DE102013101454B4 - Culture media for inokolum production and spore production in the context of an in-vitro mass production of VA mycorrhizal spores and process for their preparation - Google Patents

Abstract

Culture media for inokolum production and / or spore production in the context of an in-vitro mass production of VA mycorrhizal spores consisting at least of the macroelements calcium chloride, magnesium sulfate, potassium nitrate and potassium hydrogen phosphate, the microelements zinc sulphate, copper sulphate, boric acid, sodium molybdate, the vitamins thiamine, nicotinic acid, pyridoxine, one Potassium compound, NaFeEDTA, sucrose, calcium nitrate and a gelling agent, characterized in that it contains as further vitamins biotin, cyanocobalmin and calcium panthothenate and at least one phytohormone or more phytohormones and as potassium compound potassium chloride, manganese sulfate, traces of ammonium molybdate and an increased proportion of calcium chloride.

Description

The invention relates to culture media for inokolum production and spore production in the context of an in-vitro mass production of VA mycorrhizal spores and a method for preparing the culture media.

The use of the symbiosis between mycorrhizal fungi and higher plants has long been known and is becoming increasingly important, especially in the areas of reforestation, recultivation, organic farming, horticulture and horticulture.

Conventional VA mycorrhiza production is based on in vivo methods on plants by fixing the VA mycorrhizal spores on support materials.

The inocula thus produced contain up to 200 infection units / ml vehicle, of which about one third are spores or spore groups. Expanded clay, vermiculite or other porous granular materials may be considered as carriers. In this methodology, whole plants, e.g. Corn, dressed in beds. The plants root through the expanded clay or similar. and the inoculated VA mycorrhiza multiplies in the root-expanded clay mixture. The expanded clay is harvested, dried, optionally ground and then used as an inoculum.

The disadvantage of this in vivo procedure is the required amounts of plant and carrier materials. In addition, there is a high contamination of the acquired mycorrhiza spores with ubiquitous microorganisms.

Methods have already been proposed for in vitro production of VA mycorrhizal spores, but these are not suitable for mass production.

The nutrient or culture media required for the cultivation or production of roots, inoculum and spores are subject to special requirements, also for reasons of economy.

The media should be inexpensive and suitable for further at least secondary utilization. Essential factors for mass production are, in particular, such factors as spore germination, root length growth, root branching, hyphal growth, number of secondary spores (spore yield).

Not to be underestimated is the suitability of the medium for a cost-effective spore harvest. So far, no nutrient or culture medium is known that meets these requirements. A method is known for the monoxenic in vitro cultivation of VA mycorrhizal fungi ( WO2005 / 000008 A2 ) with the process of growing the roots and the inoculum, inoculating and growing the spore root culture, wherein the cultivation phase of the inoculum in the presence of a rhizogenic callus takes place under sterile conditions. Inoculum, nutrient medium and rhizogenic callus are prepared under sterile culture conditions.

For in vitro production of VA mycorrhizal spores, the use of a gel-containing MSR medium (Strullu Romand medium) is known as nutrient medium ( WO2005 / 000008 A2 and EP 1 231 854 B1 ). In addition to the gelling agent agar, this contains macroelements, microelements, vitamins, hormones and sucrose. With regard to the specific composition of this nutrient medium, reference is made to the information WO A2.

From the US 6,576,457 B1 For the production of VA mycorrhizal spores, a culture medium with the following composition is known:
300 to 500 mg / l hydrated Ca (NO ₃ ) ₂ , 60 to 90 mg / l CaCl ₂ × 2H ₂ O, 450 to 600 mg / l MgSO ₄ × 7H ₂ O, 80 to 120 mg / l KNO ₃ , 2 to 5 mg / l KH ₂ PO ₄ , 5 to 20 mg / l Ca phytate, 6 to 10 mg / l NaFeEDTA, 3 to 6 mg / l MnCl ₂ × 4H ₂ O, 2 to 3 mg / l ZnSO ₄ × 7H ₂ O, 0.05 to 0.15 mg / l CuSO ₄ × 5H ₂ O, 0.5 to 0.8 mg / l KJ, 1 to 2 mg / l H ₃ BO ₃ , 0.003 to 0.006 mg / l Na ₂ MoO ₄ x 2H ₂ O, 20 to 50 mg / l myoinositol, 0.1 to 0.4 mg / l thiamine, 0.5 to 1.0 mg / l nicotinic acid, 0.1 to 0.5 mg / l Pyridoxine, 2 to 4 mg / l glycine, 20 to 80 mg / l carbenicillin, 7.5 to 10 g / l sucrose and 3 to 5 g gelling agent.

Phosphorus is contained in the medium in the range of 0.022 to 0.065 mmol.

The disadvantage of the known culture media is that the process of rooting (root mass, branching of the roots) and their mycorrhization are too slow. In addition, the spore formation process is too slow and the spore yield too low.

For these reasons, the known culture or nutrient media are not suitable for economical mass production of in vitro VA mycorrhizal spores.

The invention has for its object to provide culture media for inokolumproduktion and spore production, which are characterized by very good root growth, rapid spore germination and rapid mycorrhization and allow for relatively low root mass very good fungal growth and high spore yield. According to the invention the object is achieved by the features specified in claim 1. Advantageous embodiments and further developments of the culture media are the subject of claims 2 to 9. The preparations of the culture media according to the invention are the subject of claim 10. Claim 11 relates to a further embodiment of features of claim 10.

The culture media according to the invention contain as further constituents as vitamins biotin, cyanocobalmin and calcium panthothenate and at least one or more phytohormones and as potassium compound potassium chloride, manganese sulfate, traces of ammonium molybdate and an increased proportion of calcium chloride.

An increased level of calcium chloride is several times greater than the amount of 60 to 90 mg / l known per se, e.g. at least 400 to 500 mg / l.

Experiments have shown that the addition of vitamins in combination with the phytohormones significantly increases the formation of root mass and increases the degree of branching of the root.

Preferred phytohormones are indolebutyric acid (IBA), indoleacetic acid (IAA), abscisic acid (ABA), naphthylacetic acid (NAA), either individually or in combination.

The concentration range for IBA should be between 0.1 mg / l and 1 mg / l. Improvements in root growth and spore production have been identified in this area. The optimal concentration is 0.5 mg / l. The improved root growth is reflected by the doubling of the root diameter and a reduction of the average distance to the next branching between the roots by a factor of 5. IBA also causes an initialization of spore germination. This is reflected in a faster start of hyphal and spore regeneration. In experiments, twice the number of spores could be achieved compared to known nutrient media.

Experiments with IAA showed in the concentration range between 0.01 mg / l and 0.15 mg / l a significant improvement in rooting. The optimal concentration is 0.1 mg / l.

For the production of in-vitro VA mycorrhizaspores, it was found that adding the phytohormones indolebutyric acid (IBA) and indoleacetic acid (IAA) significantly accelerates the spore formation process and increases the spore yield in the stated concentrations.

The preferred concentration range for ABA is between 0.1 mg / L and 0.75 mg / L. Root growth was significantly improved. The optimal concentration is 0.15 mg / l. It was found 3 to 4 times the increase of root mass.

The addition of NAA should be between 0.1 and 0.2 mg / l. NAA leads to strong branching of the roots.

The phytohormones can be added either individually or in combination to the respective culture medium.

The culture media of the invention now meet all requirements for mass production of in vitro VA mycorrhizal spores. By mass production is meant an annual production of at least about 1 trillion in vitro VA mycorrhizal spores.

Preferably, culture media with the following compositions are used:

Macro elements:
700 to 900 mg / l CaCl ₂ x 2 H ₂ O, 400 to 700 mg / l MgSO ₄ × 7H ₂ O 65 to 115 mg / l KNO _3, 5.5 to 8 mg / l KH ₂ PO ₄ and 60 up to 80 mg / l KCl;

Microelements:
1.5 to 2.5 mg / l MnSO ₄ .4H ₂ O, 0.2 to 1.5 mg / l ZnSO ₄ .7H ₂ O, 0.15 to 0.3 mg / l CuSO ₄ .5H ₂ O , 0.6 to 1.5 mg / l H ₃ BO ₃ , 0.002 to 0.0035 mg / l Na ₂ MoO ₄ x 2H ₂ O, 0.025 to 0.035 mg / l (NH ₄ ) ₂ Mo ₇ O ₂₄ x 4H ₂ O;

vitamins:
0.4 to 1 mg / l thiamin, 0.4 to 1.2 mg / l nicotinic acid, 0.4 to 0.9 mg / l pyridoxine, 0.1 to 0.5 mg / l cyanocobalmin, 0.0004 to 0.0009 mg / l biotin, 0.3 to 1.1 mg / l calcium pantothenate;

phytohormones:
0.2 to 0.7 mg / l indolebutyric acid and / or 0.01 to 0.15 mg / l indoleacetic acid;
NaFeEDTA: 4 to 8 mg / l,
Sucrose: 8 to 10 g / l and
Gelling agent: 2 to 4 g / l phytagel.

It has proved to be advantageous if the culture media are adjusted to a pH of 5.6. The adjustment is made e.g. with KOH.

For inokolum production a culture medium with the following composition was found, which is characterized by particularly good performance properties:
800 mg / l CaCl ₂ × 2 H ₂ O, 400 mg / l MgSO ₄ × 7H ₂ O, 110 mg / l KNO ₃ , 7 mg / l KH ₂ PO ₄ , 5 mg / l NaFeEDTA, 1.8 mg / 1 MnSO ₄ .4H ₂ O, 1 mg / l ZnSO ₄ .7H ₂ O, 0.2 mg / l CuSO ₄ .5H ₂ O, 70 mg / l KCl, 0,9 mg / l H ₃ BO ₃ , 0 , 0029 mg / l Na ₂ MoO ₄ × 2H ₂ O, 0.03 mg / l (NH ₄ ) ₂ Mo ₇ O ₂₄ × 4H ₂ O, 0.5 mg / l thiamine, 0.5 mg / l nicotinic acid, 0.45 mg / L pyridoxine, 0.2 mg / L cyanocobalmin, 0.00045 mg / L biotin, 0.45 mg / L calcium pantothenate, 0.5 mg / L indole butyric acid, 0.1 mg / L indole acetic acid, 8 to 10 mg / l sucrose and 2.5 g / l phytagel as gelling agent.

For spore production, a culture medium with the following composition was found, which is characterized by particularly good performance properties:
800 mg / l CaCl ₂ × 2 H ₂ O, 400 mg / l MgSO ₄ × 7H ₂ O, 110 mg / l KNO ₃ , 7 mg / l KH ₂ PO ₄ , 5 mg / l NaFeEDTA, 1.8 mg / 1 MnSO ₄ .4H ₂ O, 1 mg / l ZnSO ₄ .7H ₂ O, 0.2 mg / l CuSO ₄ .5H ₂ O, 70 mg / l KCl, 0,9 mg / l H ₃ BO ₃ , 0 , 0029 mg / l Na ₂ MoO ₄ × 2H ₂ O, 0.03 mg / l (NH ₄ ) ₂ Mo ₇ O ₂₄ × 4H ₂ O, 70 mg / l myoinositol, 0.5 mg / l thiamine, 0, 5 mg / l nicotinic acid, 0.45 mg / l pyridoxine, 0.2 mg / l cyanocobalmin, 0.00045 mg / l biotin, 0.45 mg / l calcium pantothenate, 0.5 mg / l indolebutyric acid, 9.5 g / l sucrose and 2.5 g / l phytagel as gelling agent.

After completion of the growth and propagation phases, the culture media can be converted back into a liquid consistency by mechanical comminution and addition of a citrate buffer. This is of considerable importance for economic mass production.

The culture media, in particular those for spore production, may contain 50 to 80 mg / l, preferably 70 mg / l, myoinositol. This substance serves as a growth factor for microorganisms and has a vitamin character. It has a beneficial effect on spore production.

By compared to known nutrient or culture media significantly higher levels of calcium chloride and potassium hydrogen phosphate, an increased spore and hyphae formation could be found.

The use of a special vitamin complex (Ca-pantothenate, cyanocobalamin and biotin) has a positive influence on the sporulation process.

The aim was to avoid the use of an antibiotic, such as carbenicillin, in the medium for spore production in order to prevent inhibition of root growth and sporulation.

The preparation of the culture media is carried out as follows:
First, separate mixtures of the intended groups of macroelements (magnesium sulfate, potassium nitrate, potassium chloride and potassium hydrogen phosphate and calcium chloride), microelements (manganese sulfate, zinc sulfate, boric acid, copper sulphate, sodium molybdate, ammonium molybdate) and vitamins (pantothenic acid, biotin, nicotinic acid, thiamine, cyanocobalamin, Pyridoxine).

The prepared mixture of macroelements and the amount of sugar used are dissolved in demineralized water and heated to a temperature of 80 ° C. To this mixture is added with stirring the gelling agent Phytagel and dissolved. Subsequently, the prepared mixture of microelements and the components NaFeEDTA, calcium nitrate, and the mixture of vitamins added. Finally, the addition of one or both phytohormones then takes place.

The finished culture or nutrient medium is sterilized in a heat exchanger and filled under clean room conditions in metered quantity in sterilized culture vessels.

The invention will be explained below by way of example of a culture medium for in-line production and an example of a culture medium for producing spores.

I. Culture media for inokolum production

Example 1:

A culture medium with the following composition, based on an amount of 1.5 l, is used:

Macro elements:
800 mg CaCl ₂ × 2H ₂ O, 600 mg MgSO ₄ × 7H ₂ O, 165 mg KNO ₃ , 105 mg KCl, 10.5 mg KH ₂ PO ₄ ;

Microelements:
2.7 mg of MnSO ₄ .4H ₂ O, 1.5 mg of ZnSO ₄ .7H ₂ O, 0.3 mg of CuSO ₄ .5H ₂ O, 1.35 mg of H ₃ BO ₃ , 0.0043 mg of Na ₂ MoO ₄ × 2H ₂ O, 0.0675 mg (NH ₄ ) ₂ Mo ₇ O ₂₄ × 4H ₂ O;

vitamins:
0.75 mg thiamin, 0.75 mg nicotinic acid, 0.675 mg pyridoxine, 0.3 mg cyanocobalmin, 0.000675 mg biotin and 0.675 mg calcium pantothenate;

phytohormones:
0.75 mg indolebutyric acid (IBA), 0.15 mg indoleacetic acid (IAA);
NaFeEDTA: 7.5 mg;
Sucrose: 14.25 g;
gelling agent:
3.75 g phytagel.

With KOH the pH is adjusted to 5.6.

For preparation of 1.5 liters of culture medium, the ingredients are mixed in the amounts mentioned above as follows:
The macroelements (magnesium sulfate, potassium nitrate, potassium chloride and potassium hydrogen phosphate and calcium chloride) and sugar are dissolved in demineralized water, adjusted to pH 5.6 with KOH and heated to a temperature of 80 ° C.

While stirring, phytagel is added and dissolved. Subsequently, the nutrient media groups microelements (manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, ammonium molybdate), NaFeEDTA and vitamins (pantothenic acid, biotin, nicotinic acid, thiamine, cyanocobalamin, pyridoxine) are added. Finally, the phytohormones IBA and IAA are added in pre-dissolved state.

The nutrient or culture medium is sterilized at 121 ° C. for 20 minutes in a heat exchanger and then filled in a clean room environment from the heat exchanger into an already sterilized culture vessel. The culture vessel consists of a cuboid container made of polycarbonate Caliber. After cooling, a gel of 1.5 liters volume is formed.

The height of the gel layer formed is 3 cm (filling height).

The inoculation is carried out on the nutrient media surface with approximately 30 root pieces (length 3 cm) of Daucus carota and 300 in vitro spores of Glomus intraradices. After 3 weeks incubation at room temperature (26 ° C) and darkness, the roots are mycorrhizal and harvestable.

The results are given at the end of the examples.

Comparative Example 1a

A first comparison is carried out with a known nutrient medium, a gel-containing MSR medium (Strullu Romand medium) having the following composition, based on an amount of 1.5 l:

Macro elements:
552 mg MgSO ₄ × 7H ₂ O, 454.5 mg KNO ₃ , 66 mg KH ₂ PO ₄ ;

Microelements:
3.345 mg of MnSO ₄ .4H ₂ O, 0.435 mg of ZnSO ₄ .7H ₂ O, 0.36 mg of CuSO ₄ .5H ₂ O, 2.745 mg of H ₃ BO ₃ , 0.0525 mg of (NH ₄ ) ₂ Mo ₇ O ₂₄ 4H ₂ O;

vitamins:
1.5 mg thiamin, 1.5 mg nicotinic acid, 1.5 mg pyridoxine, 0.75 mg cyanocobalmin, 0.015 mg biotin, and 1 mg calcium pantothenate;
NaFeEDTA: 97.5 mg;
Sucrose: 15 g;
CaNO ₃ × 4 H ₂ O: 525 mg;
Gelling agent: 4.5 g.

The pH value is set to 5.5.

For the preparation of 1.5 liters of MSR medium, the amounts indicated above are used, wherein for exact dosage in each case individual stock solutions of the components macroelements; Calcium nitrate; vitamins; NaFeEDTA and microelements are produced.

From the macro elements, a stock solution is prepared in 100-fold concentration in 1.5 liters of demineralized water and stored at 4 ° C.

A stock solution of calcium nitrate is prepared in 100-fold concentration in 1.5 liters of demineralized water and stored at 4 ° C.

A stock solution is prepared from the vitamins by dissolving the individual vitamins in 500 ml demineralized water and storing them in 10 ml portions at -20 ° C.

The component FeNaEDTA is dissolved in 1 liter of demineralized water and stored at 4 ° C.

In order to obtain one liter of the stock solution microelements, the microelements are dissolved individually in small volumes in demineralized water, combined and made up to 1000 ml and stored at 4 ° C.

For the preparation of 1.5 liter of MSR medium, 10 ml each of the stock solutions macroelements, calcium nitrate and vitamins, and 15 ml each of the stock solutions microelements and NaFeEDTA are mixed in 750 ml demineralized water. Subsequently, 15 g of sucrose are added and the pH is adjusted to 5.5 with 1 M NaOH. Thereafter, the addition of the gelling agent. The resulting solution is supplemented to 1500 ml and autoclaved at 121 ° C for 15 min. After cooling to 60 ° C, the medium is poured into a culture vessel, which corresponds to that according to Example 1.

The inoculation is carried out on the nutrient media surface with approximately 30 root pieces (length 3 cm) of Daucus carota and 300 in vitro spores of Glomus intraradices. After 3 weeks incubation at room temperature (26 ° C) and darkness rooting and mycorrhization of the roots are assessed.

The results are given at the end of the examples.

Comparative Example 1b

A second comparison will be made with one from the U.S. Patent 6,576,457 B1 known nutrient medium having the following composition, based on an amount of 1.5 l, carried out:

Macro elements:
111 mg CaCl ₂ x 2 H ₂ O, 739.5 mg MgSO ₄ · 7H ₂ O, 150 mg _KNO3, 4.5 mg KH ₂ PO _4;

Microelements:
3 mg ZnSO ₄ .7H ₂ O, 0.15 mg CuSO ₄ .5H ₂ O, 2.25 mg H ₃ BO ₃ , 09 mg KJ, 0.0075 mg
Na ₂ MoO ₄ × 2H ₂ O

vitamins:
Thiamine 0.3 mg, nicotinic acid 0.75 mg, pyridoxine 0.15 mg;
NaFeEDTA: 12 mg;
Ca-phytate: 15 mg;
Myoinositol: 45 mg;
Glycine 45 mg;
Carbenicillin: 75 mg;
MnCl ₂ × 4 H ₂ O: 6 mg;
Sucrose: 12 g and
Gelling agent: 6 g

The pH was adjusted to 6.0.

The individual components are prepared in the specified amounts as a solution.

The solution is autoclaved at 121 ° C for 15 minutes. After cooling to 60 ° C, the medium is poured into a culture vessel, which corresponds to that according to Example 1.

The inoculation is carried out on the nutrient media surface with approximately 30 root pieces (length 3 cm) of Daucus carota and 300 in vitro spores of Glomus intraradices. After 3 weeks incubation at room temperature (26 ° C) and darkness rooting and mycorrhization of the roots are assessed.

The results are given at the end of the examples.

II. Culture media for spore production

Example 2:

A culture medium (amount 1.5 l) is used which differs in comparison to the medium specified in Example 1 only in that it contains no indole acetic acid (IAA).

The preparation of this medium is carried out analogously as in the culture medium specified in Example 1. Also, the culture vessel used does not differ from that according to Example 1.

The inoculation takes place on the nutrient media surface with approximately 30 root pieces (length 3 cm) of Daucus carota which are mycorrhizal with Glomus intraradices. After 90 days incubation at room temperature (26 ° C) and darkness, the formation of VA mycorrhizal spores occurred and the culture was harvestable.

Comparative Example 2a

For a first comparison, a culture medium is used which is identical in its composition and preparation with the culture medium according to Example 1a. The inoculation is carried out analogously as indicated above for Example 2.

Comparative Example 2b

For a second comparison, a culture medium is used which is identical in its composition and preparation to the culture medium according to Example 1b. The inoculation is carried out analogously as indicated above for Example 2.

III. Results

As part of the inoculum production and spore production experiments, root growth (length growth, branching, mass) and spore production were evaluated visually according to the following evaluation scheme after completion of the experiments in the usual practice: category Customer rating No growth 0 Bad growth 1 Bad to moderate growth 2 Moderate growth 3 Moderate to good growth 4 Good growth 5 Good to very good growth 6 Very good growth 7

The evaluations led to the following results: medium root growth hyphal spore production example 1 7 7 7 Example 2 7 7 7 Comparative Example 1a 5 5 5 Comparative Example 1b 4 5 4 Comparative Example 2a 5 5 5 Comparative Example 2b 4 5 4

The test results show that the addition of phytohormones in the indicated amounts significantly improves root growth. Compared to test results with the two known media (MSR and according to U.S. Patent 6,578,457 B1 ) a doubling of the root diameter and a reduction of the average distance to the next branching between the roots by a factor of 5 can be determined. The addition of indole acetic acid (IAA) in the indicated amount causes a significant increase in mycorrhizal roots in inoculum production.

IBA causes an initialization of spore germination. This is reflected in a faster start of hyphal and spore regeneration. It was achieved 2 times the number of spores.

In addition, it was possible to demonstrate in experiments that the combination of the vitamins used according to the invention, in particular in the amounts indicated, positively influences spore production.

In the in-vitro mass production of VA mycorrhizaspores, the efficacy of this vitamin complex is maintained in the technology stage of nutrient media sterilization by lower heat load (vitamin addition at 80 ° C. and nutrient medium sterilization in the heat exchanger).

Claims (11)

Culture media for inokolum production and / or spore production in the context of an in-vitro mass production of VA mycorrhizal spores consisting at least of the macroelements calcium chloride, magnesium sulfate, potassium nitrate and potassium hydrogen phosphate, the microelements zinc sulphate, copper sulphate, boric acid, sodium molybdate, the vitamins thiamine, nicotinic acid, pyridoxine, one Potassium compound, NaFeEDTA, sucrose, calcium nitrate and a gelling agent, characterized in that this as further vitamins biotin, cyanocobalmin and calcium panthothenate and at least one phytohormone or more Phytohormone and as potassium compound contains potassium chloride, manganese sulfate, traces of ammonium molybdate and an increased proportion of calcium chloride. Culture media according to claim 1, characterized in that the phytohormones are selected from indolebutyric acid, indoleacetic acid, abscisic acid, naphthylacetic acid. Culture media according to one of Claims 1 or 2, characterized in that the amounts of indolebutyric acid employed are from 0.2 to 0.7 mg / l, indole acetic acid from 0.01 to 0.15 mg / l, abscisic acid 0.1 mg / l and 0.75 mg / l and naphthylacetic acid 0.1 to 0.2 mg / l, each alone or in combination. Culture media according to one of Claims 1 to 3, characterized in that they contain as macroelements 700 to 900 mg / l CaCl ₂ .2H ₂ O, 400 to 700 mg / l MgSO ₄ .7H ₂ O, 65 to 115 mg / l KNO ₃ , 5.5 to 8 mg / l KH ₂ PO ₄ , and 60 to 80 mg / l KCl; as microelements 1.5 to 2.5 mg / l MnSO ₄ .4H ₂ O, 0.2 to 1.5 mg / l ZnSO ₄ .7H ₂ O, 0.15 to 0.3 mg / l CuSO ₄ .5H ₂ O, 0.6 to 1.5 mg / l H ₃ BO ₃ , 0.002 to 0.0035 mg / l Na ₂ MoO ₄ x 2H ₂ O, 0.025 to 0.035 mg / l (NH ₄ ) ₂ Mo ₇ O ₂₄ × 4H ₂ O; as vitamins 0.4 to 1 mg / l thiamin, 0.4 to 1.2 mg / l nicotinic acid, 0.4 to 0.9 mg / l pyridoxine, 0.1 to 0.5 mg / l cyanocobalmin, 0, 0004 to 0.0009 mg / l biotin, and 0.3 to 1.1 mg / l calcium pantothenate; and 4 to 8 mg / l NaFeEDTA, 8 to 10 g / l sucrose and 2 to 4 g / l phytagel as gelling agent. Culture media according to one of claims 1 to 4, characterized in that they contain 50 to 80 mg / l myoinositol. Culture media according to one of claims 1 to 5, characterized in that they are adjusted to a pH of 5.6. Culture media of any of claims 1 to 6, characterized in that the 800 mg / l CaCl ₂ x 2 H ₂ O, 400 mg / l MgSO ₄ × 7H ₂ O, 110 mg / l KNO _3, 7 mg / l KH ₂ PO ₄ , 5 mg / l NaFeEDTA, 1.8 mg / l MnSO ₄ × 4H ₂ O, 1 mg / l ZnSO ₄ × 7H ₂ O, 0.2 mg / l CuSO ₄ × 5H ₂ O, 70 mg / l KCl, 0.9 mg / l H ₃ BO ₃ , 0.0029 mg / l Na ₂ MoO ₄ x 2H ₂ O, 0.03 mg / l (NH ₄ ) ₂ Mo ₇ O ₂₄ x 4H ₂ O, 0, 5 mg / l thiamin, 0.5 mg / l nicotinic acid, 0.45 mg / l pyridoxine, 0.2 mg / l cyanocobalmin, 0.00045 mg / l biotin, 0.45 mg / l calcium pantothenate, 0.5 mg / l indolebutyric acid, 0.1 mg / l indole acetic acid, 8 to 10 mg / l sucrose and 2.5 g / l phytagel as gelling agent. Culture media of any of claims 1 to 6, characterized in that the 800 mg / l CaCl ₂ x 2 H ₂ O, 400 mg / l MgSO ₄ × 7H ₂ O, 110 mg / l KNO _3, 7 mg / l KH ₂ PO ₄ , 5 mg / l NaFeEDTA, 1.8 mg / l MnSO ₄ × 4H ₂ O, 1 mg / l ZnSO ₄ × 7H ₂ O, 0.2 mg / l CuSO ₄ × 5H ₂ O, 70 mg / l KCl, 0.9 mg / L H ₃ BO ₃ , 0.0029 mg / L Na ₂ MoO ₄ × 2H ₂ O, 0.03 mg / L (NH ₄ ) ₂ Mo ₇ O ₂₄ × 4H ₂ O, 70 mg / l myoinositol, 0.5 mg / l thiamine, 0.5 mg / l nicotinic acid, 0.45 mg / l pyridoxine, 0.2 mg / l cyanocobalmin, 0.00045 mg / l biotin, 0.45 mg / l Calcium pantothenate, 0.5 mg / l indolebutyric acid, 9.5 g / l sucrose and 2.5 g / l phytagel as gelling agent. Culture media according to one of claims 1 to 8, characterized in that they can be converted into a liquid consistency after completion of the growth and propagation phases by mechanical comminution and addition of a citrate buffer. Process for the preparation of culture media according to any one of Claims 1 to 9, characterized in that the macroelements (magnesium sulphate, potassium nitrate, potassium chloride and potassium hydrogenphosphate and calcium chloride), microelements (manganese sulphate, zinc sulphate, boric acid, copper sulphate, sodium molybdate, ammonium molybdate) and vitamins (pantothenic acid, Biotin, nicotinic acid, thiamine, cyanocobalamin, pyridoxine) are each formed as separate mixtures, and the mixture of macroelements and sugars are dissolved in demineralized water and heated to a temperature of 80 ° C, phytagel is added with stirring and dissolved, and then the mixture are added to microelements and the components NaFeEDTA, calcium nitrate, and the mixture of vitamins and finally the addition of the phytohormones takes place. A method according to claim 10, characterized in that the culture or nutrient medium is sterilized in a heat exchanger and filled under clean room conditions in metered quantity in sterilized culture vessels.