Method for rapidly screening saline-alkaline tolerant single alfalfa plants

Abstract

The invention is suitable for the technical field of plant breeding, and provides a method for rapidly screening a saline-alkaline tolerant single plant of alfalfa, which comprises the following steps: (1) seed selection: selecting 20 different alfalfa plant species; (2) disinfection: rinsing the seeds with 75% alcohol for 60 seconds, then washing the seeds with sterile water once, then putting the seeds into 50% sulfuric acid, enabling the sulfuric acid to submerge the seeds, soaking the seeds for 5-10 minutes, pouring out the sulfuric acid, washing the alfalfa seeds with a large amount of sterile water for 5-10 times, then adding 50% sodium hypochlorite, putting the alfalfa seeds on a shaking table, lightly shaking and disinfecting for 20 minutes, finally washing the alfalfa seeds with sterile water for 5-10 times, and taking out the seeds; the alfalfa seeds are placed on sterile filter paper to be aired. Then, performing germination and saline-alkaline tolerance screening to obtain saline-alkaline tolerance plants, and detecting and identifying the saline-alkaline tolerance plants through saline-alkaline tolerance gene expression. According to the method for rapidly screening the saline-alkaline tolerant single plant of the alfalfa, saline-alkaline tolerance evaluation and identification can be carried out on different alfalfa seedlings, and a new material is provided for saline-alkaline tolerant breeding of the alfalfa.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a method for rapidly screening salt-alkali tolerant alfalfa plants. Background Art

[0002] Alfalfa is an important forage and green manure crop, widely distributed throughout the world. However, due to the prevalence of saline-alkali soils, the growth of alfalfa in these environments is greatly restricted. Salt-alkali stress mainly increases the ion concentration in the soil, especially sodium ions (Na+) and bicarbonate ions (HCO3-), leading to damage to cell membranes, obstruction of photosynthesis, and absorption and transport barriers of nutrient elements, thereby inhibiting the normal physiological processes of plants.

[0003] At present, there are very few alfalfa varieties planted and promoted on saline-alkali soils in arid and semi-arid areas, which restricts the planting and promotion of alfalfa in this area. Therefore, screening out salt-alkali tolerant individual plants will provide a basis for cultivating salt-alkali tolerant alfalfa varieties for planting and promotion in saline-alkali soils. It will also help to understand the molecular mechanism of salt-alkali tolerance of alfalfa and provide candidate genes for variety improvement. This will help to solve the limitations of saline-alkali soils on agricultural production and promote sustainable agricultural development.

[0004] Most studies on alfalfa's stress resistance are conducted through field selection trials, which are time-consuming and inefficient. Summary of the invention

[0005] The invention provides a method for rapidly screening alfalfa salt-alkali tolerant individual plants, aiming to solve the problem that alfalfa stress resistance research is mostly conducted through field selection tests, which is time-consuming and inefficient.

[0006] To solve the above problems, the present invention is achieved in this way: a method for quickly screening alfalfa salt-alkali tolerant individual plants, comprising the following steps:

[0007] (1) Seed selection

[0008] Choose from 20 different alfalfa plant species;

[0009] (2) Disinfection

[0010] Rinse the seeds with 75% alcohol for 60 seconds, then rinse once with sterile water, then put the seeds into 50% sulfuric acid so that the sulfuric acid covers the seeds, soak for 5-10 minutes, pour out the sulfuric acid, rinse the alfalfa seeds with a large amount of sterile water for 5-10 times, then add 50% sodium hypochlorite and place on a shaker for 20 minutes to disinfect, finally rinse the alfalfa seeds with sterile water for 5-10 times, and place the alfalfa seeds on sterile filter paper to dry;

[0011] (3) Germination

[0012] Evenly place the dried alfalfa seeds in a germination box and place them in a constant temperature culture room for growth;

[0013] (4) Seedling cultivation

[0014] Rehydrate the germination box once a day, adding 2-3 ml of sterile water each time. After six days, transfer the seedlings that grow consistently in the germination box to a hydroponic box containing 0.1× Hoagland nutrient solution. Replace the nutrient solution every two days to ensure that the hydroponic box has sufficient oxygen. After seven days of growth in the hydroponic box, begin to treat with saline-alkali solution.

[0015] (5) Pre-selection of saline-alkali solution for testing

[0016] Through comparative experiments, 0.1× Hoagland nutrient solution containing 100mM Na+ was selected as the appropriate concentration for screening alfalfa salt-alkali tolerant plants, where NaCl:NaHCO3=1:1, PH=8.5;

[0017] (6) Screening of salt-alkali tolerant plants of different alfalfa varieties

[0018] Twenty commercial seedlings grown in a hydroponic box for 7 days were treated with the 0.1× Hoagland nutrient solution containing 100 mM Na+ selected in step (5), with three replicates for each variety, and 30 seedlings in each replicate. The number of seedlings standing upright was counted after 48 hours, 60 hours, and 72 hours of saline-alkali solution treatment. After 72 hours of treatment, the seedlings were transferred to a hydroponic box containing 0.1× Hoagland nutrient solution for recovery treatment, and the number of seedlings standing upright was also counted;

[0019] (7) Verification

[0020] The alfalfa samples and salt-tolerant plants were separated by treatment with 0.1× Hoagland nutrient solution containing 100 mM Na+ for six hours. The salt-alkali tolerance of the plants was evaluated after they grew in vermiculite for four months. Samples were taken before and 24 hours after the salt-alkali treatment, and physiological indicators such as relative water content and electrolyte permeability were tested.

[0021] Preferably, the comparative experiment in step (5) comprises the following steps:

[0022] In the preliminary experiment, at least two different alfalfa seedlings were selected as experimental materials, and treated with 0.1× Hoagland nutrient solution containing 0mM, 100mM, 150mM, 200mM, and 250mM Na+ for 72 hours respectively. The lodging state of the seedlings in each period was compared, and it was found that the seedlings of the two varieties in the 150mM, 200mM, and 250mM treatment groups all lodged, and the three solution concentrations could not distinguish the salt-alkali tolerance between the varieties;

[0023] Based on the comparison of the lodging conditions of alfalfa seedlings, the seedlings that are more resistant to salt-alkali stress were identified, and 0.1× Hoagland nutrient solution containing 100mM Na+ was determined to be the appropriate concentration for screening salt-alkali resistant alfalfa plants, where NaCl:NaHCO3=1:1, PH=8.5.

[0024] Preferably, the germination box in step (3) is made of transparent plastic material, and the ambient temperature of the constant temperature culture room is 15°C-25°C.

[0025] Compared with the related art, the method for rapid screening of alfalfa salt-alkali tolerant individual plants provided by the present invention has the following beneficial effects:

[0026] Compared with the existing technology, the method for rapid screening of alfalfa salt-alkali tolerant individual plants provided by this scheme can screen the salt-alkali tolerance of different alfalfa seedlings cultivated under the same environment at a single time, and obtain the final result by controlling the variable method, which is easy to operate and can ensure the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a growth state diagram of alfalfa seeds in the hydroponic box of the present invention;

[0028] Figure 2 This is a diagram showing the growth state of alfalfa seedlings in an acid-base solution in the present invention;

[0029] Figure 3 This is a schematic diagram of the expression of salt-tolerant candidate genes in the present invention;

[0030] Figure 4 Schematic diagram of the relative expression of salt-alkali tolerance gene in the present invention Figure 1 ;

[0031] Figure 5 Schematic diagram of the relative expression of salt-alkali tolerance gene in the present invention Figure 2 ;

[0032] Figure 6 Schematic diagram of the relative expression of salt-alkali tolerance gene in the present invention Figure 3 ;

[0033] Figure 7 Schematic diagram of the relative expression of salt-alkali tolerance gene in the present invention Figure 4 ;

[0034] Figure 8 This is a diagram of the upright state of the seedlings in the present invention;

[0035] Fig. 9 Statistical diagram of the upright state of the sample in the present invention Figure 1 ;

[0036] Fig.10 Statistical diagram of the upright state of the sample in the present invention Figure 2. DETAILED DESCRIPTION

[0037] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0038] The embodiment of the present invention provides a method for quickly screening alfalfa salt-alkali tolerant individual plants, comprising the following steps:

[0039] (1) Seed selection

[0040] Choose from 20 different alfalfa plant species;

[0041] (2) Disinfection

[0042] Rinse the seeds with 75% alcohol for 60 seconds, then rinse once with sterile water, then put the seeds into 50% sulfuric acid so that the sulfuric acid covers the seeds, soak for 5-10 minutes, pour out the sulfuric acid, rinse the alfalfa seeds with a large amount of sterile water for 5-10 times, then add 50% sodium hypochlorite and place on a shaker for 20 minutes to disinfect, finally rinse the alfalfa seeds with sterile water for 5-10 times, and place the alfalfa seeds on sterile filter paper to dry;

[0043] (3) Germination

[0044] Evenly place the dried alfalfa seeds in a germination box and place them in a constant temperature culture room for growth;

[0045] (4) Seedling cultivation

[0046] Rehydrate the germination box once a day, adding 2-3 ml of sterile water each time. After six days, transfer the seedlings that grow consistently in the germination box to a hydroponic box containing 0.1× Hoagland nutrient solution. Replace the nutrient solution every two days to ensure that the hydroponic box has sufficient oxygen. After seven days of growth in the hydroponic box, begin to treat with saline-alkali solution.

[0047] (5) Pre-selection of saline-alkali solution for testing

[0048] Through comparative experiments, 0.1× Hoagland nutrient solution containing 100mM Na+ was selected as the appropriate concentration for screening alfalfa salt-alkali tolerant plants, where NaCl:NaHCO3=1:1, PH=8.5;

[0049] (6) Screening of salt-alkali tolerant plants of different alfalfa varieties

[0050] Twenty commercial seedlings grown in a hydroponic box for 7 days were treated with the 0.1× Hoagland nutrient solution containing 100 mM Na+ selected in step (5), with three replicates for each variety, and 30 seedlings in each replicate. The number of seedlings standing upright was counted after 48 hours, 60 hours, and 72 hours of saline-alkali solution treatment. After 72 hours of treatment, the seedlings were transferred to a hydroponic box containing 0.1× Hoagland nutrient solution for recovery treatment, and the number of seedlings standing upright was also counted;

[0051] (7) Verification

[0052] The alfalfa samples and salt-tolerant plants were separated by treatment with 0.1× Hoagland nutrient solution containing 100 mM Na+ for six hours. The salt-alkali tolerance of the plants was evaluated after they grew in vermiculite for four months. Samples were taken before and 24 hours after the salt-alkali treatment, and physiological indicators such as relative water content and electrolyte permeability were tested.

[0053] In this embodiment, a variety of different alfalfa species are selected for cultivation, and comparative experiments can be carried out to select the most salt- and alkali-tolerant plants. Cultivating a variety of different alfalfa species under the same environment can reduce experimental deviations caused by different cultivation methods and environments.

[0054] In a further preferred embodiment of the present invention, at least two different alfalfa seedlings are selected as experimental materials in a preliminary experiment, and are treated with 0.1× Hoagland nutrient solution containing 0mM, 100mM, 150mM, 200mM, and 250mM Na+ for 72 hours, respectively. The lodging state of the seedlings in each period is compared, and the seedlings of the two varieties in the 150mM, 200mM, and 250mM treatment groups all lodged, and the three solution concentrations cannot distinguish the salt-alkali tolerance between the varieties;

[0055] Based on the comparison of the lodging conditions of alfalfa seedlings, the seedlings that are more resistant to salt-alkali stress were identified, and 0.1× Hoagland nutrient solution containing 100mM Na+ was determined to be the appropriate concentration for screening salt-alkali resistant alfalfa plants, where NaCl:NaHCO3=1:1, PH=8.5.

[0056] In this embodiment, different alfalfa seedlings and different solutions are selected for cultivation to increase the accuracy of the comparative experiment.

[0057] In a further preferred embodiment of the present invention, the germination box in step (3) is made of transparent plastic material, and the ambient temperature of the constant temperature culture room is 15°C-25°C.

[0058] In this embodiment, the germination box made of transparent plastic material is more convenient for personnel to observe the development status of the seed buds, and the germination rate of the seed buds can be guaranteed by appropriate cultivation temperature.

[0059] The method for rapidly screening alfalfa salt-alkali tolerant individual plants provided by the present invention has the following working steps:

[0060] First, this experiment selected 20 different alfalfa plant materials for disinfection (Table 1). First, rinse the seeds with 75% alcohol for 60s → rinse once with sterile water → treat with 50% sulfuric acid for 5-10min → rinse with sterile water 5-10 times → add 50% sodium hypochlorite and put in a shaker at 28℃, 80rpm, 20min → rinse with sterile water 5-10 times → put on sterile filter paper to dry, evenly place the dried seeds in a germination box, and grow in a constant temperature culture room. The plant materials are shown in the figure:

[0061]

[0062] For the growth management of alfalfa seeds in the germination box, it is necessary to replenish the water in the germination box once a day, adding 2-3 ml of sterile water each time. After six days, the seedlings are in a state suitable for growth in the hydroponic box ( Figure 1 ), after six days, the seedlings growing uniformly in the germination box were transferred to a hydroponic box containing 0.1× Hoagland nutrient solution, and the nutrient solution was replaced every two days to ensure that the hydroponic box had sufficient oxygen. After seven days of growth in the hydroponic box, saline-alkali solution treatment was started;

[0063] In the preliminary experiment, Zhongmu No. 1 and ZMAD-2 were selected as experimental materials (the laboratory found that ZMAD-2 had stronger salt and alkali resistance than Zhongmu No. 1 in the early stage). They were treated with 0.1× Hoagland nutrient solution (NaCl: NaHCO3 = 1: 1, PH = 8.5) containing 0mM, 100mM, 150mM, 200mM, and 250mM Na+ for 72 hours. The results showed that after 1 hour of treatment with saline-alkali solution, seedlings fell over except for the control group. After 15 hours of treatment, seedlings of both varieties in the 150mM, 200mM, and 250mM treatment groups all fell over, indicating that these three solution concentrations could not distinguish the salt and alkali resistance between varieties ( Figure 2 ,Note: Each hydroponic box is Zhongmu No. 1, ZMAD-2 from left to right. It is worth noting that in the 100mM treatment group, Zhongmu No. 1 was completely lodged, while ZMAD-2 was partially upright, indicating that ZMAD-2 was more resistant to salt-alkali stress than Zhongmu No. 1, which was consistent with the results of previous laboratory studies. Based on the above research results, 0.1× Hogland nutrient solution (NaCl: NaHCO3 = 1: 1, PH = 8.5) containing 100mM Na+ was determined to be the appropriate concentration for screening alfalfa salt-alkali resistant individual plants;

[0064] Previous studies in the laboratory found that MsSPL12 increased the salt tolerance of transgenic alfalfa plants. Transcriptome sequencing results showed that there were 1934 genes related to salt stress response, of which 32% (619 / 1934) were upregulated and 68% (1315 / 1934) were downregulated. Real-time fluorescence quantitative detection of differentially expressed genes was performed to screen out salt-tolerant candidate genes that were upregulated under salt stress conditions ( Figure 3 , Note: zr0-zr24, roots of Zhongmu No. 1 were treated with salt for different periods of time);

[0065] The salt-sensitive and salt-tolerant materials of three alfalfa varieties, Zhongmu 1, Algonquin, and Queen, were separated. After being hydroponically cultured in 0.1× Hoagland's nutrient solution for two weeks, they were treated again with 0.1× Hoagland's nutrient solution containing 100 mM Na+ (NaCl: NaHCO3 = 1:1, pH = 8.5). The roots and leaves were sampled before and at the 6th hour of treatment.

[0066] RNA was extracted from roots and leaves, and reverse transcribed into cDNA. RT-qPCR was used to detect the expression levels of Ms.gene038106, Ms.gene00225, and Ms.gene72412 (differential transcriptome genes in the laboratory in the early stage, which have been confirmed to be highly expressed under salt treatment) in roots and leaves. The results showed that the expression of these three genes in the salt-alkali sensitive type after 6 hours of salt-alkali treatment was lower than that in the tolerant type ( Figure 4 , Figure 5 , Figure 6 , Figure 7 ), which confirmed that seedling screening had a certain correlation with plant salt-alkali tolerance;

[0067] After preliminary experiments, it was determined that the concentration for screening salt-alkali tolerant plants was 0.1× Hoagland nutrient solution (NaCl: NaHCO3 = 1: 1, pH = 8.5) containing 100mM Na+. In the formal experiment, 21 commercial seedlings grown in hydroponic boxes for 7 days were treated with 0.1× Hoagland nutrient solution (NaCl: NaHCO3 = 1: 1, PH = 8.5) containing 100mM Na+. Three replicates were set for each variety, with 30 seedlings in each replicate. After 48 hours, 60 hours, and 72 hours of salt-alkali solution treatment, the number of upright seedlings was counted ( Figure 8 ), 72 hours after treatment, the seedlings were transferred to a hydroponic box containing 0.1× Hoagland nutrient solution for recovery treatment, and the number of upright seedlings was also counted;

[0068] The statistical results show that 20 alfalfa varieties ( Fig. 9 , Fig.10), the salt-alkali tolerant varieties compared with Zhongmu No. 1 include ZN-ST36 (19 years), ZN-ST36 (23 years), Xinmu No. 3, Hulunbuir Huanghua, Gongnong No. 5, Gongnong No. 1, Knight T, Longmu 801, Kangsai I, Fudi, Algonquin, BS4, Baimu 202, Dayinhe, Bara416, WL-168HQ, and Queen; the salt-alkali sensitive varieties compared with Zhongmu No. 1 include WL-SALT, Gongnong No. 1, and ZN-CT25. The final statistical results showed that a total of 182 salt-alkali tolerant plants were screened out. In summary, 0.1× Hoagland nutrient solution containing 100mM Na+ (NaCl:NaHCO3=1:1, pH=8.5) can quickly screen out salt-alkali tolerant alfalfa plants. After the salt-alkali tolerant alfalfa varieties are screened out, further breeding work is needed to improve their adaptability and yield.

[0069] In summary, compared with related technologies, this method can screen the salt-alkali tolerance of different alfalfa seedlings cultivated under the same environment at a single time, and obtain the final result by controlling the variable method, which is easy to operate and can ensure the accuracy of the test results.

[0070] In the several embodiments provided in this application, it should be understood that the disclosed device can be implemented in other ways.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A method for rapidly screening alfalfa plants tolerant to salt and alkali, characterized in that: The following steps are involved: (1) Seed selection Choose from 20 different alfalfa plant species; (2) Disinfection The seeds were rinsed with 75% alcohol for 60 seconds, and then rinsed with sterile water once, and then the seeds were placed in 50% sulfuric acid so that the sulfuric acid covered the seeds, and soaked for 5-10 minutes, and then the alfalfa seeds were taken out, and the alfalfa seeds were rinsed with sterile water for 5-10 times, and then 50% sodium hypochlorite was added to the alfalfa seeds and placed in a shaker for 20 minutes for gentle shaking. Finally, the alfalfa seeds were rinsed with sterile water for 5-10 times, and the alfalfa seeds were placed on sterile filter paper to dry; (3) Germination Evenly place the dried alfalfa seeds in a germination box and place them in a constant temperature culture room for growth; (4) Seedling cultivation Rehydrate the germination box once a day, adding 2-3 ml of sterile water each time. After six days, transfer the seedlings that grow consistently in the germination box to a hydroponic box containing 0.1× Hoagland nutrient solution. Replace the nutrient solution every two days to ensure that the hydroponic box has sufficient oxygen. After seven days of growth in the hydroponic box, begin to treat with saline-alkali solution. (5) Pre-selection of saline-alkali solution for testing Through comparative experiments, 0.1× Hoagland nutrient solution containing 100mM Na+ was selected as the appropriate concentration for screening alfalfa salt-alkali tolerant plants, where NaCl:NaHCO3=1:1, PH=8.5; (6) Screening of salt-alkali tolerant plants of different alfalfa varieties Twenty commercial seedlings grown in a hydroponic box for 7 days were treated with the 0.1× Hoagland nutrient solution containing 100 mM Na+ selected in step (5), with three replicates for each variety, and 30 seedlings in each replicate. The number of seedlings standing upright was counted after 48 hours, 60 hours, and 72 hours of saline-alkali solution treatment. After 72 hours of treatment, the seedlings were transferred to a hydroponic box containing 0.1× Hoagland nutrient solution for recovery treatment, and the number of seedlings standing upright was also counted; (7) Verification The alfalfa samples and salt-tolerant plants were separated by treatment with 0.1× Hoagland nutrient solution containing 100 mM Na+ for six hours. The salt-alkali tolerance of the plants was evaluated after they grew in vermiculite for four months. Samples were taken before and 24 hours after the salt-alkali treatment, and physiological indicators such as relative water content and electrolyte permeability were tested.

2. The method for rapidly screening alfalfa salt-alkali tolerant plants as claimed in claim 1, characterized in that: The comparative experiment in step (5) includes the following steps: In the preliminary experiment, at least two different alfalfa seedlings were selected as experimental materials, and treated with 0.1× Hoagland nutrient solution containing 0mM, 100mM, 150mM, 200mM, and 250mM Na+ for 72 hours respectively. The lodging state of the seedlings in each period was compared, and it was found that the seedlings of the two varieties in the 150mM, 200mM, and 250mM treatment groups all lodged, and the three solution concentrations could not distinguish the salt-alkali tolerance between the varieties; Based on the comparison of the lodging conditions of alfalfa seedlings, the seedlings that are more resistant to salt-alkali stress were identified, and 0.1× Hoagland nutrient solution containing 100mM Na+ was determined to be the appropriate concentration for screening salt-alkali resistant alfalfa plants, where NaCl:NaHCO3=1:1, PH=8.

5.

3. The method for rapid screening of salt-alkali tolerant alfalfa plants according to claim 1, characterized in that: The germination box in step (3) is made of transparent plastic material, and the ambient temperature of the constant temperature culture room is 15°C-25°C.

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