CN112931206B - A kind of plant culture medium without explosive compounds and its application - Google Patents

Abstract

The invention discloses a plant culture medium free of explosive compounds and an application thereof, belonging to the field of plant biotechnology. The plant culture medium without explosive compounds includes the following content of compound components that provide a large number of elements: 3500-4200 mg/L calcium ammonium nitrate, 1200-1600 mg/L potassium chloride, 370 mg/L heptahydrate sulfuric acid Magnesium and 170mg/L potassium dihydrogen phosphate. According to the needs of plants for ammonium nitrogen, 1000-1400mg/L ammonium chloride or 1000-1400mg/L ammonium sulfate can be added to supplement ammonium nitrogen. Compared with the MS medium, the medium of the present invention has little difference in composition, but the content of calcium ions is relatively high, which is conducive to promoting plant growth, has good tissue culture effect, and does not contain explosive compounds, and the composition is stable and safe. It is regulated and inexpensive, and has great application value to the tissue culture industry.

Description

A kind of plant culture medium without explosive-producing compound and application thereof

technical field

The invention belongs to the technical field of plant biology, and in particular relates to a plant culture medium that does not contain explosive compounds and its application.

Background technique

Plant culture medium is the material basis for the growth and development of in vitro materials in tissue culture, and is an essential production material for seedling production enterprises to apply biotechnology to produce seedlings. Usually plant tissue culture medium consists of inorganic nutrients, carbon sources, vitamins, retinoids and growth regulating substances. Inorganic nutrients include macroelements and trace elements; among them, macroelements refer to 6 elements that are in great demand, including nitrogen, phosphorus, potassium, calcium, magnesium and sulfur; trace elements refer to iron, boron, manganese, zinc, molybdenum, Cobalt and copper are 7 elements in low demand.

To date, almost all medium formulations contain ammonium nitrate or/and potassium nitrate to supply the nitrogen required by plant tissues. However, according to regulations such as the Regulations on the Safety Management of Hazardous Chemicals (Order No. 591 of the State Council), the Regulations on the Safety Management of Civil Explosives, and the Inventory of Predisposing Hazardous Chemicals (2017 Edition), ammonium nitrate and nitric acid Potassium is an explosive controlled chemical. With the strengthening of national supervision, the purchase, storage and use of all explosive chemicals are strictly regulated, and it is difficult to buy ammonium nitrate and potassium nitrate necessary for plant tissue culture medium on the market. At present, plant tissue culture enterprises can only purchase medium mixed powder, which increases costs and bears the additional risk of unreliable powder quality. In response to this problem, Wang Yuanhua et al. used ammonium sulfate instead of ammonium nitrate to prepare a medium not long ago, and reported success in tissue culture of apples, strawberries and grapes. However, due to the extremely high concentration of ammonium nitrogen (NH ₄ ⁺ ) and the significantly low concentration of nitrate nitrogen (NO ₃ ^- ) in the medium prepared by it, it may only be suitable for tissue culture of some plants, and Although ammonium nitrate has been replaced, potassium nitrate is still used. Therefore, it is urgent to find a tissue culture medium that does not contain ammonium nitrate and potassium nitrate and has a good effect.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a plant culture medium that does not contain explosive compounds. The plant culture medium of the invention does not contain explosive components ammonium nitrate and potassium nitrate, which is convenient for plant tissue culture enterprises to formulate the formula of plant tissue culture medium with low cost and good use effect, so as to fundamentally solve the problem of safety management departments for professional production The tedious problem of the management of the explosive ingredients ammonium nitrate and potassium nitrate that enterprises urgently need, and the high production cost and unstable quality of the powder due to the high production cost and unstable quality of the powder that the manufacturer needs to purchase the mixed culture medium.

Another object of the present invention is to provide the application of the above-mentioned plant culture medium free of explosive compounds.

The purpose of this application is achieved through the following technical solutions:

A plant culture medium that does not contain explosive compounds, comprising the following content of compound components that provide a large number of elements: 3500-4200 mg/L calcium ammonium nitrate, 1200-1600 mg/L potassium chloride, 370 mg/L magnesium sulfate heptahydrate and 170mg/L potassium dihydrogen phosphate;

The plant culture medium that does not contain explosive compounds preferably includes the following content of compounds that provide macroelements: 3872 mg/L calcium ammonium nitrate, 1401 mg/L potassium chloride, 370 mg/L magnesium sulfate heptahydrate and 170 mg/L. L potassium dihydrogen phosphate.

The plant preferably includes but is not limited to at least one of Asteraceae Echinacea and Orchidaceae Phalaenopsis; more preferably Asteraceae Echinacea.

The Asteraceae Echinacea plants include but are not limited to Echinacea.

The Orchidaceae Phalaenopsis plants include, but are not limited to, Phalaenopsis.

The plant culture medium without explosive compounds also includes other components in the MS medium except for a large number of elements: ammonium nitrate, potassium nitrate, magnesium sulfate heptahydrate, calcium chloride dihydrate and potassium dihydrogen phosphate , as follows: KI 0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ ·4H ₂ O 22.3mg/L, ZnSO ₄ ·7H ₂ O 8.6mg/L, Na ₂ MoO ₄ ·2H ₂ O 0.25 mg/L, CuSO ₄ ·5H ₂ O 0.025mg/L, CoCl ₂ ·6H ₂ O 0.025mg/L; FeSO ₄ ·7H ₂ O 27.85mg/L, Na ₂ -EDTA 37.25mg/L, Inositol 100mg/L L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L and niacin 0.5mg/L.

The compound that provides a large amount of elements in the plant medium without explosive compounds also includes ammonium chloride or ammonium sulfate.

When ammonium chloride is contained in the plant medium without explosive precursor compounds, the content of ammonium chloride is preferably 1000mg-1400mg/L; more preferably 1220mg/L.

When ammonium sulfate is contained in the plant medium without explosive compounds, the content of ammonium sulfate is preferably 1000-1400 mg/L; more preferably 1126 mg/L.

The application of the plant culture medium without explosive compounds in tissue culture.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The plant culture medium formula of the present invention does not contain explosive compounds, and the necessary NO ₃ ^- and NH ₄ ⁺ in the culture medium are provided with calcium ammonium nitrate, which is freely circulated in the market, has a low price, and is stable and safe in physical and chemical properties. It contains ammonium nitrate and potassium nitrate, the explosive ingredients that are currently banned in the medium, which greatly reduces social security risks, and also facilitates the purchase and storage of medium raw materials for production and use of enterprises, and reduces the preparation cost of the medium.

(2) the plant culture medium formula of the present invention that does not contain explosive compounds, it can be applied to the plant tissue culture of Echinacea of the genus Asteraceae Echinacea, and the proliferation and rooting effects of tissue culture seedlings are equivalent or better than MS culture medium (The most widely used medium at present).

(3) Compositae is one of the three major plant families (the other two are Orchidaceae and Cruciferae), and Echinacea is one of the three major medicinal materials in circulation in the world, with recognized significant antibacterial, anti-inflammatory and immunomodulatory effects (the other two are for enhancing physical fitness) ginseng and anti-aging ginkgo). The success of tissue culture from Echinacea suggests that the medium formulation of the present invention has a wider range of applied plants.

(4) The plant culture medium formula of the present invention does not contain explosive compounds, and in the formula, calcium ammonium nitrate and potassium chloride are used to replace ammonium nitrate, potassium nitrate and calcium chloride dihydrate. Compared with the MS medium, the medium of the present invention has little difference in composition compared with the MS medium, but the calcium ion content is relatively high, which is more conducive to the effect of plant tissue on other elements (nitrogen, phosphorus, potassium, magnesium, manganese, zinc, boron, copper, etc.). Elements) absorption, promote plant growth and biomass accumulation, improve plant resistance, good tissue culture effect, and do not contain explosive compounds, stable and safe ingredients, unregulated, low price, have a greater impact on the tissue culture industry. Value.

(5) In the plant medium of the present invention that does not contain explosive compounds, NH ₄ ⁺ is only provided by calcium ammonium nitrate, thereby greatly reducing the content of NH ₄ ⁺ in the formula. The accumulation of ammonium in plants has toxic effects on many plants, and reducing ammonium nitrogen is also more suitable for plants that prefer nitrate nitrogen. In addition, in the medium of the present invention, NH ₄ ⁺ is only provided by calcium ammonium nitrate, which simplifies the formulation of the medium, thereby making the formulation of the medium more convenient.

(6) Calcium ammonium nitrate (5Ca(NO ₃ ) ₂ ·NH ₄ NO ₃ ·10H ₂ O) is a modified product of ammonium nitrate. As a new type of compound fertilizer, it can simultaneously provide NO ₃ ^- , NH ₄ ⁺ and Water-soluble calcium, its nutrients are more comprehensive than ammonium nitrate, especially the application and storage and transportation are very safe.

Description of drawings

Fig. 1 is the result graph of the adventitious bud proliferation of Echinacea purpurea on three kinds of plant media without explosive compounds and MS medium; wherein, Fig. A is the result graph of the adventitious bud proliferation of Echinacea purpurea on MS medium; Fig. B is NECC1 Figure C is the result of adventitious bud proliferation of Echinacea on NECC2 medium; Figure D is the result of adventitious bud proliferation of Echinacea on NECC3 medium.

Fig. 2 is the result graph of the growth and rooting situation of Echinacea on three kinds of plant mediums without explosive compounds and MS medium; wherein, Fig. A is the result graph of the growth and rooting of Echinacea on MS medium; Fig. B is NECC1 The growth and rooting results of Echinacea on the medium; Figure C is the growth and rooting results of Echinacea on NECC2 medium; Figure D is the growth and rooting results of Echinacea on NECC3 medium.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The organic solvents used in the examples are all analytical grade organic solvents unless otherwise specified. Unless otherwise specified, the reagents or raw materials used in the examples can be obtained through purchase.

Non-explosive-compounds-containing medium formula NECC1: 3872 mg of calcium ammonium nitrate, 1401 mg of potassium chloride, 370 mg of magnesium sulfate heptahydrate and 170 mg of potassium dihydrogen phosphate per 1 L of medium;

Medium formula NECC2: Each 1L medium contains 3872mg calcium ammonium nitrate, 1220mg ammonium chloride, 1401mg potassium chloride, 370mg magnesium sulfate heptahydrate and 170mg potassium dihydrogen phosphate;

Medium formulation NECC3: 3872 mg of calcium ammonium nitrate, 1126 mg of ammonium sulfate, 1401 mg of potassium chloride, 370 mg of magnesium sulfate heptahydrate and 170 mg of potassium dihydrogen phosphate per 1 L of medium.

The composition of macroelements in normal MS medium is: KNO ₃ 1900mg/L, NH ₄ NO ₃ 1650mg/L, KH ₂ PO ₄ 170mg/L, MgSO ₄ 7H ₂ O 370mg/L, CaCl ₂ 2H ₂ O 440mg/L L.

In the above-mentioned culture medium, according to the needs of the specific culture purpose, add the same other components: KI 0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ 4H ₂ O 22.3mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L, Na ₂ MoO ₄ ·2H ₂ O 0.25mg/L, CuSO ₄ ·5H ₂ O 0.025mg/L, CoCl ₂ ·6H ₂ O 0.025mg/L; FeSO ₄ ·7H ₂ O 27.85mg/L L, Na ₂ -EDTA 37.25mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L and niacin 0.5mg/L.

The comparison results of the ion concentrations (mmol/L) of various elements and the total and ratio of nitrate nitrogen (NO ₃ ^- ) and ammonium nitrogen (NH ₄ ⁺ ) in NECC1, NECC2, NECC3 and normal MS medium are shown in the table below 1 shown.

Table 1:

Example 1: Proliferation and culture of adventitious buds of Echinacea

(1) Obtaining aseptic adventitious bud material of Echinacea purpurea: Echinacea purpurea aseptic adventitious bud is obtained from young petiole through induction culture and regeneration. The height of adventitious bud is about 2cm, including terminal bud and 2-3 tender leaves.

(2) Preparation of proliferation medium: 30g/L of sucrose, 0.4mg/L of 6-BA and 0.01mg/L of NAA, 4.8g/L of agar, and 4.8g/L of agar were added to the formulations of NECC1, NECC2, NECC3 and MS medium respectively. The value is adjusted to 6.0.

(3) Culture method: The above-mentioned adventitious buds were inoculated (vertically inserted) into the above-prepared NECC1, NECC2, NECC3 and MS medium. The cells were cultured for 45 days under the conditions of culture temperature of 25±2℃, light intensity of 2000Lx and light time of 12h/d.

Culture results: The proliferation folds of adventitious shoots of Echinacea on NECC1, NECC2 and NECC3 and MS medium are shown in Table 2 below.

Table 2:

culture medium multiplication factor 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, compared with MS medium, NECC1 and NECC2 significantly increased the proliferation fold of Echinacea adventitious buds, while NECC3 had no significant difference with MS medium. The growth vigor of propagating seedlings on NECC1, NECC2 and NECC3 was better than that on MS medium, as shown in Figure 1.

Example 2: Growth and rooting of adventitious shoots of Echinacea purpurea

(1) Obtainment of aseptic adventitious bud material of Echinacea purpurea: the same as the step (1) of Example 1.

(2) Preparation of growth and rooting medium: On the basis of NECC1, NECC2, NECC3 and MS medium, sucrose 30 g/L, NAA 0.02 mg/L, and agar 4.8 g/L were added, and the pH value was adjusted to 6.0.

(3) Culture method: Inoculate the adventitious buds obtained in step (1) into the NECC1, NECC2, NECC3 and MS medium prepared above. The cells were cultured for 45 days under the conditions of culture temperature of 25±2℃, light intensity of 2000Lx and light time of 12h/d.

(4) Transplanting method for rooted seedlings: Unscrew the cap of the culture bottle and move it to natural light. After 3 days, carefully remove the plants from the medium, wash the medium on the roots, soak in tap water for 12 hours, and plant them in a seedling tray containing moist seedling soil (peat soil: fir fine sawdust = 2:1). , under the conditions of room temperature and scattered light, cover with moisturizing and hardening; open the cover of the seedling tray after 3 days, and check the survival status of the seedlings after 10 days.

(5) The culture results are shown in Table 3 below.

table 3:

From Table 3 and Figure 2, it can be seen that NECC1, NECC2 and NECC3 and MS medium can all root the tissue culture seedlings, but in general, tissue culture grown on NECC1, NECC2 and NECC3 medium relative to MS medium The seedlings were stronger, the plants were taller, and the leaf area was larger, as shown in Figure 2. Judging from the specific conditions of each medium, tissue cultured seedlings in NECC1 medium have significantly improved main root length, main root diameter, plant height, weight per plant and transplant survival rate; in addition to longer main roots, tissue cultured seedlings in NECC2 medium There were no major differences in other aspects; the NECC3 medium tissue cultured seedlings had significant improvements in both main root length and plant height.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection 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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