Method suitable for temperature treatment and germ control in corn germination period under heat stress

Abstract

The invention discloses a method suitable for temperature treatment and germ control in a corn germination period under heat stress. The method comprises the following steps: screening the most suitable temperature of the heat stress in the germination period; disinfecting the seeds before sowing; and pathogen prevention and control measures are taken in the germination process. According to the method, the appropriate temperature for corn heat stress treatment is explored, and the problem that mold is easy to breed at high temperature in the seed germination period is effectively solved through appropriate treatment on the growth environment of the seeds before sowing and the seeds after sowing. The method is clear in condition, high in replicability and capable of saving a large amount of trial and error time of people encountering similar problems. And the problem of germ breeding of a large number of materials under high-temperature stress can be effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of maize molecular biology and relates to a method for temperature control and pathogen control during the germination period of maize under heat stress. Background Technology

[0002] The frequent occurrence of high-temperature events caused by global warming has made heat stress one of the key factors restricting high and stable maize yields. Studying the mechanism of maize response to heat stress and creating heat-resistant germplasm are urgent needs to cope with climate change and stabilize food production capacity.

[0003] Due to limitations in population size, the tall stature of mature maize plants, and the complexity of heat stress conditions, current methods often involve high-temperature treatment of a small number of materials in a limited space within a laboratory artificial climate chamber. This method, due to the small number of materials, makes it difficult to effectively subject all populations to heat stress, and therefore hinders the objective screening of heat-resistant germplasm from a large pool of materials. Seed germination requires less space, allowing for initial heat stress screening of a large number of materials during the germination period. Ensuring robust and full seedling emergence provides an irreplaceable starting point for high and stable maize yields. However, high temperatures during germination promote mold growth, severely impacting seedling growth and subsequent phenotypic analysis.

[0004] Therefore, exploring a suitable temperature for heat stress treatment of maize while also being able to treat a large quantity of materials with heat stress is of great significance. The inventors of this invention have learned that treating seeds with sodium hypochlorite before sowing and then watering with a certain concentration of hymexazol after sowing can significantly reduce mold growth, thereby ensuring the success rate of the experiment. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a method suitable for large-scale heat stress treatment of maize, which solves the dilemma of the difficulty in achieving heat stress in maize population materials and the problem of large-scale mold growth in seeds during the germination period when exposed to high temperatures. It is of great significance for downstream molecular biology experiments on maize response to high temperatures and screening of heat-resistant germplasm.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical means:

[0007] This invention first discloses a method suitable for temperature control and pathogen control during the germination period of maize under heat stress, comprising the following steps:

[0008] 1. Heat stress temperature screening: Germination experiments were conducted using multiple maize hybrids and inbred lines, including Xianyu 335 and B73, at 28℃, 35℃, 40℃, 42℃, and 45℃. Results showed that all materials failed to germinate above 40℃, and the seeds showed a tendency to mold and die. High-temperature treatment results on a natural population containing 325 inbred lines showed that at 40℃, 70% of the materials had a germination rate of less than 40%; after treatment at 39℃, 83% of the materials achieved a germination rate of 50% or higher, but significant differences were observed in root and aboveground phenotypes (see details). Figure 1 (All materials failed to germinate above 40℃, and the seeds showed a tendency to mold and die). Therefore, we recommend setting the screening temperature for extremely heat-resistant materials to 40℃, and for population analysis, setting the heat stress treatment temperature to 39℃.

[0009] 2. Seed treatment before sowing: Select plump, uniform, and consistent-sized corn seeds for each material and place them in a paper cup. Rinse them 2-3 times with purified water and stir with a glass rod. Then, sterilize them with a 5% sodium hypochlorite solution for 15 minutes. Discard the solution and rinse them 2-3 times with purified water. Set aside for use.

[0010] 3. Seed planting: Vermiculite culture method is adopted, and the temperature of the incubator is set at 39℃ or 40℃ (depending on the research object and purpose); first, a layer of vermiculite is laid in the planting cell, the corn seed embryo is placed in the cell, and finally a thin layer of vermiculite is covered.

[0011] 4. Watering management: For the first watering, use a 1 / 500 solution of hymexazol to thoroughly water the plant. Then, water twice more with the same hymexazol solution. After that, water normally with clean water. Stop watering the day before the morphological identification.

[0012] 5. Phenotypic Identification: Seed emergence was recorded on the fourth day of cultivation, and again on the seventh day, with phenotypic identification conducted. The seed roots were rinsed to remove vermiculite, ensuring the root system remained intact. Root length, hypocotyl length, and total aboveground length were measured. Seeds were removed with a scalpel, and the fresh weight of roots and aboveground parts was weighed using an analytical balance. Samples were placed in an oven and treated at 120℃ for 30 minutes, then cooled to 50℃ and dried for 3 days. The dry weight of roots, aboveground parts, and seeds was weighed. The heat resistance of the material was comprehensively evaluated based on the heat resistance coefficients of each trait.

[0013] The present invention has the following beneficial effects:

[0014] This invention provides a method for heat stress treatment of large quantities of maize, which has the following advantages:

[0015] 1) This method explored the appropriate temperature for heat stress treatment of maize.

[0016] 2) This method effectively solves the problem of mold growth during seed germination due to high temperatures by appropriately treating the seed growth environment before and after sowing.

[0017] 3) This method has clear conditions, is highly replicable, and can save a lot of trial and error time for people who encounter similar problems. Attached Figure Description

[0018] Figure 1 Germination performance of different materials on the fourth day after sowing at different temperatures;

[0019] Figure 2 A comparison of mold growth with and without seed treatment.

[0020] Figure 3 The image shows a Manhattan plot for GWAS analysis of heat resistance, where: RFWT represents root fresh weight heat resistance; SFWT represents seedling fresh weight heat resistance; RLT represents root length heat resistance; SDWT represents seedling dry weight heat resistance; RDWT represents root dry weight heat resistance; and HLT represents hypocotyl length heat resistance. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1

[0023] GWAS analysis and heat resistance evaluation were performed on natural populations of 298 inbred lines (including B73) using a method suitable for large-scale maize heat stress treatment.

[0024] 1. Select plump and uniformly sized corn seeds for each maize inbred line and place them in a paper cup. Rinse with purified water 2-3 times and stir with a glass rod. Then, sterilize with a 5% sodium hypochlorite solution for 15 minutes. Discard the solution and rinse with purified water 2-3 times. Set aside for use.

[0025] 2. The vermiculite culture method was adopted, with two temperature groups of 28℃ and 39℃. First, a layer of vermiculite was laid in the planting grid, the corn seeds were placed with the embryo facing up, and finally a layer of vermiculite was covered.

[0026] 3. For the first watering, use a 1 / 500 solution of hymexazol to thoroughly water the plant. Then, water twice more with the same hymexazol solution. After that, water normally with plain water. Stop watering the day before the morphological identification.

[0027] 4. Seed emergence was recorded on the fourth day of cultivation, and again on the seventh day, followed by phenotypic identification. The seed roots were rinsed to remove vermiculite, ensuring the root system remained intact. Root length, hypocotyl length, and total aboveground length were measured. Seeds were removed with a scalpel, and the fresh weight of roots and aboveground parts was weighed using an analytical balance. Samples were placed in an oven at 120℃ for 30 minutes, then cooled to 50℃ and dried for 3 days. The dry weight of roots, aboveground parts, and seeds was weighed. Based on the heat resistance coefficients (39℃ / 28℃) of each trait, principal component analysis was used to screen for the heat-resistant material HCL499 and the sensitive material HCL102. Combined with the population's genotype data, GWAS analysis was performed to screen for SNPs significantly associated with heat resistance. Specific results are shown below. Figure 3 As shown, SNPs above the threshold line are significantly associated with heat resistance.

[0028] according to Figure 2 The results show that appropriate sterilization treatment before and after sowing can significantly improve the growth of mold.

[0029] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method suitable for temperature management and pathogen control during the germination period of maize under heat stress, comprising the following steps: 1) Screening for heat stress temperature: Germination experiments were conducted on maize hybrid materials and inbred lines at 28℃, 35℃, 40℃, 42℃ and 45℃. The results showed that all materials failed to germinate above 40℃ and the seeds showed a tendency to mold and die. Therefore, the heat stress temperature was determined to be 40℃. 2) Seed treatment before sowing: Select plump, uniform, and consistent-sized corn seeds for each material and place them in a paper cup. Rinse them 2-3 times with purified water and stir with a glass rod. Then sterilize them and rinse them 2-3 times with purified water. Set aside for use. 3) Seed planting: Vermiculite culture method is adopted, and the heat stress temperature of the incubator is set at 40℃; first, a layer of vermiculite is laid in the planting cell, the corn seed embryo is placed in the cell, and finally a thin layer of vermiculite is covered. 4) Watering management: For the first watering, use a 1 / 500 solution of hymexazol to thoroughly water the soil. Then, use the same hymexazol solution to water the soil once on the third and fourth days. After that, use clean water to water the soil once a day. Stop watering the day before the phenotypic identification. 5) Recording and identification: Record the seed germination status on the fourth day of cultivation, and record the germination status again on the seventh day and conduct phenotypic identification.

2. The method according to claim 1, wherein: Step 1) The maize hybrids mentioned include: Xianyu 335, Zhengdan 958, Baiyu No. 1, and Baiyu No. 2; The inbred lines include: B73, 2011, PH6WC, and Zheng58.

3. The method according to claim 1, wherein: Step 2) Sterilization treatment includes: sterilization treatment with 5% sodium hypochlorite solution for 15 minutes.

4. The method according to claim 1, wherein: Step 5) The trait identification includes: Rinse the seed roots to remove vermiculite, keeping the root system intact, and measure the root length, hypocotyl length, and total length of the aboveground parts. Remove the seeds with a scalpel and weigh the fresh weight of the roots and aboveground parts using an analytical balance. Place the samples in an oven and treat at 120℃ for 30 minutes, then cool to 50℃ and dry for 3 days. Weigh the dry weight of the roots, aboveground parts, and seeds.

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