WO2022033527A1 - Plant tissue culture medium and preparation method - Google Patents

Abstract

Provided are a plant tissue culture medium, and a preparation method therefor and an application thereof. According to the present invention, a nitrogen source in calcium ammonium nitrate (5Ca(NO₃)₂.NH₄NO₃.10H₂O) is used for replacing nitrogen sources provided by ammonium nitrate (NH₄.NO₃) and potassium nitrate (NH₄.NO₃) in an MS culture medium, and moreover, due to the introduction of calcium ammonium nitrate, the original concentrations of macro elements and calcium ions are changed, so that proper ammonium sulfate, potassium chloride and potassium sulfate are introduced during preparation; the ratio of the macro elements to calcium elements is consistent.

Description

A kind of plant tissue culture medium and preparation method thereof technical field

The invention belongs to the biological field, and in particular relates to a plant tissue culture medium and a preparation method.

Background technique

MS medium was designed by Murashige and Skoog in 1962 for tobacco cell culture. It is characterized by high concentration of inorganic salts and ions, and is a relatively stable ion-balanced solution. It has high nitrate content and suitable amounts and proportions of nutrients. It can meet the nutritional and physiological needs of plant cells, so it has a wide range of applications.

MS medium is currently the most commonly used medium in the world. It has a high concentration of inorganic salts, which can ensure the mineral nutrition required for tissue growth and the normal growth of tissue cultured explants. MS solid medium can be used to induce callus, and can also be used for the culture of embryos, stem segments, shoot tips and anthers, and its liquid medium can be used for cell suspension culture with obvious success. The quantity and ratio of inorganic nutrients in MS medium are suitable enough to meet the nutritional and physiological needs of plant cells. Therefore, under normal circumstances, there is no need to add organic additional ingredients such as amino acids, casein hydrolyzate, yeast extract and coconut water. Compared with the basic components of other media, MS medium has high content of nitrate, potassium and ammonium, which is its distinguishing feature.

Nitrogen comes from potassium nitrate and ammonium nitrate. Potassium nitrate provides nitrate nitrogen, and ammonium nitrate provides both ammonium and nitrate nitrogen. Ammonium nitrogen is a reduced state and is a cation; nitrate nitrogen is an oxidized state and an anion. Crops have different preferences for ammonium nitrogen and nitrate nitrogen, but the two have their own functions as suppliers of plant nitrogen sources and do not replace each other. In soil, in addition to being directly absorbed by plants, ammonium ions can also be converted into nitrite nitrogen by nitrifying bacteria, and then nitrite nitrogen can be converted into nitrate nitrogen by nitrifying bacteria. , which can meet the nitrogen requirements for plant growth. However, in tissue culture, nitrifying bacteria and nitrosifying bacteria do not exist, and ammonium nitrogen and nitrate nitrogen must be provided at the same time, otherwise, it is difficult to maintain the nutritional requirements of plant tissue culture.

Nitrates, such as ammonium nitrate, potassium nitrate, sodium nitrate, etc., are all listed in my country's "List of Explosion-Producing Hazardous Chemicals (2011 Edition)." In recent years, the state has strengthened the management and control of hazardous chemicals. On January 26, 2012, the State Council of the People's Republic of China issued Order No. 344. On February 16, 2011, the 144th executive meeting of the State Council revised and passed the "Hazardous Chemical Safety". Management Regulations. Nitrates cannot be purchased without relevant documents, and businesses that use nitrates are also required to have relevant storage facilities. Nitrates are cheap and low-profit, and most reagent companies are reluctant to handle cumbersome documents for low profits.

technical problem

When ammonium nitrate is difficult to buy and use, and the needs of plant tissue culture are not met, some manufacturers have introduced finished MS media, including solid powder MS media and liquid MS media. Solid powder MS medium is expensive and not suitable for factory breeding. Buying finished liquid MS medium is more problematic. First, transportation is more difficult and production costs are increased; second, the storage period is short. Because the liquid MS medium contains calcium ions, it is easy to cause precipitation, and the organic matter contained is easy to acidify. Both of these conditions will cause the composition of the medium to change. . Therefore, it is imperative to explore a medium raw material for factory tissue culture to replace ammonium nitrate and potassium nitrate.

Incident: On the evening of August 4, 2020, a violent explosion of 2,700 tons of ammonium nitrate in the port area of Beirut, the capital of Lebanon, has killed at least 100 people, injured more than 4,000 people, made 300,000 homeless, and lost as much as $3 billion. On August 9, the State Council Office of the Safety Committee carried out a special inspection of ammonium nitrate and other explosive key controlled chemicals, requiring strict implementation of relevant national regulations; on August 10, the Ministry of Industry and Information Technology issued a notice that it is strictly forbidden to sell or otherwise transfer ammonium nitrate to individuals and other units. The competent departments of the civil explosive industry at the provincial level should strictly control the approval of ammonium nitrate sales licenses, strengthen the safety supervision of ammonium nitrate sales, and ensure that the accounts and documents are consistent, the accounts and materials are consistent, and the flow can be traced and traced. In fact, explosions and fires caused by ammonium nitrate and potassium nitrate have occurred from time to time. Products that use chemicals such as ammonium nitrate and potassium nitrate as raw materials must find alternatives to continue.

technical solutions

The invention proposes a new formulation and preparation method of MS culture medium. The MS culture medium is prepared by using calcium ammonium nitrate instead of ammonium nitrate and potassium nitrate to achieve the same effect as the MS culture medium prepared with ammonium nitrate and potassium nitrate.

The technical scheme of the present invention is realized as follows:

The application of calcium ammonium nitrate in preparing plant tissue culture medium is characterized in that: calcium ammonium nitrate is used to replace ammonium nitrate and potassium nitrate in MS culture medium.

A plant tissue culture medium comprising, per liter of medium, components of the following qualities: calcium ammonium nitrate (5Ca(NO ₃ ) ₂ ∙ NH ₄ NO ₃ ∙ 10H ₂ O) 0.982-4.911 g, ammonium sulfate ((NH ₄ ) ) ₂ SO ₄ ) 0.721-6.486 g, potassium chloride (KCl) 0.2-2 g, potassium sulfate (K ₂ SO ₄ ) 0-1.5 g, magnesium sulfate (MgSO ₄ ∙7H ₂ O) 0.1-0.5 g; phosphoric acid Potassium dihydrogen (KH ₂ PO ₄ ) 0.1-0.3 g, trace elements 0.3064-0.04656 g, iron salts 0.06-0.07 g, organic components 0.077-0.131 g.

Further, each liter of the medium includes the following components: calcium ammonium nitrate (5Ca(NO ₃ ) ₂ ∙ NH ₄ NO ₃ ∙ 10H ₂ O) 3.87 g, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 1.123 g, potassium chloride (KCl) 1.4 g, magnesium sulfate (MgSO ₄ ∙7H ₂ O) 0.37 g; potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.17 g, trace elements 0.03823 g, iron salt 0.0651 g, organic Composition 0.104 g.

Further, each liter of the medium includes the following components: calcium ammonium nitrate (5Ca(NO ₃ ) ₂ ∙ NH ₄ NO ₃ ∙ 10H ₂ O) 3.87 g, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 1.123 g, potassium chloride (KCl) 0.4473 g, potassium sulfate (K ₂ SO ₄ ) 1.1152 g, magnesium sulfate (MgSO ₄ ∙7H ₂ O) 0.37 g; potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.17 g, Trace elements 0.03823 g, iron salts 0.0651 g, organic components 0.104 g.

Further, the trace elements include components in the following mass ratios, manganese sulfate: zinc sulfate: boric acid: potassium iodide: sodium molybdate: copper sulfate: cobalt chloride=20-25:5-10:5-10:0.5- 1:0.1-0.5:0.02-0.03:0.02-0.03.

Further, the iron salt includes components in the following mass ratios, ferrous sulfate: Na ₂ -EDTA=5-6:7-8.

Further, the organic components include components in the following mass ratios, glycine: thiamine hydrochloride: pyridoxine hydrochloride: niacin: inositol=0.2-0.6:0.05-1.5:0.1-0.3:0.05-0.15:15 -25.

A preparation method of plant tissue culture medium, comprising the following steps:

(1) According to the quality of each material in the formula, weigh each component respectively, and dissolve calcium ammonium nitrate in distilled water to make No. 1 component; take the mixed solution of ammonium sulfate, potassium sulfate and potassium chloride as 2. No. Component; prepare a mixed solution of magnesium sulfate and potassium dihydrogen phosphate as No. 3 component; trace elements, iron salts and organic components are respectively prepared into solutions;

(2) First, mix the No. 1 component and No. 2 according to the volume ratio of 1:1, stir well, let stand, take the supernatant, and then add No. 3 component, the supernatant and No. 3 component. The volume ratio is 2:1; finally, add trace element solution, iron salt solution and organic component solution, and add distilled water to make up.

Application of the above-mentioned plant tissue culture medium in plant tissue culture.

Further, the plant is sugarcane.

beneficial effect

Calcium ammonium nitrate is a kind of plant fertilizer, which is white round granulation, soluble in water, a new type of high-efficiency compound fertilizer containing nitrogen and quick-acting calcium. Meet the needs of ammonium nitrogen and nitrate nitrogen. Calcium ammonium nitrate is easy to buy in the market, and its purity can reach 99%, which can meet the needs of medium preparation. It has been verified by experiments that each sugarcane variety grows well in the medium of the present invention, and the proliferation rate is not significantly different from that of the original MS medium, so it can replace the ammonium nitrate and potassium nitrate of the original MS medium to solve the problem. The source of raw materials for MS medium was discussed.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

Figure 1 shows the growth of Zhongtang No. 1 in formula medium No. 1 and No. 2 and in the original MS medium (CK);

Figure 2 shows the growth of Zhongtang No. 4 in formula medium No. 1 and No. 2 and in the original MS medium (CK);

Figure 3 shows the growth of Zhongtang No. 5 in formula medium No. 1 and No. 2 and in the original MS medium (CK);

Figure 4 shows the growth of Zhongtang No. 6 in formula medium No. 1 and No. 2 and in the original MS medium (CK);

Figure 5 shows the growth of Guitang No. 55 in formula medium No. 1 and No. 2 and in the original MS medium (CK);

Figure 6 shows the growth of Guiliu 05136 in No. 1 and No. 2 formula medium and in the original MS medium (CK);

Figure 7 shows the growth of Guiliu 07150 in No. 1 and No. 2 formula medium and in the original MS medium (CK).

Figure 8 is a bar graph of the proliferation rate of each sugarcane variety.

Embodiments of the present invention

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In order to facilitate the operation, in the process of preparing the culture medium, firstly, according to a certain concentration multiple, the concentrated mother liquors are respectively prepared and stored separately. When the plant tissue culture medium needs to be prepared, the corresponding volume of the mother liquor can be taken according to the concentration times.

Example 1

A plant tissue culture medium, the concentration and amount of each component are shown in Table 1 (hereinafter referred to as No. 1 formula medium).

Table 1 No. 1 formula medium mother liquor and preparation reference

 

  For example, if it is necessary to prepare 1L of plant culture medium, firstly, each component is weighed according to the preparation and weighing amount of the mother liquor, and prepared into each mother liquor. Then mix the No. 1 component mother liquor and No. 2 component mother liquor with an equal volume of 30-40ml each at a volume ratio of 1:1, mix, stir well, let stand for 10-15 minutes, take 40ml of the supernatant, and then take No. 3 20ml of component mother liquor was added, and then according to the preparation dosage, 1ml of trace element mother liquor, 5ml of iron salt mother liquor, 5ml of organic component mother liquor, and distilled water to 1L were respectively measured.

Example 2

A plant tissue culture medium, the concentration and dosage of each component are shown in Table 2 (hereinafter referred to as No. 2 formula medium).

Table 2 No. 2 formula medium mother liquor and preparation reference

The preparation method is the same as that of Example 1.

Example 3

A plant tissue culture medium, the concentration and dosage of each component are shown in Table 3.

Table 3 Medium stock solution and preparation reference

 

Example 4

A plant tissue culture medium, the concentration and dosage of each component are shown in Table 4.

Table 4 Medium stock solution and preparation reference

Investigation on the effect of different proportions of medium

The sugarcane variety Zhongtang No.5 was selected as the experimental variety, and different concentrations of medium were prepared respectively to compare the growth and proliferation rate of the original sugarcane seedlings. MS medium and no N element were used as controls. The results are shown in Table 5. The remaining elements were used in MS medium.

Proliferation rate = the number of seedlings after proliferation / the number of seedlings inserted

Table 5 Proliferation rate of sugarcane seedlings in different concentrations of medium

The investigation of the tissue culture effect of the medium of the present invention

(1) Experimental design

The sugarcane varieties Zhongtang No. 1, Zhongtang No. 4, Zhongtang No. 5, Zhongtang No. 6, Guitang No. 55, Guiliu 05136 and Guiliu 07150 were selected as test varieties, and the No. 1 formula medium and No. 2 formula medium were respectively connected. In the formula medium and MS medium, 30 bottles of each variety were connected, and each bottle was connected to two proliferating seedlings of about 1 cm ² size. After 15 days, the subcultures were transferred; subcultured 5 times, and the proliferation was counted after each subculture. The number of bottles and blocks, and the proliferation rate was calculated.

(2) Experimental results

After comparing the growth conditions of each sugarcane variety in different media, it was found that the proliferative seedlings of different sugarcane were able to grow normally in No. 1, No. 2 medium and MS medium, and there was no significant difference in growth conditions. The proliferation rate of each sugarcane variety in No. 1 and No. 2 formula medium was not significantly different from that of the original MS medium. Therefore, the original MS medium can be replaced by the plant tissue culture medium of the present invention. The specific results are shown in Table 6 and Figures 1-8.

Table 6 Proliferation rates of various sugarcane varieties in different media

Variety	No. 1 formula medium	No. 2 formula medium	original MS medium
Medium Sugar No. 1	2.4	2.5	2.4
Medium Sugar No. 4	2.5	2.6	2.5
Medium Sugar No. 5	2.65	2.45	2.5
Medium Sugar No. 6	2.97	3.1	3.1
Cinnamon Sugar No. 55	2.1	2.4	2.25
Gui Liu 05136	2.69	2.65	2.67
Gui Liu 07150	2.3	2.25	2.37

 

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (10)

1. The application of calcium ammonium nitrate in preparing plant tissue culture medium is characterized in that: calcium ammonium nitrate is used to replace ammonium nitrate and potassium nitrate in MS culture medium.
2. A plant tissue culture medium comprising the following qualities per liter of medium: Calcium ammonium nitrate (5Ca(NO ₃ ) ₂ ∙NH ₄ NO ₃ ∙10H ₂ O ) 0.982-4.911 g, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 0.721-6.486 g, potassium chloride (KCl) 0.2-2 g, potassium sulfate (K ₂ SO ₄ ) 0-1.5 g, magnesium sulfate (MgSO 4 ) ₄ ∙7H ₂ O) 0.1-0.5 g; potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.1-0.3 g, trace elements 0.3064-0.04656 g, iron salts 0.06-0.07 g, organic components 0.077-0.131 g.
3. A plant tissue culture medium according to claim 4, characterized in that: each liter of culture medium comprises the following components: calcium ammonium nitrate 3.87 g, ammonium sulfate 1.123 g, potassium chloride 1.4 g, Magnesium sulfate 0.37 g; potassium dihydrogen phosphate 0.17 g, trace element 0.03823 g, iron salt 0.0651 g, organic component 0.104 g.
4. A plant tissue culture medium as claimed in claim 4, characterized in that: each liter of culture medium comprises the following components: calcium ammonium nitrate 3.87 g, ammonium sulfate 1.123 g, potassium chloride 0.4473 g, Potassium sulfate 1.1152 g, magnesium sulfate 0.37 g; potassium dihydrogen phosphate 0.17 g, trace element 0.03823 g, iron salt 0.0651 g, organic component 0.104 g.
5. A plant tissue culture medium according to claim 4, characterized in that: the trace elements comprise components in the following mass ratios: manganese sulfate: zinc sulfate: boric acid: potassium iodide: sodium molybdate: copper sulfate: chloride Cobalt = 20-25:5-10:5-10:0.5-1:0.1-0.5:0.02-0.03:0.02-0.03.
6. A plant tissue culture medium according to claim 4, characterized in that: the iron salt comprises components in the following mass ratios, ferrous sulfate: Na ₂ -EDTA=5-6:7-8.
7. A plant tissue culture medium according to claim 4, characterized in that: the organic components comprise components in the following mass ratios, glycine: thiamine hydrochloride: pyridoxine hydrochloride: niacin: inositol=0.2 -0.6:0.05-1.5:0.1-0.3:0.05-0.15:15-25.
8. The preparation method of a kind of plant tissue culture medium as described in any one of claim 3-8, comprises the following steps:

   (1) According to the quality of each material in the formula, weigh each component separately, prepare an aqueous solution of calcium ammonium nitrate to make component No. 1; prepare a mixed aqueous solution of ammonium sulfate, potassium sulfate and potassium chloride as component No. 2 ; Prepare a mixed aqueous solution of magnesium sulfate and potassium dihydrogen phosphate as component No. 3; trace elements, iron salts and organic components are dissolved in water and prepared into an aqueous solution;

   (2) First, mix the No. 1 component and No. 2 component according to the volume ratio of 1:1, stir well, let stand, take the supernatant, and then add No. 3 component, the supernatant and the No. 3 group. The volume ratio of the ingredients is 2:1; finally, add the trace element aqueous solution, the iron salt aqueous solution and the organic component aqueous solution, and add distilled water to make up.
9. The application of the plant tissue culture medium according to any one of claims 3-8 in plant tissue culture.
10. The application according to claim 9, wherein the plant is sugarcane.