CN112931206B - Plant culture medium free of easily-exploding compound and application thereof - Google Patents
Plant culture medium free of easily-exploding compound and application thereof Download PDFInfo
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- CN112931206B CN112931206B CN202110241099.4A CN202110241099A CN112931206B CN 112931206 B CN112931206 B CN 112931206B CN 202110241099 A CN202110241099 A CN 202110241099A CN 112931206 B CN112931206 B CN 112931206B
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- 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
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a plant culture medium free of easily-exploding compounds and application thereof, and belongs to the technical field of plant biology. The plant culture medium without the easily explosive compound comprises the following components of a compound for providing macroelements in content: 3500-4200 mg/L of calcium ammonium nitrate, 1200-1600 mg/L of potassium chloride, 370mg/L of magnesium sulfate heptahydrate and 170mg/L of monopotassium phosphate. According to the requirement of the plant for ammonium nitrogen, 1000-1400 mg/L ammonium chloride or 1000-1400 mg/L ammonium sulfate can be added to supplement the ammonium nitrogen. Compared with an MS culture medium, the culture medium has small component difference, but has relatively high calcium ion content, is favorable for promoting the growth of plants, has good tissue culture effect, does not contain easily-exploding compounds, has stable and safe components, is not controlled, has low cost and has greater application value in the tissue culture industry.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a plant culture medium free of easily-exploding compounds and application thereof.
Background
The plant culture medium is the material basis for the growth and development of the isolated material in tissue culture and is an essential production material for seedling production enterprises to apply biotechnology to produce seedlings. Typically, plant tissue culture media consist of inorganic nutrients, carbon sources, vitamins, retinoids, and growth regulating substances. Inorganic nutrients including macro-elements and trace elements; wherein, the macroelements refer to 6 elements with larger demand of nitrogen, phosphorus, potassium, calcium, magnesium and sulfur; the trace elements are 7 elements with small demand, such as iron, boron, manganese, zinc, molybdenum, cobalt and copper.
To date, almost all media formulations contain ammonium nitrate or/and potassium nitrate to supply the nitrogen elements required by plant tissues. However, according to the regulations and regulations such as "regulations on safety management of hazardous chemicals" (No. 591 in the state-of-affairs' academy), "regulations on safety management of civil explosive materials", and "famous records of hazardous chemicals prone to explosion" (2017 edition), ammonium nitrate and potassium nitrate are both controlled chemicals prone to explosion. With the enhancement of national regulation, the purchase, storage and use of all explosive chemicals are strictly controlled, and ammonium nitrate and potassium nitrate which are necessary for plant tissue culture media are difficult to buy in the market. At present, plant tissue culture enterprises can only purchase culture medium mixed powder, so that the cost is increased and the powder quality is additionally borneRisk of unreliable volume. To solve this problem, ammonium sulfate was used in place of ammonium nitrate to prepare a culture medium, and success in tissue culture of apple, strawberry and grape was reported. However, due to the ammonium Nitrogen (NH) of the culture medium prepared therefrom4 +) Extremely high concentration, and significantly lower nitrate Nitrogen (NO)3 -) The concentration imbalance may only be suitable for tissue culture of part of plants, and although ammonium nitrate is replaced, potassium nitrate is still used. Therefore, a tissue culture medium which does not contain ammonium nitrate and potassium nitrate and has good use effect needs to be found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a plant culture medium which does not contain easily exploded compounds. The plant culture medium does not contain the easily-exploding components of ammonium nitrate and potassium nitrate, is convenient for plant tissue culture enterprises to prepare the plant tissue culture medium formula with low cost and good use effect, and fundamentally solves the complex problem of management of the easily-exploding components of ammonium nitrate and potassium nitrate which are urgently needed by safety management departments for professional production enterprises, and the high production cost and high risk problem of unstable powder quality due to the need of purchasing mixed culture medium powder by the production enterprises.
It is another object of the present invention to provide the use of the above plant culture medium without the easily explodable compound.
The purpose of the application is realized by the following technical scheme:
a plant culture medium free of easily explodable compounds comprising macroelement providing compound components in the following amounts: 3500-4200 mg/L of calcium ammonium nitrate, 1200-1600 mg/L of potassium chloride, 370mg/L of magnesium sulfate heptahydrate and 170mg/L of monopotassium phosphate;
the plant culture medium without the easily explosive compound preferably comprises the following macroelement-providing compound components in the following contents: 3872mg/L of calcium ammonium nitrate, 1401mg/L of potassium chloride, 370mg/L of magnesium sulfate heptahydrate and 170mg/L of monopotassium phosphate.
The plant preferably includes but is not limited to at least one of Echinacea plant of Compositae and Phalaenopsis plant of Orchidaceae; more preferably, it is a plant of Echinacea of Compositae.
The Echinacea plant of Compositae includes but is not limited to Echinacea purpurea.
The plant of the genus phalaenopsis in the family of orchidaceae includes, but is not limited to, phalaenopsis.
The plant culture medium without the easily-exploding compound also comprises the following components except macroelements in the MS culture medium: the components except ammonium nitrate, potassium nitrate, magnesium sulfate heptahydrate, calcium chloride dihydrate and potassium dihydrogen phosphate are as follows: KI 0.83mg/L, H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L；FeSO4·7H2O 27.85mg/L、Na237.25mg/L EDTA, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride and 0.5mg/L nicotinic acid.
The compound for providing macroelements in the plant culture medium without the easily exploding compound also comprises ammonium chloride or ammonium sulfate.
When the plant culture medium without the easy-explosion compound contains ammonium chloride, the content of the ammonium chloride is preferably 1000 mg-1400 mg/L; more preferably 1220 mg/L.
When the plant culture medium without the easily explosive compound contains ammonium sulfate, the content of the ammonium sulfate is preferably 1000-1400 mg/L; more preferably 1126 mg/L.
The application of the plant culture medium without the easy explosion-producing compound in tissue culture.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the plant culture medium formula without the easily-exploding compound uses the calcium ammonium nitrate which is freely circulated in the market, low in price, stable and safe in physicochemical property to provide necessary NO in the culture medium3 -And NH4 +Does not contain the easily-exploded components of ammonium nitrate and potassium nitrate which are usually prohibited to circulate in the culture medium at present, and is extremely largeThe method reduces social security risk, also facilitates the purchase and storage of culture medium raw materials of enterprises for production and use, and reduces the preparation cost of the culture medium.
(2) The plant culture medium formula without the easily exploding compound can be applied to the plant tissue culture of echinacea purpurea of echinacea of Compositae, and the proliferation and rooting effects of tissue culture seedlings are equivalent to or superior to those of an MS culture medium (the most widely applied culture medium at present).
(3) The Compositae is one of 3 families (the other two families are orchidaceae and cruciferae), and the Echinacea purpurea is an international 3-family circulating medicinal material, and has well-known remarkable antibacterial, anti-inflammatory and immunoregulatory effects (the other two families are ginseng for strengthening physique and anti-aging ginkgo). The success of the echinacea tissue culture suggests that the culture medium formula of the invention has wider application plant range.
(4) The plant culture medium formula without the easily-exploding compound has the advantages that ammonium nitrate, potassium nitrate and calcium chloride dihydrate are replaced by calcium ammonium nitrate and potassium chloride. Compared with an MS culture medium, the culture medium has small component difference, but relatively high calcium ion content, is more favorable for plant tissues to absorb other elements (nitrogen, phosphorus, potassium, magnesium, manganese, zinc, boron, copper and other elements), promotes plant growth and biomass accumulation, improves plant resistance, has good tissue culture effect, does not contain easily explosive compounds, has stable and safe components, is not controlled, has low cost and has greater application value to the tissue culture industry.
(5) In the plant culture medium of the invention, which does not contain the easily exploding compound, NH is supplied only by calcium ammonium nitrate4 +Thereby greatly reducing NH in the formula4 +The content of (a). The accumulation of ammonium in plants has a toxic effect on many plants, and the reduction of ammonium nitrogen is also more suitable for plants that prefer nitrate nitrogen. In addition, NH is supplied to the medium of the present invention solely by calcium ammonium nitrate4 +The formula of the culture medium is simplified, so that the preparation of the culture medium is more convenient.
(6) Calcium ammonium nitrate (5Ca (NO)3)2·NH4NO3·10H2O) is a modified product of ammonium nitrate as oneA novel compound fertilizer can provide NO simultaneously3 -、NH4 +And water-soluble calcium, the nutrient of which is more comprehensive than that of ammonium nitrate, and the fertilizer is particularly safe to apply, store and transport.
Drawings
FIG. 1 is a graph showing the results of adventitious bud proliferation of Echinacea purpurea on three plant media and MS media without easily exploding compounds; wherein, the picture A is a result picture of the proliferation of echinacea adventitious buds on the MS culture medium; FIG. B is a graph showing the result of proliferation of an echinacea adventitious bud on the NECC1 medium; FIG. C is a graph showing the result of proliferation of an adventitious bud of Echinacea purpurea on the NECC2 medium; FIG. D is a graph showing the result of proliferation of an adventitious bud of Echinacea purpurea in the NECC3 medium.
FIG. 2 is a graph showing the results of the growth and rooting of Echinacea on three plant media and MS media without the easily explosive compound; wherein, the picture A is a picture of the growth and rooting results of the echinacea purpurea on the MS culture medium; FIG. B is a graph showing the results of Echinacea growth and rooting on NECC1 medium; FIG. C is a graph showing the results of Echinacea growth and rooting on NECC2 medium; panel D is a graph of Echinacea growth and rooting results on NECC3 medium.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The organic solvents used in the examples are, unless otherwise specified, analytical grade organic solvents. Unless otherwise specified, reagents or raw materials used in examples are commercially available.
Plant medium (non-productive-compounds-containing medium) formulation NECC1 without a prohibitively explosive compound: each 1L of the culture medium contains 3872mg of calcium ammonium nitrate, 1401mg of potassium chloride, 370mg of magnesium sulfate heptahydrate and 170mg of monopotassium phosphate;
medium formulation NECC 2: each 1L of the culture medium contains 3872mg of calcium ammonium nitrate, 1220mg of ammonium chloride, 1401mg of potassium chloride, 370mg of magnesium sulfate heptahydrate and 170mg of potassium dihydrogen phosphate;
medium formulation NECC 3: each 1L of the medium contained 3872mg of calcium ammonium nitrate, 1126mg of ammonium sulfate, 1401mg of potassium chloride, 370mg of magnesium sulfate heptahydrate, and 170mg of potassium dihydrogen phosphate.
The macroelement composition in normal MS medium is: KNO3 1900mg/L、NH4NO3 1650mg/L、KH2PO4170mg/L、MgSO4·7H2O 370mg/L、CaCl2·2H2O 440mg/L。
The above media, depending on the needs of the specific culture purpose, are supplemented with the same other components: KI 0.83mg/L, H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L；FeSO4·7H2O 27.85mg/L、Na237.25mg/L EDTA, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride and 0.5mg/L nicotinic acid.
Ion concentrations (mmol/L) of various elements in NECC1, NECC2, NECC3 and normal MS medium, and nitrate Nitrogen (NO)3 -) And ammonium Nitrogen (NH)4 +) The results of comparing the total amount and the ratio are shown in table 1 below.
Table 1:
example 1: multiplication culture of echinacea adventitious bud
(1) Obtaining an echinacea purpurea sterile adventitious bud material: echinacea purpurea sterile adventitious bud is obtained by inducing, culturing and regenerating tender petiole, wherein the adventitious bud is about 2cm in height, and comprises terminal bud and 2-3 tender leaves.
(2) Preparing a proliferation culture medium: sucrose 30g/L, 6-BA 0.4mg/L, NAA 0.01mg/L and agar 4.8g/L are added on the basis of NECC1, NECC2, NECC3 and MS culture medium formula respectively, and the pH value is adjusted to 6.0.
(3) The culture method comprises the following steps: the adventitious buds were inoculated (vertically inserted) into the prepared NECC1, NECC2, NECC3 and MS medium described above. Culturing for 45d under the conditions of culture temperature of 25 + -2 deg.C, illumination intensity of 2000Lx, and illumination time of 12 h/d.
And (3) culture results: proliferation fold of echinacea adventitious buds on NECC1, NECC2, and NECC3 and MS medium is shown in Table 2 below.
Table 2:
| culture medium | Fold of proliferation |
| MS | 1.5±0.17b |
| NECC1 | 1.97±0.29a |
| NECC2 | 2.06±0.1a |
| NECC3 | 1.5±0.3b |
As can be seen from Table 2, NECC1 and NECC2 significantly increased the proliferation fold of Echinacea adventitious bud compared to MS medium, while NECC3 did not differ significantly from MS medium. The growth of the seedlings on NECC1, NECC2 and NECC3 was better than that on MS medium, as shown in FIG. 1.
Example 2: growth and rooting culture of echinacea adventitious bud
(1) Obtaining an echinacea purpurea sterile adventitious bud material: same as in step (1) of example 1.
(2) Preparing a growth and rooting culture medium: sucrose 30g/L, NAA 0.02.02 mg/L and agar 4.8g/L were added to NECC1, NECC2, NECC3 and MS medium, and the pH was adjusted to 6.0.
(3) The culture method comprises the following steps: inoculating the adventitious bud obtained in step (1) into the prepared NECC1, NECC2, NECC3 and MS medium. Culturing for 45d under the conditions of culture temperature of 25 + -2 deg.C, illumination intensity of 2000Lx, and illumination time of 12 h/d.
(4) The method for transplanting the rooted seedlings out of the bottles comprises the following steps: the cover of the culture bottle is unscrewed and the culture bottle is moved to the natural light. After 3 days, carefully taking out the plants from the culture medium, cleaning the culture medium on the roots, soaking the plants in tap water for 12 hours, planting the plants in a seedling raising tray containing moist seedling raising soil (peat soil: China fir wood sawdust is 2:1), and covering the seedling raising tray with a cover to keep moisture and harden the seedlings under the conditions of room temperature and scattered light; and 3d, opening the seedling raising tray cover, and checking the survival condition of the seedlings after 10 days.
(5) The culture results are shown in Table 3 below.
Table 3:
as can be seen from table 3 and figure 2, NECC1, NECC2 and NECC3 all rooted tissue culture seedlings but in general, tissue culture seedlings grown on NECC1, NECC2 and NECC3 media were stronger, more tall and larger in leaf area, relative to MS media, as shown in figure 2. From the specific situation of each culture medium, the tissue culture seedling of the NECC1 culture medium is obviously improved in the aspects of length of a main root, diameter of the main root, plant height, single plant weight and transplanting survival rate; the tissue culture seedlings of the NECC2 culture medium have no great difference in other aspects except that the main roots are longer; the tissue culture seedling of the NECC3 culture medium has obviously improved main root length and plant height.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (1)
1. The application of the plant culture medium without the easy explosion-producing compound in the echinacea tissue culture is characterized in that,
the plant culture medium without the easily explosive compound comprises the following components:
3872mg/L calcium ammonium nitrate, 1220mg ammonium chloride, 1401mg/L potassium chloride, 370mg/L magnesium sulfate heptahydrate, 170mg/L monopotassium phosphate, 0.83mg/L KI, 6.2mg/L H3BO3、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·6H2O、27.85mg/L FeSO4·7H2O、37.25mg/L Na2EDTA, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride and 0.5mg/L nicotinic acid.
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