CN108812313B - Basic culture medium for plant tissue culture - Google Patents
Basic culture medium for plant tissue culture Download PDFInfo
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- CN108812313B CN108812313B CN201810623423.7A CN201810623423A CN108812313B CN 108812313 B CN108812313 B CN 108812313B CN 201810623423 A CN201810623423 A CN 201810623423A CN 108812313 B CN108812313 B CN 108812313B
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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 discloses a plant tissue culture basic culture medium ZS, and relates to the field of plant propagation, wherein each 1000mL of the ZS basic culture medium comprises 3075mg of potassium nitrate, 578mg of ammonium phosphate, 491mg of calcium nitrate, 160mg of ammonium chloride, 222mg of magnesium nitrate, 396mg of ammonium sulfate, 0.83mg of potassium iodide, 6.2mg of boric acid, 22.3mg of manganese sulfate, 8.6mg of zinc sulfate, 0.25mg of sodium molybdate, 0.025mg of copper sulfate, 0.025mg of cobalt chloride, 100mg of inositol, 0.1mg of thiamine hydrochloride (VB1), 0.5mg of pyridoxine hydrochloride (VB6), 0.5mg of nicotinic acid, 37.3mg of disodium ethylenediaminetetraacetate, and 27.8mg of ferrous sulfate. The basic culture medium can obtain the same tissue culture effect as an MS culture medium under the condition of not using ammonium nitrate.
Description
Technical Field
The invention relates to the field of plant tissue culture and rapid propagation, in particular to a basic culture medium for plant tissue culture.
Background
Murshige and Skoog medium (MS, 1962) are widely used plant tissue culture media, and ammonium nitrate is an important component of the culture medium. Ammonium nitrate is a strong oxidant, and can promote the fire when meeting with the fire of combustible materials; mixing with combustible powder, and reacting violently to explode; explosion can also occur when subjected to intense shock or rapid heating. It can be mixed with reducing agent, organic matter, inflammable matter such as sulfur, phosphorus or metal powder to form explosive mixture, and is used as raw material for producing explosive. Because of the huge potential safety hazard, in recent years, national supervision of such articles is more and more strict, the market circulation is limited by relevant national departments, the difficulty of purchasing ammonium nitrate in laboratories is high, and even ammonium nitrate cannot be purchased, so that inconvenience is brought to scientific research and production. However, ammonium nitrogen and nitrate nitrogen exist in ammonium nitrate molecules at the same time, which are necessary for plant growth and widely used in plant tissue culture. Therefore, there is an urgent need for a minimal plant tissue culture medium that can maintain the properties of the original MS culture medium and avoid the use of ammonium nitrate. At present, CN102224802A strawberry multiplication culture medium discloses a culture medium capable of replacing ammonium nitrate, but the culture medium is only suitable for strawberry multiplication culture medium, has no universality and is not suitable for popularization.
Disclosure of Invention
Aiming at the defects in the prior art, the invention discloses a basic culture medium for plant tissue culture, which can be suitable for culturing various common plant tissues and can obtain the same or similar effect as an MS culture medium.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a basic culture medium ZS for plant tissue culture, wherein each 1000mL of the culture medium contains: 3075mg potassium nitrate, 578mg ammonium phosphate, 491mg calcium nitrate, 160mg ammonium chloride, 222mg magnesium nitrate, 396mg ammonium sulfate, 0.83mg potassium iodide, 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 100mg inositol, 0.1mg thiamine hydrochloride (VB1), 0.5mg pyridoxine hydrochloride (VB6), 0.5mg nicotinic acid, 37.3mg disodium ethylenediaminetetraacetate, and 27.8mg ferrous sulfate.
The specific invention process of the invention is as follows:
(1) calculating the ion concentration of each element (table 2) according to the content of macroelements in the MS culture medium (table 1);
TABLE 1 composition and content of macroelements in MS Medium
TABLE 2 various ion concentrations of macroelements in MS medium
| Ion(s) | Ion concentration (mmol/L) |
| K + | 20.04 |
| NO 3 - | 39.40 |
| NH 4 + | 20.61 |
| PO 4 3- | 1.25 |
| Ca 2+ | 2.99 |
| Mg 2+ | 1.50 |
| SO 4 2- | 1.50 |
| Cl - | 3.00 |
(2) Removal of ammonium Nitrate (NH)
4NO
3) Finding suitable chemical agents for combinatorial substitution (table 3);
the invention maintains NO
3 -、NH
4 +、Ca
2+、Mg
2+、Cl
-The concentration of the plasma is not changed, and the K is properly improved
+、PO
4 3-、SO
4 2-The concentration of the ions (table 4), which is the key point of the present invention. The inventor shows through a large number of experiments that: the ZS culture medium provided by the invention has the components and the contents, and can obtain the effects similar to those of an MS culture medium in the aspects of callus induction, bud differentiation, rooting of tobacco, test-tube plantlet culture of potatoes and the like.
TABLE 3 ZS Medium macroelement composition and content of the invention
| Chemical reagent | Chemical formula | Molecular weight | Dosage (mg/L) | Reagent concentration mmol/L |
| Potassium nitrate | KNO 3 | 101.11 | 3075 | 30.42 |
| Ammonium phosphate | (NH 4) 3PO 4 | 149.09 | 578 | 3.88 |
| Calcium nitrate | Ca(NO 3) 2·4H 2O | 236.18 | 707 | 2.99 |
| Ammonium chloride | NH 4Cl | 53.49 | 160 | 3.00 |
| Magnesium nitrate | Mg(NO 3) 2·6H 2O | 256.40 | 384 | 1.50 |
| Ammonium sulfate | (NH 4) 2SO 4 | 132.14 | 396 | 3.00 |
TABLE 4 various ion concentrations of macroelements in ZS Medium
| Ion(s) | Ion concentration (mmol/L) |
| K + | 30.42* |
| NO 3 - | 39.40 |
| NH 4 + | 20.61 |
| PO 4 3- | 3.88* |
| Ca 2+ | 2.99 |
| Mg 2+ | 3.00 |
| SO 4 2- | 3.00* |
| Cl - | 3.00 |
Note: is the changed ion concentration.
(3) The other elements and contents in the MS culture medium are unchanged.
The invention fully considers the factors of N, P, K proportion, nitrate nitrogen and ammonium nitrogen proportion, pH value of culture medium, demand of plants on elements such as chlorine, sulfur and the like, and realizes the regulation and control of ion concentration, reasonable proportion among ions, charge number and the like by changing the composition and content of chemical substances, thereby having higher difficulty. The inventor optimizes the chemical substances for dozens of times to ensure that the final culture medium achieves the same or similar effect as the MS culture medium.
The invention has the beneficial effects that:
1. the basic culture medium for plant tissue culture disclosed by the invention can be suitable for culturing various common plant tissues and can obtain the same or similar effect as an MS culture medium.
2. The culture medium avoids using ammonium nitrate, so the culture medium has the characteristic of good safety and is convenient for large-scale popularization and use.
Drawings
FIG. 1 is a graph showing the comparative effect of tobacco callus induction and shoot differentiation on ZS and MS media, A: inducing tobacco leaf callus; b: differentiating adventitious buds of tobacco;
FIG. 2 is a graph showing the comparative effect of tobacco tissue culture seedling growth and root differentiation on both ZS and MS media, A: growing tobacco tissue culture seedlings; b: rooting the tobacco tissue culture seedling;
FIG. 3 is a graph showing the comparative effect of growth of potato test-tube plantlets on both MS and ZS media.
Detailed Description
The present invention will be further described with reference to examples, but the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
The following table shows the composition and content of the ZS minimal medium described in the present application. (the balance being distilled water, pH 5.5 to 6.0)
TABLE 5 ZS minimum Medium composition Table (mg/L)
Example 1: tobacco leaf callus induction and bud regeneration culture
The culture medium for the callus induction and the bud regeneration of the tobacco leaves comprises the following components: ZS minimal medium +6-BA 3mg/L + NAA0.2mg/L + sucrose 30g/L + agar powder 8g/L, pH 5.8. The composition of the ZS minimal medium is shown in Table 5 (the balance is distilled water, and the pH is 5.5-6.0).
The preparation method comprises the following steps: respectively adding 6-Benzyladenine (BA), naphthylacetic acid (NAA), sucrose and agar powder based on a ZS basic culture medium, uniformly mixing, and adjusting the pH to 5.8 by using 1mol/L KOH or 1mol/L HCl; 3mg of 6-BA, 0.2mg of NAA, 30g of cane sugar and 8g of agar powder are added into each 1L of ZS minimal medium.
The plant tissue culture medium is used for inducing tobacco callus and differentiating buds, and specifically comprises the following steps: sterilizing tobacco leaf with 70% ethanol for 30 s, and adding 0.1% HgCl
2Sterilizing for 8-10 min, washing with sterile water for 4 times, and cutting into small pieces of about 1cm square; placing the cut tobacco leaves on a culture medium for culturing; the culture conditions were: illumination is carried out for 16 hours, the illumination intensity is 2000-3000 LX, and the temperature is 25-26 ℃; the color is dark for 8 hours, and the temperature is 22-23 ℃; illumination and dark culture were performed alternately.
The results were: after 20 days of inoculation, the leaves are enlarged and thickened, and a large amount of callus is generated around the leaves; after further subculture for 20 days, a large number of adventitious buds are generated on the leaves, the multiplication times are 5-10 times, the buds are strong, the buds are light green, and the leaves are stretched. As shown in fig. 1.
Comparative example 1, a ZS minimal medium was replaced with a conventional MS medium, and the procedure was otherwise exactly the same as in example 1.
The results were: after 20 days of inoculation, the leaves are enlarged and thickened, and a large amount of callus is generated around the leaves; after further subculture for 20 days, a large number of adventitious buds are generated on the leaves, the multiplication times are 5-10 times, the buds are strong, the buds are light green, and the leaves are stretched. Basically similar to the ZS culture medium. As shown in fig. 1.
Example 2: rooting of tobacco tissue culture seedling
The rooting culture medium of the tobacco tissue culture seedling comprises: 1/2ZS minimal medium, 20g/L of cane sugar and 8g/L of agar powder, and the pH value is 5.8.
The preparation method comprises the following steps: respectively adding sucrose and agar powder based on 1/2ZS minimal medium, mixing well, and adjusting pH to 5.8 with 1mol/L KOH or 1mol/L HCl; 20g of sucrose and 8g of agar powder were added to 1L of 1/2ZS minimal medium. The composition of the ZS minimal medium is shown in Table 5 (the balance is distilled water, and the pH is 5.5-6.0).
The plant tissue culture medium is used for rooting the tobacco tissue culture seedling, and specifically comprises the following steps: when the regeneration bud of the tobacco grows to 1cm high, cutting and inoculating the regeneration bud into the rooting culture medium for culture; the culture conditions were: illumination is carried out for 16 hours, the illumination intensity is 2000-3000 LX, and the temperature is 25-26 ℃; the color is dark for 8 hours, and the temperature is 22-23 ℃; illumination and dark culture were performed alternately.
The results were: the regeneration bud is inoculated to a rooting culture medium for 30 days and can grow into a complete plant with the plant height of 8-10 cm; the plants are strong, the leaves are extended, the color of the leaves is dark green, and the root system is developed. As shown in fig. 2.
Comparative example 2, a ZS minimal medium was replaced with a conventional MS medium, and the procedure was otherwise exactly the same as in example 2.
The results were: the regeneration bud is inoculated to a rooting culture medium for 30 days and can grow into a complete plant with the plant height of 8-10 cm; the plants are strong, the leaves are extended, the color of the leaves is dark green, and the root system is developed. Basically similar to the ZS culture medium. As shown in fig. 2.
Example 3: potato test-tube plantlet culture
The culture medium of the potato test-tube plantlet is as follows: ZS minimal medium, 30g/L of white sugar, 6g/L of agar powder and pH of 5.8.
The preparation method comprises the following steps: respectively adding white sugar and agar powder based on a ZS basic culture medium, uniformly mixing, and adjusting the pH to 5.8 by using 1mol/L KOH or 1mol/L HCl; 30g of white sugar and 6g of agar powder are added into each 1L of the ZS minimal medium. The composition of the ZS minimal medium is shown in Table 5 (the balance is distilled water, and the pH is 5.5-6.0).
The method for culturing the potato test-tube plantlet by using the plant tissue culture medium comprises the following steps:
selecting vigorous potato virus-free seedlings, cutting stem segments with relatively consistent sizes on a superclean bench, inoculating 1-2 leaf buds to a culture medium, inoculating 15 stem segments to each culture bottle, and culturing in a culture room (incubator); the culture conditions were: illumination is carried out for 14 hours, the illumination intensity is 2000-3000 LX, and the temperature is 25-26 ℃; dark for 10 hours at a temperature of 22-23 ℃; illumination and dark culture were performed alternately.
The results were: after 20 days of culture, the potato virus-free test-tube plantlet grows vigorously, leaves are large and dark green, stems are thick and strong, root systems are developed, and the plant height is 7-8 cm. As shown in fig. 3 and in table 6.
Comparative example 3, a ZS minimal medium was replaced with a conventional MS medium, and the procedure was otherwise exactly the same as in example 3.
The results were: after 15 days of culture, the potato virus-free test-tube plantlet grows vigorously, leaves are large and dark green, stems are thick and strong, root systems are developed, and the plant height is 7-8 cm. The virus-free seedlings are basically similar to the ZS culture medium in plant height, leaf number, root number, average root length, stem thickness, effective node number and the like. As shown in fig. 3 and table 6.
TABLE 6 Effect of ZS and MS Medium on growth of Potato test-tube plantlets
| Culture medium | Plant height/cm | Number of blades/sheet | Root number/strip | Average root length/cm | Thickness of stem/mm | Effective number of nodes/node |
| ZS | 7.62±0.25 | 8.33±0.22 | 2.92±0.15 | 10.85±0.32 | 1.08±0.15 | 5.25±0.24 |
| MS | 7.59±0.31 | 8.30±0.27 | 2.89±0.17 | 9.97±0.29 | 1.06±0.12 | 5.16±0.28 |
Although the preferred media formulations and preferred embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended that the claims be limited thereto. It should be understood by those skilled in the art that various modifications or changes may be made without inventive efforts based on the technical solution of the present invention and still fall within the protective scope of the present invention.
Claims (3)
1. The application of a basic culture medium ZS in the induction and bud differentiation of tobacco callus is characterized in that each 1000mL of the ZS basic culture medium only contains the following raw materials in parts by mass: 3075mg potassium nitrate, 578mg ammonium phosphate, 491mg calcium nitrate, 160mg ammonium chloride, 222mg magnesium nitrate, 396mg ammonium sulfate, 0.83mg potassium iodide, 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 100mg inositol, 0.1mg thiamine hydrochloride (VB1), 0.5mg pyridoxine hydrochloride (VB6), 0.5mg nicotinic acid, 37.3mg disodium ethylenediaminetetraacetate, and 27.8mg ferrous sulfate.
2. The application of a basic culture medium ZS in the growth and root differentiation of tobacco tissue culture seedlings is characterized in that each 1000mL of the ZS basic culture medium only contains the following raw materials in parts by mass: 3075mg potassium nitrate, 578mg ammonium phosphate, 491mg calcium nitrate, 160mg ammonium chloride, 222mg magnesium nitrate, 396mg ammonium sulfate, 0.83mg potassium iodide, 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 100mg inositol, 0.1mg thiamine hydrochloride (VB1), 0.5mg pyridoxine hydrochloride (VB6), 0.5mg nicotinic acid, 37.3mg disodium ethylenediaminetetraacetate, and 27.8mg ferrous sulfate.
3. The application of the ZS in the growth of potato test-tube plantlets is characterized in that each 1000mL of the ZS minimal medium only contains the following raw materials in parts by mass: 3075mg potassium nitrate, 578mg ammonium phosphate, 491mg calcium nitrate, 160mg ammonium chloride, 222mg magnesium nitrate, 396mg ammonium sulfate, 0.83mg potassium iodide, 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 100mg inositol, 0.1mg thiamine hydrochloride (VB1), 0.5mg pyridoxine hydrochloride (VB6), 0.5mg nicotinic acid, 37.3mg disodium ethylenediaminetetraacetate, and 27.8mg ferrous sulfate.
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| CN110754366B (en) * | 2019-12-04 | 2022-03-08 | 西南大学 | Eurytopic plant tissue culture medium and 1/2 culture medium |
| CN112136688A (en) * | 2020-08-12 | 2020-12-29 | 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 thereof |
| CN111937745A (en) * | 2020-08-13 | 2020-11-17 | 江苏宝德农业科技有限公司 | Tissue culture method for rapid propagation of tissue culture seedlings for field planting in production process of detoxified miniature potatoes |
| CN112352679A (en) * | 2020-11-19 | 2021-02-12 | 广州市卉通农业科技有限公司 | Culture medium for efficient production of double-line arrowroot tissue culture seedlings and preparation method thereof |
| CN112931206B (en) * | 2021-03-04 | 2022-07-05 | 华南农业大学 | Plant culture medium free of easily-exploding compound and application thereof |
| CN114916443B (en) * | 2022-05-27 | 2023-03-17 | 广西特色作物研究院 | Method for differentiating cluster buds from excellent single-plant callus of Guangxi sweet tea |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
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| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
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