A method for large-scale rapid screening of salt tolerance in millet seedlings and its application

Abstract

This invention discloses a method and application for large-scale rapid screening of salt tolerance in millet seedlings, belonging to the field of agricultural science and technology. The method includes the following steps: (1) Before sowing, select plump, undamaged seeds as experimental materials, sow the experimental materials in a seed box, and add nutrient solution; (2) Place the seed box in a regular light incubator for cultivation, and subject the seedlings to salt stress treatment when they reach 2 leaves and 1 bud; (3) Rinse the leaves of the salt-stressed millet seedlings with deionized water, and dispense 3,3-diaminobenzidine staining solution into containers, add the pretreated plant tissue, and perform staining; (4) Boil the stained leaves in ethanol fixative, cool to room temperature, transfer the leaves to fresh fixative, and observe overnight at room temperature. The method for screening salt tolerance in millet seedlings according to this invention can complete the salt tolerance screening of a large number of millet varieties in a short period of time. It is time-efficient, easy to operate, low-cost, and yields reliable results.

Description

Technical Field

[0001] This invention relates to the field of agricultural science and technology, and in particular to a method and application for large-scale rapid screening of salt tolerance in millet seedlings. Background Technology

[0002] my country has over 500 million mu (approximately 33 million hectares) of saline-alkali wasteland and saline-alkali barrier arable land, of which 200 million mu (approximately 133 million hectares) are saline marshland with agricultural potential, accounting for about 10% of the country's arable land area. Under the severe pressure of decreasing arable land and insufficient freshwater resources, cultivating salt-tolerant crop varieties and developing saline-alkali agriculture by utilizing large areas of saline-alkali and desertified land and abundant saline water resources have become major issues of global concern.

[0003] In saline soils, large amounts of salt enter plant cells, causing sodium... + Toxicity and cellular dehydration disrupt the distribution of ions within cells, causing ion stress, osmotic stress, and secondary stress, primarily oxidative stress, in plants. Salt stress is a major environmental factor limiting crop growth and yield. Excessive salt content in the soil can create reverse osmotic pressure, displacing water from the roots and causing root tips to turn brown or dry out. Fluctuations in soil moisture can further exacerbate the problem of excessive soluble salt content, severely damaging the plant's root system and preventing it from absorbing water and nutrients, leading to symptoms such as wilting, yellowing, tissue necrosis, or stunted growth. High soil salinity also increases the incidence of root rot caused by cottony rot fungi. Therefore, soil salinization significantly reduces the yield of most crops.

[0004] Screening and planting salt-tolerant varieties is one of the effective methods to mitigate the harm of soil salinization. The screening of salt-tolerant crop varieties and the cultivation of new salt-tolerant varieties can not only fundamentally change the agricultural planting structure of saline-alkali land but also alter the ecological environment. Therefore, studying the physiological responses of different crops under salt stress, understanding the impact of salt stress on crop growth, and screening salt-tolerant crops are of great significance for improving land utilization in my country, improving saline-alkali land, and thus enhancing the productivity and economic benefits of saline-alkali land.

[0005] However, most crops are currently sweet soil plants that are sensitive to salinity, making it impractical to cultivate existing varieties in these saline soils. Millet (Setaria italica L.), a traditional and advantageous crop in my country, is a dual-purpose food and forage crop. Due to its robust root system, strong salt and drought tolerance, it has always been an important strategic reserve crop. In addressing future global warming and water scarcity, it plays a crucial role in ensuring food security and production in arid and infertile regions, thus holding significant strategic importance.

[0006] Millet, a grain crop belonging to the Poaceae family, has a long history of cultivation in my country, dating back to the Paleolithic era, primarily in the middle and upper reaches of the Yellow River. my country possesses abundant millet germplasm resources, especially numerous local varieties with wide genetic variation. However, current research on salt tolerance evaluation and key indicator screening of millet germplasm from different regions is scarce, severely hindering the widespread application of millet in saline-alkali lands. Therefore, establishing a large-scale, rapid screening method for salt-tolerant millet germplasm is of paramount importance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of traditional screening methods and provide a method suitable for laboratory culture conditions that can rapidly screen large quantities of salt-tolerant millet seedlings. This method can complete the salt tolerance screening of a large number of millet varieties in a short period of time, is time-saving, easy to operate, low in cost, and provides reliable results.

[0008] The technical solution adopted by this invention to solve its technical problem is as follows:

[0009] A method for large-scale rapid screening of salt tolerance in millet seedlings is provided, comprising the following steps:

[0010] (1) Before sowing, select plump, undamaged seeds as experimental materials, sow the experimental materials in the seed box, and add nutrient solution;

[0011] (2) Place the seed box in an ordinary light incubator for cultivation, and subject the seedlings to salt stress treatment when they grow to 2 leaves and 1 bud.

[0012] (3) Rinse the leaves of the grain seedlings after salt stress treatment with deionized water, and dispense the 3,3-diaminobenzidine staining solution into containers, add the pretreated plant tissue, and stain.

[0013] (4) Place the stained leaves in ethanol fixative, boil for 10 minutes, cool to room temperature, transfer the leaves to fresh fixative and leave at room temperature overnight, then observe.

[0014] Furthermore, in step (1), the experimental material is sown in a standardized 96-hole, 40*60*15cm seeding box, and 1 / 2 of the seeding box is filled with nutrient solution.

[0015] Furthermore, in step (1), the nutrient solution is composed of hydroponic nutrient solutions I, II, and III and iron salts;

[0016] The raw material composition and concentration of the hydroponic nutrient solution I are as follows:

[0017] NH4NO3 90-95g / L;

[0018] CaCl₂·2H₂O 4.5-5.5 g / L;

[0019] The raw material composition and concentration of the hydroponic nutrient solution II are as follows:

[0020] NaH2PO4·2H2O 70-75g / L;

[0021] K2SO4 85-90g / L;

[0022] The raw material composition and concentration of the hydroponic nutrient solution III are as follows:

[0023]

[0024] The raw material composition and concentration of the iron salt are as follows:

[0025] FeSO4·7H2O 10-14 g / L;

[0026] Na2EDTA·2H2O 14-16g / L;

[0027] The solvents for the hydroponic nutrient solutions I, II, III and the iron salts are all water.

[0028] Furthermore, in step (2), the culture conditions are 28°C, 16 hours of light, 8 hours of darkness, and 70% humidity.

[0029] Furthermore, in step (2), NaCl is used for salt stress treatment. The concentration of NaCl in the nutrient solution is 200 mmol / L, and the treatment time is 2 days.

[0030] Furthermore, in step (3), the concentration of the 3,3-diaminobenzidine staining solution is 5 mg / mL.

[0031] Furthermore, in step (3), the staining is carried out for 12-24 hours under conditions of 28°C, protection from light, and vacuum.

[0032] Furthermore, the vacuuming time is 0.5-1 hour.

[0033] Application of a method for rapid screening of salt tolerance in millet seedlings at a large scale in the identification, screening and breeding of salt-tolerant millet varieties.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention presents a method for large-scale rapid screening of salt tolerance in millet seedlings. It employs histochemical staining to identify the salt tolerance of millet germplasm in the laboratory. The method has a short experimental cycle, is not limited by external climatic conditions, and is inexpensive. It preliminarily establishes a relatively economical, convenient, and rapid method for screening salt-tolerant millet germplasm, which is of great significance for expanding the planting range of millet and developing and utilizing saline-alkali land. Furthermore, it provides salt-resistant and sensitive materials for millet cultivation and provides a basis for mining salt-tolerant genes from millet. Attached Figure Description

[0036] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0037] Figure 1 The images show DAB staining patterns of different varieties of millet, where (A) is a salt-tolerant variety control; (B) is a salt-sensitive variety control; (C) is a salt-tolerant variety treated with 200mM NaCl; and (D) is a salt-sensitive variety treated with 200mM NaCl.

[0038] Figure 2 The graph shows the determination of stress-related physiological indicators for different millet varieties. Detailed Implementation

[0039] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0041] I. Salt tolerance test of millet seedlings

[0042] (1) Selection of plants:

[0043] Before sowing, select plump, undamaged seeds as experimental materials, sow the experimental materials in a standardized 96-well seeding box (40*60*15cm), and add nutrient solution;

[0044] In this embodiment, the selected grain varieties are shown in Table 1.

[0045] Table 1. Test Material Numbers and Names

[0046]

[0047]

[0048] Hydroponic nutrient solutions I, II, III and iron salts were prepared according to the formulas in Tables 2, 3, 4 and 5.

[0049] Table 2 800× Nutrient Solution Stock Solution I Formula

[0050]

[0051] Table 3 800× Nutrient Solution Stock Solution II Formula

[0052]

[0053] Table 4. 800× Nutrient Solution Stock Solution III Formula

[0054]

[0055]

[0056] Table 5 400× Iron Salt Formula

[0057]

[0058] (2) Material cultivation

[0059] The seeding boxes were placed in a regular light incubator and cultured under the following conditions: 28°C, 16 hours of light, 8 hours of darkness, and 70% humidity for 2 weeks.

[0060] (3) Salt stress treatment

[0061] When the seedlings grew to 2 leaves and 1 bud, they were subjected to salt stress treatment. The treatment group was treated with 200 mmol·L⁻¹ salt solution prepared with 1 / 2 nutrient solution. - 1 Treatment with NaCl solution for 2 days, while the control group was still cultured normally in 1 / 2 nutrient solution;

[0062] (4) Preparation of staining solution

[0063] The preparation method for 3,3-diaminobezidine (DAB) is as follows:

[0064] Preparation of 200mM Na2HPO4·12H2O solution: Weigh 7.1628g of disodium hydrogen phosphate solid into a 100ml Erlenmeyer flask, add ddH2O and bring the volume to 100ml;

[0065] Preparation of DAB staining solution 1 mg / ml: Weigh 0.1 g DAB into a 100 ml beaker, add 90 ml H2O to dissolve, add 5 μl Tween 20 solution and 5 ml 200 mM Na2HPO4·12H2O solution, and adjust the pH to 3.0 with NaOH.

[0066] (5) Histochemical staining analysis

[0067] For both control and salt-treated millet seedlings, leaves were quickly rinsed with deionized water to remove dust or soil. The liquid was then blotted dry with lint-free paper. 5 mg / mL of 3,3-diaminobezidine (DAB) staining solution was dispensed into containers, and the pretreated plant tissue was added. The mixture was then evacuated using a vacuum pump and stained at 28°C in the dark for 12-24 hours. The stained leaves were then placed in ethanol fixative, boiled for 10 minutes, cooled to room temperature, and transferred to fresh fixative for overnight incubation at room temperature. The leaves were then photographed and observed.

[0068] Salt stress often leads to O 2- The accumulation of O2 can lead to oxidative damage. We investigated the O2 content in the leaves of various millet varieties through staining. 2- Accumulation analysis was conducted to detect the accumulation of peroxides in each material. In the presence of peroxidase, DAB is oxidized by H2O2, producing a reddish-brown precipitate. Based on the coloring after staining, we evaluated the salt tolerance of the varieties and screened for salt-tolerant millet varieties. Salt-tolerant varieties had light brown, colorless, or plant-derived pigments in their leaves, while salt-sensitive varieties had dark brown leaves. Therefore, we screened and obtained 10 salt-tolerant materials and 11 salt-sensitive materials.

[0069] II. Results and Analysis

[0070] Staining results as follows Figure 1 As shown, the darker the reddish-brown, the more O it represents. 2- The more O it accumulates; the lighter the reddish-brown color, the more O it represents. 2- The less accumulation, the better. Under normal growth conditions, there is no significant difference in coloring among varieties; after salt stress treatment, salt-tolerant millet varieties show lighter or almost no coloring, and the O2 content in the leaves is lower. 2- The accumulation of peroxides was relatively low, and the coloring was not significantly different from the control group, indicating that it has a strong peroxide scavenging ability under salt stress, making it a potential salt-tolerant variety; while the salt-sensitive millet variety stained darker, with leaves mostly turning dark brown, indicating that it had a lower peroxide scavenging ability under salt stress. 2- The variety has accumulated a relatively large amount of ROS and has a weak ability to scavenge ROS, making it a potentially salt-sensitive variety. This is consistent with our previous results on the salt tolerance of millet varieties, proving the feasibility of this method.

[0071] Through analysis of antioxidant staining under salt stress, 10 salt-tolerant varieties were screened: Qiu Mao Yu, Xiao Zao Gu, Qian Chuan Zi, Huang Su (Jin Yin Bao), Kuan Jing Zao Ai 3-1-1, Zhu Sha Gu, Hong Xiao Gu Zi, Ji Ai 9, Hong Miao Xiao Bai Mi, and Ji Gu 20. Eleven salt-sensitive varieties were also identified: 584, Bai Gu, Da Hei Gu, Xia Mi Yao, Ba Dou Zi, Liu Tiao Qing, Shi Li Xiang, E Si Zhu, 223, Da Luo Chui Gu, and Jing Gu 29. These results provide a reference for the cultivation of salt-tolerant millet varieties in the seedling stage and for research on the mechanisms of salt stress.

[0072] III. Verification of Coercion-Related Indicators

[0073] Materials with significant staining differences were used to measure different stress-related physiological indicators, and the results were consistent with the antioxidant staining screening results. For example... Figure 2 The figure shows the determination of stress-related physiological indicators for different millet varieties.

[0074] Salt tolerance of crops was assessed by measuring changes in physiological and biochemical indicators during plant physiological metabolism, such as conductivity, malondialdehyde (MDA), antioxidant enzyme activity (POD), and glutathione S-transferase (GST). We used a conductivity meter to measure the effect of stress on electrolyte extravasation from plant tissues; the thiobarbituric acid colorimetric method was used to determine changes in MDA content in millet under salt stress; and reagent kits provided by Suzhou Keming Biotechnology Co., Ltd. were used to measure the activities of antioxidant enzymes such as peroxidase (POD) and glutathione S-transferase (GST) in the aboveground parts of millet.

[0075] Different stress-related physiological indicators were measured using materials with significant staining differences. The results showed that the salt-tolerant varieties, Zhusha Valley and Hongmiao Xiaobai Rice, had lower conductivity and MDA content at the three selected time points than the sensitive varieties, while their GST and POD antioxidant enzyme activities were higher, indicating stronger salt tolerance. These results validated the results of the antioxidant staining screening.

[0076] Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-mentioned technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-mentioned technical features or their equivalent features without departing from the inventive concept. For example, technical solutions formed by substituting the above-mentioned features with technical features disclosed in this application (but not limited to) that have similar functions.

Claims

1. A method for large-scale rapid screening of salt tolerance of millet seedling stage, characterized in that, The method comprises the following steps: (1) before sowing, select full and undamaged seeds as the test material, sow the test material in a sowing box, and add nutrient solution; (2) place the sowing box in a common light incubator for culture, and perform salt stress treatment when the seedlings grow to 2 leaves and 1 core; (3) rinse the leaves of the cereal seedlings after salt stress treatment with deionized water, and divide 3,3-diaminobenzidine staining solution into containers, add the pretreated plant tissues, and perform staining; (4) place the stained leaves in ethanol fixing solution, boil for 10 min, cool to room temperature, transfer the leaves into fresh fixing solution at room temperature overnight, and then observe; In step (2), NaCl is used for salt stress treatment, the concentration of NaCl in the nutrient solution is 200 mmol / L, and the treatment time is 2 days; In step (3), the concentration of 3,3-diaminobenzidine staining solution is 5 mg / mL.

2. The method for screening salt tolerance of foxtail millet at seedling stage on a large scale according to claim 1, characterized in that, In step (1), the test material is sowed in a uniform standard 96-hole, 40*60*15 cm sowing box, and 1 / 2 of the nutrient solution is added to the sowing box.

3. The method for screening salt tolerance of foxtail millet at seedling stage on a large scale according to claim 1, characterized in that, In step (1), the nutrient solution is composed of water culture nutrient solution I, II, III and iron salt; The raw material composition and concentration of the water culture nutrient solution I are as follows: NH4NO3 90-95 g / L; CaCL2·2H2O 4.5-5.5 g / L; The raw material composition and concentration of the water culture nutrient solution II are as follows: NaH2PO4·2H2O 70-75 g / L; K2SO4 85-90 g / L; The raw material composition and concentration of the water culture nutrient solution III are as follows: The raw material composition and concentration of the iron salt are as follows: FeSO4·7H2O 10-14 g / L; Na2EDTA·2H2O 14-16 g / L; The solvent of the water culture nutrient solution I, II, III and iron salt is water.

4. The method for screening salt tolerance of foxtail millet at seedling stage on a large scale according to claim 1, characterized in that, In step (2), the culture conditions are 28°C, 16 h light, 8 h darkness, and the humidity is 70%.

5. The method for screening salt tolerance of foxtail millet at seedling stage on a large scale according to claim 1, characterized in that, In step (3), the staining is performed at 28°C, in the dark, under vacuum conditions for 12-24 h.

6. The method for screening salt tolerance of foxtail millet at seedling stage on a large scale according to claim 5, characterized in that, The vacuum time is 0.5-1 h.

7. The method according to any one of claims 1-6 in the identification, screening and cultivation of salt-tolerant varieties of millet.

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