CN112544371B - Method for identifying low temperature resistance of maize inbred line in germination period - Google Patents
Method for identifying low temperature resistance of maize inbred line in germination period Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a method for identifying low-temperature resistance characteristics of a maize inbred line in a germination period, and belongs to the technical field of crop identification methods. The method for identifying the low temperature resistance of the maize inbred line in the germination period comprises the following steps: sowing the selfing line corn seeds; treating under low-temperature stress conditions; counting the germination rate, and identifying the low-temperature resistant characteristic of the maize inbred line in the germination period; the low temperature stress condition includes a light low temperature stress condition and a dark low temperature stress condition. The method provided by the invention is simple to operate, field remote repeated identification is not needed, conditions are easy to control, the screened low-temperature-resistant maize inbred line in the germination period is consistent with the field planting result, the identification result is accurate, and the method is suitable for popularization.
Description
Technical Field
The invention belongs to the technical field of crop identification methods, and particularly relates to a method for identifying low-temperature resistance characteristics of a maize inbred line in a germination period.
Background
Low temperature chilling injury is a key limiting factor affecting corn seed germination and yield. In the production practice of corn in northeast China, barrier type cold damage, particularly low-temperature cold damage in the seed germination period, often causes great yield reduction of corn, and seriously threatens the safe production of corn in China.
The cold resistance characteristic of the maize in the germination period is researched, a set of simple and efficient maize inbred line germination period identification method is established, low-temperature-resistant maize germplasm resources suitable for precision sowing are excavated and screened, the cultivation of low-temperature-resistant maize varieties to resist the adverse effect caused by low-temperature cold damage is an effective way to ensure the safe production economy of the maize, and meanwhile, the method has important reference significance for mastering the influence and change rule of temperature on the vitality of the seeds, reducing the storage cost of the seeds and prolonging the storage life.
The low temperature resistance evaluation is an important method for screening low temperature resistance germplasm of corn and related genetic research, and comprises two evaluation systems of field identification and indoor identification. In general, field identification utilizes visual morphological indexes to evaluate materials, such as: the low temperature resistance of the material is evaluated according to the characters of emergence rate, seedling growth speed, plant morphology and the like. The indoor identification is mainly to evaluate the low temperature resistance of the corn by low temperature stress by means of temperature control systems such as a phytotron, and the evaluation indexes comprise: germination rate, germination vigor, germination index, root weight, root length, relative root length, leaf area, etc. However, the low temperature resistance identification cost in the field is high, the identification needs to be repeatedly carried out in different places, the influence of the change of climatic conditions among years is easily caused, and the result is not accurate enough; according to the indoor identification method, the difference between the processing temperature, the processing conditions and the like and the actual field situation is large, the difference between the indoor identification method and the indoor identification method is obvious when the indoor identification method is used for planting the materials in the field and the low temperature resistant corn materials in the germination period is low, and the low temperature resistant corn materials in the germination period cannot be screened really. Therefore, a method for identifying the low-temperature-resistant corn material in the germination period, which has accurate identification result and simple operation, is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides a method for identifying the low temperature resistance of the maize inbred line in the germination period, which is simple to operate and suitable for popularization, and has accurate identification result which is consistent with the field planting result.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for identifying the low temperature resistance of a maize inbred line in the germination period, which comprises the following steps: sowing the selfing line corn seeds; treating under low-temperature stress conditions; counting the germination rate, and identifying the low-temperature resistant characteristic of the maize inbred line in the germination period;
the low-temperature stress condition comprises a light low-temperature stress condition and a dark low-temperature stress condition; the illumination low-temperature stress condition is that the illumination intensity is 18000-20000 Lux, the illumination time is 14-16 h, the temperature is 11-13 ℃, and the humidity is 70-80%; the dark low-temperature stress condition is that the dark time is 8-10 h, the temperature is 6-8 ℃, and the humidity is 70-80%; the illumination low-temperature stress condition and the dark low-temperature stress condition are alternately carried out; the processing time of the low-temperature stress is 18-21 d;
the maize inbred line with the germination rate of [ 85%, 100% ] is a low-tolerance inbred line; the maize inbred line with the germination rate of (50 percent, 85 percent) is an intermediate inbred line; the maize inbred line with the germination rate of 0 percent and 50 percent is a non-low temperature resistant inbred line.
Preferably, the inbred corn grain is a full grain.
Preferably, the inbred corn kernels are harvested and aired until the water content of the kernels is below 17%.
Preferably, the selfing line corn kernel is pretreated before seeding, and the pretreatment steps are as follows: and soaking the selfing line corn kernels in a sodium hypochlorite solution with the mass volume fraction of 3% -5% for 20-30 min, cleaning and airing.
Preferably, the matrix for seeding the selfing line corn kernels comprises vermiculite, nutrient soil and water, and the mass ratio of the vermiculite to the nutrient soil to the water is (0.5-1): 0.5.
Preferably, the mass ratio of the number of the sown inbred line corn grains to the matrix is 20-30: 200 g.
Preferably, the germination rate is a ratio of the number of seeds that germinate after low temperature stress to the number of sown seeds.
Preferably, the maize inbred line comprises a northern maize inbred line.
Preferably, the northern corn inbred line comprises a northeast corn inbred line and an inbred line in Huang-Huai-Hai region.
Has the advantages that:
the invention provides a method for identifying the low temperature resistance of a maize inbred line in the germination period, which comprises the following steps: sowing the selfing line corn seeds; treating under low-temperature stress conditions; and (4) counting the germination rate, and identifying the low-temperature resistance characteristic of the maize inbred line in the germination period. According to the method, the corn inbred line is treated by adopting a reasonable temperature-changing stress condition, the stress on corn seeds is severer, the low-temperature-resistant corn inbred line and the intermediate-type corn inbred line can be well distinguished, the screened low-temperature-resistant corn inbred line in the germination period is consistent with the field planting result, the identification result is more accurate, the method is simple to operate, the field remote repeated identification is not needed, the condition is easy to control, and the method is very suitable for popularization.
Detailed Description
The invention provides a method for identifying the low temperature resistance of a maize inbred line in the germination period, which comprises the following steps: sowing the selfing line corn seeds; treating under low-temperature stress conditions; counting the germination rate, and identifying the low-temperature resistant characteristic of the maize inbred line in the germination period;
the low-temperature stress condition comprises a light low-temperature stress condition and a dark low-temperature stress condition; the illumination low-temperature stress condition is that the illumination intensity is 18000-20000 Lux, the illumination time is 14-16 h, the temperature is 11-13 ℃, and the humidity is 70-80%; the dark low-temperature stress condition is that the dark time is 8-10 h, the temperature is 6-8 ℃, and the humidity is 70-80%; the illumination low-temperature stress condition and the dark low-temperature stress condition are alternately carried out; the processing time of the low-temperature stress is 18-21 d;
the maize inbred line with the germination rate of [ 85%, 100% ] is a low-tolerance inbred line; the maize inbred line with the germination rate of (50 percent, 85 percent) is an intermediate inbred line; the maize inbred line with the germination rate of 0 percent and 50 percent is a non-low temperature resistant inbred line.
The method for identifying the low temperature resistance of the maize inbred line in the germination period is simple to operate, field remote repeated identification is not needed, conditions are easy to control, the screened low temperature resistance maize inbred line in the germination period is consistent with field planting results, identification results are accurate, and the method is suitable for popularization.
In the invention, the inbred line corn kernel is preferably full kernel, and is further preferably full kernel in the current year, so that the difference of identification results caused by natural aging can be avoided, and the identification accuracy is improved. After the inbred line corn kernel is harvested, the inbred line corn kernel is preferably aired until the water content of the kernel is less than 17%, more preferably 13-15%, and even more preferably 14%, so that the activity of the seed can be improved, and the seed is stable and is easier to store. In the invention, the selfing line corn kernel is preferably pretreated before seeding, and the pretreatment steps are preferably as follows: soaking the inbred line corn kernels in a sodium hypochlorite solution with the mass volume fraction of 3% -5% for 20-30 min, cleaning and airing; the concentration of the sodium hypochlorite solution is further preferably 4%, and the soaking time is further preferably 25 min; the corn inbred line grains are pretreated to sterilize bacteria and viruses on the surfaces of the grains and prevent the grains from mildewing and losing vigor in the low-temperature stress treatment process to influence the identification result. In the invention, the matrix for sowing the inbred line corn kernels preferably consists of vermiculite, nutrient soil and water, the matrix is preferably sterilized before use, the sterilization mode is preferably sterilization in an autoclave, and the matrix is sterilized to prevent bacteria breeding in the matrix from polluting seeds and causing the seeds to mildew and lose vigor. In the invention, the mass ratio of the vermiculite, the nutrient soil and the water is preferably (0.5-1): 0.5, more preferably (0.6-0.9): 0.5, more preferably 0.8: 0.8: 0.5. the matrix has good moisture retention and air permeability, and provides necessary conditions for seed germination. In the invention, the mass ratio of the number of the sown inbred line corn grains to the matrix is preferably 20-30 grains: 200g, more preferably 30: 200g, reasonable seeding density is guaranteed by setting the mass ratio, and guarantee can be provided for subsequent experiments.
After the inbred line corn kernels are sown, the low-temperature stress condition treatment is carried out on the sown inbred line corn kernels. In the present invention, the low temperature stress condition includes a light low temperature stress condition and a dark low temperature stress condition; the illumination low-temperature stress condition is that the illumination intensity is 18000-20000 Lux, the illumination time is 14-16 h, the temperature is 11-13 ℃, and the humidity is 70-80%, and the illumination intensity is 20000Lux, the illumination time is 16h, the temperature is 12 ℃, and the humidity is 75%; the dark low-temperature stress condition is that the dark time is 8-10 hours, the temperature is 6-8 ℃, the humidity is 70-80%, and further the dark time is 8 hours, the temperature is 7 ℃, and the humidity is 75%. In the present invention, the light low-temperature stress condition and the dark low-temperature stress condition are alternately performed. The invention has no special requirements on the sequence of the low-temperature stress of illumination and the low-temperature stress of darkness. The treatment time of the low-temperature stress is 18-21 d, and the treatment time is preferably 21 d. The low-temperature stress treatment is preferably carried out in an intelligent low-temperature illumination incubator, so that the low-temperature stress condition can be accurately controlled, and the identification result is more accurate and stable. The low-temperature stress condition of the invention enables the temperature and the illumination to change periodically, is harsher compared with the constant-temperature low-temperature stress condition, and can well distinguish a low-temperature resistant corn inbred line and an intermediate corn inbred line; in addition, the moisture in the matrix is ensured to be sufficient through controlling the humidity, sufficient moisture is provided for seed germination, manual water supplement is not needed in the low-temperature stress treatment process, the workload is reduced, meanwhile, the influence of water supplement on the low-temperature stress condition is avoided, the anti-interference capability is stronger, and the identification result is more accurate.
After low-temperature stress condition treatment, the method provided by the invention counts the germination rate and identifies the low-temperature resistance characteristic of the maize inbred line in the germination period. In the present invention, the germination rate is the ratio of the number of seeds that germinate after low temperature stress to the number of sown seeds. In the invention, the maize inbred line with the germination rate of [ 85%, 100% ] is a low-tolerance inbred line; the maize inbred line with the germination rate of (50 percent, 85 percent) is an intermediate inbred line; the maize inbred line with the germination rate of 0 percent and 50 percent is a non-low temperature resistant inbred line. The invention has no special requirement on the type of the maize inbred line to be identified, and the maize inbred lines which are commonly sold in the field can be identified. The method is particularly suitable for identifying the northern corn inbred line, and the northern corn inbred line further preferably comprises a northeast corn inbred line and a Huang-Huai-Hai region inbred line.
To further illustrate the present invention, the following examples are provided to describe the method for identifying the low temperature resistance of the inbred line of northeast maize in germination stage in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
A method for identifying low temperature resistance of a maize inbred line in a germination period comprises the following steps:
1) taking full inbred line corn seeds harvested in autumn in the same year, airing the inbred line corn seeds until the water content of the seeds is below 14%, soaking the inbred line corn seeds in a 4% sodium hypochlorite solution for 25min, then washing the inbred line corn seeds with distilled water for three times, naturally airing the inbred line corn seeds, and sealing the inbred line corn seeds with a10 x 15cm gauze bag for marking for later use.
2) Packing and sealing vermiculite and nutrient soil with a fresh-keeping bag, putting the packed vermiculite and nutrient soil into an autoclave for sterilization, then putting the packed vermiculite and nutrient soil into a 40 ℃ oven for drying, and then putting the packed vermiculite and nutrient soil into room temperature for later use. Respectively taking vermiculite (2mm), nutrient soil and distilled water according to a mass ratio of 1: 1: mixing and stirring at the ratio of 0.5. And (3) putting 200g of uniformly stirred matrix into a culture dish, uniformly sowing 30 selfing line corn grains in the culture dish, and then covering the culture dish with the matrix.
3) And placing the sown inbred line corn grains into an intelligent low-temperature illumination incubator for cultivation so as to screen the inbred line corn grains under the low-temperature stress condition. Before the intelligent low-temperature illumination incubator (GXZ430D, Ningbo southeast instruments Co., Ltd.) is started, an ultraviolet sterilizing lamp is used for sterilizing for 10 hours, and then 75% alcohol is used for secondary sterilization, so that cleanness, tidiness and sterility in the incubator are guaranteed. Setting the low-temperature stress condition of the intelligent low-temperature illumination incubator as 20000Lux illumination intensity, 16h illumination, 12 ℃ temperature, 75% humidity, 8h darkness, 7 ℃ temperature and 75% humidity, quickly putting the seeded culture into a box body after the low-temperature stress condition meets the requirement, and carrying out low-temperature treatment for 21 d.
4) And taking out the culture dish from the incubator after low-temperature treatment, counting the germination rate, and performing germination period low-temperature resistance characteristic identification on the maize inbred line. The calculation formula of the germination rate is as follows: the germination rate is G/Nx 100%. G represents the number of seeds germinated after low temperature stress, and N represents the total amount of seeds per treatment.
The maize inbred line with the germination rate of 85 percent and 100 percent is a low-tolerance inbred line; the maize inbred line with the germination rate of (50 percent, 85 percent) is an intermediate inbred line; the maize inbred line with the germination rate of 0 percent and 50 percent is a non-low temperature resistant inbred line.
Example 2
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as that in the example 1, except that the low temperature treatment time is 18 d.
Example 3
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and is different in that the low temperature stress condition of the intelligent low temperature illumination incubator is 20000Lux in illumination intensity, 16h in illumination, 13 ℃ in temperature and 75% in humidity, 8h in darkness, 7 ℃ in temperature and 75% in humidity.
Example 4
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and is different in that the low temperature stress conditions of the intelligent low temperature illumination incubator are 19000Lu' x in illumination intensity, 16h in illumination, 12 ℃ in temperature, 80% in humidity, 8h in darkness, 6 ℃ in temperature and 80% in humidity.
Example 5
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and the difference is that the low temperature stress condition of the intelligent low temperature illumination incubator is illumination intensity 18000Lux, illumination for 16h, temperature 11 ℃, humidity 70%, darkness for 8h, temperature 8 ℃ and humidity 70%.
Comparative example 1
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as that in the example 1, except that the low temperature treatment time is 3 d.
Comparative example 2
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as that in the example 1, except that the low temperature treatment time is 7 d.
Comparative example 3
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as that in the example 1, except that the time for low-temperature treatment is 12 days.
Comparative example 4
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and the difference is that the low temperature stress condition of the intelligent low temperature illumination incubator is illumination culture, the illumination intensity is 16000Lux, the temperature is 14 ℃, the humidity is 95%, and the low temperature treatment is carried out for 21 d.
Comparative example 5
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and the difference is that the low temperature stress condition of the intelligent low temperature illumination incubator is dark culture, the temperature is 10 ℃, the humidity is 85 percent, and the low temperature treatment is carried out for 21 d.
Comparative example 6
The method for identifying the low temperature resistance of the maize inbred line in the germination period is the same as the method in the embodiment 1, and is different in that the low temperature stress conditions of the intelligent low temperature illumination incubator are illumination intensity of 15000Lux, illumination of 16h, temperature of 10 ℃, humidity of 90%, darkness of 8h, temperature of 4 ℃ and humidity of 90%.
Application example 1
A low-temperature-resistant inbred line JiA 1035, a sensitive inbred line 12-156 and an intermediate inbred line JiA 65 are used as test materials, the three materials are purchased from corn research institute of agricultural academy of Jilin province, 30 seeds are taken from each material, low-temperature stress condition tests are carried out according to the methods of examples 1-5 and comparative examples 1-6, the germination rates of treatment groups are counted, the identification performances of different identification methods are investigated, and the detection results are shown in table 1.
TABLE 1 identification results of different identification methods
As can be seen from the results in table 1, the methods for identifying the low temperature resistance characteristics of the maize inbred lines in the germination period in examples 1 to 5 can well distinguish the low temperature resistance maize inbred line from the sensitive inbred line, and can also clearly distinguish the low temperature resistance inbred line from the intermediate inbred line; in comparative examples 1 to 3, since the stress time was too short, the low temperature resistant inbred line, the intermediate inbred line and the sensitive inbred line could not be distinguished; the results of comparative examples 4 to 6 were able to distinguish between the low temperature resistant inbred line and the sensitive inbred line, but the intermediate inbred line and the low temperature resistant inbred line could not be distinguished significantly under the stress condition.
Application example 2
In order to further verify the accuracy of the method for identifying the low temperature resistance characteristic of the maize inbred line in the germination period, 60 parts of maize inbred lines (purchased from corn research institute of agriculture academy of Jilin province) are identified by using the method of example 1, and 30 seeds are taken from each part of material for identification; in addition, field tests were conducted to investigate the consistency of the method of the invention with field tests.
The field test scheme is as follows: seeding is carried out on the test field of the princess ridge for 3 consecutive years in 4 months and 15 days (half a month earlier than conventional seeding and lower outdoor temperature) in 2018 to 2020, the planting density is 8 ten thousand plants/hectare, the plant spacing is 10cm, each material comprises 100 seeds, 50 seeds/bag, 2 bags, 2 dibbling, 1 bag and 1 row, and 2 rows. The field management refers to national regional test standards. And investigating the germination rate at 20-30 days in 5 months, identifying the maize inbred line, taking the result of at least two times of consistent identification results as a final identification result, and referring to the specification of national standard GB4401.1-1996 by the identification standard, wherein the detection result is shown in Table 2.
TABLE 260 identification results of 3-year field of maize inbred line and germination period low temperature resistance characteristics of example 1
As can be seen from Table 2, the result of the low temperature resistant maize inbred line identified by the method of the embodiment 1 of the invention is consistent with the field identification result, and the identification accuracy is good.
According to the results of the embodiment and the application example, the method for identifying the low temperature resistance of the maize inbred line in the germination period is simple to operate, field remote repeated identification is not needed, conditions are easy to control, the screened low temperature resistance maize inbred line in the germination period is consistent with the field planting result, the identification result is accurate, and the method is suitable for popularization.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (8)
1. A method for identifying the low temperature resistance of a maize inbred line in the germination period is characterized by comprising the following steps: sowing the selfing line corn seeds; treating under low-temperature stress conditions; counting the germination rate, and identifying the low-temperature resistant characteristic of the maize inbred line in the germination period;
the low-temperature stress condition is a light low-temperature stress condition and a dark low-temperature stress condition; the illumination low-temperature stress condition is that the illumination intensity is 18000-20000 Lux, the illumination time is 14-16 h, the temperature is 11-13 ℃, and the humidity is 70-80%; the dark low-temperature stress condition is that the dark time is 8-10 h, the temperature is 6-8 ℃, and the humidity is 70-80%; the illumination low-temperature stress condition and the dark low-temperature stress condition are alternately carried out; the processing time of the low-temperature stress is 18-21 d;
the maize inbred line with the germination rate of [ 85%, 100% ] is a low-temperature resistant inbred line; the maize inbred line with the germination rate of (50 percent, 85 percent) is an intermediate inbred line; the maize inbred line with the germination rate of 0 percent and 50 percent is a non-low temperature resistant inbred line;
the germination rate is the ratio of the number of germinated seeds to the number of sown seeds after low-temperature stress.
2. The method of claim 1, wherein the inbred corn grain is a filled grain.
3. The method of claim 1, wherein the inbred corn kernel is air-cured after harvest to a kernel moisture content of less than 17%.
4. The method of claim 1, wherein the inbred corn grain is pretreated prior to seeding, the pretreatment comprising the steps of: and soaking the selfing line corn kernels in a sodium hypochlorite solution with the mass volume fraction of 3% -5% for 20-30 min, cleaning and airing.
5. The method of claim 1, wherein the matrix for seeding the inbred corn kernels comprises vermiculite, nutrient soil and water, and the mass ratio of the vermiculite to the nutrient soil to the water is (0.5-1): 0.5.
6. The method of claim 5, wherein the mass ratio of the number of sown inbred corn kernels to the matrix is 20-30: 200 g.
7. The method of claim 1, wherein said maize inbred line comprises a northern maize inbred line.
8. The method of claim 7, wherein the northern corn inbreds comprise a northeast corn inbred and a Huang-Huai sea area inbred.
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