A method for identifying and evaluating the cold tolerance of Brassica napus during the flowering period

Abstract

The present invention belongs to the technical field of rapeseed planting, and discloses a method for identifying and evaluating the cold tolerance of Brassica napus during the flowering period, which comprises the following steps: First, when Brassica napus is in the flowering period, it is subjected to sub-zero low-temperature stress treatment, and after the treatment is completed, the plants are moved back to the greenhouse to resume growth; Then, 5 days after the plants resume growth, the cold damage phenotypic symptoms of the upper stem leaves, flower buds, petals, main inflorescence, branched inflorescence, fruit stalks, young siliques, and long siliques of the plants are collected, and the corresponding phenotypic symptoms are scored; Finally, the low-temperature tolerance level of the plant is determined according to the total score. Starting from the changes in the phenotypic symptoms of Brassica napus plants under low-temperature stress, the present invention visually observes the phenotypic traits of Brassica napus, and constructs an evaluation system for Brassica napus under low-temperature stress during the flowering period; The operating conditions are easy to meet, easy to operate, convenient and fast. Through field production tests, the results are accurate and reliable, and can provide germplasm reference for the breeding of rapeseed varieties with cold tolerance during the flowering period.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapeseed cultivation, and particularly relates to a method for phenotypic identification and evaluation of cold tolerance during the flowering period of Brassica napus L. Background Art

[0002] Rapeseed is one of the most important oil crops in China. Vigorously developing rapeseed production using winter fallow fields in the south is of great significance for ensuring the security of edible oil supply. At present, there are more than 64 million mu of winter fallow fields in the Yangtze River Basin in the south of China that can be used for "rice-rapeseed" and "rice-rice-rapeseed" production. Under the "rice-rapeseed" and "rice-rice-rapeseed" rotation production models, early-maturing rapeseed varieties need to be planted. However, early-maturing rapeseed varieties have a fast growth process, an earlier flowering period, and weak cold tolerance in spring. Once they encounter freezing or continuous low-temperature and rainy weather, it will lead to serious yield losses. Therefore, improving the cold tolerance of rapeseed varieties during the flowering period is the key to solving the problem of yield decline caused by low-temperature stress.

[0003] At present, the identification of cold tolerance in Brassica napus L. by predecessors mainly focuses on the germination stage and seedling stage, and has established methods for identifying and evaluating low-temperature tolerance based on morphological and growth and development indicators such as germination rate, seedling establishment rate, leaf frost damage index, and overwintering rate, as well as physiological and biochemical indicators related to the cell membrane system, protective enzyme system, osmotic regulation system, and photosynthesis. However, there is no report on the identification method for cold tolerance during the flowering period of rapeseed. Establishing a technical standard for identifying cold tolerance during the flowering period is a prerequisite for genetic improvement of cold tolerance during the flowering period. Therefore, it is of great significance to establish a method for identifying and evaluating cold tolerance during the flowering period of Brassica napus L. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art and provide a method for identifying and evaluating cold tolerance during the flowering period of Brassica napus L.

[0005] To solve the above technical problem, the technical solution proposed by the present invention is as follows:

[0006] A method for identifying and evaluating cold tolerance during the flowering period of Brassica napus L. includes the following steps:

[0007] (1) When Brassica napus L. is in the flowering period, perform sub-zero low-temperature stress treatment. After the treatment is completed, move the plants back to the greenhouse to resume growth;

[0008] (2) After the plants resume growth for 5 days, collect the cold damage phenotypic symptoms of the upper stem leaves, flower buds, petals, main inflorescence, branch inflorescence, fruit stalks, young siliques, and long siliques of the plants, and score according to the corresponding phenotypic symptoms. The specific cold damage phenotypic symptoms and scoring criteria are as follows:

[0009] Upper stem leaves: When the stem leaves are stretched, it is scored 2 points; when part of them is wilted and drooping, it is scored 1 point; when the whole leaf is wilted and drooping, it is scored 0 point;

[0010] Flower bud: If the flower bud is normal, it is scored 2 points; if the flower bud is closed or dead, it is scored 0 points;

[0011] Petal: If the petal is normal, it is scored 2 points; if the petal margin is involute, it is scored 1 point; if the petal is withered, it is scored 0 points;

[0012] Main inflorescence: If the main inflorescence elongates normally, it is scored 2 points; if the main inflorescence is bent but can resume growth, it is scored 1 point; if the main inflorescence droops and cannot resume, it is scored 0 points;

[0013] Branch inflorescence: If the branch inflorescence elongates normally, it is scored 2 points; if the branch inflorescence is bent but can resume growth, it is scored 1 point; if the branch inflorescence droops and cannot resume, it is scored 0 points;

[0014] Fruit stalk: If the fruit stalk is normal, it is scored 2 points; if the fruit stalk is withered or wilts and droops, it is scored 0 points;

[0015] Young silique: If the young silique is normal, it is scored 2 points; if the young silique turns yellow, wilts or withers, it is scored 0 points;

[0016] Long silique: If the silique elongates and the embryo is normal, it is scored 2 points; if the silique elongates and the embryo is browned and shriveled, it is scored 0 points;

[0017] (3) Determine the low-temperature tolerance level of the plant according to the total score. The determination criteria are as follows: If the total score ≥ 15 points, it is classified as a strong cold-resistant level; if the total score is 12 - 14 points, it is classified as a cold-resistant level; if the total score is 6 - 11 points, it is classified as a sensitive level; if the total score ≤ 5 points, it is classified as an extremely sensitive level.

[0018] For the above method for identifying and evaluating the cold tolerance during the flowering period of Brassica napus, preferably, in step (1), the flowering period is the period after 7 days of the initial flowering stage of Brassica napus, when the branches are unfolded and young siliques are formed.

[0019] Preferably, in step (1), the temperature for the low-temperature stress treatment is -1°C to -3°C, and the treatment time is more than 12 hours.

[0020] Preferably, in step (1), the day and night conditions in the greenhouse are 24°C / 14h and 16°C / 10h respectively.

[0021] Preferably, in step (3), the phenotypic symptom description of the strong cold-resistant level is: the leaves are not deformed or damaged by freezing, the leaves are unfolded; the flower buds are normal; the flowers are unfolded, and the inflorescence is straight; the fruit stalks are normal; the young siliques are green, and the long siliques can continue to elongate.

[0022] The phenotypic symptom description of the cold-resistant level is: the leaves are not deformed or damaged by freezing, the leaves are unfolded; the flower buds are normal; the inflorescence is slightly bent but can resume upward growth after the temperature rises; the fruit stalks are normal; the young siliques turn yellow, and the long siliques can continue to elongate.

[0023] The phenotypic symptoms of the described sensitivity level are described as follows: the leaves have frost damage and part of the leaves droop; the inflorescence is slightly bent but can resume upward after the temperature rises; the flower buds are dead or closed; the flowers are deformed, and the fruit stalks are partially or completely wilted and drooping; the young siliques turn yellow, wither, and fall off, and the long siliques can continue to elongate.

[0024] The phenotypic symptoms of the described extremely sensitive level are described as follows: the leaves have frost damage, wilt, and the whole leaf droops; the main inflorescence or branch inflorescence droops, and the flower buds are dead or closed; the flowers wither and die, the fruit stalks are partially or completely wilted and drooping; the young siliques turn yellow, wither, and fall off, and the long siliques no longer elongate.

[0025] In the present invention, during the flowering stage of rapeseed, after treatment at -1°C to -3°C and recovery for 5 days, the phenotype is observed. It is statistically found that low temperature can cause the inflorescence of Brassica napus to bend. Sensitive materials cannot recover and gradually wither and die, while tolerant materials will gradually recover, showing a "Z" shape upward or even upright upward. Low temperature can cause the flower buds to be dead or closed, the petals to shrink, the flower shape to become smaller, the flower organs to develop incompletely or deform, or even wither, significantly reducing the pollen viability; it can also cause the leaves to turn yellow, wilt, and droop; the fruit stalks are partially or completely wilted, withered, and even drooping. The young siliques turn yellow, wither, and the long siliques have embryo browning and death. Starting from the changes in the phenotypic symptoms of Brassica napus plants under low temperature stress, the discoloration and deformation of leaves, inflorescences, flowers, flower buds, fruit stalks, siliques, etc. of Brassica napus can be observed more intuitively, and an evaluation system for Brassica napus under low temperature stress during the flowering stage is constructed; by adopting this evaluation system, Brassica napus resources with low temperature tolerance during the flowering stage can be identified, providing germplasm reference for the breeding of cold-tolerant rapeseed varieties during the flowering stage.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] In the present invention, rapeseed is cultivated to the flowering stage, and a low temperature treatment chamber is used to simulate low temperature stress treatment at -1°C to -3°C. Starting from the changes in the phenotypic symptoms of Brassica napus plants under low temperature stress, the discoloration and deformation of leaves, inflorescences, flowers, flower buds, fruit stalks, siliques, etc. of Brassica napus can be observed intuitively, and an evaluation system for Brassica napus under low temperature stress during the flowering stage is constructed; the operating conditions are easy to meet, easy to operate, convenient and fast. Through field production tests, the results are accurate and reliable, and can provide germplasm reference for the breeding of cold-tolerant rapeseed varieties during the flowering stage; it provides an accurate and reliable technical standard and technical platform for the identification and evaluation of the low temperature resistance of Brassica napus during the flowering stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is the typical phenotypic characteristics of Brassica napus under -2°C low temperature stress during the flowering period. The phenotypes of Brassica napus from left to right are: (A) normal leaves; partially wilted leaves; completely wilted and drooping leaves; (B) normal fruit stalks; wilted fruit stalks; drooping fruit stalks; (C) normal young siliques; yellowing young siliques; (D) normal long siliques, with normal silique walls and ovules; abnormal long siliques, with normal silique walls and dried-up ovules; (E) normal flower buds; dead flower buds; closed flower buds. (F) normal petals; wrinkled petal edges; dried-up petals; (G) normal inflorescences; inflorescences bent into a Z shape; wilted and bent inflorescences; Bar = 1 cm;

[0030] Figure 2 It is the cluster analysis of cold tolerance scores of different rapeseed germplasms;

[0031] Figure 3 It is the temperature trend in January 2023 in Changsha County;

[0032] Figure 4 It is the field performance of cold-sensitive (B) and cold-tolerant (A) germplasms of Brassica napus under natural low temperature stress. Detailed implementation manners

[0033] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in combination with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0034] Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0035] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0036] Embodiment:

[0037] This embodiment provides a method for identifying and evaluating the cold tolerance of Brassica napus during the flowering period. Using 49 early-maturing rapeseed germplasms with different genetic backgrounds as experimental materials, they were treated at -2°C during the flowering period, and the morphological symptom characteristics of Brassica napus plants under low-temperature stress were collected. A method for scoring phenotypic symptom characteristics of Brassica napus under low-temperature stress during the flowering period was established, and an evaluation system for low-temperature tolerance of Brassica napus germplasm resources materials was constructed to identify the cold tolerance of early-maturing rapeseed, providing a germplasm basis for further analyzing the cold tolerance mechanism of early-maturing rapeseed germplasm. The specific steps are as follows:

[0038] 1 Experimental design

[0039] 1.1 Experimental materials

[0040] The 49 rapeseed germplasms participating in the test are all Brassica napus, and the experimental materials are from the Hunan Crop Research Institute.

[0041] The field experimental materials are shown in Table 1 below:

[0042] Table 1 Tested Brassica napus resources

[0043] Test Number Resource Number Test Number Resource Number Test Number Resource Number 1 2w008 18 2w107 35 2w179 2 2w009 19 2w108 36 2w189 3 2w015 20 2w109 37 2w191 4 2w024 21 2w110 38 2w194 5 2w051 22 2w129 39 2w242 6 2w053 23 2w134 40 2w271 7 2w054 24 2w135 41 2w274 8 2w057 25 2w136 42 2w276 9 2w058 26 2w137 43 2w277 10 2w060 27 2w138 44 2w292 11 2w062 28 2w144 45 2w295 12 2w070 29 2w154 46 2w313 13 2w084 30 2w157 47 2w317 14 2w093 31 2w158 48 2w322 15 2w103 32 2w161 49 2w336 16 2w104 33 2w173 17 2w106 34 2w178

[0044] 1.2 Experimental method

[0045] When the experimental materials were seedling-raised in plug trays to the 3-4 leaf stage, they were transplanted into flower pots, with 2 plants per pot. They were continued to be cultured in a glasshouse. When the materials were cultured to the flowering period, 7 days after the initial flowering period, after the branches unfolded and young pods formed, they were subjected to a low-temperature treatment of -2°C (0, 12 h), with 4 plants per treatment. After the low-temperature treatment ended, the plants were moved back to the greenhouse (24°C / 14 h, 16°C / 10 h) to resume growth, and the phenotypic changes of rapeseed were observed 5 days later.

[0046] According to the phenotypic differences, investigations and statistical analyses were carried out on the traits of flower buds, petals, main inflorescences, branches, stem leaves, and pods. According to the freezing injury degree of each phenotype, a description of the traits of rapeseed low-temperature freezing injury and its scoring standard were formulated. The comprehensive scores of the germplasms were sorted and cluster-analyzed, and the cold tolerance grades were divided based on this. Finally, through field observations, the reliability of the method for identifying and evaluating cold tolerance during the flowering period was verified.

[0047] 1.3 Statistically analyze the typical phenotypic traits of Brassica napus under low-temperature stress

[0048] (1) Effects on leaves

[0049] Treatment at -2℃ can cause the color of rapeseed leaves to fade, and the longer the low-temperature treatment, the greater the color change. Low temperature can also cause the leaves to wilt and droop, but the degree of wilting and drooping of leaves varies among different materials. Some materials have partially wilted and drooping leaves, while some have completely wilted and drooping leaves. Among the 49 tested Brassica napus resources, 29 resources showed no obvious frost damage in the cauline leaves, with normal leaf color and normal upward lifting, accounting for 59.2%; 12 resources showed frost damage, with partially wilted and drooping leaves, accounting for 24.5%; 8 resources showed significant frost damage, with overall wilted and drooping leaves, accounting for 16.3%.

[0050] (2) Effects on the fruit stalk

[0051] Normal fruit stalks generally show a horizontal or upward posture. Under low-temperature stress, the fruit stalks of rapeseed are partially or completely wilted, dried up, and even drooped. The fruit stalks of most materials show a healthy horizontal or upward posture. Among the 49 Brassica napus resources, 42 resources showed no obvious frost damage in the fruit stalks, accounting for 85.7%; 7 resources had partially or completely wilted, dried up, and even drooped fruit stalks, accounting for 14.3%.

[0052] (3) Effects on the siliques

[0053] Low temperature also has a certain impact on the siliques. Young siliques are more sensitive and mainly show yellowing, drying up, bending, and wrinkling of young siliques, and the embryos turn brown and die. The already elongated siliques generally show the ability to continue to elongate, but the embryos of most materials turn brown and die. The young siliques of most materials are vulnerable to low-temperature frost damage. Among the 49 Brassica napus resources, 21 resources showed no obvious frost damage in the young siliques, accounting for 42.9%; 28 resources had yellowed and dried up young siliques, accounting for 57.1%.

[0054] (4) Effects on the flower buds of rapeseed

[0055] Treatment at -2℃ can cause some flower buds to close or die. Among the 49 Brassica napus resources, 44 resources showed no obvious frost damage in the flower buds, with normal flower buds, accounting for 89.8%; 5 resources had closed flower buds, accounting for 10.2%.

[0056] (5) Effects on the rapeseed flowers

[0057] Petals are more sensitive to frost damage. Most petals show inward curling and wrinkling at the edges, and severely, the petals dry up. Among the 49 Brassica napus resources, 6 resources showed no obvious frost damage in the flowers, with the petals unfolding normally, accounting for 12.2%; 25 resources had petals showing inward curling and wrinkling at the edges, accounting for 51.0%; 18 resources showed dried-up flowers, accounting for 36.7%.

[0058] (6) Effects on the racemes of rapeseed

[0059] After treatment at -2°C, the inflorescences of the tested materials were all frozen and bent. After 5 days of recovery, the inflorescences of the low-temperature tolerant materials could recover to an upward state. The intermediate materials could recover, but showed an obvious "Z" shape. The main inflorescences of the low-temperature sensitive materials could not recover and showed a bent and withered state, and their flower buds and flowers gradually withered and died, and the inflorescences no longer elongated. Most inflorescences still showed an upright state, and the freeze injury was manifested as inflorescence bending, and severely as inflorescence drooping. Among the 49 rapeseed resources, the inflorescences of 39 resources could stand upright normally, accounting for 79.6%; the inflorescences of 7 resources showed bending, accounting for 14.3%; the inflorescences of 3 resources showed drooping, accounting for 6.1%.

[0060] 1.4 Establish the trait description and scoring standard for low-temperature freeze injury of rapeseed

[0061] The 49 tested materials were respectively treated at -2°C for 0 h and 12 h, and the typical traits of the phenotypes of each material were counted. Five representative and significantly distinguishable phenotypes such as leaf shape, flower bud, flower type, fruit stalk, and silique were selected as the evaluation phenotypes for freeze injury identification (see Figure 1 ), and scores were assigned according to the symptoms for the freeze injury level, as shown in Table 2.

[0062] Table 2 Score table for the phenotypic symptoms of cold injury of Brassica napus

[0063]

[0064] 1.5 Cluster analysis of phenotypic scores for cold tolerance of different Brassica napus

[0065] Using the total scores of each Brassica napus germplasm for systematic cluster analysis (the similarity scale uses Euclidean distance, and the clustering method uses the sum of squared deviations (WARD)), the 49 rapeseed varieties can be divided into 4 categories at the 5-classification distance. The results of the systematic cluster analysis are as Figure 2The clustering results are shown in Table 3 and Table 4. According to the cluster analysis results, we can divide the rapeseed germplasm into four cold resistance grades: strong resistance, resistance, sensitivity, and extreme sensitivity, and use 20B as the control variety. Among them, JD008, JD010, and JD017 belong to the first level, which are strong cold resistance germplasm, with a score of ≥15 points. The phenotype under low temperature stress is that the leaves are not deformed or frozen, and the leaves are stretched; the flower buds are normal; the flowers are stretched, the inflorescence is normally straightened; the fruit stalks are normal; the young siliques are green, and the long siliques can continue to stretch. JD004, JD006, JD014 and other twelve materials belong to the second level, which are cold resistance germplasm, with a score of 12 to 14 points. The phenotype under low temperature stress is that the leaves are not deformed or frozen, and the leaves are stretched; the flower buds are normal; the inflorescence is slightly bent but can recover upward after the temperature rises; the fruit stalks are normal; the young siliques turn yellow, and the long siliques can continue to stretch. 30 germplasms, including JD024, JD026, and JD030, belong to level III and are low-temperature sensitive germplasms with a score of 6 to 11 points. The phenotypes under low-temperature stress are as follows: leaves are damaged by freezing, and some of the leaves droop; the inflorescence is slightly bent but can recover when the temperature rises; the flower buds are dead or closed; the flowers are deformed, and the fruit stalks are partially or completely wilted and drooped; the young siliques turn yellow, dry up, and fall off. The long siliques can continue to elongate. Four germplasms, including JD036, JD038, JD041, and JD044, are extremely sensitive germplasms with a score of ≤5 points. The phenotypes under low-temperature stress are as follows: leaves are damaged by freezing, wilted, and the whole leaf droops; the main inflorescence or branch inflorescence droops, the flower buds are dead or closed; the flowers are dry and dead, and the fruit stalks are partially or completely wilted and drooped; the young siliques turn yellow, dry up, and fall off. The long siliques no longer elongate.

[0066] Table 3 Low temperature tolerance grades and phenotypic symptom descriptions of Brassica napus

[0067]

[0068] Table 4 Clustering of cold tolerance of flowering period of rapeseed germplasms tested

[0069]

[0070] 1.5 Field cold tolerance of different early-maturing Brassica napus germplasms

[0071] From January 25 to 29, 2023, Changsha experienced continuous low temperatures ranging from -1℃ to -3℃. Figure 3 The early-maturing germplasm in the field encountered low temperature stress during the flowering period. On April 3, the main inflorescence fruiting of the above-mentioned low-temperature sensitive and low-temperature tolerant materials was observed, and it was found that the low-temperature sensitive materials had serious segmented fruiting and radish corners. Figure 4 (B) As shown; the main inflorescence of the cold-resistant material developed normally, without obvious segmented fruiting phenomenon, and the fruiting property was significantly better than that of the low-temperature sensitive material, such as Figure 4 (A) shown.

[0072] Therefore, a potted plant experiment was conducted to perform indoor low-temperature treatment on Brassica napus germplasms during the flowering stage, and an evaluation system for identifying the cold tolerance of Brassica napus germplasms during the flowering stage through phenotypic scoring has a high credibility and can be used as a method for identifying and evaluating the cold tolerance of early-flowering rapeseed during the flowering stage. By adopting this evaluation system, 15 Brassica napus germplasm resources with low-temperature tolerance during the flowering stage were identified, which can provide germplasm references for the breeding of rapeseed varieties with cold tolerance during the flowering stage.

Claims

1. An identification and evaluation method for cold tolerance during the flowering period of Brassica napus, characterized in that, It includes the following steps: (1) When Brassica napus is in the flowering stage, carry out low-temperature stress treatment at sub-zero temperature. After the treatment is over, move the plants back to the greenhouse to resume growth. (2) 5 days after the plants resume growth, collect the cold damage phenotypic symptoms of the upper cauline leaves, flower buds, petals, main inflorescence, branch inflorescence, fruit stalks, young siliques, and long siliques of the plants, and score according to the corresponding phenotypic symptoms. The specific cold damage phenotypic symptoms and scoring criteria are as follows: Upper cauline leaves: The cauline leaves are unfolded, scored 2 points; partially wilted and drooping, scored 1 point; the whole leaf is wilted and drooping, scored 0 point. Flower buds: The flower buds are normal, scored 2 points; the flower buds are closed or dead, scored 0 point. Petals: The petals are normal, scored 2 points; the edges of the petals are involuted, scored 1 point; the petals are dry, scored 0 point. Main inflorescence: The main inflorescence elongates normally, scored 2 points; the main inflorescence is bent and can resume growth. Scored 1 point; the main inflorescence droops and cannot resume, scored 0 point. Branch inflorescence: The branch inflorescence elongates normally, scored 2 points; the branch inflorescence is bent and can resume growth, scored 1 point; the branch inflorescence droops and cannot resume, scored 0 point. Fruit stalks: The fruit stalks are normal, scored 2 points; the fruit stalks are dry or wilted and drooping, scored 0 point. Young siliques: The young siliques are normal, scored 2 points; the young siliques turn yellow, wilt, and dry, scored 0 point. Long siliques: The siliques elongate and the embryos are normal, scored 2 points; the siliques elongate and the embryos are browned and shriveled, scored 0 point. (3) Determine the low-temperature tolerance level of the plant according to the total score. The determination criteria are as follows: If the total score ≥ 15 points, it is classified as a strong cold-tolerant level; if the total score is 12 - 14 points, it is classified as a cold-tolerant level; if the total score is 6 - 11 points, it is classified as a sensitive level; if the total score ≤ 5 points, it is classified as an extremely sensitive level.

2. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (1), the flowering stage is the period 7 days after the initial flowering stage of Brassica napus, when the branches are unfolded and young siliques are formed.

3. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (1), the temperature of the low-temperature stress treatment is -1°C to -3°C, and the treatment time is more than 12 hours.

4. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (1), the day and night conditions of the greenhouse are 24°C / 14h and 16°C / 10h respectively.

5. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (3), the phenotypic symptoms description of the strong cold-tolerant level is: The leaves have no deformation or frost damage, the leaves are unfolded; the flower buds are normal; the flowers are unfolded, and the inflorescence is straight; the fruit stalks are normal; the young siliques are green, and the long siliques can continue to elongate.

6. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (3), the phenotypic symptoms description of the cold-tolerant level is: The leaves have no deformation or frost damage, the leaves are unfolded; the flower buds are normal; the inflorescence is slightly bent but can resume upward after the temperature rises; the fruit stalks are normal; the young siliques turn yellow, and the long siliques can continue to elongate.

7. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (3), the phenotypic symptoms description of the sensitive level is: The leaves have frost damage and part of the leaves droop; the inflorescence is slightly bent but can resume upward after the temperature rises; the flower buds are dead or closed; the flowers are deformed, the fruit stalks are partially or completely wilted and drooping; the young siliques turn yellow, dry, and fall off, and the long siliques can continue to elongate.

8. The identification and evaluation method for cold tolerance during the flowering period of Brassica napus according to claim 1, characterized in that, In step (3), the phenotypic symptoms description of the extremely sensitive level is: The leaves have frost damage, wilt, and the whole leaf droops; the main inflorescence or branch inflorescence droops, the flower buds are dead or closed; the flowers dry and die, the fruit stalks are partially or completely wilted and drooping; the young siliques turn yellow, dry, and fall off, and the long siliques no longer elongate.

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