CN108812313A - A kind of minimal medium of Plant Tissue Breeding - Google Patents

Abstract

The invention discloses a basic medium ZS for plant tissue culture, and relates to the field of plant propagation. The basic medium ZS contains 3075 mg of potassium nitrate, 578 mg of ammonium phosphate, 491 mg of calcium nitrate, chlorine Ammonium chloride 160mg, magnesium nitrate 222mg, ammonium sulfate 396mg, potassium iodide 0.83mg, boric acid 6.2mg, manganese sulfate 22.3mg, zinc sulfate 8.6mg, sodium molybdate 0.25mg, copper sulfate 0.025mg, cobalt chloride 0.025mg, inositol 100mg , Thiamine hydrochloride (VB1) 0.1mg, pyridoxine hydrochloride (VB6) 0.5mg, niacin 0.5mg, edetate disodium 37.3mg, ferrous sulfate 27.8mg. Using this basic medium can obtain the same tissue culture effect as MS medium without using ammonium nitrate.

Description

A basic medium for plant tissue culture

technical field

The invention relates to the field of plant tissue culture rapid propagation, in particular to a basic medium for plant tissue culture.

Background technique

Murshige and Skoog medium (MS, 1962) is a plant tissue culture medium widely used at present, and ammonium nitrate is an important component of the medium. Ammonium nitrate is a strong oxidizing agent, which can fuel the fire when combustibles catch fire; when mixed with combustible powder, it can react violently and explode; it can also explode when subjected to strong vibration or rapid heating. It can form an explosive mixture when mixed with reducing agents, organic substances, flammable substances such as sulfur, phosphorus or metal powder, and is the raw material for making explosives. Because of the huge potential safety hazards, in recent years, the national supervision of such items has become more and more strict, and the market circulation has been restricted by the relevant state departments. Production is inconvenient. However, there are both ammonium nitrogen and nitrate nitrogen in the ammonium nitrate molecule, which is necessary for plant growth and is widely used in plant tissue culture. Therefore, there is an urgent need for a plant tissue basic medium that can maintain the performance of the original MS medium and avoid the use of ammonium nitrate. At present, CN102224802A strawberry proliferation medium discloses a medium that can replace ammonium nitrate, but this medium is only suitable for strawberry proliferation medium, has no versatility, and is not suitable for promotion.

Contents of the invention

Aiming at the deficiencies in the prior art, the invention discloses a basic medium for plant tissue culture, which can be applied to various common plant tissue cultures, and can achieve the same or similar effect as MS medium.

In order to solve the above technical problems, the technical solution of the present invention is:

The invention provides a basic culture medium ZS for plant tissue culture, containing in every 1000mL culture medium: 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, Boric acid 6.2mg, manganese sulfate 22.3mg, zinc sulfate 8.6mg, sodium molybdate 0.25mg, copper sulfate 0.025mg, cobalt chloride 0.025mg, inositol 100mg, thiamine hydrochloride (VB1) 0.1mg, pyridoxine hydrochloride ( VB6) 0.5 mg, niacin 0.5 mg, edetate disodium 37.3 mg, ferrous sulfate 27.8 mg.

Concrete invention process of the present invention is as follows:

(1) Calculate the ion concentration of each element (Table 2) according to the content of macroelements (Table 1) in the MS medium;

Table 1 Composition and content of macroelements in MS medium

Table 2 Various ion concentrations of macroelements in MS medium

ion Ion concentration (mmol/L) K ⁺ 20.04 NO ₃ - 39.40 _NH4 ⁺ 20.61 PO ₄ ^3- 1.25 Ca ²⁺ 2.99 Mg ²⁺ 1.50 SO ₄ ^2- 1.50 ^Cl- 3.00

(2) Remove ammonium nitrate (NH ₄ NO ₃ ) and find suitable chemical reagents for combined substitution (Table 3);

The present invention keeps the concentrations of NO ₃ ^- , NH ₄ ⁺ , Ca ²⁺ , Mg ²⁺ , and Cl ^- ions unchanged, and appropriately increases the concentrations of K ⁺ , PO ₄ ^3- , SO ₄ ^2- ions (Table 4) , which is the key point of the present invention. The inventors have shown through a large number of experiments that the composition and content of the ZS medium provided by the present invention can achieve effects similar to those of the MS medium in terms of tobacco callus induction, bud differentiation, rooting and potato plantlet cultivation.

Table 3 ZS culture medium macroelement composition and content of the present 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 ) ₃ PO ₄ 203.13 578 3.88 calcium nitrate Ca(NO ₃ ) ₂ 4H ₂ O 236.18 707 2.99 ammonium chloride NH ₄ Cl 53.49 160 3.00 magnesium nitrate Mg(NO ₃ ) ₂ 6H ₂ O 256.40 384 1.50 ammonium sulfate (NH ₄ ) ₂ SO ₄ 132.14 396 3.00

Table 4 Various ion concentrations of macroelements in ZS medium

ion Ion concentration (mol/L) K ⁺ 30.42* NO ₃ ^- 39.40 _NH4 ⁺ 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) Other elements and contents in MS medium remained unchanged.

The present invention fully considers the ratio of N, P and K, the ratio of nitrate nitrogen and ammonium nitrogen, the pH value of the culture medium, the demand of plants for elements such as chlorine and sulfur, and realizes ionization by changing the composition and content of chemical substances. It is difficult to control the concentration, the reasonable ratio between ions, and the number of charges. The inventors have optimized the chemical substances dozens of times to achieve the same or similar effect as the MS medium in the final medium.

The beneficial effects of the present invention are:

1. The basal medium for plant tissue culture disclosed in the present invention can be applied to a variety of common plant tissue cultures, and can achieve the same or similar effects as MS medium.

2. The culture medium of the present invention avoids the use of ammonium nitrate, so it has the characteristics of good safety and is convenient for large-scale popularization and use.

Description of drawings

Figure 1 is the comparative effect of tobacco callus induction and bud differentiation on ZS and MS two media, A: tobacco leaf callus induction; B: tobacco adventitious bud differentiation;

Fig. 2 is the comparison effect diagram of tobacco tissue culture seedling growth and root differentiation on ZS and MS two kinds of media, A: tobacco tissue culture seedling growth; B: tobacco tissue culture seedling rooting;

Fig. 3 is a comparison effect diagram of the growth of potato plantlets in test tubes on MS and ZS two media.

Detailed ways

The present invention will be further described in conjunction with the examples, and the following descriptions are only for explaining the present invention, not limiting its content.

The following table is the composition and content of the ZS basic medium described in this application. (The rest is distilled water, pH 5.5-6.0)

Table 5 ZS basic medium composition list (mg/L)

Example 1: Tobacco leaf callus induction and bud regeneration culture

Tobacco leaf callus induction and shoot regeneration medium is: ZS basic medium + 6-BA 3mg/L + NAA0.2mg/L + sucrose 30g/L + agar powder 8g/L, pH 5.8. The composition of ZS basic medium is shown in Table 5 (the rest is distilled water, pH 5.5-6.0)

The production method is as follows: Based on the ZS basic medium, add 6-benzyladenine (BA), naphthaleneacetic acid (NAA), sucrose and agar powder respectively, mix well, and use 1mol/L KOH or 1mol/L HCl Adjust the pH to 5.8; add 3 mg 6-BA, 0.2 mg NAA, 30 g sucrose and 8 g agar powder to each 1 L of ZS basic medium.

Utilize above-mentioned plant tissue culture medium of the present invention to carry out the differentiation of tobacco callus tissue and bud, be specifically: get tobacco leaf, sterilize with 70% ethanol for 30 seconds, then sterilize with 0.1% _HgCl for 8-10 minutes, use without After washing with bacterial water for 4 times, cut into small pieces about 1 cm square; the cut tobacco leaves are placed on the culture medium for cultivation; the cultivation conditions are: 16 hours of light, light intensity of 2000-3000LX, and temperature of 25-26°C; 8 hours of darkness, the temperature is 22 ~ 23 ℃; light and dark culture alternately.

The results were: 20 days after inoculation, the leaves increased and thickened, and a large number of calluses were produced around the leaves; after further subculture for 20 days, a large number of adventitious buds were produced on the leaves, with a multiplication factor of 5 to 10 times, the buds were strong, and the buds were light green. The blade stretches. As shown in Figure 1A, B.

Comparative Example 1. The ZS basic medium was replaced by conventional MS medium, and the rest were completely the same as in Example 1.

The results were: 20 days after inoculation, the leaves increased and thickened, and a large number of calluses were produced around the leaves; after further subculture for 20 days, a large number of adventitious buds were produced on the leaves, with a multiplication factor of 5 to 10 times, the buds were strong, and the buds were light green. The blade stretches. Basically similar to ZS medium. As shown in Figure 1A, B.

Embodiment 2: Tobacco tissue culture seedling takes root

The rooting medium of tobacco tissue culture seedlings is: 1/2ZS basic medium + 20g/L sucrose + 8g/L agar powder, pH is 5.8.

The production method is as follows: based on 1/2ZS basic medium, add sucrose and agar powder respectively, mix well, adjust the pH to 5.8 with 1mol/L KOH or 1mol/L HCl; every 1L of 1/2ZS basic medium Add 20g sucrose and 8g agar powder. The composition of ZS basic medium is shown in Table 5 (the rest is distilled water, pH 5.5-6.0)

Utilize above-mentioned plant tissue culture medium of the present invention to carry out the rooting of tobacco tissue culture seedling, specifically: when the regenerated bud of tobacco grows to 1cm high, cut off and inoculate in the above-mentioned rooting medium and cultivate; culture condition is: 16 hours light , the light intensity is 2000-3000LX, the temperature is 25-26°C; 8 hours of darkness, the temperature is 22-23°C; light and dark cultivation are carried out alternately.

The result is: after the regenerated shoots are inoculated into the rooting medium for 30 days, they can grow into a complete plant with a plant height of 8-10 cm; the plant is robust, the leaves are stretched, the leaves are dark green, and the root system is developed. as shown in picture 2.

Comparative example 2, the conventional MS medium was used to replace the ZS basic medium, and the rest were completely the same as in Example 2.

The result is: after the regenerated shoots are inoculated into the rooting medium for 30 days, they can grow into a complete plant with a plant height of 8-10 cm; the plant is strong, the leaves are stretched, the leaves are dark green, and the root system is developed. Basically similar to ZS medium. as shown in picture 2.

Embodiment 3: Potato test-tube plantlet cultivation

Potato test-tube seedling medium is: ZS basic medium + white sugar 30g/L + agar powder 6g/L, pH 5.8.

The production method is as follows: based on the ZS basic medium, add sugar and agar powder respectively, mix well, adjust the pH to 5.8 with 1mol/L KOH or 1mol/L HCl; add 30g white sugar to every 1L of ZS basic medium and 6g agar powder. The composition of ZS basic medium is shown in Table 5 (the rest is distilled water, pH 5.5-6.0)

Utilize above-mentioned plant tissue culture medium of the present invention to carry out the cultivation of potato plantlet in test tube, specifically:

Select the virus-free potato seedlings that are growing vigorously. On the ultra-clean workbench, cut the stem segments to be relatively consistent in size, each stem segment has 1-2 leaf buds, and insert them into the medium. Each culture bottle is connected to 15 stem segments. Placed in a culture room (incubator) for cultivation; the cultivation conditions are: 14 hours of light, light intensity 2000-3000LX, temperature 25-26°C; 10 hours of darkness, temperature 22-23°C; light and dark cultivation are carried out alternately.

The results were: after 20 days of cultivation, the virus-free potato seedlings grew vigorously, with large leaves and dark green leaves, thick stems, well-developed root systems, and a plant height of 7-8 cm. As shown in Figure 3 and Table 6.

Comparative example 3, the conventional MS medium was used to replace the ZS basic medium, and the rest were completely the same as in Example 3.

The result was: after culturing for 15 days, the virus-free potato plantlets grew vigorously, with large leaves and dark green leaves, thick stems, well-developed root systems, and a plant height of 7-8 cm. Virus-free seedlings were basically similar to ZS medium in terms of plant height, number of leaves, number of roots, average root length, stem diameter, and number of effective nodes. As shown in Figure 3 and Table 6.

Table 6 Effects of ZS and MS medium on the growth of potato tube plantlets

culture medium Plant height/cm Number of blades/piece Rooting number/piece Average root length/cm Stem thickness/mm Effective number of sections/section 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 above descriptions have been made in conjunction with the drawings to describe the best medium formula and several preferred specific embodiments of the present invention, they should not be construed as limitations on the claims. Those skilled in the art should understand that on the basis of the technical solutions of the present invention, various modifications or deformations that those skilled in the art can make without creative efforts are still within the protection scope of the present invention.

Claims (5)

1. a kind of minimal medium ZS of Plant Tissue Breeding, which is characterized in that contain in the ZS minimal medium of every 1000mL The raw material of following mass parts：Potassium nitrate 3075mg, ammonium phosphate 578mg, calcium nitrate 491mg, ammonium chloride 160mg, magnesium nitrate 222mg, ammonium sulfate 396mg, potassium iodide 0.83mg, boric acid 6.2mg, manganese sulfate 22.3mg, zinc sulfate 8.6mg, sodium molybdate 0.25mg, copper sulphate 0.025mg, cobalt chloride 0.025mg, inositol 100mg, thiamine hydrochloride (VB1) 0.1mg, puridoxine hydrochloride (VB6) 0.5mg, niacin 0.5mg, disodium ethylene diamine tetraacetate 37.3mg, ferrous sulfate 27.8mg.

2. application of the minimal medium ZS described in claim 1 in plant tissue culture.

3. application according to claim 2, which is characterized in that the application is in tobacco healing tissue's induction, bud differentiation In application.

4. application according to claim 2, which is characterized in that the application is in the growth of tobacco tissue-cultured seedling and Furcation defects Using.

5. application according to claim 2, which is characterized in that the application is the application in Potato Plantlets in vitro.