A method for creating drought-resistant germplasm of feed rye

Abstract

This invention provides a method for creating drought-resistant rye germplasm for feed, relating to the field of agricultural technology. The method involves selecting rye seeds from areas with winter and spring rainfall less than 150 mm, drying them, burying them in sand at low temperatures, screening out necrotic seeds, fumigating them with ethylene, and then subjecting them to 15% PEG stress for germination. Subsequently, drought stress treatment is applied from the start of tillering until the jointing stage, and continues after flowering until harvest. The harvested seeds are then further screened through dry field planting. This invention overcomes the shortcomings of existing technologies and can effectively screen new rye lines that possess strong drought resistance, early maturity, and stable yield, providing favorable conditions for large-scale rye cultivation in arid regions.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a method for creating drought-resistant germplasm of rye for feed. Background Technology

[0002] rye( Secale cereale L Rye is an important dual-purpose forage and food crop in cold regions worldwide, boasting abundant foliage, soft stems, rich nutrition, and high palatability for cattle and sheep. Its extreme cold tolerance makes it irreplaceable in the extremely cold regions of North America and Northeast Europe. Introduced to my country in the late 1950s and early 1960s, rye is currently mainly used for winter and spring forage production in the Huang-Huai-Hai Plain and cold mountainous areas of southern China, with cold-resistant varieties being a key focus of breeding. The Yunnan-Guizhou Plateau region experiences scarce rainfall during winter and spring (November to April of the following year), with normal dry season rainfall less than 200mm, resulting in significant seasonal drought. This places relatively low demands on the cold tolerance of autumn-sown forage, but extremely high demands on drought tolerance. Existing domestic rye varieties perform poorly in Yunnan due to drought resistance issues. The Dian-Northeast rye variety, widely cultivated in Yunnan for over 60 years, exhibits excellent drought tolerance, but long-term self-seeding has led to significant varietal degeneration, resulting in low forage and seed yields. Therefore, cultivating new drought-resistant rye varieties suitable for planting on fallow dry slopes in the Yunnan-Guizhou Plateau is of great significance for promoting forage production on fallow dry farmland in Yunnan during winter.

[0003] Currently, existing breeding methods for forage grasses mainly include traditional hybridization breeding and systematic selection, mutation breeding, and molecular breeding. While traditional hybridization breeding is relatively simple to operate, it suffers from long breeding cycles, low efficiency in screening target traits, and difficulty in aggregating superior traits. Mutation breeding, although capable of rapidly generating mutations, suffers from random mutation directions, low rates of high-quality mutations, and a massive workload for subsequent screening. Molecular breeding has high technical barriers and costs, and its operation is difficult. Therefore, how to rapidly induce and screen drought-resistant rye materials to obtain new drought-resistant rye germplasm is a major research direction at present. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for creating drought-resistant rye germplasm for feed, which can effectively screen out new rye lines that combine strong drought resistance, early maturity, and stable yield, providing favorable conditions for large-scale rye cultivation in arid regions.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for creating a drought-resistant germplasm of rye for feed, the method comprising the following steps: S1. Seed pretreatment: Select rye seeds, dry the seeds to a moisture content of 8%-12%, then bury them in river sand, control the burial temperature at 2-4℃, treat for 10-12 hours, then raise the temperature to 8-10℃, continue burying for 5-7 days, then take them out, float them in clean water and then disinfect them to obtain pretreated seeds. S2, Stress germination: Pretreated seeds were placed in a sealed environment for ethylene fumigation, and then placed in a petri dish containing 15% PEG-6000 for germination treatment and culture for 8-10 days to obtain germinated seedlings. S3. Transplanting Management: After rinsing the above-mentioned germinated seedlings with clean water, transplant them into seedling pots for cultivation. When the plants begin to tiller, drought stress treatment is applied, i.e., the relative soil moisture content is controlled at 40%-50%, and the stress is continued until the jointing stage. The stress is relieved after the jointing stage. After the plants enter the flowering stage, the relative soil moisture content is controlled at 40%-50% until the harvest period. The greenhouse is kept well-ventilated and well-lit. The fruiting ears are harvested and reserved for seed. S4. Screening: The above-mentioned seeds are planted in areas with an altitude of 2000-2300m and dry and rainless autumn and winter. They are planted continuously for 2 years. The first 20% of the ears that mature each year and have a seed setting rate of more than 50% are harvested. After the second sowing and harvest, the ears with the top 10% seed setting rate are screened and mixed for sowing as drought-resistant and stable-yielding new materials.

[0006] Preferably, the rye seeds selected in step S1 are obtained from areas where rye is traditionally grown, and where the winter and spring rainfall over three years is less than 150 mm.

[0007] Preferably, the moisture content of the river sand buried in step S1 is controlled at 30%-60%.

[0008] Preferably, in step S1, the disinfection method involves immersing the seeds in a 0.1%-0.2% potassium permanganate solution for 10-30 minutes.

[0009] Preferably, the concentration of ethylene fumigation in step S2 is 100-150 ppm, and the fumigation time is 4-6 h.

[0010] Preferably, in step S2, the culture dish containing 15% PEG-6000 is prepared by placing filter paper at the bottom of the culture dish, and then adding 15% PEG-6000 solution to the filter paper to make the filter paper wet but without water accumulation.

[0011] Preferably, the germination treatment conditions in step S2 are a light / dark period of 12h / 12h per day, a temperature of 25℃, and a relative humidity of 80%.

[0012] Preferably, in step S3, the temperature for transplanting the seedlings into the seedling pot for cultivation is 20-25℃, and the relative soil moisture content is controlled at 70%-80% before the plants begin to tiller.

[0013] Preferably, the way to relieve stress in step S3 is to first replenish water to the soil relative moisture content of 55%-65%, maintain it for 5 days, and then replenish water to 70%-80%.

[0014] This invention provides a method for creating drought-resistant germplasm of forage rye, which has the following advantages compared with the prior art: 1. This invention involves drying and then burying the initial rye seeds in low-temperature sand, effectively screening the seeds and removing those that are not cold-hardy and prone to water loss and death. Subsequent germination-inducing treatment using ethylene fumigation combined with 15% PEG drought stress further ensures that the resulting seedlings have good drought resistance and subsequent growth vigor. Further screening under drought stress during key growth stages in pots and comprehensive evaluation of drought resistance and agronomic traits in the field are then conducted. This systematically yields new rye varieties with strong drought resistance, early maturity, and stable yield. This invention focuses on agricultural production in drought-prone areas. This invention utilizes a targeted approach to collect varieties, enabling the acquisition of germplasm resources with natural drought-resistant genetic backgrounds. This lays a solid foundation for subsequent drought-resistant breeding and addresses the issues of insufficient germplasm diversity and limited drought-resistant potential in existing breeding practices. Furthermore, this invention establishes a multi-dimensional drought-resistant identification system combining early seed drying and low-temperature sand burial with ethylene fumigation, PEG simulation, and pot evaluation. Covering the germination and critical growth stages, the identification results are more comprehensive and accurate, effectively screening for drought-resistant materials with dormancy characteristics or strong mutation potential, thus improving the screening efficiency and reliability of drought-resistant germplasm. By employing stress training during critical growth stages (germination and tillering stages) and scientific stress relief cultivation methods, the drought adaptability of germplasm materials can be directionally strengthened, resulting in germplasm with stronger drought-resistant stability.

[0015] 2. The new strain obtained by this invention exhibits outstanding comprehensive traits and high production application value, organically combining three key agronomic traits: drought resistance, growth period (early maturity), and forage production performance (stable yield). Through comprehensive agronomic trait evaluation of materials surviving in harsh environments, and by selecting the 10% of panicles with the best overall performance for mixed planting, it is ensured that the selected new strain is a superior population with synergistic trait improvement, rather than a low-yielding material with only drought resistance. This strain is particularly suitable for rainfed agricultural areas or seasonally arid regions in southwestern my country's plateau and mountainous areas. It can effectively utilize natural autumn and winter rainfall to complete its growth cycle and can be harvested in April, providing valuable and high-quality forage for local livestock farming.

[0016] 3. This invention realizes the potential for production and application in barren and arid lands, which is conducive to promotion and application. The entire screening process was carried out under conditions of no irrigation or simulated dry land. The drought-resistant materials obtained through screening were propagated and their trait stability verified in the target environment (sparsely rained mountainous areas of northeastern Yunnan) for multiple rounds (two years or more). Finally, the materials were successfully planted and harvested in the sparsely rained mountainous areas of northeastern Yunnan during autumn and winter. This effectively ensured the stability and consistency of the genetic traits of the new strains, laying a solid foundation for subsequent large-scale demonstration and promotion, and is of great value for ensuring the supply of forage and agricultural development in these areas. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the germination status of rye seeds in groups 1-9 of Example 2 of the present invention on the 7th day of germination treatment; Figure 2 This is a schematic diagram showing the growth of germinating seedlings of rye seeds in Group 1 after 10 days of cultivation in Example 2 of the present invention; Figure 3 This is a schematic diagram showing the growth of germinating seedlings of rye seeds in group 2 after 10 days of cultivation in Example 2 of the present invention; Figure 4 This is a schematic diagram showing the growth of germinating seedlings of rye seeds in group 3 after 10 days of cultivation in Example 2 of the present invention; Figure 5 This is a schematic diagram showing the growth of germinating seedlings of rye seeds in group 9 after 10 days of cultivation in Example 2 of the present invention; Figure 6 This is a schematic diagram showing the growth of each group of plants in the pot experiment of Embodiment 3 of the present invention to the point where tillering begins; Figure 7 This is a schematic diagram of the growth of the potted plants in Group 1 of Example 3 of the present invention before drought stress, at the jointing stage, and at the harvest stage; Figure 8 This is a schematic diagram showing the growth of the drought-resistant new strain (left) and strain number 4 (right) obtained in Example 3 of the present invention on April 2, 2025. Figure 9 This is a schematic diagram of the ear filling situation after harvesting of the drought-resistant new variety in Example 3 of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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.

[0019] Example 1: Seed selection for experiments: Nineteen rye samples from different ecological environments were collected in the mountainous areas of Yunnan, where rye is traditionally grown. Seed germination experiments were conducted, and germination rate and germination potential were recorded. The specific results are shown in Table 1 below: Table 1: Germination status of 19 collected resources

[0020] Based on the table above, 10 rye materials with good germination (material numbers 1-10) were selected. These 10 selected rye materials and two commercial varieties (Winter Mulberry 70 and Green Forage Rye) were used as research subjects. Under dry season field planting conditions, the overall performance of the tested materials was evaluated in the field. No irrigation was provided throughout the entire growth period. Agronomic traits (plant height, ear length, ear width, ear-leaf distance, flag leaf length, flag leaf width, number of stem nodes, lodging length, stem diameter, tillering) and production performance (number of ears per plant, awn length, leaf weight per plant, stem weight per plant, ear weight per plant) were measured at the milk stage and compared. The experimental results are shown in Tables 2 and 3. The data in the tables show that Green Forage Rye had a shorter plant height, Winter Mulberry 70 had more tillers, and the selected rye farmyard materials (Nos. 1, 2, 4, 5, and 6) grew well under no-irrigation conditions, exhibiting excellent yield indicators such as plant height, stem diameter, and weight per plant.

[0021] Table 2: Evaluation of the adaptability of 12 major rye cultivars to the dry season in Yunnan (morphological indicators)

[0022] Table 3: Adaptability evaluation (production performance) of 12 major rye cultivars to the dry season in Yunnan.

[0023] Based on Tables 2 and 3, principal component analysis was performed on the 15 phenotypic indicators and production performance. The top three rye varieties in terms of comprehensive performance were No. 4, No. 5, and No. 8. It should be noted that the excellent materials selected above all came from mountainous areas in Yunnan where traditional rye is grown and the cultivation environment is relatively closed. The annual winter and spring rainfall in these areas is usually less than 150 mm, which gives them a natural drought-resistant genetic background.

[0024] Example 2: The rye seeds numbered 4 above were selected as experimental materials for the following screening experiment: 1. Pretreatment of rye seeds: A1: Rye seeds were dried to a moisture content of 10%, then buried in river sand (the moisture content of the river sand was controlled at 30%-60% during burial), and the burial temperature was controlled at 4℃. After 10 hours of treatment, the temperature was raised to 10℃ and the burial treatment continued for 6 days. After that, the seeds were taken out and floated in clean water, and then immersed in a 0.2% potassium permanganate solution for disinfection for 20 minutes. After rinsing with clean water, the pretreated seeds A1 were obtained.

[0025] A2: Rye seeds were buried in river sand (the moisture content of the river sand was controlled at 30%-60% during burial), and the burial temperature was controlled at 4℃. After 10 hours of treatment, the temperature was raised to 10℃ and the burial treatment continued for 6 days. After that, the seeds were taken out and floated with clean water, and then immersed in a 0.2% potassium permanganate solution for 20 minutes for disinfection. After rinsing with clean water, the pretreated seeds A2 were obtained.

[0026] A3: Dry rye seeds to a moisture content of 10%, then bury them in river sand (controlling the moisture content of the river sand to 30%-60% during burial), control the burial temperature at 4℃, treat for 10 hours, and then continue to bury at room temperature for 6 days. After that, take them out and float them in clean water, then soak them in a 0.2% potassium permanganate solution for 20 minutes for disinfection. Take them out and rinse them with clean water to obtain pretreated seeds A3.

[0027] A4: Dry rye seeds to a moisture content of 10%, then bury them in river sand (controlling the moisture content of the river sand to 30%-60% during burial), control the burial temperature at 10℃, and bury for 6 days. After burial, remove the seeds, float them in clean water, and then immerse them in a 0.2% potassium permanganate solution for 20 minutes for disinfection. Remove the seeds, rinse them with clean water, and you will get pretreated seeds A4.

[0028] A5: Rye seeds are floated in clean water and then soaked in a 0.2% potassium permanganate solution for 20 minutes for disinfection. After being taken out and rinsed with clean water, pretreated seeds A5 are obtained.

[0029] 2. Stress-induced budding treatment: B1: The pretreated seeds were fumigated in a closed environment with an ethylene concentration of 120 ppm for 5 hours, and then placed in a petri dish (using filter paper as a pad at the bottom of the petri dish, and then adding 15% PEG-6000 solution to the filter paper to make the filter paper moist but without water accumulation). The culture temperature was set at 25℃, the relative humidity at 80%, and the light / dark time was 12h / 12h per day for culture.

[0030] B2: Place the pretreated seeds in a petri dish (use filter paper to line the bottom of the petri dish, then add 15% PEG-6000 solution to the filter paper to make the filter paper moist but without water accumulation), set the culture temperature to 25℃, the relative humidity to 80%, and the light / dark time to 12h / 12h per day for culture.

[0031] B3: Place the pretreated seeds in a petri dish (use filter paper to line the bottom of the petri dish, then add distilled water to the filter paper to make the filter paper moist but without water accumulation), set the culture temperature to 25℃, the relative humidity to 80%, and the light / dark time to 12h / 12h per day for culture.

[0032] 3. Results of the germination experiment: Different experimental groups were set up according to different pretreated seeds and stress germination treatment methods selected according to Table 4 below: Table 4: Germination Experiment Conditions

[0033] On the 7th day of germination treatment, the number of germinated seeds in each group was counted at the same time (e.g., Figure 1 Germination is considered complete when the radicle elongates to half the seed length. Germination status for each group is as follows: Figure 1 And as shown in Table 5 below: Table 5 Results of germination experiments in each group

[0034] Groups with a germination rate ≥85% (Groups 1, 2, 3, and 9) were selected, and seedlings were observed after 10 days of cultivation. Details are as follows: Figure 2-5 As shown, the seedlings in group 9 grew the best, the seedlings in groups 1 and 2 also grew well, and the seedlings in group 3 grew poorly.

[0035] Example 3: 1. Potted plant stress experiment: After 10 days of cultivation, the germinated seedlings from groups 1, 2, 3, and 9 in Example 2 were rinsed with clean water and transplanted into seedling pots for pot cultivation. The cultivation substrate for the pots was a mixture of 0-30 cm topsoil and perlite (in a 4:1 ratio), with approximately 4.1 kg of the substrate added to each pot. Three seedlings were transplanted evenly into each pot. The soil was irrigated thoroughly during transplanting, and the relative soil moisture content was controlled at 70%-80%. The temperature was maintained at 20-25℃ throughout the pot cultivation period.

[0036] When the plants in each group have grown to the point where tillering begins (plant height approximately 20cm, such as...) Figure 6 (As shown) Drought stress treatment was applied, i.e., the relative soil moisture content was controlled at 40%-50% and the stress was maintained until the jointing stage; the stress was relieved in the late jointing stage (first watering was added until the relative soil moisture content was 55%-65%, maintained for 5 days, and then watering was added again to 70%-80%); after the plants entered the flowering stage, the relative soil moisture content was controlled at 40%-50% until the harvest period, the greenhouse was kept well-ventilated and light-permeable, the fruiting ears were harvested, and the average number of ears / pot and the number of seeds / pot for each group were recorded. The specific results are shown in Table 6 below: Table 6 Results of the pot experiment

[0037] Group 1 showed the best effect after stress. The potted plants of Group 1 before drought stress, at the jointing stage, and at harvest are as follows: Figure 7 As shown.

[0038] 2. Planting and screening experiment: Seeds from the fruiting ears in group 1 were retained for subsequent planting. They were planted in the 2300-meter altitude area of ​​Huize County, Yunnan Province and the 2100-meter altitude area of ​​Malong District, Yunnan Province (both are arid and low-rainfall areas). The planting was carried out for two consecutive years. The first 20% of the ears that matured each year and had a seed setting rate of more than 50% were harvested. After the second sowing and harvest, the ears with the top 10% seed setting rate were selected and mixed for sowing as seeds for the new drought-resistant variety.

[0039] 3. Comparative experiment: Rye seeds (number 4) and the aforementioned drought-resistant new variety seeds were sown together on October 21, 2024, at an altitude of 2300 meters in Huize County, Yunnan Province. The drought-resistant new variety plants entered the harvest period on April 2, 2025 (at this time, the growth of the drought-resistant new variety plants and the number 4 plants were compared as follows). Figure 8 As shown, the drought-resistant new variety has more tillers and a significantly earlier growth period, while plant number 4 is sparser and has a later growth period. Plant number 4 entered the harvest period on May 1, 2025. Furthermore, the seed setting rate of the single ear of the new variety is 72% (e.g., ...). Figure 9 The seed setting rate of single spikelets of variety No. 4 was 48%, and the seed setting rate of the new variety was significantly improved, and it was also early-maturing and drought-resistant.

[0040] The above methods can be used to obtain new drought-resistant and early-maturing rye varieties.

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for creating a drought-resistant germplasm of rye for feed, characterized in that, The creation method includes the following steps: S1. Seed pretreatment: Select rye seeds, dry the seeds to a moisture content of 8%-12%, then bury them in river sand, control the burial temperature at 2-4℃, treat for 10-12 hours, then raise the temperature to 8-10℃, continue burying for 5-7 days, then take them out, float them in clean water, and then disinfect them to obtain pretreated seeds; the rye seeds are obtained from areas where rye is traditionally grown, and the winter and spring rainfall in these areas is less than 150mm for more than three years; S2, Stress germination: Pretreated seeds were placed in a sealed environment for ethylene fumigation, and then placed in a petri dish containing 15% PEG-6000 for germination treatment and culture for 8-10 days to obtain germinated seedlings. S3. Transplanting Management: After rinsing the above-mentioned germinated seedlings with clean water, transplant them into seedling pots for cultivation. When the plants begin to tiller, drought stress treatment is applied, i.e., the relative soil moisture content is controlled at 40%-50%, and the stress is continued until the jointing stage. The stress is relieved after the jointing stage. After the plants enter the flowering stage, the relative soil moisture content is controlled at 40%-50% until the harvest period. The greenhouse is kept well-ventilated and well-lit. The fruiting ears are harvested and reserved for seed. S4. Screening: The above-mentioned seeds are planted in areas with an altitude of 2000-2300m and dry and rainless autumn and winter. The seeds are planted continuously for 2 years. The first 20% of the ears that mature each year and have a seed setting rate of more than 50% are harvested. After the second sowing and harvest, the ears with the top 10% seed setting rate are selected and mixed for sowing as new drought-resistant and stable-yielding materials.

2. The creation method according to claim 1, characterized in that: The moisture content of the river sand buried in step S1 is controlled at 30%-60%.

3. The creation method according to claim 1, characterized in that: In step S1, the seeds are disinfected by immersing them in a 0.1%-0.2% potassium permanganate solution for 10-30 minutes.

4. The creation method according to claim 1, characterized in that: In step S2, the concentration of ethylene fumigation is 100-150 ppm, and the fumigation time is 4-6 hours.

5. The method of creation according to claim 1, characterized in that: In step S2, the culture dish containing 15% PEG-6000 is prepared by placing filter paper at the bottom of the culture dish, and then adding 15% PEG-6000 solution to the filter paper to make the filter paper wet but without water accumulation.

6. The method of creation according to claim 1, characterized in that: The germination treatment conditions in step S2 are: 12h / 12h light / dark time per day, 25℃ temperature, and 80% relative humidity.

7. The method of creation according to claim 1, characterized in that: In step S3, the temperature for transplanting seedlings into seedling pots for cultivation is 20-25℃, and the relative soil moisture content is controlled at 70%-80% before the plants begin to tiller.

8. The method of creation according to claim 1, characterized in that: The method to relieve stress in step S3 is to first replenish water to the soil relative moisture content of 55%-65%, maintain it for 5 days, and then replenish water to 70%-80%.

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