CN112136688A - Plant tissue culture medium and preparation method thereof - Google Patents
Plant tissue culture medium and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to the field of biology, and particularly relates to a plant tissue culture medium and a preparation method thereof. The invention adopts calcium ammonium nitrate (5Ca (NO)3)2.NH4NO3.10H2O) nitrogen source instead of ammonium Nitrate (NH) in MS medium4.NO3) And potassium Nitrate (NH)4.NO3) The nitrogen source provided, because of introducing ammonium calcium nitrate, make original macroelement and calcium ion concentration change, therefore introduced appropriate ammonium sulfate, potassium chloride and potassium sulfate while formulating, make its macroelement and proportion of calcium element unanimous. Experiments prove that the growth condition of each sugarcane variety in the culture medium is good, the proliferation rate of the sugarcane variety is not obviously different from that of the original MS culture medium, and the problem of the source of the MS culture medium is solved.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a plant tissue culture medium and a preparation method thereof.
Background
The MS culture medium is designed for tobacco cell culture in 1962 by Murashige and Skoog, and is characterized by higher inorganic salt and ion concentration, more stable ion balance solution, high nitrate content, proper nutrient quantity and proportion, and capability of meeting the nutritional and physiological requirements of plant cells, so that the MS culture medium has wider application range, and most plant tissues are cultured and rapidly propagated to be used as the basic culture medium of the culture medium.
MS medium is currently the most commonly used medium worldwide. It has a high inorganic salt concentration and can ensure mineral nutrition required for tissue growth and normal growth of explants cultured by the tissue. The MS solid culture medium can be used for inducing callus, and can also be used for culturing embryos, stem segments, stem tips and anthers, and the liquid culture medium can obtain obvious success when used for cell suspension culture. The amount and proportion of the inorganic nutrients in the MS culture medium are proper enough to meet the needs of plant cells in nutrition and physiology. Therefore, in general, no additional organic ingredients such as amino acids, casein hydrolysate, yeast extract and coconut water are added. The high content of nitrate, potassium and ammonium in the MS medium compared to the basic components of other media is a significant feature.
Wherein the nitrogen is derived from potassium nitrate and ammonium nitrate. Potassium nitrate supplies nitrate nitrogen, and ammonium nitrate supplies both ammonium nitrogen and nitrate nitrogen. The ammonium nitrogen is in a reduction state and is a cation; nitrate nitrogen is in an oxidized state and is anionic. Crops have different preferences for ammonium nitrogen and nitrate nitrogen, but both have functions of being used as suppliers of plant nitrogen sources and are not replaced by each other. In soil, besides being directly absorbed by plants, ammonium ions can be converted into nitrite nitrogen through nitrite bacteria, and then the nitrite nitrogen is converted into nitrate nitrogen through nitrobacteria, so that the nitrogen requirement of plant growth can be met. However, in tissue culture, nitrifying bacteria and nitrifying bacteria are not present, and ammonium nitrogen and nitrate nitrogen must be supplied at the same time, otherwise, the nutrient requirement of plant tissue culture is difficult to maintain.
Nitrates, such as ammonium nitrate, potassium nitrate and sodium nitrate, are listed in the records of dangerous chemicals prone to explosion (2011 edition) in China. The national regulations on dangerous chemicals have been strengthened in recent years, and the 144 nd routine meeting revision of national academy of people's republic of China, No. 344 of national academy of republic of China, No. 2 and 16 of 2011 in 1 and 26 of 2012 passes the regulations on safety management of dangerous chemicals. Nitrate cannot be purchased without holding the associated certificate, and businesses that use nitrate also require the presence of associated storage facilities. Nitrate is cheap and has low profit, and most reagent companies are reluctant to process complicated certificates for low profit.
When ammonium nitrate is difficult to purchase and use, the requirements of plant tissue culture are not met, and some manufacturers provide finished product MS culture media, including solid powder MS culture media and liquid MS culture media. The solid powder MS culture medium is high in price and is not suitable for industrial breeding. But the problem of purchasing finished product liquid MS culture medium is more. Firstly, the transportation difficulty is increased, and the production cost is increased; secondly, the storage period is short, the liquid MS culture medium contains calcium ions, so that precipitates are easy to generate, and organic matters contained in the liquid MS culture medium are easy to acidify, so that the components of the culture medium are changed under the two conditions. Therefore, it is imperative to search for a raw material for a culture medium for industrial tissue culture, which substitutes ammonium nitrate and potassium nitrate.
Event: in 8 months and 4 nightfall in 2020, at least 100 people are in distress, 4000 people are injured, 30 ten thousand people can not return home, and the loss is as high as $ 30 hundred million due to severe explosion of 2700 tons of ammonium nitrate in the Hongbao first-capital Belut port area. 8, 9 months, the security commission of the state department initiates special checks of explosive key control chemicals such as ammonium nitrate and the like, and the national relevant regulations are required to be strictly executed; the 8/10/month department of industry and informatization issues notices that it is strictly prohibited to sell or otherwise transfer ammonium nitrate to individuals and other entities. The competent departments in the provincial civil explosion industry need to strictly approve the ammonium nitrate sale permission, strengthen the ammonium nitrate sale safety supervision, ensure the conformity of account certificates and account objects and ensure the flow direction to be checked and traced. In fact, explosion and fire caused by ammonium nitrate and potassium nitrate occur occasionally, and national regulations on dangerous goods such as ammonium nitrate and potassium nitrate are more and more strict. The production products using chemicals such as ammonium nitrate, potassium nitrate and the like as raw materials must be searched for alternative products.
Disclosure of Invention
The invention provides a novel formula and a preparation method of an MS culture medium, wherein the MS culture medium is prepared by adopting calcium ammonium nitrate to replace ammonium nitrate and potassium nitrate, and the same effect as the MS culture medium prepared by using ammonium nitrate and potassium nitrate can be realized.
The technical scheme of the invention is realized as follows:
the application of calcium ammonium nitrate in preparing plant tissue culture medium is characterized by comprising the following steps: calcium ammonium nitrate is used to replace ammonium nitrate and potassium nitrate in MS culture medium.
A plant tissue culture medium comprises the following components in parts by mass per liter of culture medium: calcium ammonium nitrate (5Ca (NO)3)2·NH4NO3·10H2O)0.982-4.911g, ammonium sulfate ((NH)4) 2SO4)0.721-6.486g, 0.2-2g of potassium chloride (KCl), potassium sulfate (K)2SO4)0-1.5g, magnesium sulfate (MgSO)4·7H2O)0.1 to 0.5 g; potassium dihydrogen phosphate (KH)2PO4)0.1-0.3g, 0.3064-0.04656g of trace elements, 0.06-0.07g of iron salts and 0.077-0.131g of organic components.
Further, each liter of culture medium comprises the following components by mass: calcium ammonium nitrate (5Ca (NO)3)2·NH4NO3·10H2O)3.87g, ammonium sulfate ((NH)4)2SO4)1.123 g, potassium chloride (KCl)1.4g, magnesium sulfate (MgSO)4·7H2O)0.37 g; potassium dihydrogen phosphate (KH)2PO4)0.17g, 0.03823g of trace elements, 0.0651g of iron salt and 0.104g of organic components.
Further, each liter of culture medium comprises the following components by mass: calcium ammonium nitrate (5Ca (NO)3)2·NH4NO3·10H2O)3.87g, ammonium sulfate ((NH)4)2SO4)1.123 g, potassium chloride (KCl)0.4473g, potassium sulfate (K)2SO4)1.1152g, magnesium sulfate (MgSO)4·7H2O)0.37 g; potassium dihydrogen phosphate (KH)2PO4)0.17g, 0.03823g of trace elements, 0.0651g of iron salt and 0.104g of organic components.
Further, the trace elements comprise manganese sulfate which is a component in the following mass ratio: 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 comprises the following components in percentage by mass, ferrous sulfate: na (Na)2-EDTA＝5-6：7-8。
Further, the organic component comprises the following components in percentage by mass: thiamine hydrochloride: pyridoxine hydrochloride: nicotinic acid: inositol 0.2-0.6:0.05-1.5: 0.1-0.3: 0.05-0.15:15-25.
A preparation method of a plant tissue culture medium comprises the following steps:
(1) weighing the components according to the mass of the materials in the formula, and dissolving calcium ammonium nitrate in distilled water to prepare a No. 1 component; taking a mixed solution of ammonium sulfate, potassium sulfate and potassium chloride as a No. 2 component; preparing a mixed solution of magnesium sulfate and potassium dihydrogen phosphate as a No. 3 component; preparing the trace elements, the ferric salt and the organic components into solutions respectively;
(2) firstly, mixing the component No. 1 and the component No. 2 according to the volume ratio of 1:1, stirring uniformly, standing, taking supernatant, adding the No. 3 component, wherein the volume ratio of the supernatant to the No. 3 component is 2: 1; and finally adding the trace element solution, the ferric salt solution and the organic component solution, and adding distilled water to complement.
The application of the plant tissue culture medium in plant tissue culture.
Further, the plant is sugarcane.
The invention has the beneficial effects that:
calcium ammonium nitrate is a plant fertilizer, is white round granulated, is soluble in water, is a novel high-efficiency compound fertilizer containing nitrogen and quick-acting calcium, has the characteristics of quick fertilizer effect and quick nitrogen supplement, and can meet the requirements of ammonium nitrogen and nitrate nitrogen at the same time. The calcium ammonium nitrate is easy to purchase in the market, the purity can reach 99 percent, and the requirement of culture medium preparation can be met. Experiments prove that each sugarcane variety has good growth condition in the culture medium, and the proliferation rate has no obvious difference with that of the original MS culture medium, so that the sugarcane can replace ammonium nitrate and potassium nitrate of the original MS culture medium, and the problem of source of raw materials of the MS culture medium is solved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the growth of Medium sugar No. 1 in formula 1, 2 and in Primary MS Medium (CK);
FIG. 2 shows the growth of Medium sugar No. 4 in formula 1, 2 and in Primary MS Medium (CK);
FIG. 3 shows the growth of Medium sugar No. 5 in formula 1, 2 and in Primary MS Medium (CK);
FIG. 4 shows the growth of Medium sugar No. 6 in formula 1, 2 and in Primary MS Medium (CK);
FIG. 5 shows the growth of Guise No. 55 in formula 1 and 2 media and in original MS medium (CK);
FIG. 6 shows the growth of Osmanthus fragrans 05136 in formula 1 and formula 2 medium and in original MS medium (CK);
fig. 7 shows growth of cassia twig 07150 in formula 1 and formula 2 medium and in original MS medium (CK).
FIG. 8 is a bar graph of the proliferation rate of each sugarcane variety.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to facilitate the operation, concentrated mother liquor is prepared according to a certain concentration multiple in the process of preparing the culture medium and is stored respectively. When the plant tissue culture medium needs to be prepared, the mother liquor with the corresponding volume is measured according to the concentration multiple.
Example 1
A plant tissue culture medium, wherein the concentrations and amounts of the respective components are shown in Table 1 (hereinafter referred to as formula 1 medium).
Formula culture medium mother liquor and preparation reference in Table 11
For example, if 1L of plant culture medium is required to be prepared, the components are weighed according to the preparation weighing amount of the mother liquor to prepare each mother liquor. And then, respectively taking 30-40mL of equivalent amount of the No. 1 component mother liquor and 30-40mL of equivalent amount of the No. 2 component mother liquor according to the volume ratio of 1:1, uniformly stirring, standing for 10-15 minutes, taking 40mL of supernatant, adding 20mL of the No. 3 component mother liquor, then respectively taking 1mL of trace element mother liquor, 5mL of ferric salt mother liquor and 5mL of organic component mother liquor according to the preparation dosage, and fixing the volume to 1L by using distilled water.
Example 2
A plant tissue culture medium, wherein the concentrations and amounts of the respective components are shown in Table 2 (hereinafter referred to as formula 2 medium).
Culture medium mother solution and preparation reference of formula No. 22 in Table
The formulation method was the same as in example 1.
Example 3
A plant tissue culture medium comprising the components in the concentrations and amounts shown in Table 3.
TABLE 3 culture Medium mother liquors and formulation references
Example 4
A plant tissue culture medium comprising the components in the concentrations and amounts shown in Table 4.
TABLE 4 culture Medium mother liquors and formulation references
Investigation of effect of culture medium with different proportions
Selecting sugar No. 5 in sugarcane varieties as experimental varieties, respectively preparing culture media with different concentrations, and respectively comparing the growth conditions and the proliferation rates of sugarcane original seedlings. MS culture medium and no N element added are used as control. The results are shown in tables 5 and 1. The rest elements are used in MS culture medium.
Proliferation rate is the number of seedlings after proliferation/number of inoculated seedlings
TABLE 5 proliferation rates of sugarcane seedlings in media of different concentrations
Investigation of culture Medium tissue culture Effect (first) Experimental design
Selecting sugar No. 1, sugar No. 4, sugar No. 5, sugar No. 6 and sweet osmanthus sugar No. 55 from sugarcane varietiesThe cassia twig 05136 and the cassia twig 07150 are experimental varieties and are respectively inoculated into a No. 1 formula culture medium, a No. 2 formula culture medium and an MS culture medium, each variety is respectively inoculated into 30 bottles, and each bottle is inoculated into about 1cm2Subculturing and transferring two large proliferated seedlings after 15 days; and (5) subculture, counting the number of proliferation bottles and the number of blocks after each subculture, and calculating the proliferation rate.
(II) results of the experiment
After the growth conditions of the sugarcane varieties in different culture media are compared, the accessed propagation seedlings of different sugarcanes can grow normally in the No. 1 culture medium, the No. 2 culture medium and the MS culture medium, and the growth conditions have no obvious difference. The proliferation rate of each sugarcane variety in the culture medium with the formula No. 1 and the culture medium with the formula No. 2 is not obviously different from that of the original MS culture medium. Thus, the plant tissue culture medium of the present invention can be used in place of the original MS medium. The results are shown in Table 6 and FIGS. 1-8.
TABLE 6 proliferation rates of various sugarcane varieties in different media
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The application of calcium ammonium nitrate in preparing plant tissue culture medium is characterized by comprising the following steps: calcium ammonium nitrate is used to replace ammonium nitrate and potassium nitrate in MS culture medium.
2. A plant tissue culture medium comprises the following components in parts by mass per liter of culture medium: the culture medium comprises the following components in parts by mass per liter: calcium ammonium nitrate (5Ca (NO)3)2·NH4NO3·10H2O)0.982-4.911g, ammonium sulfate ((NH)4)2SO4)0.721-6.486g, 0.2-2g of potassium chloride (KCl), potassium sulfate (K)2SO4)0-1.5g，Magnesium sulfate (MgSO)4·7H2O)0.1 to 0.5 g; potassium dihydrogen phosphate (KH)2PO4)0.1-0.3g, 0.3064-0.04656g of trace elements, 0.06-0.07g of iron salts and 0.077-0.131g of organic components.
3. A plant tissue culture medium according to claim 4, wherein: the culture medium comprises the following components in parts by mass per liter: 3.87g of calcium ammonium nitrate, 1.123g of ammonium sulfate, 1.4g of potassium chloride and 0.37g of magnesium sulfate; 0.17g of monopotassium phosphate, 0.03823g of trace elements, 0.0651g of iron salt and 0.104g of organic components.
4. A plant tissue culture medium according to claim 4, wherein: the culture medium comprises the following components in parts by mass per liter: 3.87g of calcium ammonium nitrate, 1.123g of ammonium sulfate, 0.4473g of potassium chloride, 1.1152g of potassium sulfate and 0.37g of magnesium sulfate; 0.17g of monopotassium phosphate, 0.03823g of trace elements, 0.0651g of iron salt and 0.104g of organic components.
5. A plant tissue culture medium according to claim 4, wherein: the trace elements comprise the following components in percentage by mass, 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.
6. A plant tissue culture medium according to claim 4, wherein: the iron salt comprises the following components in percentage by mass, ferrous sulfate: na (Na)2-EDTA＝5-6：7-8。
7. A plant tissue culture medium according to claim 4, wherein: the organic component comprises the following components in percentage by mass: thiamine hydrochloride: pyridoxine hydrochloride: nicotinic acid: inositol 0.2-0.6:0.05-1.5: 0.1-0.3: 0.05-0.15:15-25.
8. A method of preparing a plant tissue culture medium according to any one of claims 3 to 8, comprising the steps of:
(1) weighing the components according to the mass of the materials in the formula respectively to prepare a calcium ammonium nitrate water solution to prepare a No. 1 component; preparing a mixed aqueous solution of ammonium sulfate, potassium sulfate and potassium chloride as a No. 2 component; preparing a mixed aqueous solution of magnesium sulfate and potassium dihydrogen phosphate as a No. 3 component; dissolving microelements, iron salt and organic components in water respectively to prepare aqueous solution;
(2) firstly, mixing the component No. 1 and the component No. 2 according to the volume ratio of 1:1, mixing, stirring uniformly, standing, taking supernatant, adding the component No. 3, wherein the volume ratio of the supernatant to the component No. 3 is 2: 1; and finally, adding a trace element aqueous solution, an iron salt aqueous solution and an organic component aqueous solution, and adding distilled water to complement.
9. Use of a plant tissue culture medium according to any one of claims 3 to 8 for plant tissue culture.
10. The use of claim 9, wherein: the plant is sugarcane.
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| CN202010807635.8A CN112136688A (en) | 2020-08-12 | 2020-08-12 | Plant tissue culture medium and preparation method thereof |
| PCT/CN2021/112099 WO2022033527A1 (en) | 2020-08-12 | 2021-08-11 | Plant tissue culture medium and preparation method |
| ZA2023/02409A ZA202302409B (en) | 2020-08-12 | 2023-02-23 | Plant tissue culture medium and preparation method |
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| CN202010807635.8A CN112136688A (en) | 2020-08-12 | 2020-08-12 | Plant tissue culture medium and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112931206A (en) * | 2021-03-04 | 2021-06-11 | 华南农业大学 | Plant culture medium free of easily-exploding compound and application thereof |
| WO2022033527A1 (en) * | 2020-08-12 | 2022-02-17 | 中国热带农业科学院热带生物技术研究所 | Plant tissue culture medium and preparation method |
| CN116034875A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | Cherry stock tissue culture seedling culture medium and tissue culture seedling culture method thereof |
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| CN116034875A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | Cherry stock tissue culture seedling culture medium and tissue culture seedling culture method thereof |
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| WO2022033527A1 (en) | 2022-02-17 |
| ZA202302409B (en) | 2023-05-31 |
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