Method for preparing macro-element culture medium from purified nitro compound fertilizer extract

Abstract

This invention relates to the field of biological culture media and their preparation methods, and particularly to a method for preparing macro-element culture solutions for plant culture media from purified nitro compound fertilizer extracts. The macro-element culture solution in this application uses nitro compound fertilizer produced by modern technology. Through purification processes, the stability and effectiveness of its main nutrient components and proportions are ensured. Nitro-ammonia-containing nitro compound fertilizer is used as a reagent raw material to prepare reagent-grade biological culture media. The raw materials used in biological culture media are widely available and inexpensive, the purification methods are simple and clear, and the application prospects are broad and the solution is safe and reliable. The macro-element culture solution can be used to prepare various reagent-grade culture media containing ammonium nitrate, and can be widely used in laboratory microbial culture, plant tissue culture propagation, and the cultivation of virus-free seedlings.

Description

Technical Field

[0001] This invention relates to the field of biological culture media and their preparation methods, and more particularly to a method for preparing a macro-element culture medium for plant culture media from purified nitro compound fertilizer extracts. Background Technology

[0002] Plant tissue culture technology, developed since the mid-20th century, has become an important means of rapid plant propagation, virus-free seedling cultivation, germplasm resource preservation, and genetic improvement. The composition of the culture medium directly determines the growth, differentiation, and metabolic activity of the cultured material. Among numerous culture medium formulations, those containing ammonium nitrate (such as Murashige and Skoog (MS) medium and N69 medium) are the most widely used due to their unique chemical composition. Taking MS medium as an example, its formulation contains a high concentration of inorganic salts and ions, forming a stable ion balance system. Basic MS medium contains ammonium nitrate per liter of working solution (… 1650 mg, potassium nitrate ( 1900 mg, calcium chloride dihydrate ( 440 mg, magnesium sulfate heptahydrate ( 370 mg, potassium dihydrogen phosphate ( The formula contains 170 mg of nitrates, as well as trace elements (potassium iodide (0.83 mg), boric acid (6.2 mg), manganese sulfate (22.3 mg), zinc sulfate (8.6 mg), sodium molybdate (0.25 mg), copper sulfate (0.025 mg), and cobalt chloride (0.025 mg). Iron salts consist of ferrous sulfate (27.8 mg) and disodium EDTA (37.3 mg), and organic matter (inositol (100 mg), glycine (2 mg), thiamine hydrochloride (vitamin B1, 0.1 mg), pyridoxine hydrochloride (vitamin B6, 0.5 mg), and niacin (0.5 mg). Sugars are typically added as sucrose. This formula has a high nitrate content, a suitable ratio of ammonium nitrogen to nitrate nitrogen, and the absolute and relative content of nutrients can fully meet the needs of plant cells for nitrogen, potassium, calcium, magnesium, phosphorus, and other major elements, while maintaining pH stability and osmotic pressure balance. Therefore, MS and its derived media are widely used for callus induction, adventitious shoot differentiation, rooting culture and virus-free seedling propagation in various plants such as rice, tobacco, Arabidopsis, potato and citrus, and have become one of the most classic basic media in the field of plant tissue culture.

[0003] However, ammonium nitrate-containing culture media face significant technical bottlenecks and safety risks in practical applications. Ammonium nitrate ( Ammonium nitrate is the sole source of ammonium nitrogen and one of the main sources of nitrate nitrogen in the aforementioned culture media. However, its chemical properties are extremely unique: ammonium nitrate is a strong oxidizing agent and is prone to violent decomposition or even explosion when heated, impacted, or in contact with reducing agents. Therefore, it is listed in the strict control catalog of civilian explosives in various countries. In my country, the production, sale, storage, transportation, and use of ammonium nitrate are all subject to the "Regulations on the Safety Management of Civil Explosives" and special supervision by the public security department. No individual or enterprise may store or sell ammonium nitrate without permission; even if research institutions or biotechnology companies need to purchase small quantities for culture media preparation, they must go through a strict filing procedure, entrust a special vehicle with dangerous goods transportation qualifications for transportation, and complete the handover and short-term storage under the supervision of the public security department. This series of control measures results in a long acquisition cycle, cumbersome procedures, and high transportation costs for ammonium nitrate. Furthermore, the user unit must be equipped with explosion-proof storage facilities, which further increases the cost of culture media preparation.

[0004] Due to the aforementioned policy and safety factors, the vast majority of domestic biochemical companies are unable to directly use industrial-grade or reagent-grade ammonium nitrate to prepare MS media. Currently, the MS media products containing ammonium nitrate circulating in the market mainly rely on imported pre-prepared ammonium nitrate powder, which domestic companies then repackage and resell. This model is not only limited by the stability and price fluctuations of import channels but also keeps the final price of the media high. Meanwhile, companies and research teams in the agricultural seedling field are attempting to circumvent the use of ammonium nitrate through formulation improvements. For example, they are trying to replace ammonium nitrate with single or compound nitrogen sources such as calcium nitrate, sodium nitrate, ammonium sulfate, urea, and ammonium dihydrogen phosphate, while adjusting the concentrations of other ions to maintain balance. However, existing alternatives generally suffer from the following problems: (1) the alternative nitrogen source cannot simultaneously provide a suitable ratio of ammonium nitrogen and nitrate nitrogen, resulting in slow plant cell growth, decreased differentiation rate, or exacerbated vitrification; (2) changes in ionic strength or pH buffering capacity cause precipitation of the culture medium or degradation of its components after sterilization; (3) different plant species or explants have large differences in adaptability to the alternative formulation, poor reproducibility, and are difficult to use as a universal culture medium. To date, no mature formulation has emerged that can completely replace ammonium nitrate while maintaining the original stability, broad spectrum, and culture effect of MS culture medium. This seriously restricts the localization and promotion of plant tissue culture technology and the industrialization of large-scale agricultural seedling production.

[0005] Therefore, there is an urgent need to develop a safe, readily available, low-cost method to effectively replace reagent-grade ammonium nitrate for preparing culture media that meet the requirements of plant tissue culture. In recent years, nitro-compound fertilizers (made with ammonium nitrate as the base nutrient and the addition of phosphorus, potassium, calcium, magnesium, etc. through physical or chemical processes) widely used in modern agriculture contain a high proportion of nitrate nitrogen and ammonium nitrogen, and the proportions of macronutrients are controllable. However, nitro-compound fertilizers are agricultural-grade products containing certain amounts of impurities, anti-caking agents, fillers, and heavy metal ions, making them unsuitable for direct use in the preparation of reagent-grade culture media with strict purity requirements. If harmful impurities in nitro-compound fertilizers can be removed through appropriate purification processes while retaining their effective nitrogen source and accompanying macronutrients, and if their final ionic composition can match the equivalent contribution of ammonium nitrate and other salts required by classic culture media such as MS, it is hoped that a safe, low-cost, and large-scale alternative to ammonium nitrate-containing culture media can be achieved. This technical route not only avoids the hazardous and explosive control issues associated with ammonium nitrate but also significantly reduces the production cost of culture media, providing a reliable material basis for the sustainable development of plant tissue culture and agricultural biotechnology. Summary of the Invention

[0006] In order to provide a safe, low-cost, widely available and unregulated ammonium nitrate-containing composition and plant culture medium, this application provides a method for preparing a macro-element culture medium from purified nitro compound fertilizer extract.

[0007] In a first aspect, this application provides a method for preparing a plant culture medium from a purified nitro compound fertilizer, comprising the following steps:

[0008] 1) Determine the composition and content of ions in the target culture medium and nitro compound fertilizer containing ammonium nitrate;

[0009] 2) Purification of nitro compound fertilizer;

[0010] 3) Use the ammonium nitrate in the purified nitro compound fertilizer to match the ammonium nitrate requirements in the target culture medium, calculate the required mass of compound fertilizer, and set it as X;

[0011] 4) Verify whether the other ions brought by the compound fertilizer of mass X are within the concentration range required by the target culture medium; if they are within the range required by the target culture medium, calculate and supplement the amount of other ions, and the concentration of each ion must not exceed the maximum concentration allowed by the target culture medium; if they are not within the range required by the target culture medium, adjust the concentration of ions exceeding the range in the compound fertilizer so that the ammonium nitrate in the compound fertilizer matches the minimum ammonium nitrate requirement of the target culture medium, and the concentration of ions formed by other elements is within the maximum concentration allowed by the target culture medium. Calculate and supplement the amount of other ions to obtain a macro-element culture solution.

[0012] The concentrations of each ion in the macro-element culture medium fluctuate by less than 3% compared to the corresponding element content in the target culture medium.

[0013] The calculation method for the minimum ammonium nitrate requirement in macronutrient culture medium is as follows: the total amount of ammonium nitrate in the target culture medium minus the sum of ammonium nitrate that can be obtained from the target culture medium by resolving the compounds of the other macronutrients besides ammonium nitrate.

[0014] Macroelements refer to elements that are present in high concentrations (usually 1000-1000 mg / L) in the culture medium. to (mol / L) and are a class of mineral nutrients essential for the growth, cell division and differentiation of organisms; including nitrogen (corresponding compounds: potassium nitrate, ammonium nitrate), phosphorus (corresponding compounds: sodium dihydrogen phosphate or potassium dihydrogen phosphate), potassium (corresponding compounds: potassium nitrate, potassium dihydrogen phosphate), calcium (corresponding compound: calcium chloride), magnesium (corresponding compound: magnesium sulfate), and sulfur (corresponding compound: magnesium sulfate).

[0015] The target culture medium can be an existing culture medium or an improvement on an existing culture medium according to actual needs.

[0016] Preferably, the concentrations of various ions in the macro-element culture medium are greater than or equal to the corresponding ion concentrations in the target culture medium.

[0017] Furthermore, the content of ammonium nitrate in the nitro compound fertilizer is greater than 30 wt%.

[0018] Furthermore, the nitro compound fertilizer containing nitrate nitrogen contains three macronutrient elements: nitrogen, phosphorus, and potassium. Its nitrogen component includes nitrate nitrogen. The total phosphorus nutrient of the product is calculated as phosphorus pentoxide, and the total potassium nutrient is calculated as potassium oxide. The mass ratio of nitrate nitrogen to total phosphorus nutrient calculated as phosphorus pentoxide in the product is greater than 3:5, and the mass ratio of nitrate nitrogen to total potassium nutrient calculated as potassium oxide is greater than 100:169. Based on the total mass of the compound fertilizer, the mass percentage of ammonium sulfate is less than 20%.

[0019] Furthermore, the heavy metal content (calculated as lead) in the purified nitro compound fertilizer is ≤ 0.35 mg / L, of which arsenic is ≤ 0.07 mg / L; the water-insoluble matter content is less than 10 ppm.

[0020] Furthermore, the purification process for nitro compound fertilizer includes the following steps:

[0021] Dissolution and initial filtration: Dissolve the nitro compound fertilizer in deionized water and filter it through multi-stage filter paper to remove insoluble particles;

[0022] Adsorption and impurity removal: Activated carbon is added to the filtrate for adsorption treatment, and the activated carbon is removed by filtration;

[0023] Oxidation and pH adjustment: Add an oxidant to oxidize ferrous iron to ferric iron, oxidize arsenic trivalent to arsenic pentavalent, and adjust the pH to weakly acidic. After standing, filter.

[0024] Arsenic removal by ion exchange: The filtrate passes at a low speed through an ion exchange resin loaded with zirconium compounds;

[0025] Fine filtration and volume determination: Filtration is carried out using μm-level filter membranes.

[0026] Furthermore, the purification process for nitro compound fertilizer is as follows:

[0027] 1) Weigh 5.3-17.70 grams of nitro compound fertilizer and pour it into 50-200 ml of deionized water, stir and mix to prepare a compound fertilizer solution;

[0028] 2) Filter the compound fertilizer solution sequentially through 5 μm filter paper and 1 μm filter paper, and collect the filtrate;

[0029] 3) Weigh 1-2 grams of acid-washed powdered activated carbon, slowly pour it into the filtrate, and stir for 15-20 minutes;

[0030] 4) Filter the solution through 1 μm filter paper, collect the filtrate, and ensure that there are no tiny black carbon particles at the bottom of the cup. Then filter it through 0.45 μm filter paper and collect the clear solution.

[0031] 5) Adjust the pH to 5.5-6.5 with 0.1-1.0 mol / L potassium hydroxide solution, stir, and let stand; add acid-washed activated carbon powder to remove organic impurities;

[0032] 6) Filter using 0.45 μm filter paper and collect the filtrate;

[0033] 7) Add an iminodiacetic acid-type chelating resin or a zirconium-based ion exchange resin column to allow most of the residual metal ions to be adsorbed by the resin; after filtration, use deionization to clean the resin, and pour the cleaning solution into the filtrate.

[0034] 8) Confirm that the pH value is within the range of 5.5-6.5;

[0035] 9) Filter using a 0.22-0.45 μm microporous membrane until a clear solution is obtained;

[0036] 10) If the purity of the nitro compound fertilizer extract does not meet the requirements of claim 4, repeat any one or more of steps 3) to 8).

[0037] Preferably, in step 4), the solution is filtered using 0.45 μm filter paper. After collecting the clear solution, the pH of the solution is adjusted to between 6 and 0.2. 0.1-0.3 mol / L ammonium sulfide solution is added while stirring until no new precipitate is formed. Then, the addition is stopped, and the solution is allowed to stand before proceeding to step 5.

[0038] Preferably, in step 4), the solution is filtered using 0.45 μm filter paper, and after collecting the clear solution, 0.3-3% hydrogen peroxide is added. The amount added is 0.5-3.0 mL per 100 mL of filtrate, and the mixture is stirred for 10-30 min.

[0039] Adding hydrogen peroxide oxidizes excess sulfur, oxidizes ferrous iron to ferric iron, and oxidizes arsenic trivalent to arsenic pentavalent.

[0040] Preferably, the amount of iminodiacetic acid chelating resin used is 5-20 g per 100 mL of solution.

[0041] Secondly, this application provides a macro-element culture medium obtained by the preparation method of this application for use in culture media. The macro-element culture medium is used to prepare culture media for laboratory plant cell culture, plant tissue culture propagation culture media, and plant culture media for virus-free seedling cultivation.

[0042] Furthermore, a macro-element culture medium was used to prepare MS medium, comprising a composition of nitro compound fertilizer 30-4-0, potassium nitrate, potassium dihydrogen phosphate, calcium chloride dihydrate, calcium nitrate tetrahydrate, potassium sulfate, and magnesium chloride in a mass ratio of 820-2530:800-2000:10-170:0-440:0-706:0-174:0-143.

[0043] Furthermore, the macro-element culture medium is a macro-element culture medium used to prepare N69 culture medium, comprising, by weight, a composition of potassium nitrate, nitro compound fertilizer 30-6-0, potassium dihydrogen phosphate, monoammonium phosphate, ammonium chloride, magnesium nitrate hexahydrate, and calcium nitrate tetrahydrate in a mass ratio of 444-1455:500-1208:0-1700-144:0-192:0-267.

[0044] The ammonium nitrate-based compound fertilizer containing nitrate nitrogen contains three macronutrient elements: nitrogen, phosphorus, and potassium. Its nitrogen component includes nitrate nitrogen. The total phosphorus nutrient content of the product is calculated as phosphorus pentoxide, and the total potassium nutrient content is calculated as potassium oxide. The mass ratio of nitrate nitrogen to total phosphorus nutrient content calculated as phosphorus pentoxide in the product is greater than 3:5, and the mass ratio of nitrate nitrogen to total potassium nutrient content calculated as potassium oxide is greater than 100:169. Furthermore, based on the total mass of the compound fertilizer, the mass percentage of ammonium sulfate is less than 20%.

[0045] Thirdly, this application provides a plant culture medium, including a macro-element culture solution obtained by the preparation method described in this application or a macro-element culture solution as described in this application.

[0046] The basic concept of this application is as follows: When cultivating plants using MS medium, plant cells meet their growth and development needs by absorbing various ionic nutrients provided by the MS medium nutrient solution; therefore, ensuring that the concentration ratio of various ions in the prepared solution is as consistent as possible with the original formula is crucial for success. Figure 1 shows the mass and molar concentration of each macronutrient in one liter of MS medium working solution.

[0047] Table 1. Macroelement composition and ion concentration of MS culture medium

[0048] ;

[0049] As can be seen from Table 1, most elements in MS medium ultimately exist in ionic form in the solution. The overall amount of ammonium nitrate used can be reduced by breaking down some elements and increasing the amount of some elements. For example, 2.99 mM calcium nitrate and 5.98 mM ammonium chloride can be used to obtain 2.99 mM calcium chloride and 5.98 mM ammonium nitrate. Even so, considering the overall ion balance, content consistency, and consistency of ionic strength or pH buffering capacity, it is impossible to completely replace all the ammonium nitrate in the original formula.

[0050] To find a suitable substitute, and taking into full consideration the overall ion balance, content consistency, and consistency of ionic strength or pH buffering capacity, the purified nitro-compound fertilizer in each liter of MS culture medium nutrient solution prepared in this application must meet the following standards: 10.37mM ≤ ammonium nitrate ≤ 20.6mM, with other nutrients not exceeding the required concentration, and the raw material source must be safe and widely available; at the same time, as one of the raw materials for the culture medium, its high purity meets the requirements for heavy metal content in GB / T 659-2011 "Chemical Reagents Ammonium Nitrate": heavy metals ≤ 10mg / KG, water-insoluble matter ≤ 50mg / KG; and batch differences should be reduced, with arsenic ≤ 2mg / KG.

[0051] Beneficial effects: 1. The macro-element culture medium in this application uses nitro compound fertilizer produced by modern technology. Through purification process, its main nutrient components and proportions are kept stable and effective. Nitro compound fertilizer (commercially available product) containing more than 30% ammonium nitrate is used as reagent raw material to prepare reagent-grade biological culture medium. The raw materials used in biological culture medium are widely available and inexpensive. The purification method is simple and clear. It has broad application prospects and is safe and reliable. The macro-element culture medium can be used to prepare various reagent-grade culture medium raw materials containing ammonium nitrate. It can be widely used in laboratory microbial culture, plant tissue culture propagation and virus-free seedling cultivation. Attached Figure Description

[0052] Figure 1 This is a control diagram of Arabidopsis seed growth in Group A 1 / 2MS medium in Example 4 of this invention;

[0053] Figure 2 This is a control diagram of Arabidopsis seed growth in Group B 1 / 2MS medium in Example 4 of this invention;

[0054] Figure 3 This is a control diagram of Arabidopsis seed growth in group C 1 / 2MS medium in Example 4 of this invention;

[0055] Figure 4 This is a control diagram of Arabidopsis seed growth in Group D 1 / 2MS medium in Example 4 of this invention. Detailed Implementation

[0056] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] This application uses high-tower melt granulation to produce nitro compound fertilizer (ammonium nitrate content greater than 30%, and all fertilizer manufacturers that use ammonium nitrate as the main raw material have similar products in production and sales, excluding products that use urea as the raw material): N, P, K ratio is (e.g., 30-4-0, 32-4-0, 30-5-0, 32-5-0, 32-6-0, etc.), and the heavy metal content must comply with the national standard GB 38400-2019.

[0058] This application uses commercially available fertilizer (nitro compound fertilizer 30-4-0) as raw material to prepare MS culture medium nutrient solution: the fertilizer is a compound fertilizer made by high tower melting process of ammonium nitrate, ammonium sulfate, monoammonium phosphate and a small amount of magnesium sulfate (Table 2, the feed ratio table of ammonium compound fertilizer model of this fertilizer plant is 30:4:0, white) provided by KLCX Company.

[0059] The composition of the nitro compound fertilizer used in this application is shown in Table 2.

[0060] Table 2. Composition list of nitro compound fertilizer 30-4-0 used in this application

[0061]

[0062] Theoretically, by dissolving 1.5 grams of this fertilizer in 1 liter of water, the concentrations of various ions can be approximately obtained as follows: 18.41mM 14.45 mM 1.47 mM 1.24 mM : 0.11 mML.

[0063] According to the relevant national standard for compound fertilizers in my country, GB 15063-2020 "Compound Fertilizers", the deviations of each raw material fully meet the requirements for use as material for plant tissue culture; however, the fertilizer production process is complex, and the proportions and purity of the raw materials may be slightly adjusted according to the actual situation, resulting in some deviations in each batch of finished product. The component test results of the nitro compound fertilizer 30-4-0 used in this application are shown in Table 3.

[0064] Table 3. List of elemental contents of nitro-compound fertilizer 30-4-0

[0065]

[0066] Table 3 shows that this compound fertilizer (produced through a high-tower melt granulation process, with the addition of monoammonium phosphate, ammonium sulfate, and magnesium sulfate to modify ammonium nitrate, thus altering the crystal structure of ammonium nitrate and reducing friction between particles during granulation) is sold at agricultural fertilizer stations across various regions, and the manufacturer holds a production license and has legally registered the fertilizer. Therefore, it can be judged to be safer than MS medium mixed powder (containing no sugar or agar, with ammonium nitrate and potassium nitrate accounting for approximately 75%). Tests show that the main elements of this nitro fertilizer are highly pure, with the total amount of conventional harmful heavy metals less than 15 mg / KG, and water-soluble phosphorus accounting for 80% of available phosphorus.

[0067] Table 4. List of Heavy Metal Detection Tests for Nitro Compound Fertilizer 30-4-0

[0068]

[0069] Preparation method of macro-element culture medium:

[0070] 1) Determine the composition and content of ions in the target culture medium and nitro compound fertilizer containing ammonium nitrate;

[0071] 2) Purification of nitro compound fertilizer;

[0072] 3) Use the ammonium in the purified nitro compound fertilizer to match the ammonium requirements in the target culture medium, calculate the required mass of compound fertilizer, and set it as X;

[0073] 4) Verify whether the other ions brought by the compound fertilizer of mass X are within the concentration range required by the target culture medium; if they are within the range required by the target culture medium, calculate and supplement the amount of other ions, and the concentration of each ion must not exceed the maximum concentration allowed by the target culture medium; if they are not within the range required by the target culture medium, adjust the concentration of ions exceeding the range in the compound fertilizer so that the ammonium nitrate in the compound fertilizer matches the minimum ammonium nitrate requirement of the target culture medium, and the concentration of other ions is within the maximum concentration allowed by the target culture medium. Calculate and supplement the amount of other ions to obtain a macro-element culture solution.

[0074] Example 1: Preparation of MS medium using macro-element culture medium:

[0075] Using 17.70 grams of nitro-compound fertilizer 30-4-0 (the dosage is calculated according to Table 3), insoluble impurities were removed and soluble impurities were reduced through dissolution, filtration, and adsorption. The purified product replaced ammonium nitrate and some chemical elements in the MS medium's macronutrient solution. Combined with other deficient elements, a 20-fold concentrated MS macronutrient stock solution was prepared. After a 20-fold dilution, the concentrations of various ions in the prepared MS medium macronutrient stock solution were essentially consistent with the concentrations of the standard MS medium macronutrient working solution (Table 1).

[0076] 1. Purification of nitro compound fertilizer:

[0077] 1.1 Weigh 17.70 g of nitro compound fertilizer 30-4-0 and pour it into a beaker containing 80 ml of deionized water. Stir gently to avoid foaming and ensure the compound fertilizer is completely dissolved. The liquid temperature will drop significantly. Let it stand for 5 minutes until the temperature returns to room temperature, and ensure the outer wall of the beaker is dry. Pour the solution into a clean Erlenmeyer flask lined with disposable 5 μm No. 2 qualitative filter paper. After pouring, rinse the inner wall of the beaker and the glass rod repeatedly with 20 ml of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. Finally, rinse the filter paper three times with 10 ml of deionized water, collect the residual solution, and combine the filtrate into a clean beaker. Rinse the inner wall of the Erlenmeyer flask repeatedly with 15 ml of deionized water, and combine the rinsing water into the beaker.

[0078] 1.2 Pour the filtrate from the beaker into a clean Erlenmeyer flask funnel containing disposable 1 μm glass fiber filter paper for filtration. After pouring, rinse the inner wall of the beaker and the glass rod repeatedly with 20 mL of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. Rinse the filter paper three times with 10 mL of deionized water. Combine the collected filtrate and rinsing solution and pour them into a 400 mL clean beaker. After pouring, rinse the inner wall of the Erlenmeyer flask repeatedly with 15 mL of deionized water. Combine the rinsing water and pour it into the beaker.

[0079] 1.3 Place the beaker on a magnetic stirrer and stir at 150 rpm. Adjust the pH of the solution to 5.5 ± 0.2 with 0.1 mol / L potassium hydroxide (KOH) under the detection of a pH meter, and record the amount of potassium hydroxide used.

[0080] 1.4 Weigh 1.5 grams of acid-washed coconut shell activated carbon powder (to remove organic impurities), slowly pour it into a beaker containing filtrate, and adjust the magnetic stirrer to 250 rpm and stir for 45 minutes.

[0081] 1.5 Pour the liquid in the beaker into a disposable 0.45 μm filter paper flask funnel for vacuum filtration. After filtration, rinse the beaker and glass rod with 20 mL of deionized water. Pour all the rinsing water into the funnel for vacuum filtration. Then rinse the carbon powder and filter paper three times with 15 mL of deionized water to recover the filtrate. After ensuring that there are no tiny black carbon particles at the bottom of the beaker, pour the collected liquid into a clean 500 mL beaker. After pouring, rinse the flask with 20 mL of deionized water. Combine the rinsing liquid and pour it into the beaker.

[0082] 1.6 Inject the filtrate from the beaker into a column containing 5 g of zirconium-based ion exchange resin at a flow rate of 0.5 mL / min. After the filtrate has been injected, rinse the beaker repeatedly with 20 mL of deionized water. Continue to inject the rinsing solution into the exchange column. Finally, rinse the exchange column with 3 BV of deionized water and collect the filtrate in a 500 mL beaker. Confirm that the pH value is within the range of 5.8 ± 0.2.

[0083] 1.7 The filtrate in the beaker is filtered through a clean Erlenmeyer flask funnel fitted with a 0.22 μm disposable microporous filter membrane. After filtration, the inner wall of the beaker and the glass rod are repeatedly rinsed with 20 mL of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. The filter paper is then rinsed three times with 10 mL of deionized water. The collected filtrate and rinsing solution are combined, and finally, the filtrate is poured into a dry and clean beaker. The inner wall of the Erlenmeyer flask is then rinsed with 20 mL of deionized water (to remove impurities and sterilize; the volume can be adjusted to 400 mL before concentration testing).

[0084] 1.8 Finally, dilute the solution to approximately 400 ml. At this point, each 40 ml solution contains a mixture of 20.6 mmol of ammonium ions, 1.8 mmol of nitrate ions, 0.09 mmol of potassium ions, 1.13 mmol of dihydrogen phosphate ions (without deducting trace losses from ion exchange), 0.79 mmol of sulfate ions, and approximately 0.01 mmol of magnesium ions. Label the solution, seal it, and it can be stored at room temperature for a long period of time.

[0085] When obtaining the solution from step 1.8 (for high-standard physiological and biochemical research), the content of each major ion (ammonium ion, nitrate ion, sulfate ion, phosphate ion, magnesium ion, potassium ion, chloride ion, iron ion, etc.) should be detected using instruments such as ion chromatography. Then, the required volume should be determined in reverse (the solution should be clear and free of visible impurities, the nutrient composition should meet the preparation requirements, and the heavy metal ion threshold should be determined according to the needs of the target plant culture to ensure it meets the standards). Steps 1.3-1.7 can be repeated.

[0086] The above steps are the purification process for compound fertilizer, which can basically completely remove the anti-caking powder and anti-caking oil in the fertilizer, and effectively remove some metals. It fully meets the basic standards of most plants for heavy metals in MS medium macronutrient 20 times mother liquor: lead ≤0.35 mg / L, arsenic ≤0.07 mg / L, and water-insoluble matter less than 10 ppm (reagent ammonium nitrate chemically pure GB / T659-2011). Thus, the purification of nitro compound fertilizer, one of the raw materials for MS medium macronutrients, is completed.

[0087] 2. Using the purified nitro compound fertilizer extract described above, a macro-element culture medium for MS medium was prepared. The raw materials used are shown in Table 5, and the preparation process is as follows:

[0088] 2.1 Weigh 18600 mg of potassium nitrate and 210 mg of potassium dihydrogen phosphate using a high-precision electronic scale, add them to the beaker in step 1.9, and stir to dissolve using a magnetic stirrer at 250 rpm; then bring the volume up to 500 ml to form mother liquor 1, label it, and store it in a sealed container at room temperature.

[0089] 2.2 Weigh 2210 mg of calcium chloride dihydrate, 3520 mg of calcium nitrate tetrahydrate, and 1420 mg of magnesium chloride and pour them into a beaker containing 300 ml of deionized water. Stir until completely dissolved, and bring the volume up to 500 ml to form mother liquor 2. Label the mother liquor, seal it, and store it at room temperature.

[0090] 2.3 When preparing 1 liter of MS medium working solution, take 50 ml of solution from each of the mother solutions 1 and 2, stir them separately and slowly pour them into a container containing 500 ml of deionized water. This solution is used as the macro-element solution for preparing MS medium, which is the macro-element culture medium for MS medium.

[0091] 3. Prepare 1 / 2 MS medium using the above-mentioned macro-element culture medium:

[0092] 3.1 Add the micronutrients and organic matter required for MS medium to the prepared 500 ml macronutrient mixed solution, then add sucrose and other materials as needed, and make up to 1000 ml to complete the preparation of 1 liter of MS medium.

[0093] 3.2 As shown in Table 9 (Preparation of 1 / 2 MS medium with half the amount of macroelements): Take 25 ml of each of stock solution 1 and stock solution 2, pour them into a container of 500 ml of deionized water, add the full amount of trace elements and organic matter, and add sugar and other materials as needed, and make up to 1000 ml to prepare 1 liter of 1 / 2 MS medium.

[0094] Table 5. List of macro-element culture medium formulations for MS medium

[0095] Example 2: Preparation of N69 culture medium using macro-element culture medium:

[0096] This embodiment uses nitro compound fertilizer 30-6-0 (raw materials are ammonium nitrate, ammonium sulfate, monoammonium phosphate, and a small amount of magnesium sulfate). The heavy metal content is unknown, and it is labeled as a fully water-soluble fertilizer. The compound fertilizer factory's self-inspection data shows: total nitrogen 29.78%, available phosphorus 6.48%, nitrate nitrogen 12.32%, and free water and water-insoluble matter 0.3%. Based on this, we obtained the following composition ratio for this fertilizer: ammonium nitrate 70.4%, ammonium sulfate 17.8%, monoammonium phosphate 10.5%, and magnesium sulfate approximately 1%. The macro-element scheme for preparing N69 culture medium using 30-6-0 fertilizer is as follows: use fertilizer stock solution + dry powder. This culture medium can also be prepared using Case 1 30-4-0 (Table 8).

[0097] The plant culture medium in this embodiment is a modified MS medium, mainly suitable for in vitro culture of woody plants such as pear trees. Tobacco is its earliest and classic application, especially in anther culture and haploid breeding. Among the macroelements, ammonium nitrate of 725 mg / L (that is, the raw material requires ammonium nitrate to provide 4.05 mmol / L-9.06 mmol / L, and ensures that there are no additional nutrients, and the other effective components must not exceed the specified values ​​of N69 medium standard) has higher threshold requirements for heavy metals in in vitro culture.

[0098] Table 6: List of components of existing conventional N69 culture medium

[0099]

[0100] First, the 30-6-0 nitro compound fertilizer was purified. Then, it was used to replace part of the high-purity chemical reagents in the original N69 culture medium, and other necessary chemical reagents were added. Finally, 500 ml of 20-fold concentrated nitro compound fertilizer extract was prepared. After 20-fold dilution, the concentrations of all macro-elements (ammonium, nitrate, potassium, calcium, magnesium, phosphate, chloride, sulfate, etc.) in this nitro compound fertilizer extract were basically consistent with the macro-element ratios in the N69 culture medium formula (as shown in Table 6).

[0101] 1. Purification of nitro compound fertilizer:

[0102] 1.1 Weigh 5.4 g of ammonium nitrate phosphate N:P:K compound fertilizer with a ratio of 30-6-0 and pour it into a beaker containing 80 ml of deionized water. Stir gently to avoid foaming and ensure the compound fertilizer is completely dissolved. The liquid temperature will drop significantly. Let it stand for 5 minutes until the temperature returns to room temperature and ensure the outer wall of the beaker is dry. Pour the solution into a clean Erlenmeyer flask lined with disposable 5 μm No. 2 qualitative filter paper and filter. After pouring, rinse the inner wall of the beaker and the glass rod repeatedly with 20 ml of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. Finally, rinse the filter paper three times with 10 ml of deionized water, collect the residual solution, and combine the filtrate into a clean beaker. Rinse the inner wall of the Erlenmeyer flask repeatedly with 15 ml of deionized water and combine the rinsing water into the beaker.

[0103] 1.2 Pour the filtrate from the beaker into a clean Erlenmeyer flask funnel lined with disposable 1 μm glass fiber filter paper for filtration. After pouring, rinse the inner wall of the beaker and the glass rod repeatedly with 20 mL of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. Rinse the filter paper three times with 10 mL of deionized water. Combine the collected filtrate and rinsing solution and pour them into a 400 mL clean beaker. After pouring, rinse the inner wall of the Erlenmeyer flask repeatedly with 15 mL of deionized water, and combine the rinsing water and pour it into the beaker. Adjust the pH to 5.8 ± 0.2 with 1.0 mol / L potassium hydroxide solution, stir, and let stand.

[0104] 1.3 Weigh 2 grams of acid-washed coconut shell activated carbon powder (to remove organic impurities and possible trace amounts of chlorine), slowly pour it into a beaker containing the filtrate, place the beaker on a magnetic stirrer, and stir at a speed of 250 rpm for 45 minutes, starting slowly and then increasing the speed.

[0105] 1.4 Pour the liquid in the beaker into a disposable 0.45 μm filter paper flask funnel for vacuum filtration. After filtration, rinse the beaker and glass rod with 20 mL of deionized water. Pour all the rinsing water into the funnel for vacuum filtration. Then rinse the carbon powder and filter paper three times with 15 mL of deionized water to recover the filtrate. After ensuring that there are no tiny black carbon particles at the bottom of the beaker, pour the collected liquid into a clean 500 mL beaker. After pouring, rinse the flask with 20 mL of deionized water. Combine the rinsing liquid and pour it into the beaker.

[0106] 1.5 Transfer the beaker containing the liquid to a fume hood, check the pH value of the solution to ensure it is between 6 and 0.2, place it on a magnetic stirrer and stir at a constant speed of 400 rbm. Heat the solution to 45 degrees Celsius, add ammonium sulfide (0.1 mol / L) solution, not exceeding 0.2 mL each time (record the amount of ammonium sulfide used), observe the formation of precipitate, and stop adding precipitate when no new precipitate is formed. Let it stand for 45 minutes.

[0107] 1.6 Use a suction flask equipped with a 0.45 μm disposable PTFE hydrophilic filter membrane. After filtration, rinse the beaker and glass rod repeatedly with deionized water. Pour the rinse water into a funnel for suction filtration, rinse the filter membrane again, and pour the collected filtrate into a clean beaker. Rinse the Erlenmeyer flask with deionized water and pour the rinse water into the beaker as well. Ensure that the filtered residue and filter paper are kept moist. Pack them in a dedicated leak-proof and airtight container, affix a label, and hand them over to a professional hazardous waste company for disposal.

[0108] 1.7 Place the beaker on a magnetic stirrer and stir at 500 rpm, using a solution of 0.3% hydrogen peroxide (… Add hydrogen peroxide to the beaker in four portions, 3 mL each time, with a 5-minute interval between each addition. Stir the mixture for 30 minutes to fully oxidize excess sulfur. After the reaction is complete, use hydrogen peroxide test paper to check the solution and ensure that the reaction is complete.

[0109] 1.8 Remove the beaker from the fume hood. Adjust the pH of the solution to 6±0.2 using 0.1mol / L potassium hydroxide (KOH) under the detection of a pH meter, and record the amount of potassium hydroxide used. Then, filter the solution again using an Erlenmeyer flask equipped with a disposable 0.45 μm PTFE hydrophilic filter membrane. Rinse the beaker and glass rod repeatedly with deionized water. Pour the rinse water into a funnel for filtration. Rinse the filter membrane again and pour the collected filtrate into a clean beaker. Rinse the Erlenmeyer flask with deionized water and pour the rinse water into the beaker containing the filtrate.

[0110] Add 5 g of iminodiacetic acid-type chelating resin to the clear solution collected in beaker 1.9. Shake horizontally at 30 °C and 150 rbm for 1.5 h to allow most of the residual metal ions to be adsorbed by the resin. After standing for 10 minutes, filter the solution and then filter the Erlenmeyer flask again using 0.45 μm filter paper. Rinse the beaker and glass rod repeatedly with deionized water. Pour the rinse water into a funnel and filter again. Rinse the resin and filter membrane, and collect the filtrate in a clean beaker. Rinse the Erlenmeyer flask with deionized water, and pour the rinse water into the beaker containing the filtrate.

[0111] 1.10 Confirm that the pH value is within the range of 5.8±0.2. If not, adjust the obtained filtrate to the range of 5.8±0.2 using 0.1 mmol / L potassium hydroxide. Then, inject the filtrate in the beaker into an exchange column containing 5 g of zirconium-based ion exchange resin at a flow rate of 0.5 mL / min. After the filtrate is injected, rinse the beaker repeatedly with 20 mL of deionized water. Continue to inject the rinsing solution into the exchange column. Finally, rinse the exchange column with 3 BV of deionized water and collect the filtrate in a 500 mL beaker.

[0112] 1.11 Final sterilization and impurity removal: Use a clean Erlenmeyer flask funnel fitted with a 0.22μm disposable PTFE hydrophilic filter membrane for filtration. After pouring, repeatedly rinse the inner wall of the beaker and the glass rod with 20 ml of deionized water, ensuring that all the rinsing water is transferred to the funnel for filtration. Rinse the filter paper three times with 10 ml of deionized water. Combine the collected filtrate and washing solution, and finally pour the filtrate into a dry and clean beaker. Rinse the inner wall of the Erlenmeyer flask with 20 ml of deionized water (after sterilization and impurity removal, the volume can be adjusted to 400 ml before concentration testing).

[0113] 1.12. Add deionized water to dilute and bring the volume to 500 ml. The amount of each ion obtained per 50 ml solution is: 4.75 mmol of nitrate, 6.72 mmol of ammonium, 0.49 mmol of dihydrogen phosphate, and 0.75 mmol of sulfate, which is a purified nitro compound fertilizer.

[0114] The final 20-fold dilution of nitro compound fertilizer mother liquor met the chemical purity requirements of "Reagent Ammonium Nitrate GB / T 659-2011". Thus, the purified nitro compound fertilizer, one of the raw materials for macronutrients in N69 culture medium, was completed. Depending on the needs of the cultured organisms, instruments such as ion chromatography can be used to detect and record the content of various major ions (ammonium ions, nitrate ions, sulfate ions, phosphate ions, magnesium ions, potassium ions, chloride ions, iron ions, and heavy metals, etc.). Based on the test results, it can be determined whether the nutrient composition meets the requirements for N69 macronutrient formulation, and whether the heavy metal ion threshold meets the standard based on the needs of the target plant (steps 1.2-2.2 can be repeated).

[0115] 2. The purified nitro compound fertilizer was used to prepare the macro-element working solution for N69 culture medium. The raw materials used are shown in Table 7.

[0116] Table 7. List of macro-element culture medium formulations for N69 medium

[0117]

[0118] The preparation process of the macro-element culture medium for N69 medium is as follows:

[0119] 2.1 Take 50 ml of the extract containing nitro compound fertilizer to prepare a 20-fold concentration N69 mother liquor, and pour it into a beaker containing 500 ml of deionized water;

[0120] 2.2 According to the data in Table 7, use a precision weigher (accuracy 0.001 g) to weigh out 1020 mg of potassium nitrate, 82 mg of potassium dihydrogen phosphate, 9 mg of monoammonium phosphate, 121 mg of ammonium chloride, 192 mg of magnesium nitrate hexahydrate, and 267 mg of calcium nitrate tetrahydrate, respectively. Stir and add them sequentially to a mixed solution containing 50 ml of purified nitro compound fertilizer solution, and set aside for later use.

[0121] The above is a mixed solution of macro-elements working solution for N69 plant culture medium.

[0122] 3. Prepare N69 plant culture medium using the above-mentioned macro-element culture solution:

[0123] 3.1 Place the macro-element mixed solution prepared in 2.2 on a magnetic stirrer and stir. Then add the trace elements manganese sulfate hydrate: 18.94 mg, boric acid: 10.00 mg, sodium molybdate (dihydrate): 0.25 mg, zinc sulfate (heptahydrate): 10.00 mg, copper sulfate (pentahydrate): 0.025 mg, ferrous sulfate (heptahydrate): 27.85 mg, disodium EDTA (dihydrate): 37.25 mg, organic matter, inositol: 10.00 mg, thiamine hydrochloride: 0.50 mg, pyridoxine hydrochloride: 0.50 mg, niacin: 5.00 mg, folic acid: 0.50 mg, biotin: 0.05 mg, glycine: 2.00 mg, and sucrose: 20000 mg (i.e., 2%), ensuring that all elements are completely dissolved.

[0124] 3.2 Add deionized water to the completely dissolved solution and bring the volume to one liter;

[0125] 3.3 Finally, adjust the pH value to 5.8±0.1.

[0126] Table 8: Macro-element formulation of N69 culture medium prepared according to the purified nitro fertilizer extract in Implementation Case 1

[0127]

[0128] This invention calculates the usage of different formulations of nitro compound fertilizer not only by prioritizing ammonium nitrate, but also by prioritizing dihydrogen phosphate, sulfate, nitrate, and potassium ions (if any). That is, if the maximum requirement for ammonium nitrate is met, and one or more of the other four ions exceed the maximum usage concentration, the fertilizer usage should be calculated based on the ion with the highest excess. It is then necessary to verify whether the ammonium nitrate ion requirement is met. If the minimum requirement cannot be met, other methods must be used to add it. The calculation method used in this invention also includes, after determining the ion ratio of the purified nitro compound fertilizer, introducing new elements using ion balance to maintain the ion balance of the target culture medium. Each formulation is not limited to the elements already listed in the table, but also includes methods using this invention to introduce other elements to achieve overall balance, and then using the prepared culture medium for plant cultivation.

[0129] The purification technique of this invention achieves high purity, making it suitable for use in mid-to-high-end plant culture media. Compared to existing purification methods, this application uses ammonia instead of potassium hydroxide to adjust the pH value, reducing the addition of unnecessary ions from other chemicals. It also uses acid-washed activated carbon instead of food-grade activated carbon, reducing the risk of incorporation of unknown metal ions.

[0130] Example 4: Prepare 1 / 2 MS medium, wherein the ratio of each ion of macroelements in group B is the same as the ratio of each ion in the macroelement culture medium prepared in Example 1, and the formula used is shown in Table 9.

[0131] Table 9. List of 1 / 2MS culture medium formulations

[0132]

[0133] Table 9 shows the composition of each component of the 1 / 2MS medium working solution: Group A is the formula table of each nutrient element published by commercial 1 / 2MS medium; the molar concentration ratio of ions in the macronutrient part of Group B is consistent with the macronutrient culture solution formula of Example 1 (half of the formula concentration of macronutrient culture solution in Table 5); Group C is different from Group B in that it has removed nitro compound fertilizer; Group D is different from Group B in that the macronutrient culture solution has not been purified, and the organic components, vitamins, sugars, agar, and antibacterial agents prepared by open culture are all consistent, with a pH value of 5.7±0.1.

[0134] Example 5, Plant Cultivation

[0135] Arabidopsis seeds were cultured using 1 / 2 MS medium from the AC group to confirm growth. The target plant was selected as Arabidopsis seed WT (Col-0), Colombian wild type; the culture environment was set as follows: temperature 20.5-23.5 degrees Celsius, humidity 55-65%, and light intensity 16 hours.

[0136] Performance testing

[0137] The equipment and instruments used included: beakers, No. 2 qualitative filter paper / 1 μm glass fiber filter paper, vacuum filtration flasks, acid-washed coconut shell activated carbon powder (iodine value above 1000), iminodiacetic acid chelating resin, zirconium-based resin ion exchange column, 0.45 μm and 0.22 μm PTFE hydrophilic filter membranes, magnetic stirrer, etc.; 0.1 mol / L potassium hydroxide and nitric acid standard solutions; pH meter; high-precision electronic balance, etc.; inductively coupled plasma spectrometer; ion chromatograph, etc.; and YL-01 chlorophyll meter.

[0138] Comparison of cultivation effects: Table 10 and Figures 1 to 4 :

[0139] Table 10. List of Arabidopsis thaliana seed cultured using Group A-D / 2MS medium.

[0140]

[0141] Example 5, the cost comparison of 1 / 2MS culture medium for AC group in Table 8 is shown in Table 10.

[0142] Table 11. Cost Comparison of 1 / 2MS Culture Medium for AC Group

[0143]

[0144] The retail purchase cost of nitro compound fertilizer is approximately 3.5 yuan / KG, and the wholesale price is 2.5-3 yuan / KG; the retail cost in Table 10 already includes the cost of purification. Commercial purchases are based on small-scale procurement prices (Group A procurement price: 210 yuan / kg). If purchased by the ton, current investigations show the lowest price per ton (25KG / barrel) is 80 yuan / KG for MS culture medium powder (sugar-free and gel-free) (industrial grade). Domestically renowned brands labeling similar AR-grade products cost approximately 500-800 yuan / KG, and their macro-element mother liquor is 20 times more concentrated, resulting in a working solution cost of approximately 10-30 yuan / liter. The self-prepared cost of this invention, calculated per ton of dry powder, is 15-20 yuan / KG. The MS macro-element mother liquor can be prepared to a 100-fold concentration, sealed, and stored at room temperature for extended periods, completely eliminating the use of hazardous chemicals such as potassium nitrate and calcium nitrate involved in dry powder preparation. The working solution cost is approximately 0.1-0.2 yuan / liter. Directly purchasing ammonium nitrate for self-preparation incurs extremely high compliance costs.

[0145] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a macro-element culture medium from a purified nitro compound fertilizer, characterized in that, Includes the following steps: 1) Determine the composition and content of ions in the target culture medium and nitro compound fertilizer containing ammonium nitrate; 2) Purification of nitro compound fertilizer; 3) Use the ammonium nitrate in the purified nitro compound fertilizer to match the ammonium nitrate requirements in the target culture medium, calculate the required mass of compound fertilizer, and set it as X; 4) Verify whether the other ions brought by the compound fertilizer of mass X are within the concentration range required by the target culture medium; if they are within the range required by the target culture medium, calculate and supplement the amount of other ions, and the concentration of each ion must not exceed the maximum concentration allowed by the target culture medium; if they are not within the range required by the target culture medium, adjust the concentration of ions exceeding the range in the compound fertilizer so that the ammonium nitrate in the compound fertilizer matches the minimum ammonium nitrate requirement of the target culture medium, and the concentration of ions formed by other elements is within the maximum concentration allowed by the target culture medium. Calculate and supplement the amount of other ions to obtain a macro-element culture solution.

2. The method for preparing a macro-element culture medium from a purified nitro compound fertilizer according to claim 1, characterized in that, The content of ammonium nitrate in the nitro compound fertilizer is greater than 30 wt%.

3. The method for preparing a macro-element culture medium from a purified nitro compound fertilizer according to claim 1 or 2, characterized in that, The nitro compound fertilizer contains nitrate nitrogen and three macronutrient elements: nitrogen, phosphorus, and potassium. Its nitrogen component includes nitrate nitrogen. The total phosphorus nutrient content of the product is calculated as phosphorus pentoxide, and the total potassium nutrient content is calculated as potassium oxide. The mass ratio of nitrate nitrogen to total phosphorus nutrient content calculated as phosphorus pentoxide in the product is greater than 3:5, and the mass ratio of nitrate nitrogen to total potassium nutrient content calculated as potassium oxide is greater than 100:

169. Based on the total mass of the compound fertilizer, the mass percentage of ammonium sulfate is less than 20%.

4. The method for preparing a macro-element culture medium from a purified nitro compound fertilizer according to claim 3, characterized in that, The purified nitro compound fertilizer contains ≤ 0.35 mg / L of heavy metals (calculated as lead), of which ≤ 0.07 mg / L of arsenic; and the water-insoluble matter content is less than 10 ppm.

5. The method for preparing a macro-element culture medium from a purified nitro compound fertilizer according to claim 4, characterized in that, The process of purifying nitro compound fertilizer includes the following steps: Dissolution and initial filtration: Dissolve the nitro compound fertilizer in deionized water and filter it through multi-stage filter paper to remove insoluble particles; Adsorption and impurity removal: Activated carbon is added to the filtrate for adsorption treatment, and the activated carbon is removed by filtration; Arsenic removal by ion exchange: The filtrate is passed at low speed through an ion exchange resin loaded with metal compounds or an iminodiacetic acid-type chelating resin. Fine filtration and volume determination: Micron-level filter membrane is used for filtration.

6. The method for preparing a macro-element culture medium from a purified nitro compound fertilizer according to claim 5, characterized in that, The process for purifying nitro compound fertilizer is as follows: 1) Weigh 5.3-17.70 grams of nitro compound fertilizer and pour it into 50-200 ml of deionized water, stir and mix to prepare a compound fertilizer solution; 2) Filter the compound fertilizer solution sequentially through 5 μm filter paper and 1 μm filter paper, and collect the filtrate; 3) Weigh 1-2 grams of acid-washed powdered activated carbon, slowly pour it into the filtrate, and stir for 15-20 minutes; 4) Filter the solution through 1 μm filter paper, collect the filtrate, and ensure that there are no tiny black carbon particles at the bottom of the cup. Then filter it through 0.45 μm filter paper and collect the clear solution. 5) Adjust the pH to 5.5-6.5 with 0.1-1.0 mol / L potassium hydroxide solution, stir, and let stand; Add acid-washed activated carbon powder to remove organic impurities; 6) Filter using 0.45 μm filter paper and collect the filtrate; 7) Add an iminodiacetic acid-type chelating resin or a zirconium-based ion exchange resin column to allow most of the residual metal ions to be adsorbed by the resin; after filtration, use deionization to clean the resin, and pour the cleaning solution into the filtrate. 8) Confirm that the pH value is within the range of 5.5-6.5; 9) Filter using a 0.22-0.45 μm microporous membrane until a clear solution is obtained; 10) If the purity of the nitro compound fertilizer extract does not meet the requirements of claim 4, repeat any one or more of steps 3) to 8).

7. A macro-element culture medium for a culture medium obtained by the method according to any one of claims 1-6, characterized in that, Macro-element culture medium is used to prepare culture media for laboratory plant cell culture, plant tissue culture propagation media, and plant culture media for virus-free seedling cultivation.

8. The macro-element culture medium according to claim 7, characterized in that, The MS medium is a composition of nitro compound fertilizer 30-4-0, potassium nitrate, potassium dihydrogen phosphate, calcium chloride dihydrate, calcium nitrate tetrahydrate, potassium sulfate, and magnesium chloride, in a mass ratio of 820-2530:800-2000:10-170:0-440:0-706:0-174:0-143.

9. The macro-element culture medium according to claim 7 or 8, characterized in that, The medium used to prepare N69 culture medium comprises a composition of potassium nitrate, nitro compound fertilizer 30-6-0, potassium dihydrogen phosphate, monoammonium phosphate, ammonium chloride, magnesium nitrate hexahydrate, and calcium nitrate tetrahydrate in a mass ratio of 444-1455:500-1208:0-700:0-50:0-144:0-192:0-267.

10. A plant culture medium, characterized in that, Includes the macroelement culture medium obtained by the method according to any one of claims 1-6 or the macroelement culture medium according to any one of claims 7-9.

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