A method for screening cold-tolerant germplasm of Brassica napus

Abstract

The invention discloses a screening method for low-temperature-resistant germplasm of Brassica napus, which comprehensively evaluates the low-temperature resistance of each germplasm by combining the seedling index of an indoor experiment and the low-temperature resistance grade of an outdoor field experiment. The higher the low-temperature resistance grade of the outdoor field experiment and the higher the seedling index of the indoor experiment, the better the low-temperature resistance of the germplasm, and vice versa. The screening method for low-temperature-resistant germplasm of Brassica napus of the invention comprehensively evaluates the low-temperature resistance of each germplasm by combining the seedling index of an indoor experiment and the low-temperature resistance grade of an outdoor field experiment, and the obtained result is more scientific and accurate, has strong application value, has strong innovation, and the conclusion can provide important reference value for cold-resistant breeding of rapeseed.

Description

Technical Field

[0001] The invention belongs to the technical field of crop germplasm screening, and particularly relates to a method for screening low-temperature-resistant germplasm of Brassica napus. Background Art

[0002] Brassica napus L. is widely planted in winter. Due to the promotion of the double-cropping system of "late rice and rapeseed" and the triple-cropping system of "rice, rice and rapeseed", the harvest time of the previous rice crop is getting later and later, and the demand for direct seeding and late seeding of Brassica napus is increasing. With the postponement of the sowing date, the period when direct-seeded rapeseed seeds experience low temperatures in the field environment is also advanced, which often leads to a decrease in the germination rate and seedling rate of rapeseed, slow growth of seedlings, and difficulty in overwintering. Therefore, it is of great theoretical and application value to establish a simple, rapid and accurate method for screening rapeseed late-tolerant sowing quality.

[0003] The invention application with publication number CN107018707A discloses a method for identifying the low temperature resistance of rapeseed seed germination and seedling formation, and proposes to use filter paper as a germination bed, measure the relevant indicators of rapeseed germination and seedling formation after low temperature treatment, and thus select rapeseed cold-resistant germplasm. However, filter paper germination cannot fully represent the whole process of seed development in the soil, and whether the low temperature-resistant germplasm selected by the indoor incubator can withstand late sowing in field production has not been verified.

[0004] Patent application with publication number UA133841U discloses a method for evaluating the low temperature tolerance of winter rapeseed, which involves treating the swollen seeds at a low temperature of -10±1°C for 60 hours, keeping them at +2±1°C for 6 hours, and then germinating them at +25±1°C for two days, and comparing the number of germinated seeds in the experimental sample and the control sample.

[0005] In the above-mentioned prior art, the research on the low temperature resistance of rapeseed seeds is carried out indoors, mainly conducting low temperature germination and seedling tests on rapeseed seeds on filter paper or culture medium, which cannot well reflect the actual low temperature resistance of rapeseed in the field. Summary of the invention

[0006] The present invention aims at the above-mentioned deficiencies in the prior art and provides a method for screening low-temperature-resistant germplasm of Brassica napus. The present invention combines indoor experiments with outdoor field production, screens low-temperature-resistant germplasm by soil cultivation indoors, and identifies and verifies it through direct seeding and delayed seeding experiments in the field. The proposed standard is more scientific and accurate, has strong application value, and is highly innovative.

[0007] A method for screening low-temperature-resistant germplasm of Brassica napus, comprising the following steps:

[0008] (1) Indoor experiments were conducted. The seeds were planted in soil pots and the periodic temperature conditions were set as follows: 10°C for 12 hours of light and 5°C for 12 hours of darkness. The seedling growth status of the seeds was recorded every day for 14 consecutive days, and the seedling index was calculated. Among them, e i is the number of seeds that have grown into seedlings on the i-th day, t i is the i-th day after sowing, m is the number of seeds in each soil pot, and n is the number of statistical days14;

[0009] (2) Conduct outdoor field experiments, select a time and place where the minimum temperature can be below 10°C, record the seedling formation and growth of the seeds two weeks after sowing, and evaluate the low temperature resistance level of each germplasm based on the seedling formation rate, cotyledon and true leaf length, and whether the cotyledons and true leaves shrink after low temperature stress. If the seedling formation rate is high, the cotyledons and true leaves grow fast, and the cotyledons and true leaves do not shrink after low temperature stress, the low temperature resistance level of the germplasm is high, otherwise the low temperature resistance level is low;

[0010] (3) The low-temperature resistance of each germplasm was evaluated by combining the seedling index of the indoor experiment with the low-temperature resistance grade of the outdoor field experiment. The higher the low-temperature resistance grade of the outdoor field experiment and the higher the seedling index of the indoor experiment, the better the low-temperature resistance of the germplasm, otherwise the poorer the low-temperature resistance.

[0011] Preferably, when conducting outdoor field experiments, the northern Zhejiang region is selected, and the time is selected from October to November. Specifically, it can be Changxing County, Huzhou, northern Zhejiang (E119.7°, N39.4°), and the time is selected from October to November, when low temperatures of about 10°C often occur.

[0012] Preferably, an artificial climate chamber is used when conducting indoor experiments.

[0013] Preferably, when conducting indoor experiments, a rectangular plant planting pot with a width of 8 cm, a length of 8 cm, and a depth of 8 cm is selected, peat soil and vermiculite are mixed in a volume ratio of 1:1 to obtain simulated soil, and the plant planting pot is filled with the simulated soil to 90% full.

[0014] More preferably, the number of seeds sown in each plant planting pot is 15 to 25. Each germplasm can be sown in one pot or multiple pots, with each pot serving as one replicate.

[0015] Preferably, during illumination, the intensity of the illumination is set to reach 325.15 μmol / m 2 / s, its light wavelength is mainly distributed between 420 and 780 nanometers, the ratio of red light to blue light is close to 3 to 1, and the color temperature is 3800K.

[0016] Preferably, the seedling standards for both indoor and outdoor field experiments are: the seedlings are upright, and the cotyledons turn green and flatten. The seedling rate of each pot is calculated by dividing the number of seeds that have grown into seedlings by the total number of seeds.

[0017] Preferably, the outdoor field experiment is carried out three times of sowing in early October, late October and early November, the field plots for sowing each germplasm material are of uniform size, 1 to 2 meters in length and 1 to 2 meters in width, and the number of seeds sown for each material is 50 to 200.

[0018] Preferably, the low temperature resistance level of outdoor field experiments is divided into five levels from low to high.

[0019] Field Grade 1 is extremely intolerant to low temperatures: 2 weeks after sowing, field statistics show that the seedling rate is 0%≤<20%, ​​the length of a single cotyledon is 3mm≤<5mm, and the cotyledons are obviously shrunken and damaged by low temperatures;

[0020] Field Grade 2 is not cold-resistant: 2 weeks after sowing, the field statistics show that the seedling rate is 20%≤<40%, the true leaves have not grown or the true leaves have just emerged, the length of a single cotyledon is 5mm≤<8mm, and the cotyledons are shrunken after being exposed to low temperatures;

[0021] Field grade 3 is weak cold resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 40%≤<60%, the cotyledons are fully developed, 8mm≤single cotyledon length<12mm, the seedlings grow true leaves, 5mm≤the first true leaf length<8mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperature;

[0022] Field grade 4 is low temperature resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 60%≤<80%, the cotyledons are fully developed, the length of a single cotyledon is 8mm≤<12mm, the length of the first true leaf is similar to that of the cotyledon, both ≥8mm and <12mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperatures;

[0023] Field level 5 is strong resistance to low temperature: 2 weeks after sowing, the field statistics show that the seedling rate is 80%≤≤100%, the cotyledons are fully developed, the length of a single cotyledon is 8 mm≤<12 mm, and the length of the first true leaf is greater than the length of the cotyledon, the length of the first true leaf is ≥12 mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperature.

[0024] More preferably, when comprehensively evaluating the low temperature resistance of each germplasm based on the seedling index of the indoor experiment and the low temperature resistance grade of the outdoor field experiment, the comprehensive low temperature resistance index of the rapeseed germplasm is divided into the following grades:

[0025] The seedling index is less than 0.28, the field is level 1, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 1-1;

[0026] 0.28≤seedling index<0.62, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-2;

[0027] 0.62≤seedling index<1.23, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-3;

[0028] 0.28≤seedling index<0.62, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-1;

[0029] 0.62≤seedling index<1.23, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-2;

[0030] 0.28≤seedling index<0.62, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-1;

[0031] 0.62≤seedling index<1.23, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-2;

[0032] 1.23≤seedling index<1.61, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-3;

[0033] 1.61≤seedling index<1.80, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-4;

[0034] 1.23≤Seedling index<1.61, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-1;

[0035] 1.61≤Seedling index<1.80, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-2;

[0036] The seedling index is ≥1.80, the field is level 4, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 4-3;

[0037] 1.61≤seedling index<1.80, field grade 5, rapeseed germplasm low temperature resistance comprehensive index is grade 5-1;

[0038] Seedling index ≥1.80, field grade 5, rapeseed germplasm low temperature resistance comprehensive index is 5-2,

[0039] The low temperature resistance of rapeseed germplasm gradually increases according to the comprehensive index of 1-1, 1-2, 1-3, 2-1, 2-2, 3-1, 3-2, 3-3, 3-4, 4-1, 4-2, 4-3, and 5-1.

[0040] The screening method for low-temperature-resistant germplasm of Brassica napus of the present invention comprehensively evaluates the low-temperature resistance of each germplasm by combining the seedling index of indoor experiments and the low-temperature resistance grade of outdoor field experiments. The obtained results are more scientific and accurate, have strong application value, and are highly innovative. The conclusions drawn can provide important reference value for cold-resistant breeding of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the spectrum of LED lighting sources for artificial climate chambers.

[0042] Figure 2 This is a comparison of the results of low-temperature-resistant germplasm seedlings under artificial climate chamber conditions and field conditions. The upper and middle rows are the results under artificial climate chamber conditions, and the lower row is the results under field conditions.

[0043] Figure 3 This is a temperature change range chart in Changxing area of ​​Zhejiang from mid-October to mid-November 2022.

[0044] Figure 4 This is the result of repeatability analysis of screening low-temperature-tolerant germplasm under artificial climate chamber conditions and field conditions.

[0045] Figure 5 This is a graph showing the field growth results of representative materials with different low-temperature resistance levels in the field, where the white scale bar is 1 cm.

[0046] Figure 6 This is a chart of the seedling index of representative materials of different levels of field low-temperature resistance in the incubator.

[0047] Figure 7 This is the corresponding relationship between the indoor experiment seedling index and the outdoor field experiment low temperature resistance grade. DETAILED DESCRIPTION

[0048] Example 1

[0049] 1. Indoor experiment.

[0050] The materials from the Brassica napus germplasm resource bank were used by sowing in plant pots and then conducting low-temperature screening in an artificial climate chamber.

[0051] A cubic plant planting pot with a width of 8 cm, a length of 8 cm and a depth of 8 cm was selected, peat soil and vermiculite were mixed in a volume ratio of 1:1 to obtain simulated soil, and the plant planting pot was filled with the simulated soil to 90% full.

[0052] Water the planting soil thoroughly with tap water. Screen the seeds to be sown by appearance and select seeds of uniform size and appearance. Sow 20 seeds evenly on the surface of the planting soil in each plant pot, and gently press the seeds halfway into the soil, but you can still see the structure of the upper hemisphere of the seeds.

[0053] The seeds were planted in soil pots and subjected to low temperature treatment. The periodic temperature conditions were set as follows: 10°C light for 12 hours and 5°C dark for 12 hours. During illumination, the light intensity was set to reach 325.15 μmol / m at 10 cm below the light board. 2 / s, its light wavelength is mainly distributed between 420 and 780 nanometers, the ratio of red light to blue light is close to 3 to 1, and the color temperature is 3800K. Figure 1 This is the spectrum of LED lighting sources for artificial climate chambers.

[0054] The standard for seedlings is: the seedlings are upright and the cotyledons turn green and flat. The seedling status of the seeds is recorded every day for 14 consecutive days, and the seedling index is calculated. Among them, e i is the number of seeds that have grown into seedlings on the i-th day, t i is the ith day after sowing, m is the number of seeds in each soil pot, and n is the number of statistical days14.

[0055] In the low-temperature artificial climate chamber, the seedling index on the 14th day varied from 0 to 2.18. Among them, some low-temperature resistant and low-temperature intolerant materials were screened. The low-temperature resistant material R476x【8】had a seedling rate of 100% on the 14th day and a seedling index of 2.18; while the low-temperature intolerant material R468x had a seedling rate of 15% on the 14th day and a seedling index of 0.25;

[0056] Pass the above screening criteria and take photos ( Figure 2 The names of the germplasm materials are shown in Table 1.

[0057] Table 1

[0058] R4x5x【1】 R45x6 R484x R4x7x R4x0x R48x7 R4x8x R46x1【6】 R49x8 R4x9x R46x0 R R4x1x【2】 R46x7 R50x5 R4x3x【3】 R47x8 R50x8【9】 R42x5【4】 R47x4 R50x9

[10] R4x4x R47x8 R50x7 R4x6x R47x3【7】 R50x6 R44x9【5】 R476x【8】 R50x0

[11] R45x8 R48x5 R5x4x

[12]

[0059] 2. Outdoor field experiments.

[0060] An outdoor field experiment was conducted in Changxing County, Huzhou, northern Zhejiang (E119.7°, N39.4°). The outdoor field experiment was carried out three times in early October, late October and early November. The field plots for sowing each germplasm material were of the same size, about 1.2 meters long and 1.2 meters wide, and the number of seeds sown for each material was 100.

[0061] In the field, a low-temperature tolerance experiment was conducted by sowing at three different time periods. Through local temperature surveys, it was found that the later the material was sown, the lower the temperature it experienced, which generally had a reduced effect on the germination rate and seedling rate.

[0062] In northern Zhejiang, Si'an Town, Changxing District, Huzhou City, conducted field direct seeding and delayed seeding experiments. Three sowings were carried out on October 10, 2022, October 25, 2022, and November 11, 2022. The local temperature survey ( Figure 3 ), at this time, low temperatures of around 10°C often occur, and it is found that the later the material is sown, the lower the temperature it experiences.

[0063] Two weeks after sowing, the seedling establishment and seedling growth were recorded, and the low-temperature resistance level of each germplasm was evaluated based on the seedling establishment rate, seedling morphology, and low-temperature stress. If the seedling establishment rate is fast, the seedling growth is fast, and the low-temperature stress is light, then the low-temperature resistance level of the germplasm is high; otherwise, the low-temperature resistance level is low.

[0064] The low temperature resistance level of outdoor field experiments is divided into 5 levels from low to high.

[0065] Field Grade 1 is extremely intolerant to low temperatures: 2 weeks after sowing, field statistics show that the seedling rate is 0%≤<20%, ​​the length of a single cotyledon is 3mm≤<5mm, and the cotyledons are obviously shrunken and damaged by low temperatures;

[0066] Field Grade 2 is not cold-resistant: 2 weeks after sowing, the field statistics show that the seedling rate is 20%≤<40%, the true leaves have not grown or the true leaves have just emerged, the length of a single cotyledon is 5mm≤<8mm, and the cotyledons are shrunken after being exposed to low temperatures;

[0067] Field grade 3 is weak cold resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 40%≤<60%, the cotyledons are fully developed, 8mm≤single cotyledon length<12mm, the seedlings grow true leaves, 5mm≤the first true leaf length<8mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperature;

[0068] Field grade 4 is low temperature resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 60%≤<80%, the cotyledons are fully developed, the length of a single cotyledon is 8mm≤<12mm, the length of the first true leaf is similar to that of the cotyledon, both ≥8mm and <12mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperatures;

[0069] Field level 5 is strong resistance to low temperature: 2 weeks after sowing, the field statistics show that the seedling rate is 80%≤≤100%, the cotyledons are fully developed, the length of a single cotyledon is 8 mm≤<12 mm, and the length of the first true leaf is greater than the length of the cotyledon, the length of the first true leaf is ≥12 mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperature.

[0070] In the second week of each sowing, the growth of seedlings and young plants was counted ( Figure 2 Next row), and the low temperature resistance of rapeseed was rated and scored according to the standard. Among the materials sown for the first time on October 10, 2022, 27 materials with a low temperature resistance grade of 5 were screened, and among the materials sown for the second time on October 25, 2022, 25 materials with a low temperature resistance grade of 5 were screened. Due to the large drop in temperature in early November, the materials sown for the third time on November 11, 2022 were under strong low temperature stress, and a total of 18 materials with a low temperature resistance grade of 5 in the field were screened, as shown in Table 2.

[0071] Table 2

[0072] R4x5x【1】 R458x R4x77 R46x1【6】 R4x1x【2】 R47x3【7】 R4x3x【3】 R476x【8】 R42x5【4】 R49x0 R443x R50x8【9】 R44x9【5】 R50x9

[10] R450x R50x0

[11] R4x72 R5x4x

[12]

[0073] Since the materials sown for the third time on November 11, 2022 were severely affected by low temperature stress, the low temperature resistant materials screened out were representative. Therefore, the low temperature resistant materials and steps screened out this time were selected. Figure 4 ) By comparing the low-temperature resistant materials screened out by the artificial climate chamber, it was found that 12 materials had strong low-temperature resistance under different low-temperature environments, as shown in Table 3.

[0074] Table 3

[0075] R4x5x【1】 R47x3【7】 R4x1x【2】 R476x【8】 R4x3x【3】 R50x8【9】 R42x5【4】 R50x9

[10] R44x9【5】 R50x0

[11] R46x1【6】 R5x4x

[12]

[0076] Take photos of typical materials of various grades resistant to low temperatures in the field ( Figure 5 ) and compared with the seedling index in the incubator ( Figure 6 ), the seedling index of the typical material R5158 with field low temperature resistance level 1 in the incubator was 0.89, the seedling index of the typical material R5051 with field low temperature resistance level 2 in the incubator was 1.21, the seedling index of the typical material R4723 with field low temperature resistance level 3 in the incubator was 1.27, the seedling index of the typical material R4x8x with field low temperature resistance level 4 in the incubator was 1.83, and the seedling index of the typical material R5x4x

[12] with field low temperature resistance level 5 in the incubator was 1.96.

[0077] The overall correlation analysis was conducted on the materials sown for the third time on November 11, 2022 and the materials planted in the incubator ( Figure 7 ), there were 203 materials in total, of which 14 materials had field low temperature resistance grade 1, and the seedling index ranged from 0.07 to 0.93, with a median of 0.28; there were 40 materials with field low temperature resistance grade 2, and the seedling index ranged from 0.30 to 1.21, with a median of 0.62; there were 104 materials with field low temperature resistance grade 3, and the seedling index ranged from 0.37 to 1.67, with a median of 1.23; there were 27 materials with field low temperature resistance grade 4, and the seedling index ranged from 1.29 to 1.85, with a median of 1.61; there were 18 materials with field low temperature resistance grade 5, and the seedling index ranged from 1.61 to 2.18, with a median of 1.80.

[0078] When comprehensively evaluating the low temperature resistance of each germplasm based on the seedling index of the indoor experiment and the low temperature resistance grade of the outdoor field experiment, the comprehensive low temperature resistance index of rapeseed germplasm is divided into the following grades:

[0079] The seedling index is less than 0.28, the field is level 1, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 1-1;

[0080] 0.28≤seedling index<0.62, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-2;

[0081] 0.62≤seedling index<1.23, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-3;

[0082] 0.28≤seedling index<0.62, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-1;

[0083] 0.62≤seedling index<1.23, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-2;

[0084] 0.28≤seedling index<0.62, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-1;

[0085] 0.62≤seedling index<1.23, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-2;

[0086] 1.23≤seedling index<1.61, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-3;

[0087] 1.61≤seedling index<1.80, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-4;

[0088] 1.23≤Seedling index<1.61, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-1;

[0089] 1.61≤Seedling index<1.80, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-2;

[0090] The seedling index is ≥1.80, the field is level 4, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 4-3;

[0091] 1.61≤seedling index<1.80, field grade 5, rapeseed germplasm low temperature resistance comprehensive index is grade 5-1;

[0092] The seedling index is ≥1.80, the field index is level 5, and the comprehensive index of rapeseed germplasm resistance to low temperature is level 5-2.

[0093] The seedling index in the incubator and the low temperature resistance index in the field were jointly analyzed, and the low temperature resistance index of Brassica napus germplasm was divided into 14 levels (Table 4). The field low temperature resistance level was used as the main evaluation parameter. Therefore, the comprehensive low temperature resistance of Brassica napus germplasm was ranked from small to large as follows: 1-1, 1-2, 1-3, 2-1, 2-2, 3-1, 3-2, 3-3, 3-4, 4-1, 4-2, 4-3, 5-1, 5-2. For example, the low temperature resistance comprehensive index of the typical material R5158 with field low temperature resistance level 1 was 1-3, the low temperature resistance comprehensive index of the typical material R5051 with field low temperature resistance level 2 was 2-2, the low temperature resistance comprehensive index of the typical material R4723 with field low temperature resistance level 3 was 3-3, the low temperature resistance comprehensive index of the typical material R4x8x with field low temperature resistance level 4 was 4-3, and the low temperature resistance comprehensive index of the typical material R5x4x

[12] with field low temperature resistance level 5 was 5-2.

[0094] Table 4 Comprehensive index of low temperature resistance of Brassica napus

[0095]

[0096]

[0097] There are certain differences in the low temperature resistance of different Brassica napus germplasms in artificial climate chambers and field environments. This method can be used to comprehensively evaluate the changes in these differences and clarify the characteristics of low temperature resistant and intolerant materials.

Claims

1. A method for screening low-temperature-resistant germplasm of Brassica napus, characterized in that: The following steps are involved: (1) Indoor experiments were conducted. The seeds were planted in soil pots and the periodic temperature conditions were set as follows: 10°C for 12 hours of light and 5°C for 12 hours of darkness. The seedling growth status of the seeds was recorded every day for 14 consecutive days, and the seedling index was calculated. Among them, e i is the number of seeds that have grown into seedlings on the i-th day, t i is the i-th day after sowing, m is the number of seeds in each soil pot, and n is the number of statistical days14; (2) Conduct outdoor field experiments, select a time and place where the minimum temperature can be below 10°C, record the seedling formation and growth of the seeds two weeks after sowing, and evaluate the low temperature tolerance level of each germplasm based on the seedling rate, cotyledon and true leaf length, and whether the cotyledons and true leaves shrink after low temperature stress. If the seedling rate is high, the cotyledons and true leaves grow fast, and there is no shrinkage of cotyledons and true leaves after low temperature stress, the low temperature tolerance level of the germplasm is high, otherwise the low temperature tolerance level is low; The low temperature resistance level of outdoor field experiments is divided into five levels from low to high. Field Grade 1 is extremely intolerant to low temperatures: 2 weeks after sowing, field statistics show that the seedling rate is 0%≤<20%, ​​the length of a single cotyledon is 3mm≤<5mm, and the cotyledons are obviously shrunken and damaged by low temperatures; Field Grade 2 is not cold-resistant: 2 weeks after sowing, the field statistics show that the seedling rate is 20%≤<40%, the true leaves have not grown or the true leaves have just emerged, the length of a single cotyledon is 5mm≤<8mm, and the cotyledons are shrunken after being exposed to low temperatures; Field grade 3 is weak cold resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 40%≤<60%, the cotyledons are fully developed, 8mm≤single cotyledon length<12mm, the seedlings grow true leaves, 5mm≤the first true leaf length<8mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperature; Field grade 4 is low temperature resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 60%≤<80%, the cotyledons are fully developed, the length of a single cotyledon is 8mm≤<12mm, the length of the first true leaf is similar to that of the cotyledon, both ≥8mm and <12mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperatures; Field grade 5 is strong cold resistance: 2 weeks after sowing, the field statistics show that the seedling rate is 80%≤≤100%, the cotyledons are fully developed, the length of a single cotyledon is 8mm≤<12mm, and the length of the first true leaf is greater than that of the cotyledon, the length of the first true leaf is ≥12mm, and the cotyledons and true leaves are not wrinkled after being exposed to low temperatures; (3) Comprehensively evaluate the low temperature resistance of each germplasm by combining the seedling index of the indoor experiment and the low temperature resistance grade of the outdoor field experiment. The higher the low temperature resistance grade of the outdoor field experiment and the higher the seedling index of the indoor experiment, the better the low temperature resistance of the germplasm, otherwise the poorer the low temperature resistance; When comprehensively evaluating the low temperature resistance of each germplasm based on the seedling index of the indoor experiment and the low temperature resistance grade of the outdoor field experiment, the comprehensive low temperature resistance index of rapeseed germplasm is divided into the following grades: The seedling index is less than 0.28, the field is level 1, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 1-1; 0.28≤seedling index<0.62, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-2; 0.62≤seedling index<1.23, field level 1, rapeseed germplasm low temperature resistance comprehensive index is level 1-3; 0.28≤seedling index<0.62, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-1; 0.62≤seedling index<1.23, field grade 2, rapeseed germplasm low temperature resistance comprehensive index is grade 2-2; 0.28≤seedling index<0.62, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-1; 0.62≤seedling index<1.23, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-2; 1.23≤seedling index<1.61, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-3; 1.61≤seedling index<1.80, field grade 3, rapeseed germplasm low temperature resistance comprehensive index is grade 3-4; 1.23≤seedling index<1.61, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-1; 1.61≤Seedling index<1.80, field grade 4, rapeseed germplasm low temperature resistance comprehensive index is grade 4-2; The seedling index is ≥1.80, the field is level 4, and the comprehensive index of low temperature resistance of rapeseed germplasm is level 4-3; 1.61≤seedling index<1.80, field grade 5, rapeseed germplasm low temperature resistance comprehensive index is grade 5-1; The seedling index is ≥1.80, the field is level 5, and the comprehensive index of rapeseed germplasm resistance to low temperature is level 5-2. The low temperature resistance of rapeseed germplasm gradually increases according to the comprehensive index of 1-1, 1-2, 1-3, 2-1, 2-2, 3-1, 3-2, 3-3, 3-4, 4-1, 4-2, 4-3, and 5-1.

2. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: When conducting outdoor field experiments, choose northern Zhejiang and the time is October to November.

3. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: When conducting indoor experiments, use an artificial climate chamber.

4. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: When conducting indoor experiments, a rectangular plant planting pot with a width of 8 cm, a length of 8 cm, and a depth of 8 cm was selected, and peat soil and vermiculite were mixed in a volume ratio of 1:1 to obtain simulated soil, and the plant planting pot was filled with the simulated soil to 90% full.

5. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 4, characterized in that: The number of seeds sown in each plant pot is 15 to 25.

6. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: During illumination, the light intensity was set to 325.15 μmol / m 2 / s, its light wavelength is mainly distributed between 420 and 780 nanometers, the ratio of red light to blue light is close to 3 to 1, and the color temperature is 3800K.

7. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: The seedling standards for both indoor experiments and outdoor field experiments were: the seedlings were upright and the cotyledons turned green and flattened.

8. The method for screening low-temperature-resistant germplasm of Brassica napus according to claim 1, characterized in that: The size of the field for sowing each germplasm material is consistent, 1 to 2 meters long and 1 to 2 meters wide, and the number of seeds sown for each material is 50 to 200.

Images