Comprehensive evaluation method for salt tolerance of alfalfa

Abstract

The invention relates to the technical field of plant salt tolerance evaluation, in particular to a comprehensive evaluation method for salt tolerance of alfalfa. The salt tolerance of the alfalfa is comprehensively determined according to the salt tolerance evaluation results in the germination period and the seedling period of the alfalfa, and the accuracy of the salt tolerance evaluation results can be remarkably improved, so that accurate and rapid screening of salt-alkali tolerant varieties of the alfalfa is powerfully supported, and the method has important application value for guaranteeing high-yield, stable-yield and efficient planting of the alfalfa in the saline-alkali soil.

Description

Technical Field

[0001] The invention relates to the technical field of plant salt tolerance evaluation, and in particular to a comprehensive evaluation method for alfalfa salt tolerance. Background Art

[0002] Saline soil refers to soil that accumulates a large amount of soluble salts due to natural and human factors. At present, due to the continuous deterioration of the ecological environment and unreasonable development and utilization by people, soil salinization in arid and semi-arid areas has become increasingly serious, seriously restricting agricultural development. With the continuous use of chemical fertilizers and the development of irrigation agriculture, the area of ​​secondary saline-alkali land is still expanding.

[0003] Alfalfa is the common name for plants of the genus Medicago, which is a perennial flowering plant. The most famous of these is alfalfa (Medicago sativa), which is used as forage and livestock feed. Alfalfa is a perennial herbaceous legume and is the most widely distributed cultivated forage in the world today. It has a cultivation history of more than 2,000 years in my country, and its main production areas are in Northwest China, North China, and Northeast China. Alfalfa has a well-developed root system and strong nitrogen fixation ability. It is salt-alkali, drought-resistant, and barren-resistant. It is also rich in nutritional value and is a high-quality forage favored by all kinds of livestock and poultry. It is known as the "king of forage". Planting alfalfa in moderate and severe saline-alkali land can not only effectively solve the contradiction of "grass and grain competing for land", but also improve saline-alkali land to increase soil fertility, significantly improve the ecological environment, promote agricultural quality and efficiency and increase farmers' income, and achieve efficient utilization of saline-alkali land.

[0004] The adverse effect of excessive soluble salt in the soil on plants is called salt damage, and the tolerance of plants to salt damage is called salt tolerance. To achieve high, stable and efficient yields when planting alfalfa in moderate and severe saline-alkali land, the first thing to do is to screen varieties with strong salt-alkali tolerance. The sensitive period of alfalfa to salt-alkali stress is mainly the germination stage-seedling stage. However, there are differences in salt tolerance at different developmental stages, and the salt tolerance shown in the germination stage and the seedling stage is not completely consistent, so it is not enough to use the evaluation results of only one growth and development stage as the basis for salt tolerance. Under certain conditions, seed germination still cannot predict the salt tolerance of other developmental stages. The robustness of the plant development in the seedling stage of alfalfa is the basis for harvesting seedlings, so salt tolerance in the seedling stage is more important to production than the seed germination stage. Therefore, the seedling stage is an important period for evaluating alfalfa salt tolerance.

[0005] Chinese invention patent CN106385872 A discloses a method for evaluating the salt tolerance of grass ground cover plant seeds. The invention can evaluate the salt tolerance of alfalfa during the germination or seedling stage according to the same technical solution, and finally determine the salt tolerance of the variety. However, since the salt tolerance of alfalfa during the germination and seedling stages is not completely consistent, the method is relatively rough in evaluating the salt tolerance of alfalfa and lacks precise analysis. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a comprehensive evaluation method for salt tolerance of alfalfa.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] Provide a comprehensive evaluation method for alfalfa salt tolerance, and comprehensively determine the salt tolerance of alfalfa based on the salt tolerance evaluation results of alfalfa during the germination stage and the salt tolerance evaluation results of alfalfa during the seedling stage;

[0009] S1. Operation steps for evaluating salt tolerance during germination period:

[0010] S1-1. Place alfalfa seeds treated with distilled water and different salt concentrations in an incubator at 25°C and germinate under the conditions of 8 hours of darkness / 16 hours of light per photoperiod. Record the number of germinated alfalfa seeds after several days.

[0011] S1-2. Calculate the average number of germinations under distilled water treatment, recorded as N0, and calculate the average number of germinations under different salt concentration treatments, recorded as N i The germination rate under different salt concentration treatment conditions is recorded as G i , G i =N i / N0;

[0012] S1-3. Fitting germination rate G according to germination rate under different salt concentration treatment conditions i With salt concentration C i Univariate linear regression equation: G i =aC i +b;

[0013] S1-4. Calculation of the semi-lethal salt concentration C of alfalfa during germination by regression equation 50 , according to the semi-lethal salt concentration C of alfalfa varieties during germination 50 Classify salt tolerance levels;

[0014] S2. Operation steps for evaluating salt tolerance at the seedling stage:

[0015] S2-1. Divide alfalfa seedlings into a control group and a salt treatment group. The salt treatment group is watered with 1 / 4 Hoagland nutrient solution or distilled water containing 100 mM NaCl at an appropriate time, and the control group is watered with the same amount of 1 / 4 Hoagland nutrient solution or distilled water at the same time. Cut the first crop 8 weeks after sowing, and weigh the weight of the cut fresh grass;

[0016] S2-2, after mowing the first crop, the control group and the treatment group were watered with an appropriate amount of distilled water, and the second crop was mowed after 4 weeks of growth. During this period, the treatment group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution or distilled water with 100mM NaCl, and the control group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution or distilled water. After growing for 4 weeks, the third crop was mowed; after mowing the second and third crops, the fresh weight of alfalfa harvested by the treatment and control groups was weighed;

[0017] S2-3. Calculate the relative ratio of fresh weight of three crops of the salt treatment group and the control group R = H s / H c ; R, relative ratio of fresh weight of three crops; H s , total fresh weight of three crops in salt treatment group, H c , the total fresh weight of three crops in the control group;

[0018] S2-4. Alfalfa varieties are graded according to their R values ​​at the seedling stage.

[0019] The beneficial effects of the present invention are:

[0020] The invention comprehensively determines the salt tolerance of alfalfa according to the salt tolerance evaluation results of alfalfa at the germination stage and the seedling stage, and can significantly improve the accuracy of the salt tolerance evaluation results, thereby strongly supporting the accurate and rapid screening of salt-alkali tolerant alfalfa varieties, and has important application value for ensuring high-yield, stable-yield and efficient planting of alfalfa in saline-alkali land. DETAILED DESCRIPTION

[0021] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0022] Example

[0023] 1. Operation steps for evaluating salt tolerance during the germination period

[0024] (1) Prepare NaCl solutions with concentrations of 0, 100, 150, 200, 250, and 300 mM using distilled water;

[0025] (2) Select a culture dish with a diameter of 10 cm, place a Whatman No. 2 filter paper in it, add 4 mL of distilled water or NaCl solution to each culture dish, ensure that the filter paper is completely wet, and there are no bubbles under the filter paper. Set up 3 culture dishes for each treatment solution;

[0026] (3) Select clean and full alfalfa seeds and disinfect them with 75% alcohol for 10 minutes, then wash them with distilled water once, evenly place 100 alfalfa seeds in each culture dish, seal the culture dish with sealing film, and place it in a 25°C incubator (photoperiod: dark 8 hours / light 16 hours) for germination. After 7 days, record the number of germinated alfalfa seeds;

[0027] (4) Calculate the germination rate of alfalfa seeds under different treatment conditions: Calculate the average number of germinations under distilled water treatment (0 mM NaCl), recorded as N0, and calculate the average number of germinations under different salt treatment solutions, recorded as N i The germination rate under different salt treatment conditions is recorded as G i , G i =N i / N0;

[0028] (5) According to the germination rate under different salt concentration treatment conditions, the germination rate (G i ) and salt concentration (C i )One variable linear regression equation: G i =aC i +b;

[0029] (6) Calculate the semi-lethal rate of alfalfa during germination by regression equation (germination rate G i =50%) salt concentration C 50 According to the semi-lethality of alfalfa varieties during germination (germination rate G i =50%) salt concentration C 50 Classification of salt tolerance.

[0030] 2. Operation steps for evaluating salt tolerance at the seedling stage

[0031] (1) Culture pot: inner diameter 10 cm, height 20-30 cm; culture medium ratio: (black charcoal soil: vermiculite) = (10:1). Fill the prepared culture medium into the culture pot to 3-5 cm below the pot mouth.

[0032] (2) The temperature in the culture room was 22-25°C, and the photoperiod was 8 hours dark / 16 hours light.

[0033] (3) Pour the same amount of 1 / 4 Hoagland nutrient solution (or distilled water) into each pot until all the substrate is moistened, then plant 3-5 alfalfa seeds in each pot, cover the surface with 1 cm of culture substrate, and thin out the seedlings after 10 days of growth, leaving one seedling in good growth condition in each pot.

[0034] (4) The thinned alfalfa seedlings were divided into a control group and a salt treatment group, with more than 5 pots in each group. The salt treatment group was watered with 1 / 4 Hoagland nutrient solution (or distilled water) containing 100 mM NaCl at an appropriate time, and the control group was watered with the same amount of 1 / 4 Hoagland nutrient solution (or distilled water) at the same time. The first crop was mowed about 8 weeks after sowing, and the stubble height of each plant was 3-5 cm. The weight of the mowed fresh grass was weighed.

[0035] (5) After the first crop was cut, the control and treatment groups were watered with an appropriate amount of distilled water. After 4 weeks of growth, the second crop was cut. During this period, the treatment group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution (or distilled water) containing 100 mM NaCl, and the control group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution (or distilled water). After 4 weeks of growth, the third crop was cut. After the second and third crops were cut, the fresh weight of the alfalfa harvested by the treatment and control groups was weighed.

[0036] (6) Calculate the relative ratio of fresh weight of three crops of treatment and control group R = H s / H c R, relative ratio of fresh weight of three crops; H s , total fresh weight of three crops in salt treatment group, H c , the total fresh weight of three crops in the control group.

[0037] (7) Alfalfa varieties are classified according to their R value at the seedling stage.

[0038] 3. Comparison table of salt tolerance grades.

[0039] The salt tolerance of germination was based on the germination half-lethal concentration C 50 Grading:

[0040] Salt resistance level <![CDATA[C 50 ]]> 1 (strong) >250mM 2 (strong) 200~250mM 3 (Medium) 150~200mM 4 (weak) 125~150mM 5(weak) <125mM

[0041] The salt tolerance of seedlings is graded according to the relative ratio of fresh weight R:

[0042]

[0043]

[0044] Comprehensive evaluation of salt tolerance at germination and seedling stages:

[0045] Salt resistance level C50 R 1 (strong) >250mM >0.9 2 (strong) 200~250mM 0.8～0.9 3 (Medium) 150~200mM 0.7～0.8 4 (weak) 125~150mM 0.6～0.7 5(weak) <125mM <0.6

[0046] In summary, the present invention comprehensively determines the salt tolerance of alfalfa based on the salt tolerance evaluation results of alfalfa at the germination stage and the seedling stage, which can significantly improve the accuracy of the salt tolerance evaluation results, thereby strongly supporting the accurate and rapid screening of salt-alkali tolerant alfalfa varieties, and has important application value for ensuring high-yield, stable yield and efficient cultivation of alfalfa in saline-alkali land.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0048] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A comprehensive evaluation method for salt tolerance of alfalfa, characterized in that: The salt tolerance of alfalfa was comprehensively determined based on the salt tolerance evaluation results of alfalfa at the germination stage and the seedling stage. S1. Operation steps for evaluating salt tolerance during germination period: S1-1. Place alfalfa seeds treated with distilled water and different salt concentrations in an incubator at 25°C and germinate under the conditions of 8 hours of darkness / 16 hours of light per photoperiod. Record the number of germinated alfalfa seeds after several days. S1-2. Calculate the average number of germinations under distilled water treatment, recorded as N0, and calculate the average number of germinations under different salt concentration treatments, recorded as N i The germination rate under different salt concentration treatment conditions is recorded as G i , G i =N i / N0; S1-3. Fitting germination rate G according to germination rate under different salt concentration treatment conditions i With salt concentration C i Univariate linear regression equation: G i =aC i +b; S1-4. Calculation of the semi-lethal salt concentration C of alfalfa during germination by regression equation 50 , according to the semi-lethal salt concentration C of alfalfa varieties during germination 50 Classify salt tolerance levels; S2. Operation steps for evaluating salt tolerance at the seedling stage: S2-1. Divide alfalfa seedlings into a control group and a salt treatment group. The salt treatment group is watered with 1 / 4 Hoagland nutrient solution or distilled water containing 100 mM NaCl at an appropriate time, and the control group is watered with the same amount of 1 / 4 Hoagland nutrient solution or distilled water at the same time. Cut the first crop 8 weeks after sowing, and weigh the weight of the cut fresh grass; S2-2, after mowing the first crop, the control group and the treatment group were watered with an appropriate amount of distilled water, and the second crop was mowed after 4 weeks of growth. During this period, the treatment group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution or distilled water with 100mM NaCl, and the control group was watered with an appropriate amount of 1 / 4 Hoagland nutrient solution or distilled water. After growing for 4 weeks, the third crop was mowed; after mowing the second and third crops, the fresh weight of alfalfa harvested by the treatment and control groups was weighed; S2-3. Calculate the relative ratio of fresh weight of three crops of the salt treatment group and the control group R = H s / H c ; R, relative ratio of fresh weight of three crops; H s , total fresh weight of three crops in salt treatment group, H c , the total fresh weight of three crops in the control group; S2-4. Alfalfa varieties are graded according to their R values ​​at the seedling stage.

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