A method for identifying cold resistance of upland cotton at different growth stages

Abstract

The present invention discloses a method for identifying the cold resistance of upland cotton at different growth stages. It belongs to the technical field of upland cotton identification. The method of the present invention identifies the cold resistance of upland cotton at the germination stage, bud stage, cotyledon stage or trifoliate stage. Identification of cold resistance in the germination stage: seeds are treated at 4°C for 7 to 10 days, and then growth resumes for 4 days; identification of cold resistance in the bud stage: buds are treated at 4°C for 3 to 5 days, and then growth resumes for 10 to 14 days; identification of cold resistance in the cotyledon stage: cotyledons are treated under light and 4°C for 3 to 5 days, and then growth resumes for 10 to 14 days; identification of cold resistance in the trifoliate stage: when cultivated to the trifoliate stage under normal conditions, they are treated under light and 4°C for 3 to 5 days, and then growth resumes for 10 to 14 days. The treatment conditions of the present invention are simple and easy to obtain, and the survey indicators are simple and clear. The cold resistance identification methods for the germination and trifoliate stages are newly added, and the cold resistance identification methods for the bud and cotyledon stages are optimized.

Description

Technical Field

[0001] The present invention relates to the technical field of upland cotton identification, and more particularly to a method for identifying the cold resistance of upland cotton at different growth stages. Background Art

[0002] Upland cotton is a major cash crop, providing over 95% of the world's cotton fiber. Since 1980, cotton cultivation in my country has gradually shifted northwestward, particularly to Xinjiang. Originating in tropical and subtropical regions, upland cotton is susceptible to cold weather throughout its growth period, particularly from the germination to the seedling stage. Low-temperature stress is particularly acute in Xinjiang, where chilling damage has become a significant threat to cotton production. Over the past decade, the incidence of chilling damage in cotton-producing areas in northern Xinjiang has ranged from 10% to 30%. Cotton germplasm resources are the foundation for the creation of new materials and the basis and key to variety breeding and improvement. Cotton phenotypic traits are influenced by both genetic and environmental factors and are the result of long-term natural and artificial selection. Strengthening research on the genetic diversity of cotton germplasm resources is crucial. Therefore, identifying cold tolerance in upland cotton is essential.

[0003] The existing cold resistance investigation methods are complex and easily lead to errors in human investigations among researchers; at the same time, there is a lack of cold resistance identification methods during the germination period and the three-leaf period.

[0004] In summary, how to provide an intuitive, accurate and rapid method for identifying the cold resistance of upland cotton at different growth stages is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for identifying the cold resistance of upland cotton at different growth stages.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for identifying the cold resistance of upland cotton at different growth stages is provided, wherein the cold resistance of upland cotton at the germination stage, bud stage, cotyledon stage, and trifoliate stage is identified.

[0008] (1) Identification of cold resistance during germination: Seeds were treated at 4°C for 7–10 days and then allowed to resume growth for 4 days;

[0009] (2) Identification of cold resistance at the bud stage: buds were treated at 4°C for 3–5 days and then allowed to resume growth for 10–14 days;

[0010] (3) Identification of cold resistance at the cotyledon stage: The cotyledons were treated under light and 4°C conditions for 3–5 days, and then resumed growth for 10–14 days;

[0011] (4) Identification of cold resistance at the three-leaf stage: When upland cotton reaches the three-leaf stage, it is treated under light and 4°C conditions for 3 to 5 days, and then resumes growth for 10 to 14 days.

[0012] Furthermore, for cold resistance assessment during the germination period, seeds were treated at 4°C for 7 days and then resumed growth for 4 days.

[0013] Furthermore, the cold resistance evaluation during the germination period was performed by counting the germination rate, the rate without radicle, and the rate with radicle. The calculation formula is as follows:

[0014] Germination rate (%) = number of germinated seeds / total number of seeds × 100;

[0015] Radicle-free rate (%) = number of buds without radicles / number of sprouted buds × 100;

[0016] Radicle rate (%) = 100 - no radicle rate (%); cold resistance identification at the bud stage is based on the emergence rate, with emergence based on the growth of true leaves. The calculation formula is as follows:

[0017] Seedling emergence rate (%) = number of emerged seedlings / total number of buds × 100; both the cotyledon stage cold resistance assessment and the three-leaf stage cold resistance assessment were calculated using the following formula:

[0018] Survival rate (%) = number of surviving seedlings (trees) / total number of seedlings (trees) × 100.

[0019] Furthermore, the condition for the recovery growth is culturing at 28-30°C.

[0020] Furthermore, the lighting conditions for cold resistance identification at the cotyledon stage and the three-leaf stage were 1250LAX, and the cold resistance identification at the cotyledon stage and the three-leaf stage were normal growth before low temperature treatment, the temperature conditions were 28-30°C, and the light / dark time was 14h / 10h.

[0021] Furthermore, the survival rate of seedlings at the cotyledon stage was calculated based on the growth of new true leaves.

[0022] Furthermore, at the three-leaf stage, the survival rate of seedlings is calculated based on the fact that the growth point is still green.

[0023] Furthermore, the evaluation indicators are as follows:

[0024] Evaluation indicators during germination period:

[0025] Cold resistance index during germination period (%) = germination rate (%) × 60% + radicle rate (%) × 40%;

[0026] When the cold resistance index during the germination period is ≥70%, it is Class I cold resistance and the cold resistance type is cold resistance;

[0027] When the cold resistance index during germination is 69.9% or more and ≥50% or more, it is cold resistance level II and the cold resistance type is cold tolerant;

[0028] When 49.9% ≥ the cold resistance index during germination ≥ 20%, it is cold resistance level III and the cold resistance type is cold intolerance;

[0029] When the cold resistance index during germination is <20%, it is grade IV cold resistance and the cold resistance type is cold sensitive;

[0030] The germination rate was used as the evaluation index during the budding period;

[0031] The survival rate was used as the evaluation index at the cotyledon and three-leaf stages;

[0032] When the emergence rate / survival rate is ≥70%, it is Class I cold resistance and the cold resistance type is cold resistance;

[0033] When the emergence rate is 69.9% or higher or the survival rate is 50%, it is classified as Class II cold resistance and the cold resistance type is cold-tolerant;

[0034] When the emergence rate is 49.9% or higher and the survival rate is 20%, it is classified as Class III cold resistance and the cold resistance type is cold-intolerant;

[0035] When the emergence rate / survival rate is <20%, it is level IV cold resistance and the cold resistance type is cold sensitive.

[0036] Furthermore, the upland cotton variety is Zhong JX02, Zhong H177, Hengmian No. 3 or Yu 2067.

[0037] The above-mentioned upland cotton varieties can all be obtained from public channels. For example, Zhong JX02 and Zhong H177 are distributed free of charge by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The distribution plan is announced every year on the homepage of the website of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. Hengmian No. 3 and Yu 2067 are both varieties approved at the provincial level and can be obtained from commercial channels. A small amount of experimental seeds can also be obtained by directly requesting them from the breeding units.

[0038] Through the above technical solutions, it can be known that compared with the prior art, the beneficial effects achieved by the present invention are as follows: the cold resistance identification and investigation method of different growth periods is simple, highly operational, and more practical; the identification index of the germination period is simpler, more intuitive, more convenient and economical, which not only saves the trouble of replanting and reduces the error of investigation between researchers caused thereby, but also facilitates researchers to further sample and plant and multiply seeds, which can effectively preserve rare cotton germplasm resources and is more conducive to in-depth exploration of the deep reasons for the influence of low temperature on germination. The bud stage and cotyledon stage are respectively based on the growth of true leaves as the standard for emergence or live seedlings. This is because the cotton seedlings are considered to be truly alive and stable only after growing true leaves and can grow into healthy plants. At the same time, the index is simple and clear, and it is easy for investigators to judge, thus minimizing human errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0040] Figure 1 This is a schematic diagram of the cold resistance identification operation during the germination period of the present invention;

[0041] Figure 2 Schematic diagram of the effect of low temperature stress on upland cotton germination of the present invention, wherein A represents the effect of low temperature stress on the germination of Zhong JX02, B represents the effect of low temperature stress on the germination of Zhong H177, C represents the effect of low temperature stress on the germination of Hengmian No. 3, D represents the effect of low temperature stress on the germination of Yu 2067, E represents the length of each part of Zhong JX02 during the germination period, and F represents the length of each part of Hengmian No. 3 during the germination period;

[0042] Figure 3 This is a diagram showing symptoms of low temperature chilling injury to Hengmian No. 3 during the bud stage of the present invention;

[0043] Figure 4 Symptoms of upland cotton at the cotyledon stage at 4°C for 3 days and after growth recovery;

[0044] Figure 5 This is a symptom diagram of JX02 upland cotton at the three-leaf stage under 4°C low temperature stress for different days;

[0045] Figure 6 Symptoms of upland cotton at the three-leaf stage of the present invention at 4°C for 3 days and after growth recovery;

[0046] Figure 7 The anthocyanin content in the cotyledons of different upland cotton varieties at the cotyledon stage of the present invention, Note: Different capital letters (A-B) indicate extremely significant differences (P < 0.01), n ​​= 3;

[0047] Figure 8 The anthocyanin content in different upland cotton leaves at the three-leaf stage of the present invention is shown. Note: different capital letters (A-B) indicate extremely significant differences (P < 0.01), n ​​= 3. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0050] Example 1

[0051] 1 Materials and Methods

[0052] 1.1 Experimental materials and field planting

[0053] The test materials for this experiment are shown in Table 1. The seeds required for this experiment were harvested from our laboratory in the experimental fields of the Cotton Research Institute, Chinese Academy of Agricultural Sciences, from 2019 to 2022. They were purified through continuous self-pollination over many years. The field experiments were completed from 2021 to 2022 at the Experimental Farm of the Cotton Research Institute, Chinese Academy of Agricultural Sciences (located in Anyang County, Anyang City, Henan Province). The soil type was light yellow-brown soil.

[0054] Table 1 Sources and pedigrees of test materials

[0055]

[0056]

[0057] Upland cotton ZhongJX02 was developed in 2006 by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences' stress resistance identification team in Anyang City, Henan Province, using upland cotton XINGJ-2 as the female parent and upland cotton ZhongCotton Research Institute 12 as the male parent. Using an improved pedigree method, the hybrid was developed over eight years of selection and breeding, followed by three years of self-pollination, purification, and southern propagation. The pedigree was released in 2017. The parent combination is XINGJ-2 / ZhongCotton Research Institute 12. A new variety registration application was filed in 2022, with application number 20221000196.

[0058] 1.2 Cultivation of cotton seedlings at the cotyledon stage and the three-leaf stage

[0059] The bare seeds (if they are hairy seeds, they are rubbed) of each upland cotton material are sown in sandy soil that has been sterilized at high temperature. The moisture content of the sand is maintained at 21% to 23% (mass ratio). The soil is covered to keep it moist and placed in a constant temperature growth chamber at 28°C with 14 hours of light and 10 hours of darkness. Seedlings emerge approximately 7 days after sowing. After 7 days of emergence, the seedlings with undeveloped cotyledons, rotten seedlings, inverted seedlings, weak seedlings, and deformed seedlings are removed. 25 to 30 uniform and strong seedlings are left in each box and continue to grow for 2 days before being used for cold resistance assessment at the cotyledon stage. At this time, the cotyledons of each seedling in each treatment are in a fully extended state. When the seedlings grow to three leaves and one heart, they are used for cold resistance assessment at the three-leaf stage. Before treatment, weak seedlings and plants that have not reached the three-leaf stage are removed to ensure that the cotton seedlings in each treatment are in a consistent growth state.

[0060] 1.3 Identification of cold resistance at different growth stages

[0061] The cold resistance identification is mainly carried out at the germination stage, bud stage, cotyledon stage and trifoliate stage.

[0062] The germination period refers to the period from the time when cotton seeds begin to germinate after imbibition to the time when the length of the sprout reaches at least 1 / 2 of the seed length.

[0063] The bud stage refers to the period from the germination of cotton seeds to their emergence from the soil. Here, we used buds that had germinated for 2 days under normal conditions (28°C in the dark) and were about 1 cm long.

[0064] The cotyledon stage refers to the period from the time the cotyledons are flattened after the seeds germinate and emerge from the soil to the time the first true leaf grows out. Generally, indoors it refers to seedlings 9 to 10 days after sowing, at which time the two cotyledons are fully expanded.

[0065] The three-leaf stage refers to when the third true leaf is unfolded.

[0066] Identification of cold resistance during the germination period: 3 replicates were set up before and after treatment for each variety. 100 plump and well-developed seeds were picked for each replicate. The seeds were wrapped using a vertical filter paper roll method, with the large end of the seeds facing the marked direction. After wrapping, the seeds were left to stand for 2 hours (to allow the seeds to fully swell). Then the water was controlled to a state where no water dripped. The seeds were packed in black plastic bags to protect them from light and keep them moist. Then, the seeds were placed in a beaker with the marked direction facing upwards, so that the radicles would grow downwards after germination, which was conducive to measuring the length of the buds. In order to save manpower and financial costs, the filter paper was replaced with absorbent paper that had been cut at the time of purchase (such as Libang 200 tissue paper). This eliminated the trouble of cutting the filter paper and greatly reduced the economic cost. The absorbent paper had good water absorption and water retention conditions and could effectively meet the needs of cotton seed germination. Our experimental results show that absorbent paper can completely replace our commonly used filter paper. The seeds were treated at low temperature of 4°C for 0d, 3d, 5d, 7d, and 10d ( Figure 1 ), and then after 4 days of recovery growth in a light-free growth chamber at 28°C, the germination rate, radicle-free rate, radicle-present rate, shoot length, hypocotyl length and radicle length were investigated, and the cold resistance index during the germination period was calculated.

[0067] Germination rate (%) = number of germinated seeds / total number of seeds × 100;

[0068] Radicle-free rate (%) = number of buds without radicles / number of sprouted buds × 100;

[0069] Radicle rate (%) = 100 - no radicle rate;

[0070] Cold resistance index during germination period (%) = germination rate (%) × 60% + radicle rate (%) × 40%.

[0071] Identification of cold resistance at the bud stage: Three replicates were set up for each variety before and after treatment. Buds 2-3 days old were treated at 4°C for 3-5 days. They were then allowed to resume growth under normal conditions for 10-14 days. The emergence rate was then assessed, with emergence defined by the presence of true leaves.

[0072] Seedling emergence rate (%) = number of seedlings emerged / total number of buds × 100.

[0073] Cold resistance assessment at the cotyledon stage: Each variety was subjected to 4°C low-temperature treatment for 3, 5, and 7 days while the cotyledons were flattened. The 4°C low-temperature treatment was divided into light and dark treatments. The lighted low-temperature treatment was conducted in a CDN-260B illuminated incubator with 1250 LAX; the dark treatment was conducted in a BCD-215KADZ Haier refrigerator. After the low-temperature treatment, the plants were allowed to recover in a growth chamber at 28-30°C (16 h light / 8 h dark) for 10-14 days. The survival rate of seedlings was assessed, and survival was determined by the growth of new true leaves.

[0074] Survival rate (%) = number of surviving seedlings (trees) / total number of seedlings (trees) × 100.

[0075] Identification of cold resistance at the three-leaf stage: Each variety of upland cotton was cultured to the three-leaf stage and subjected to 4°C low-temperature treatment. The 4°C low-temperature conditions were divided into light and no-light treatments, which were the same as the low-temperature stress conditions at the cotyledon stage. Zhong JX02 was treated for 1d, 2d, 3d, 4d, 5d, and 7d, respectively. After the low-temperature stress ended, the symptoms were observed and photographed. Zhong H177 and Hengmian No. 3 were treated with low-temperature stress for 3d, 5d, and 7d, respectively, and the symptoms were compared with those of Zhong JX02. Then, they were restored in a growth chamber at 28-30°C (16h light / 8h dark) for 10-14d, and the survival rate was investigated. The survival rate was based on the growth point still being green.

[0076] Survival rate (%) = number of surviving seedlings (trees) / total number of seedlings (trees) × 100.

[0077] 1.4 Determination of anthocyanins at the cotyledon and trifoliate stages: Leaves of each variety of upland cotton were sampled when they were normally cultivated to the cotyledon and trifoliate stages. The cotyledons were sampled at the cotyledon stage, and the third leaf was sampled at the trifoliate stage to determine the anthocyanin content before low-temperature treatment.

[0078] Anthocyanin content was determined by colorimetric analysis: 0.25 g of each leaf sample was minced and added to 2 ml of a 1% (g / vol) methanolic HCl solution. The sample was then incubated at 4°C in the dark for 24 hours until the leaves completely bleached. A 1% methanolic HCl solution was used as a blank control. The absorbance of the experimental solution at 530 nm and 657 nm was determined by enzyme-linked immunosorbent assay (ELISA) to determine the anthocyanin content in the cotton leaves. Three biological replicates were performed for each sample for statistical analysis.

[0079] 1.5 Instruments and Equipment

[0080] GZP-250A intelligent light growth chamber; CDN-260B light incubator, BCD-215KADZ Haier refrigerator; HBS-1101 enzyme label analyzer.

[0081] 1.6 Data Processing

[0082] Microsoft Excel 2013 was used for general data processing, and Stata 11.0 software was used for statistical analysis.

[0083] 2 Results

[0084] 2.1 Changes in upland cotton germination in response to low temperature stress

[0085] According to existing technical reports, Hengmian No. 3 is cold-resistant during the germination period, while Yu 2067 is not cold-resistant during the germination period (Wang Junjuan et al., 2016). In this experiment, after being treated at 4°C for 3 days and then recovering to grow for 4 days under normal temperature conditions, the germination rate, hypocotyl length and radicle length of the four varieties decreased significantly, especially the germination rate of the cold-sensitive materials decreased more significantly; with the increase of the number of days of low-temperature treatment, the germination rate of the four varieties continued to decrease, the radicle-free rate continued to increase, and the hypocotyl length and radicle length decreased. Among them, the germination rate of JX02 decreased the fastest, and the radicle-free rate increased the fastest. It is the variety most sensitive to low temperature during the germination period among the four varieties (Table 2 and Figure 2 The greatest impact of low temperature on upland cotton during germination is the inhibition of seed germination and radicle elongation. Low temperature has a much greater inhibitory effect on radicles than on hypocotyls, especially for cold-sensitive materials. For JX02, after being treated with 4°C for 3, 5, 7, and 10 days, the hypocotyl length decreased by 4.17%, 23.54%, 57.71%, and 80.63%, respectively, compared with the control (germinated at normal temperature (28°C) for 4 days); while the radicle length decreased by 78.08%, 90.00%, 98.63%, and 99.18%, respectively. In the cold-tolerant variety Hengmian No. 3, both the hypocotyl and radicle lengths decreased less than those of the cold-sensitive materials. The hypocotyl also decreased less than the radicle. The hypocotyl lengths decreased by 4.06%, 11.84%, 23.26%, and 26.00%, respectively, and the radicle lengths decreased by 41.51%, 58.90%, 63.01%, and 79.45%, respectively. After 3 days of low-temperature treatment, the radicle length of the cold-sensitive material decreased significantly more than that of the cold-tolerant material. After 5 days of low-temperature treatment, the hypocotyl of the cold-sensitive material decreased significantly more than that of the cold-tolerant material. The hypocotyl of JX02 was red in the control state. As the low-temperature treatment time at 4°C increased, the color gradually became lighter, indicating that low temperature inhibited the synthesis of certain pigments such as anthocyanins. The deeper the low-temperature stress, the stronger the inhibition ( Figure 2 A).

[0086] As shown in Table 2, under control conditions (germination at normal temperature (28°C without light)), there was no significant difference in the germination rate of the four varieties (lines) after 4 days, and the germination rate was above 90.00%, and the radicle-free rate was 0.00%, indicating that the seeds were of good quality and could grow and develop normally. The changes in germination rate after stress treatment can represent the effect of low temperature stress. When treated with 4℃ low temperature for 3d and 5d, there was no significant difference between Hengmian No. 3 and Zhong H177. Compared with these two materials, the germination rate of Yu 2067 decreased significantly, and that of Zhong JX02 decreased extremely significantly. The rate of radicle absence was inversely proportional to the germination rate, indicating that the low temperature treatment of 4℃ for 3d and 5d could screen out cold-intolerant and cold-sensitive materials. When treated with 4℃ low temperature for 7d and 10d, the germination rate and rate of radicle absence among the four materials were significantly and extremely significant. Therefore, the germination rate and rate of radicle absence at 4℃ for 7d to 10d and after 4d recovery can be used as indicators to identify cold resistance during the germination period, among which the low temperature treatment of 4℃ for 7d was the best. Because the germination rate of the four materials decreased rapidly under low temperature conditions, the germination rate of the four materials decreased by an average of more than 34.2% at 4℃ for 3 days. The influence of low temperature was greater than the rate of no radicle and also greater than the rate of radicle. The average rate of embryo of the four materials decreased by 21.3%. Therefore, when calculating the cold resistance index of the germination period, the proportion of germination rate was greater than the rate of radicle, accounting for 60.0%, while the rate of radicle accounted for 40.0%. The calculation formula is as follows: Cold resistance index of germination period (%) = germination rate (%) × 60.0% + rate of radicle (%) × 40.0%. Among the four upland cotton varieties (lines), JX02, Yu 2067, Zhong H177 and Hengmian No. 3 had cold resistance indexes of 13.7%, 43.2%, 59.5% and 75.5% respectively during the germination period. The degree of cold resistance of varieties (lines) during the germination period is ranked from high to low as follows: Hengmian 3 > Zhong H177 > Yu 2067 > Zhong JX02. According to the cold resistance level and evaluation criteria for upland cotton during the germination period that we developed, it can be seen that the cold resistance of Hengmian 3 during the germination period reaches level I, which is a cold-resistant material; the cold resistance of Zhong H177 during the germination period reaches level II, which is a cold-resistant material; the cold resistance of Yu 2067 reaches level III, which is a cold-intolerant type; the cold resistance of Zhong JX02 reaches level IV, which is a cold-sensitive type. The cold resistance results of Yu 2067 and Hengmian 3 during the germination period are consistent with the results of our previous study (Wang Junjuan et al., 2016), indicating that the processing methods and results of this experiment are accurate, and our experimental method is simple and intuitive, easy to operate, labor-saving, and cost-effective.

[0087] Table 2 Effects of low temperature stress on germination of upland cotton

[0088]

[0089]

[0090] Note: Different lowercase letters (a-b) indicate significant differences (P < 0.05), n = 3; different uppercase letters (A-B) indicate extremely significant differences (P < 0.01), n ​​= 3. Same below.

[0091] 2.2 Morphological changes of upland cotton after recovery from low temperature stress at the bud stage.

[0092] Three-day-old buds of three upland cotton varieties (lines), Zhong JX02, Zhong H177, and Hengmian 3, were subjected to a 5-day low-temperature treatment at 4°C. The buds then resumed growth at normal temperatures for 10-14 days. The emergence rates of the three accessions, or the cold resistance index at the bud stage, were measured to determine their cold resistance. The results showed that the emergence rates (i.e., the cold resistance index at the bud stage) of the three accessions were 19.1%, 67.2%, and 31.3%, respectively. The cold resistance of the three accessions at the bud stage differed significantly. Based on our criteria for evaluating cold resistance at the bud stage, Zhong JX02, Zhong H177, and Hengmian 3 were classified as cold-sensitive, cold-tolerant, and cold-intolerant, respectively. Zhong JX02 was classified as cold-sensitive at the bud stage.

[0093] When upland cotton was treated with 4℃ low temperature for 5 days at the bud stage and returned to normal temperature for 7 days, the low temperature-intolerant materials showed many symptoms of low temperature damage, including many deformed seedlings, such as Figure 3 As shown, taking Hengmian No. 3 as an example, these symptoms mainly include damage to the radicle and inability to grow and develop normally, the formation of inverted root seedlings, yellowing or further rot of cotyledons and leaves, yellowing to brown hypocotyls, continued poor development of the root system, poor development of both the hypocotyl and radicle, and inability of the cotyledons and leaves to unfold normally. Under the condition of suitable light, temperature and water indoors, so many types of deformed seedlings still appear after the cotton buds recover from low temperature stress. Under natural conditions in the field, the types of deformed seedlings can only be more. These deformed seedlings are the main reason for the low emergence rate, missing seedlings and broken bunches in the field.

[0094] 2.3 Identification of cold resistance of upland cotton at the seedling stage

[0095] 2.3.1 Cold resistance assessment at the cotyledon stage

[0096] Among the three upland cotton varieties (lines), JX02, Zhong H177 and Hengmian 3, when treated with 4°C low temperature for 3d, 5d and 7d under light and no light conditions respectively at the cotyledon stage, the cotyledons drooped visually and felt soft to the touch, with no other obvious symptoms. After 10 to 14 days of recovery, the survival rate of the seedlings after 5d and 7d of 4°C low temperature treatment under no light and light conditions was close to 0.00%, indicating that upland cotton cannot tolerate the stress of 4°C low temperature for 5d or more at the cotyledon stage. When treated with 4℃ low temperature for 3 days under no light conditions and then resumed growth for 10-14 days, all three varieties (lines) grew true leaves, and the survival rate was 100.00%, with no significant difference among the three materials; when treated with 4℃ low temperature for 3 days under light conditions and then resumed growth for 10-14 days, the survival rates of Zhong JX02, Zhong H177 and Hengmian No. 3 were 71.43%, 50.02% and 27.58% respectively, and the differences in survival rate among the materials were significant or extremely significant. According to the cold resistance evaluation criteria for the cotyledon stage established by us, the cold resistance of Zhong JX02, Zhong H177 and Hengmian No. 3 reached cold resistance, cold tolerance and cold intolerance, respectively. The cold resistance at the cotyledon stage was from strong to weak, namely Zhong JX02, Zhong H177 and Hengmian No. 3. Therefore, the survival rate of seedlings treated at 4℃ for 3 days under light conditions and then recovering at 28-30℃ for 10-14 days (the survival rate is defined as the growth of true leaves) can be used as an indicator of cold resistance identification at the cotyledon stage ( Figure 4 ).

[0097] 2.3.2 Identification of cold resistance at the three-leaf stage

[0098] Figure 5 The phenotypes of JX02, a three-leaf stage plant in Upland cotton, were observed after exposure to 4°C for different days in the presence of sunlight and then 10 to 14 days of normal growth recovery. The figure shows that after one day of 4°C low temperature stress at the three-leaf stage, both the cotyledons and some true leaves of JX02 were damaged, manifesting as dehydration and wilting. After two days of stress, the true leaves suffered greater damage than the cotyledons, indicating that the cotyledons have some repair function after two days of continuous 4°C low temperature stress. After four days of 4°C low temperature stress, the survival rate of JX02 plants rapidly declined, with some cotyledons still having some vitality. The true leaves withered and died, and the epicotyls began to shrink and break. After five and seven days of 4°C low temperature stress, all leaves of JX02 plants dehydrated, wilted, and the survival rate approached 0.00%. Therefore, three to four days of 4°C low temperature stress in the presence of sunlight marks the turning point for damage at the three-leaf stage. During the entire process of 1 to 7 days of low temperature stress at 4°C, most true leaves suffered greater damage than cotyledons; except for turning black, the hypocotyls remained straight, while the epicotyls gradually softened and bent until they dehydrated and dried up as the number of days of low temperature treatment increased, indicating that the epicotyls were more sensitive to low temperatures of 4°C than the hypocotyls. It also shows that with the increase in the intensity of low temperature stress, the damage to the upland cotton organs not only damaged the leaves but also the stems.

[0099] Three varieties (lines) of upland cotton, namely JX02, Zhong H177 and Hengmian 3, were treated with 4°C low temperature for 3, 5 and 7 days in the absence of light and in the presence of light, respectively. After the three-leaf stage was treated with 4°C low temperature for 5 and 7 days and then resumed growth at normal temperature, the survival rate of JX02, Zhong H177 and Hengmian 3 was basically 0.00%. After 3 days of treatment, the survival rates of JX02, Zhong H177 and Hengmian 3 under the absence of light were 78.57%, 80.92% and 77.79%, respectively. There was no significant difference in survival rate among varieties, but their growth was significantly inhibited. The true leaves and cotyledons of the three varieties all showed varying degrees of damage symptoms. Under the presence of light, the survival rates were 71.4%, 27.6% and 50.9%, respectively. The difference in survival rate among the three materials was extremely significant ( Figure 6 ). According to our criteria for evaluating cold resistance at the three-leaf stage, Zhong JX02, Zhong H177, and Hengmian No. 3 achieved cold resistance, cold intolerant, and cold tolerant, respectively. The order of cold resistance among the three materials at the three-leaf stage is Zhong JX02, Hengmian No. 3, and Zhong H177, from strongest to weakest. Low temperature stress in the presence of light is more damaging to three-leaf cotton seedlings than low temperature stress in the absence of light. Therefore, the survival rate of seedlings treated with a 4°C low temperature for 3 days in a light-treated box followed by 10-14 days of growth at normal temperature (survival criteria based on the green growth point) can be used as an indicator for evaluating cold resistance in upland cotton at the three-leaf stage.

[0100] Depend on Figure 4 and Figure 6 Comparing the cotyledon stage with the trifoliate stage under no-light conditions reveals that upland cotton has greater cold resistance at the cotyledon stage than at the trifoliate stage. At the trifoliate stage, true leaves are more sensitive to low-temperature stress and more vulnerable to damage than cotyledons. Under light conditions, the cold resistance of different upland cotton varieties at the cotyledon stage and at the trifoliate stage varies, while others differ. For example, Zhong JX02 exhibits consistent cold resistance at both the cotyledon and trifoliate stages, reaching the cold-resistant level. Zhong H177, on the other hand, is cold-tolerant at the cotyledon stage but not at the trifoliate stage. Naihengmian 3 is cold-tolerant at the cotyledon stage but not at the trifoliate stage. This demonstrates the complexity and diversity of cold resistance in upland cotton during the seedling stage. After low temperature stress at the three-leaf stage, the cotyledons of Hengmian No. 3 were the most severely damaged among the three varieties, which is consistent with its sensitivity to low temperature at the cotyledon stage. After low temperature stress at the three-leaf stage, the epicotyl and true leaves of Zhong H177 almost dried up, while its cotyledons were the least damaged. The difference in sensitivity to low temperature between its true leaves and cotyledons is the greatest, which can be used to in-depth study the mechanism of different responses of different organs of cotton to low temperature.

[0101] 2.3.3 Comparison of anthocyanin content in leaves of different varieties (lines) at the cotyledon and trifoliate stages Figure 7It can be seen that the average anthocyanin contents in the cotyledons of the three upland cotton varieties (lines) during the cotyledon stage were 35.02U / g, 26.11U / g, and 18.03U / g, respectively. The differences in anthocyanin content in the cotyledons of the three varieties were extremely significant, and were proportional to their cold resistance during the cotyledon stage. Only when the anthocyanin content in the leaves reaches 25.00U / g or above can cotton reach the cold resistance level or above.

[0102] Depend on Figure 8 It can be seen that the average anthocyanin contents in the top three leaves of the three upland cotton varieties (lines) during the three-leaf stage were 38.35U / g, 21.11U / g, and 32.52U / g, respectively. The differences in anthocyanin contents in the top three leaves of the three varieties were extremely significant, which was proportional to their cold resistance during the three-leaf stage.

[0103] Therefore, the anthocyanin content in upland cotton leaves can be used as a biochemical indicator for screening and identifying its cold resistance in the seedling stage. Because it is visible to the naked eye in the leaves, it can also be used as an indicative indicator for selecting the cold resistance of upland cotton.

[0104] 3 Results and Discussion

[0105] 3.1 Effects of low temperature stress on cotton during germination

[0106] The main effect of low temperature stress at 4℃ under no light conditions on the germination of upland cotton is to reduce the germination rate, reduce the length of the buds, and inhibit the elongation of the hypocotyl and radicle. The inhibition on the radicle is significantly greater than that on the hypocotyl. This is different from the drought stress treatment. The inhibition of drought stress on the radicle of upland cotton is less than that on the hypocotyl. With the extension of 4℃ low temperature treatment time, the germination rate of upland cotton continued to decline, and the radicle-free rate increased, and the radicle-free rate was inversely proportional to the germination rate; when treated with 4℃ low temperature for 7-10 days, the germination rate and radicle-free rate among the four materials were significantly different and extremely significant, so the germination rate and radicle-free rate at 4℃ for 7-10 days and 4 days after recovery can be used as indicators for identifying cold resistance during the germination period. 4℃ for 7 days is the best treatment time. Using this indicator, we identified that Hengmian No. 3 reached level 1 cold resistance during the germination period, Zhong H177 reached level 2 cold resistance during the germination period, Yu 2067 was not cold-resistant, and Zhong JX02 was cold-sensitive. Among them, the cold resistance results of Yu 2067 and Hengmian No. 3 during the germination period were consistent with the results of Wang Junjuan et al.'s previous study using the cotyledon flattening rate as the germination period identification index. To (Wang Junjuan et al., 2016), it shows that our identification method is reliable. At the same time, our identification indicators are simpler, more intuitive, more convenient and economical, saving labor and effort, and more conducive to researchers' investigations. It not only saves the trouble of replanting and reduces the errors caused by the investigations between researchers, but also facilitates researchers to further sample and plant and propagate seeds, which can effectively preserve rare cotton germplasm resources and is more conducive to in-depth exploration of the deep reasons for the impact of low temperature on germination. For example, seeds with normal radicles and seeds without radicles can be further sampled to study the differences in their physiological and biochemical indicators and gene expression. They can also continue to be planted to study their respective phenotypes, or further cultivate and screen cold-resistant offspring during the germination period.

[0107] 3.2 Differences in the effects of low temperature stress on cotton under conditions of no light and light during the seedling stage

[0108] The effects of 4°C low-temperature stress on upland cotton at the cotyledon and trifoliate stages differed significantly between those under no-light conditions and those under light conditions. Low-temperature stress under light conditions resulted in greater inhibition and damage than under no-light conditions. The survival rate of seedlings (defined as true leaf development at the cotyledon stage and green growth points at the trifoliate stage) following 3 days of 4°C low-temperature stress under light conditions followed by 10–14 days of normal growth can be used as an indicator for cold tolerance at the cotyledon and trifoliate stages. Cold tolerance assessment at the cotyledon stage indicated that Hengmian 3 was not cold-tolerant at the cotyledon stage. This is consistent with the findings of Wang Junjuan et al. (2016) who used a cold tolerance index as the cotyledon stage identification method. This demonstrates that our identification method is feasible, and the criteria are simple, clear, and easy to assess, effectively reducing inter-investigational error. When Hengmian 3 was subjected to 4°C low temperature stress for three days at the three-leaf stage in the presence of light, its cotyledons suffered the most severe damage of the three varieties (lines), consistent with its sensitivity to low temperatures during the cotyledonary stage. Chloroplasts in leaves are the second temperature sensor in plants, besides the cell membrane. Under low temperature stress, the metabolic activity of various enzymes in plant leaves is inhibited to varying degrees, while the chloroplast light-harvesting system is unaffected by low temperature. This imbalance in energy transfer directly causes chloroplast damage and inhibition of the photosynthetic system, leading to leaf wilt and death during low temperature stress in the presence of light. This may be the fundamental reason why 4°C low temperature stress in the presence of light causes greater damage and inhibition to upland cotton seedlings than the same low temperature treatment in the absence of light.

[0109] 3.3 Differences in low temperature resistance of upland cotton at different growth stages

[0110] Previous studies have shown that cold resistance in different varieties (lines) of upland cotton is not always consistent across the germination, bud, and cotyledon stages. This study further confirmed this and found that not only is cold resistance not always consistent across the germination, bud, and cotyledon stages, but also between the cotyledon and trifoliate stages within the same seedling stage. Zhong JX02 showed strong cold sensitivity in both the germination and bud stages, but was relatively cold-resistant in the cotyledon and three-leaf stages; Zhong H177 showed moderate cold tolerance in the germination, bud and cotyledon stages, but was cold-sensitive in the three-leaf stage; Hengmian No. 3 had strong cold resistance in the germination stage, was cold-sensitive in the bud and cotyledon stages, and its cold resistance increased in the three-leaf stage, indicating that the cold resistance of upland cotton has very complex characteristics and rich diversity, and we need different research methods to meet different research needs; in addition, the study on the cold resistance in the three-leaf stage found that it was inconsistent with the cold resistance in the cotyledon stage, which is a new discovery of our experiment, indicating that the cold resistance of any variety in any period cannot be inferred from the cold resistance results of another period; another finding is that the difference between the insensitivity of the cotyledons of Zhong H177 to low temperature stress and the strong sensitivity of the true leaves to low temperature stress in the three-leaf stage is also worthy of our in-depth study of its mechanism. During the germination period, low temperature stress reduced pigment synthesis in the hypocotyls of the JX02 cultivar. As the degree of low temperature stress intensified, pigment synthesis became more significantly inhibited. This property allowed us to further investigate phenotypic characteristics after low temperature stress and to identify biochemical markers of low temperature stress. Upland cotton JX02 was sensitive to temperatures below 4°C during both the germination and bud stages, but showed significantly increased cold tolerance during the cotyledon and trifoliate stages. Studies have shown that purple-leafed millet exhibits greater cold tolerance due to the presence of more anthocyanins in its purple leaves, which are synthesized in greater quantities after low temperature stress. Petunia hybrida may also improve its cold tolerance by accumulating anthocyanins and osmotic regulators, thereby resisting low temperature stress. Further studies have shown that the purple-red coloration of the cotyledons and true leaves of JX02, along with its significantly higher anthocyanin content than the other two cultivars (lines), is primarily responsible for its low temperature tolerance during the cotyledon and seedling stages. The anthocyanin content of upland cotton leaves can be used as a biochemical and indicative marker for screening and identifying seedling cold tolerance.

[0111] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0112] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying cold resistance of upland cotton at different growth stages, characterized in that: Identification of cold resistance of upland cotton at the germination, bud, cotyledon and trifoliate stages: (1) Identification of cold resistance during germination: Seeds were treated at 4°C for 7–10 days and then allowed to resume growth for 4 days; (2) Identification of cold resistance at the bud stage: buds were treated at 4°C for 3–5 days and then allowed to resume growth for 10–14 days; (3) Identification of cold resistance at the cotyledon stage: The cotyledons were treated under light and 4°C conditions for 3–5 days, and then resumed growth for 10–14 days; (4) Identification of cold resistance at the three-leaf stage: When upland cotton reaches the three-leaf stage, it is treated under light and 4°C conditions for 3 to 5 days, and then resumes growth for 10 to 14 days; The cold resistance evaluation during the germination period was performed by counting the germination rate, the rate without radicle and the rate with radicle. The calculation formula is as follows: Germination rate (%) = number of germinated seeds / total number of seeds × 100; Radicle-free rate (%) = number of buds without radicles / number of sprouted buds × 100; Radicle rate (%) = 100 - no radicle rate (%); cold resistance identification at the bud stage is based on the emergence rate, with emergence based on the growth of true leaves. The calculation formula is as follows: Seedling emergence rate (%) = number of emerged seedlings / total number of buds × 100; both the cotyledon stage cold resistance assessment and the three-leaf stage cold resistance assessment were calculated using the following formula: Survival rate (%) = number of surviving seedlings (trees) / total number of seedlings (trees) × 100; The evaluation indicators are as follows: Evaluation indicators during germination period: Cold resistance index during germination period (%) = germination rate (%) × 60% + radicle rate (%) × 40%; When the cold resistance index during the germination period is ≥70%, it is Class I cold resistance and the cold resistance type is cold resistance; When the cold resistance index during germination is 69.9% or more and ≥50% or more, it is cold resistance level II and the cold resistance type is cold tolerant; When 49.9% ≥ the cold resistance index during germination ≥ 20%, it is cold resistance level III and the cold resistance type is cold intolerance; When the cold resistance index during germination is <20%, it is grade IV cold resistance and the cold resistance type is cold sensitive; The germination rate was used as the evaluation index during the budding period; The survival rate was used as the evaluation index at the cotyledon and three-leaf stages; When the emergence rate / survival rate is ≥70%, it is Class I cold resistance and the cold resistance type is cold resistance; When the emergence rate is 69.9% or higher or the survival rate is 50%, it is classified as Class II cold resistance and the cold resistance type is cold-tolerant; When the emergence rate is 49.9% or higher and the survival rate is 20%, it is classified as Class III cold resistance and the cold resistance type is cold-intolerant; When the emergence rate / survival rate is <20%, it is level IV cold resistance and the cold resistance type is cold sensitive.

2. The method for identifying cold resistance of upland cotton at different growth stages according to claim 1, wherein: For cold resistance assessment during the germination period, seeds were treated at 4°C for 7 days and then resumed growth for 4 days.

3. The method for identifying cold resistance of upland cotton at different growth stages according to claim 1, wherein: The condition for the recovery growth is culturing at 28-30°C.

4. The method for identifying cold resistance of upland cotton at different growth stages according to claim 1, wherein: The light conditions for cold resistance identification at the cotyledon stage and the three-leaf stage are 1250LAX. The plants grow normally before low temperature treatment, with a temperature of 28-30°C and a light / dark time of 14h / 10h.

5. The method for identifying cold resistance of upland cotton at different growth stages according to claim 1, wherein: During the cotyledon stage, the survival rate of seedlings is calculated based on the growth of new true leaves.

6. The method for identifying cold resistance of upland cotton at different growth stages according to claim 1, wherein: At the three-leaf stage, the survival rate of seedlings is calculated based on whether the growing point is still green.

7. A method for identifying cold resistance of upland cotton at different growth stages according to any one of claims 1 to 6, characterized in that: The upland cotton varieties are Zhong JX02, Zhong H177, Hengmian No. 3 or Yu 2067.

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