embryogenic callus from celery can reach over 99% under conventional culture conditions, the experimental design did not use high concentrations of EMS to induce significant toxic effects. Instead, a low concentration (0.04%) was chosen as the initial treatment. Under these conditions, although the callus survival rate reached 95.67%, its regeneration capacity decreased to approximately 45.67%, indicating an inhibition of its normal physiological functions by about 50%. This choice was made based on the following scientific evidence: firstly, EMS is both a chemical mutagen that causes genetic changes and significantly inhibits plant tissue growth and development; secondly, numerous studies have demonstrated that EMS exposure can significantly reduce the regeneration capacity of the target material. Increasing the EMS concentration to increase the mutation frequency would lead to further degradation of the callus or complete loss of its regeneration capacity. Afterwards, treatment with 4% PEG6000 was used to achieve a callus regeneration rate of 1.26%. Excessive use of EMS may exacerbate the effects of dual stress, making it difficult to cultivate the mutated mutants into plants that can survive and reproduce.

[0051] Selection criteria for PEG-6000 screening pressure:

[0052] This application utilizes a high-pressure gradient in the design of PEG-6000 screening conditions. Experimental results show that when the concentration of PEG-6000 is set to 4%, the callus survival rate remains at 93.33%, but the regeneration efficiency significantly decreases to 1.26%, approaching complete inhibition. This parameter setting aims to enhance the selective screening effect of external pressure on callus tissue, allowing cell populations treated with ethyl methanesulfonate (EMS), exhibiting strong stress resistance and shoot differentiation potential, to stand out from the majority of cells. This design concept is consistent with the results of some studies using high-intensity stress to induce the screening of specific functional mutant strains.

[0053] The two-factor screening strategy proposed in this application can be summarized as follows: EMS mutagenesis treatment uses "mild conditions" (low lethality, moderate regeneration inhibition), while PEG screening uses a "strict environment" (high survival threshold, almost complete regeneration inhibition). The core idea of ​​this strategy is to use EMS as a mutagen to induce gene changes, disregarding cell death and only appropriately reducing the dosage to ensure the effectiveness of the mutation, thus providing sufficient cell material for subsequent screening. In the PEG screening stage, to effectively eliminate cell populations that do not exhibit the target trait change, a treatment concentration close to complete inhibition of regeneration is set to screen for callus tissue that has acquired resistance only after EMS treatment.

[0054] Experimental results showed that the callus regeneration rate obtained after EMS treatment and screening in 4% PEG-6000 was 10.4%, which was much higher than that of the control group (1.26%), indicating that the method has good feasibility, stability and regeneration efficiency. The method of mutagenesis followed by screening not only improved the efficiency of the entire operation but also obtained the desired specific mutant strains.

[0055] Effects of different EMS mutagenic concentrations on the regeneration of loose embryogenic callus in celery

[0056]

[0057] Effects of different concentrations of PEG6000 on the regeneration of loose embryogenic callus in celery

[0058]

[0059] Loose embryogenic celery callus was treated in regeneration medium containing 0.04% EMS for 20 days, and then transferred to regeneration medium containing 4% PEG-6000 for another 30 days. Statistical results showed that the regeneration rate (mutation frequency) of the EMS-treated callus under 4% PEG-6000 selection pressure was 10.4%, significantly higher than the control group (1.26%) without EMS treatment. The obtained regenerated shoots were designated as candidate celery stress-resistant mutants.

[0060] Example 3: Hardening-off transplanting and physiological and biochemical identification of stress-resistant mutants

[0061] When the regenerated shoots obtained in Example 2 reached 3-4 cm in length, the caps of the tissue culture bottles were loosened, and the seedlings were hardened off under natural light in the culture room for 2-3 days. Then, the caps were fully opened, and hardening off continued for another 2-3 days. The plants were then removed, the culture medium was carefully washed away from the roots, and they were transplanted into a mixed substrate of peat moss:vermiculite:perlite = 3:1:1. A plastic film was used to cover the plants to retain moisture. After 7 days, the film was gradually removed for ventilation. The survival rate reached over 90%. Using ordinary celery seedlings that had not undergone mutagenesis screening as a control, the following measurements were performed on the transplanted mutants:

[0062] (1) Stomatal morphology: The lower epidermis of the leaves was observed using the nail polish imprint method. The results showed that the stomatal density of the mutant was 1.72 stomata / 10000 μm. 2 The number of samples was significantly lower than that of the control (2.20 samples / 10000μm). 2 The guard cell length was 24.63 μm, significantly longer than the control (21.12 μm).

[0063] (2) Antioxidant enzyme activity: The activities of SOD, POD and CAT were determined by the nitroblue tetrazolium method, the guaiacol method and ultraviolet spectrophotometry, respectively. The results showed that the SOD activity of the mutant was 175.93 U / g FW and the CAT activity was 42.58 U / g·min FW, both of which were significantly higher than those of the control (94.58 U / g FW and 33.50 U / g·min FW); although the POD activity increased, it did not reach a significant level.

[0064] (3) Osmotic regulation and membrane damage: The soluble sugar content was determined by the anthrone colorimetric method, and the MDA content was determined by the thiobarbituric acid method. The results showed that the soluble sugar content of the mutant was 4.71 mM / L, which was significantly higher than that of the control (3.50 mM / L); the MDA content was 10.02 μmol / g, which was lower than that of the control (13.44 μmol / g), but the difference was not significant.

[0065] (4) Cold resistance: The relative conductivity of the leaves was measured after freezing at -18℃ for 30 min. The results showed that the relative conductivity (28.56%) and damage (15.87%) of the mutant were significantly lower than those of the control (88.89% and 84.96%), indicating that its cold resistance was significantly enhanced.

[0066] Based on the above results, it is proven that the mutant obtained in Example 2 is a new celery germplasm with significantly enhanced stress resistance.

[0067] Although embodiments and drawings of the present invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.

Claims

1. A method for screening celery stress-resistant mutants, characterized in that: Includes the following steps: Step 1: Obtain sterile celery seedlings; Step 2, induction of celery callus: Cotyledonary nodes of sterile celery seedlings were used as explants and inoculated onto callus induction medium. The explants were cultured at a temperature of 25±2℃, a light intensity of 1000~1500lx, and a 24h light condition to induce callus production. Step 3, induction of loose embryogenic callus from celery: The induced callus tissue was transferred to embryogenic callus induction medium for subculture to obtain loose celery embryogenic callus tissue. Step 4, Determination of EMS mutagenesis and PEG-6000 screening pressure: The loose embryogenic callus of celery obtained in step 3 was placed on regeneration medium containing different concentrations of EMS and different concentrations of PEG-6000 and cultured. The appropriate concentrations for mutagenesis and screening were determined according to the principle of semi-regeneration inhibition. Step 5, Obtaining the celery stress-resistant mutant: The loose embryogenic callus of celery obtained in step 3 was first placed in a regeneration medium containing 0.04% EMS for mutagenesis treatment; then the mutagenized callus was transferred to a regeneration medium containing 4% PEG-6000 for directional screening culture. The regenerated shoots that survived the culture were the celery stress-resistant mutants.

2. The method for screening celery stress-resistant mutants according to claim 1, characterized in that: It also includes step 6, verification of the celery stress-resistant mutant: The celery mutants obtained in step 5 were hardened off and transplanted. Physiological and biochemical indicators of the mutants and control plants were measured in terms of leaf anatomy, stomatal morphology, antioxidant enzyme activity, osmotic regulation substance content, membrane lipid peroxidation degree and cold resistance. New celery germplasm with significantly enhanced stress resistance was screened out.

3. The method for screening celery stress-resistant mutants according to claim 1, characterized in that: The method for obtaining sterile celery seedlings in step 1 is as follows: Sterile seedlings were obtained by disinfecting celery seeds. The disinfection method was to treat the seedlings with 75% ethanol for 30 seconds and then soak them in 4% sodium hypochlorite solution for 10 minutes.

4. The method for screening celery stress-resistant mutants according to claim 1, characterized in that: The callus induction medium is MS medium supplemented with plant growth regulators, with the following formula: MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D; the embryogenic callus induction medium is MS medium supplemented with plant growth regulators, with the following formula: MS + 0.1 mg / L KT + 0.5 mg / L 2,4-D.

5. The method for screening celery stress-resistant mutants according to claim 1, characterized in that: In step 4, the loose embryogenic callus of celery obtained in step 3 is placed on regeneration medium containing different concentrations of EMS and PEG-6000 and cultured. The appropriate concentrations for mutagenesis and screening are determined according to the principle of semi-regeneration inhibition. The appropriate treatment concentration of EMS is 0.04%, and the appropriate screening concentration of PEG-6000 is 4%. The regeneration medium is MS medium with added plant growth regulators. The formula is: MS + 0.2 mg / L KT + 0.5 mg / L 2,4-D.

6. The method for screening celery stress-resistant mutants according to claim 1, characterized in that: Step 6, the method for hardening off and transplanting the celery stress-resistant mutant obtained in step 5, is as follows: when the regenerated shoots grow to 3-4 cm, first loosen the cap of the tissue culture bottle, and culture it in the culture room under natural light for 2-3 days, then fully open the cap and continue to culture for 2-3 days; then take out the plant, wash off the culture medium from the roots, and transplant it into a mixed substrate with a peat moss:vermiculite:perlite mass ratio of 3:1:1, cover it with plastic film to keep it moist, and gradually uncover the film to ventilate after 7 days.

7. The method for screening celery stress-resistant mutants according to claim 2, characterized in that: The physiological and biochemical indicators in step 6 include: stomatal density, guard cell length, superoxide dismutase activity, catalase activity, peroxidase activity, soluble sugar content, malondialdehyde content, and relative conductivity and damage under low temperature stress.

8. The method for screening celery stress-resistant mutants according to claim 7, characterized in that: The superoxide dismutase activity was compared using the nitroblue tetrazolium photoreduction method; the peroxidase activity was compared using the guaiacol method; the catalase activity was compared using ultraviolet spectrophotometry; the malondialdehyde content was compared using the thiobarbituric acid colorimetric method; the soluble sugar content was compared using the anthrone colorimetric method; and the cold resistance was compared using the conductivity method.

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