A method for rapidly screening cold-tolerant alfalfa germplasm

Abstract

The application provides a method for rapidly screening alfalfa cold-tolerant germplasm, and selects germplasm capable of germination under ABA conditions as cold-tolerant germplasm. The application is directed to the understanding of alfalfa cold-tolerant physiology and molecular mechanism, and the application of plant hormone ABA in the seed germination stage of alfalfa to screen ABA-sensitive and ABA-tolerant plants can rapidly isolate alfalfa cold-tolerant germplasm, and has the characteristics of rapidness and precision.

Description

Technical Field

[0001] This application belongs to the field of plant breeding, specifically, this application provides a method for rapidly screening cold-resistant germplasm of alfalfa. Background Technology

[0002] Alfalfa (Medicago sativa L.) is a perennial, high-quality forage grass belonging to the genus *Medicago* of the legume family. It is cross-pollinated and autotetraploid, rich in nutrients and highly palatable, earning it the title of "Queen of Forages." Currently cultivated alfalfa varieties are mostly population varieties, meaning significant genetic differences can exist between any two seeds within a single variety. In my country, the vast majority of alfalfa cultivation areas are located in the north, related to alfalfa's cool-season and heat-sensitive growth habits. However, northern my country's high latitude and altitude result in cold, long winters with low annual accumulated temperature. Alfalfa varieties with high dormancy levels and poor cold tolerance are prone to frost damage or even death, leading to poor overwintering rates and severely reduced yields. Therefore, breeding new cold-resistant alfalfa varieties and improving their cold tolerance is crucial for their breeding and production applications. The rich genetic differences within alfalfa varieties also provide a basis for further screening for more cold-resistant plants.

[0003] ABA (Abscisic Acid) is a plant hormone widely distributed in higher plants. It can cause leaf abscission and inhibit cell elongation. Studies have shown that under stress, plants accumulate more ABA in their leaves. ABA can promote stomatal closure, enhance water balance, and induce antioxidant defense systems to mitigate oxidative damage. Furthermore, ABA can activate various cellular responses in plants through a series of signal transduction pathways and the induction of HSP and CBF, promoting plant tolerance to cold stress. Therefore, ABA has a certain regulatory effect on the cold tolerance of alfalfa.

[0004] Developing highly cold-resistant alfalfa varieties is an important goal of alfalfa breeding in my country. Previous methods primarily relied on field selection after the alfalfa plants had recovered from winter. Although cold-resistant alfalfa series such as Longmu and Gongnong have been developed, the breeding cycle is long and the improvement in cold resistance is limited. Therefore, there is an urgent need to establish a rapid alfalfa cold-resistant breeding technology system. Summary of the Invention

[0005] This invention aims to understand the physiological and molecular mechanisms of alfalfa's cold resistance. It employs a specific concentration of the plant hormone ABA during alfalfa seed germination to screen for ABA-sensitive and ABA-tolerant plants, thereby isolating alfalfa germplasm with enhanced cold resistance. This method is rapid and precise.

[0006] On the one hand, this application provides a method for rapidly screening cold-resistant germplasm of alfalfa, wherein germplasm that can germinate under ABA conditions is selected as cold-resistant germplasm.

[0007] Further, the method includes

[0008] (1) Seed pretreatment steps;

[0009] (2) Steps for handling ABA conditions;

[0010] (3) Screening steps.

[0011] Further, step (2) includes placing the seeds in a germination box containing 1-2 layers of filter paper, the filter paper being moistened with 10-20 μM ABA; and culturing for 2-5 days.

[0012] Furthermore, the filter paper is impregnated with 1 μM or 10-50 μM ABA.

[0013] Furthermore, incubate in a 25°C light incubator for 3-5 days.

[0014] Furthermore, the photocycle in the light incubator is 14 hours of light / 8 hours of darkness.

[0015] Further, the seedlings that germinate on filter paper moistened with 10-50 μM ABA in step (3) are used as cold-resistant germplasm; the alfalfa seedlings that germinate on filter paper moistened with 1 μM ABA after 5 days of obvious swelling but no germination, and are transferred to moist filter paper without ABA, are used as ABA-sensitive germplasm.

[0016] Furthermore, in step (1), the alfalfa seeds are disinfected with sodium hypochlorite solution and then washed with sterile water.

[0017] Furthermore, in step (1), alfalfa seeds are disinfected with a 20% sodium hypochlorite solution for 20 minutes and washed 5 times with sterile water.

[0018] On the other hand, this application provides the application of the above-mentioned method in alfalfa breeding. Attached Figure Description

[0019] Figure 1 Experimental results for the alfalfa variety Fudi (FD): Part a: ABA screening; Part b: before 4℃ treatment; Part c: 4℃ treatment for 7 days; Part d: 4℃ gradient cooling to -7℃ for 3 hours, followed by recovery at room temperature for 7 days; Part e: photos of plants after frost damage; Part f: 4℃ treatment for 7 days; Part g: ion permeability before and after 4℃ treatment; Part h: ABA screening rate; Part I: frost damage rate of the two materials.

[0020] Figure 2The experimental results for Zhongmu No. 3 are as follows: Part a: ABA screening; Part b: before 4℃ treatment; Part c: 4℃ treatment for 7 days; Part d: 4℃ gradient cooling to -7℃ for 3 hours, followed by recovery at room temperature for 7 days; Part e: photos of plants after frost damage; Part f: 4℃ treatment for 7 days; Part g: ion permeability before and after 4℃ treatment; Part h: ABA screening rate; Part I: frost damage rate of the two materials.

[0021] Figure 3 The experimental results for Baimu 401 are as follows: Part a: ABA screening; Part b: before 4℃ treatment; Part c: 4℃ treatment for 7 days; Part d: 4℃ gradient cooling to -7℃ for 3 hours, followed by recovery at room temperature for 7 days; Part e: photos of plants after frost damage; Part f: 4℃ treatment for 7 days; Part g: ion permeability before and after 4℃ treatment; Part h: ABA screening rate; Part I: frost damage rate of the two materials.

[0022] Figure 4 The experimental results for Baimu 341 are as follows: Part a: ABA screening; Part b: before 4℃ treatment; Part c: 4℃ treatment for 7 days; Part d: 4℃ gradient cooling to -7℃ for 3 hours, followed by recovery at room temperature for 7 days; Part e: photos of plants after frost damage; Part f: 4℃ treatment for 7 days; Part g: ion permeability before and after 4℃ treatment; Part h: ABA screening rate; Part I: frost damage rate of the two materials.

[0023] Figure 5 For the results of the Saiyuan experiment: a) ABA screening, b) before 4℃ treatment, c) 4℃ treatment for 7 days, d) 4℃ gradient cooling to -7℃ for 3 hours, recovery at room temperature for 7 days, e) photos of plants after frost damage, f) 4℃ treatment for 7 days, g) ion permeability before and after 4℃ treatment.

[0024] Figure 6 For the germination performance of Zhongnong No. 2;

[0025] Figure 7 The experimental results for Zhongnong No. 2 include phenotype and ion permeability before and after treatment at 4℃, and phenotype and mortality rate before and after treatment at -7℃ for 3 hours. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0027] Example 1: Basic Technical Flow of the Method of this Application

[0028] (1) Seed disinfection treatment:

[0029] Select plump alfalfa seeds, disinfect them with a 20% sodium hypochlorite solution for 20 minutes, and wash them 5 times with sterile water.

[0030] (2) ABA screening of cold-resistant alfalfa germplasm:

[0031] Experimental apparatus: germination box, filter paper, 5ml pipette;

[0032] Experimental reagents: ABA, sterile water;

[0033] Working solutions of ABA at concentrations of 15 μM and 1 μM were prepared using a stock solution of 100 mM ABA. Two treatments were administered for each variety, with three replicates per treatment and 100 seeds per replicate. After three days of treatment with each treatment, the following seedlings were separated: those germinated from the 15 μM ABA solution were designated as ABA-insensitive germplasm (ABA-In), and those that did not germinate from the 1 μM ABA solution were designated as ABA-sensitive germplasm (ABA-S). The selected seeds were placed in germination boxes with sterile water and transplanted to a greenhouse for soil cultivation once the first true leaf appeared.

[0034] (3) Evaluation of plant tolerance to low temperature stress

[0035] 1) Evaluation of frost tolerance after cold acclimatization:

[0036] ABA-In and ABA-S alfalfa plants that have been grown in the greenhouse for 4-6 weeks were placed at 4℃ for 1 week. Starting from 4℃, the temperature was lowered by 1℃ per hour to -7℃. After maintaining this temperature for 3 hours, the temperature was increased by 1℃ per hour to 4℃. After maintaining this temperature for 24 hours, the plants were placed at 25℃ to recover for 1 week.

[0037] 2) Measurement of physiological indicators:

[0038] Electrolyte leakage rate determination before cold acclimatization: Leaves from the same location were removed and placed in pre-iced glass test tubes, which were kept on ice for 1 hour. They were then transferred to a freezer and equilibrated at 0°C for 1 hour. The temperature was decreased at a rate of 2°C per hour, maintained at each temperature for 1 hour, until it reached -8°C. Samples were removed at five fixed points between 0°C and -8°C and placed in a 4°C freezer overnight to recover. Ion permeability was then measured.

[0039] Electrolyte leakage rate determination after cold acclimatization: After the plants were cold-acclimatized in a 4℃ incubator for 7 days, leaves from the same part were taken and placed in a freezer. Starting from 0℃, the temperature was lowered at a rate of 2℃ per hour, maintained at each temperature for 1 hour, and then lowered to -14℃. Samples were taken out at 5 temperature points during the process of lowering from -6℃ to -14℃, placed in a 4℃ freezer overnight to recover, and then the ion permeability was measured.

[0040] Frost damage rate determination: A tiller is defined as one with ≥3 frost-damaged compound leaves, and each leaflet within the compound leaf has a frost damage area exceeding 50%. Frost damage rate (%) = Number of frost-damaged tillers / Total number of tillers × 100.

[0041] Example 2: Alfalfa Variety – Fudi (FD)

[0042] Breed Description: Fudi, Autumn Hibernation Grade 2.0, purchased from Beijing Baiqingyuan Animal Husbandry Technology Development Co., Ltd.

[0043] Experimental results are as follows Figure 1 As shown:

[0044] 1) ABA screening results: The germination rate of Fudi at 15 μM was 23%, which was significantly higher than the other four varieties, while the germination rate at 1 μM was 84%, which was significantly lower than the other four varieties.

[0045] 2) Results of low-temperature stress: After gradient cooling treatment of detached leaves before cold acclimatization, it was found that at -6℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 7 days of treatment at 4℃, detached leaves were subjected to low-gradient cooling treatment, and at -10℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 3 hours of treatment at -7℃, the frost damage rate of ABA-In plants was 27%, significantly lower than that of ABA-S plants. These results indicate that ABA-In plants have significantly higher tolerance to low temperatures than ABA-S plants.

[0046] Example 3: Alfalfa Variety – Zhongmu No. 3

[0047] Breed Description: Zhongmu No. 3, autumn-dormant grade 3.6, purchased from Beijing Baiqingyuan Animal Husbandry Technology Development Co., Ltd.

[0048] Experimental results are as follows Figure 2 As shown:

[0049] 1) ABA screening results: Zhongmu No. 3 had a germination rate of 3.4% at 15 μM, which was significantly lower than the other four varieties, while the germination rate at 1 μM was 88%, which was significantly higher than the other three varieties.

[0050] 2) Results of low-temperature stress: After gradient cooling treatment of detached leaves before cold acclimatization, it was found that at -6℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 7 days of treatment at 4℃, detached leaves were subjected to low-gradient cooling treatment, and at -10℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 3 hours of treatment at -7℃, the frost damage rate of ABA-In plants was 49%, significantly lower than that of ABA-S plants. These results indicate that ABA-In plants have significantly higher low-temperature tolerance than ABA-S plants.

[0051] Example 4: Alfalfa Variety – Baimu 401

[0052] Breed Description: Baimu 401, autumn-dormant grade 3.4, purchased from Beijing Baiqingyuan Animal Husbandry Technology Development Co., Ltd.

[0053] Experimental results are as follows Figure 3 As shown:

[0054] 1) ABA screening results: The germination rate of Baimu 401 at 15 μM was 16.3%, and the germination rate at 1 μM was 94%.

[0055] 2) Results of low-temperature stress: After gradient cooling treatment of detached leaves before cold acclimatization, it was found that at -6℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 7 days of treatment at 4℃, detached leaves were subjected to low-degree gradient cooling treatment, and at -10℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves. After 3 hours of treatment at -7℃, the frost damage rate of ABA-In plants was 52.6%, significantly lower than that of ABA-S plants. These results indicate that ABA-In plants have significantly higher low-temperature tolerance than ABA-S plants.

[0056] Example 5: Alfalfa Variety – Baimu 341

[0057] Breed Description: Baimu 341, autumn-dormant grade, 4.0, purchased from Beijing Baiqingyuan Animal Husbandry Technology Development Co., Ltd.

[0058] Experimental results are as follows Figure 4 As shown:

[0059] 1) ABA screening results: The germination rate of Baimu 341 at 15 μM was 11%, and the germination rate at 1 μM was 93%.

[0060] 2) Results of low temperature stress: After gradient cooling treatment of detached leaves before cold acclimatization, it was found that at -6℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves; after 7 days of treatment at 4℃, after low-degree gradient cooling treatment, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves at -10℃; after 3 hours of treatment at -7℃, the frost damage rate of ABA-In plants was 37%, which was significantly lower than that of ABA-S plants.

[0061] Example 6: Alfalfa Variety – Saiyuan

[0062] Breed Description: Saiyuan, autumn-dormant grade 4.5, purchased from Beijing Baiqingyuan Animal Husbandry Technology Development Co., Ltd.

[0063] Experimental results are as follows Figure 5 As shown:

[0064] 1) ABA screening results: The germination rate of Saiyuan at 15 μM was 11%, and the germination rate at 1 μM was 88.86%.

[0065] 2) Results of low temperature stress: After gradient cooling treatment of detached leaves before cold acclimatization, it was found that at -6℃, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves; after 7 days of treatment at 4℃, after low-degree gradient cooling treatment, the electrolyte leakage rate of ABA-In plant leaves was significantly lower than that of ABA-S plant leaves at -10℃; after 3 hours of treatment at -7℃, the frost damage rate of ABA-In plants was 40.26%, which was significantly lower than that of ABA-S plants.

[0066] Example 7: A new ABA-insensitive alfalfa material – Zhongnong No. 2

[0067] Zhongnong No. 2: This variety was bred by our research group using alfalfa seedlings (ABA-In) germinated on filter paper moistened with 50 μM ABA from materials such as Zhongmu No. 1, Gongnong No. 1, and Longmu 801 as parents, and was developed through multiple field selections over 7 years. Treatment with 10 μM ABA during the germination period revealed that Zhongnong No. 2 exhibited a higher seed germination rate and faster germination. Figure 6 After undergoing low-temperature and freeze-resistance treatment, Zhongnong No. 2 exhibited stronger cold resistance. Figure 7 ).

Claims

1. A method for rapid screening of cold tolerant germplasm of Medicago sativa L. characterized by, The method comprises: (1) a pre-treatment step of alfalfa seeds; (2) an ABA conditioning step: placing the seeds in a germination box containing 1-2 layers of filter paper, which is soaked with 10-50 μM and 1 μM ABA solution respectively, and culturing in a 25℃ light incubator for 3 days; the light cycle in the light incubator is 14h light / 8h darkness; (3) a screening step: selecting seeds germinated on filter paper soaked with 10-50 μM ABA solution as cold-tolerant germplasm.

2. The method according to claim 1, wherein the alfalfa seeds in step (1) are washed with sterile water after being treated with sodium hypochlorite solution.

3. The method according to claim 2, wherein the alfalfa seeds in step (1) are treated with 20% sodium hypochlorite solution for 20 min and washed with sterile water for 5 times.

4. Use of the method according to any one of claims 1-3 in alfalfa breeding.

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