RU2308484C2 - Method for production of plant cell biomass - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: claimed method includes preparation of solid or liquid nutrient medium containing inorganic components, substrate, carbohydrates, vitamins and growth simulators; biomass cell seeding, cultivation thereof and product producing. As inorganic components mixture of glass-like metaphosphates with alternate composition containing (mass %): phosphorous oxide 40-60; potassium oxide 10-30; calcium oxide 5-20; magnesium oxide 3-13; silicium oxide 1-6; boron oxide 1-6; oxide of other elements (Co, Cu, Fe, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2.

EFFECT: method for production of biotechnological products of increased accuracy.

18 tbl, 3 ex

Description

The invention relates to the biotechnology industry, and in particular to methods for producing plant biomass for obtaining raw materials and creating new drugs, and can also be used in the food or cosmetic industry.

Cellular biotechnologies based on in vitro cultured plant cells and tissues are an integral part of all national biotechnological programs and projects. Here are just some of the features that characterize these technologies:

- the ability to obtain cell mass (genetically modified or not) of traditionally used economically important plants in unlimited quantities,

- the ability to synthesize biologically active substances, the synthesis of which in the usual way is difficult or expensive or to transform cheap precursors into a final valuable product,

- create brand new products that are superior to traditional in quality and environmentally friendly.

Therefore, the created biotechnological production must be able to withstand competition with alternative systems for obtaining the product. The latter is largely provided by the strain of cultured cells, its productivity, the intensity of synthesis of the target product, the conditions and technology of its cultivation.

There are a large number of works in which it is shown that the accumulation of the desired product is affected by both the mineral and hormonal components of the nutrient medium, temperature, illumination, and other factors.

One of the essential components of nutrient media in biotechnology for all cultures is the presence of macro- and micro-salts of various elements, as well as the presence of growth stimulants, carbohydrates and vitamins. Moreover, for different cultures, this composition varies greatly, but it necessarily includes mineral (phosphate and nitrogen) salts of potassium, sodium, iron chelate, chromium and zinc, copper and boron and other elements that are necessary for cells for both growth and synthesis products of primary and secondary metabolism (see Butenko R.G. Culture of isolated tissues and physiology of plant morphogenesis. // Publishing House "Science", M., p. 270, F.A. Kalinin, V.V. Sarnatskaya, V. E. Polishchuk. Methods of tissue culture in the physiology and biochemistry of plants // Publishing House "Naukova Dumka", Kiev, 1980, pp. . 483). The ratio of these elements in different strains can vary significantly and is subject to strict control even at different stages of cell growth.

A known method of producing biomass of ginseng and other aralia according to the USSR Author's Certificate No. 1621510, priority 08.09.88, This method, as the closest in its technical essence, was chosen by us for the prototype.

The known method includes the preparation of a nutrient medium containing inorganic components, carbohydrates, vitamins and growth stimulants, planting biomass cells, cultivating them and obtaining a product. As inorganic components, micro- and macrosalts according to Murasiga and Skoogoo, consisting of 12 different elements, are used.

The disadvantages of the prototype is that the technological process of preparing concentrates for culture media takes a lot of time, because each salt from the standard composition must be prepared separately, in addition, the concentrates of the prepared salts have a limited shelf life, and the cost of salts is quite high.

Therefore, the ability to replace many macro and micro salts with one mineral component containing all the salts necessary for the cell and other environmentally friendly natural growth stimulants is an extremely urgent task, especially for industrial biotechnology. It is possible by selection to obtain a cell line that will grow at reduced concentrations of salts, especially nitrates, which is relevant now that some strains of plant cells, including ginseng, are proposed to be used as biologically active additives (BAA).

The process of cultivating plant cells associated with their multiple reclamation in new nutrient media in order to obtain a product or preserve producer strains is very laborious and multi-stage. Therefore, its simplification at the stage of preparation of concentrates for culture media reduces the risk of errors due to the "human" factor, improves control and increases the reliability of the production of a biotechnological product, and therefore increases its efficiency.

When creating this invention, the task was to develop a relatively cheap and safe method for the production of biomass of plant cells, which can be used in industrial production. To solve this problem, in a method for producing biomass of plant cells, including the preparation of a nutrient medium containing inorganic components, substrates, carbohydrates, vitamins and growth stimulants, planting biomass cells, cultivating them and obtaining a product, it is proposed at the stage of preparing a nutrient medium solid or liquid as inorganic components use a mixture of glassy metaphosphates of variable composition with the following content, wt.%:

phosphorus oxide 40-60 potassium oxide 10-30 calcium oxide 5-20 magnesium oxide 3-13 silicon oxide 1-6 boron oxide 1-6 oxides of other elements of the periodic table (Co, Cu, Fe, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2

moreover, the composition of metaphosphates and the concentration of trace elements should be selected based on an analysis of the ash residues of the resulting plants.

A mixture of glassy metaphosphates with the above composition is a substance known under the brand name Agrovitakva-AVA, a prolonged-release vitreous fertilizer developed by Agrovit CJSC in St. Petersburg (TU 2189-001-50032241-99). The raw materials for its production are phosphoric acid (70-75%), dolomite flour, potash solution (45-50%) and caustic magnesite powder. At the final stage of production, microelements from the oxides of elements of 3-5 groups of the Periodic Table of Elements D.I. Mendeleev are added. The concentration of trace elements is selected based on the analysis of ash residues obtained by burning plants of interest to us.

It is known that the microelement composition differs significantly during the transition from one type of plant to another. The lack of iron, cobalt, copper, and other trace elements in the soil can cause diseases and death of many plants. Excessive amounts of trace elements in the soil can also be no less harmful, which, in addition, is strictly regulated by the legislation of most countries of the world. However, most plants are tolerant to a wide range of changes in the concentration of trace elements with a low total concentration of trace elements (~ 0.1 wt.%) In the fertilizer applied.

Medicinal plants have a specificity associated with the fact that they are intended for people with certain diseases. Various organs of the human body contain trace elements in different concentrations. In this regard, the character of the plant and its medicinal purpose within the tolerance of the plant and patient to certain trace elements takes on a huge role in the selection of fertilizers. Thus, a chain is built (Soil-Fertilizer - Plant Tolerance - Human Tolerance).

Experiments in the field of plant physiology in the burning of plants and the study of ash residues made it possible to determine that fruits, plant mass and roots are characterized by significant differences in the concentration of trace elements. The problem of microdosing is especially difficult when growing plants to create drugs, because enzymatic catalysis is ensured by the combined action of several trace elements.

Knowing the beneficial properties of various plants, it is possible to establish limits on the content of various trace elements in fertilizers when growing medicinal crops on various soils according to the results of studying their ash residues. This allows you to create optimal conditions for growth and consciously control the properties of plants.

As the substrates in the proposed method can be used agar-agar, phytogel, silk, paper, propylene glycol and plant phytomass waste.

- carbohydrates: sucrose, glucose, fructose;

- vitamins: inositol, thiamine chloride, etc.

- growth stimulants: kinetin, α-naphthylacetic acid, etc.

At the stage of biomass production, cultivation is carried out on a nutrient medium of a modified composition, and to maintain the stability of the strain, a medium of known composition is used, thus, a two-stage cultivation of the strain is carried out: in the first stage, in the course of reclamation, to maintain the stability of the strain, the cultivation is carried out on a standard nutrient medium, and in the second, to obtain biomass use a modified nutrient medium, the composition of which is given in this invention.

The method is as follows.

At the first stage, a control nutrient medium according to Murasiga and Skoog is used to cultivate plant biomass. In vessels with a capacity of 250 ml, the biomass of the strain is placed in an amount of 2.0-2.5 g per 50 ml of culture medium. Next, they cultivate in the dark at a temperature of 26-27 ° C for 35 days.

At the second stage of cultivation, the grown biomass in the amount of 2.0-2.5 g is transferred into 250 ml containers containing 50 ml of the nutrient medium prepared on the basis of a mixture of glassy AVA metaphosphates. Cultivation is carried out under the same conditions as in the first stage. At the end of cultivation, the main indicators of biomass are determined.

The method is illustrated by the following examples.

Example 1

The material for these studies was a ginseng strain - Panax ginseng S.A. Meu., Cultivated at the St. Petersburg State Chemical Pharmaceutical Academy (SPHFA) since 1969 (collection code - “D”). It was one of the first biotechnological strains in Russia, which was widely introduced in many biotechnological industries of the USSR. Today, this strain remains one of the main in vitro phytobiotechnology of medicinal plants, which is not only used to solve a number of theoretical and practical problems, but also to obtain valuable medicines and dietary supplements.

Today, the question of developing biotechnology of this and many other valuable medicinal plants is very acute, some of which are barbarously destroyed in nature and their crops destroyed, for example, ginseng, while others have cultivation bases located in other countries (Ukraine, Georgia, Kazakhstan).

In addition to this ginseng strain, a selective line of ginseng strain was used, obtained in SPCPA on a nutrient-free medium containing no ammonium nitrate and a kinetin growth stimulant (cipher of the SPFCA collection “D-”).

Instead of micro- and macrosalts according to Murasiga and Skoog, a mixture of glassy metaphosphates of variable composition was used, having the Agrovitakva-AVA trademark of three options:

1. AVA-1 (code AVA-1, Wagon) of the following composition,%:

phosphorus oxide fifty potassium oxide 21 calcium oxide fifteen magnesium oxide 6 silicon oxide 1.0 boron oxide 1.8 oxides of other elements of the periodic table (Co, Cu, Fe, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2

With 0,03 Cu 0.02 Fe 0.5 Mo 0,03 Mn 0.1 Cr 0.002 Zn 0.01 Ge 0.001 Se 0.00005 V 0.001

2. AVA with humic acids (97% AVA + 3% humate) (cipher-AUA + GUM) of the following composition,%:

phosphorus oxide fifty potassium oxide 21 calcium oxide fifteen magnesium oxide 6 silica 1.0 boron oxide 1.8 nitrogen 0.8 carbon 1.3 (Co, Cu, Fe, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2

With 0,03 Cu 0.02 Fe 0.5 Mo 0,03 Mn 0.1 Cr 0.002 Zn 0.01 Ge 0.001 Se 0.00005 V 0.001

3. AVA with urea carbide (85% AVA + 15% urea carbide) (code AVA + M) of the following composition,%:

phosphorus oxide 52 potassium oxide eighteen calcium oxide fourteen magnesium oxide 5 silica 1.5 boron oxide 1.2 oxides of other elements of the periodic table (Co, Cu, Fe, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2

With 0.02 Cu 0.01 Fe 0.7 Mo 0.02 Mn 0.05 Cr 0.003 Zn 0.01 Ge 0.002 Se 0.0001 V 0.001

The remaining components of the control nutrient medium corresponded to the standard medium and contained: sugar - 3%, agar-agar - 0.6%, inositol - 0.08 g / l, casein hydrolyzate - 0.5 g / l, iron hellate - 5.0 ml / l, α-naphthylacetic acid - 2 ml / l and kinetin - 1 ml / l.

The strains were cultured under standard conditions at a temperature of 26-28 ° C on an agar medium in 250 ml flasks per 50 ml of culture medium prepared in the following options:

No. 1. Control medium - macro salts according to Murashige and Skoog - 50.0 ml / l

microsalts according to Murashige and Skoogoo - 1 ml / l

sugar - 3%, agar-agar - 0.6%

inositol - 0.08 g / l, casein hydrolyzate - 0.5 g / l

iron chelate - 5 ml / l

α-naphthylacetic acid - 2 ml / l

kinetin - 1 ml / l.

No. 2. Ginseng strain (D) medium: instead of macro- and micro-salts, AVA-1-1 g / l is given, the remaining components are listed according to the control medium, but without a kinetin growth stimulator.

Number 3. Ginseng strain (D) medium: instead of macro- and micro-salts, AVA + GUM is given - 1 g / l, the rest of the components according to the prescription of the control medium, but without the kinetin growth stimulator.

Number 4. Ginseng strain (D) medium: instead of macro- and micro-salts, AVA + M is given - 1 g / l, the rest of the components according to the prescription of the control medium, but without a kinetin growth stimulator.

No. 5. Ginseng strain (D-) medium: instead of macro- and micro-salts, AVA + GUM is given - 1 g / l, the rest of the components according to the prescription of the control medium, but without the kinetin growth stimulator.

No. 6. Ginseng strain (D-) medium: instead of macro- and micro-salts, AVA + M is given - 1 g / l, the rest of the components according to the prescription of the control medium, but without the kinetin growth stimulator.

The cultivation cycle in all cases was 35 days.

At the end of the cycle, wet and dry tissue masses, Lowry protein, water-soluble GRP polysaccharides and the total glycoside fraction — GHF — were determined.

Controlled parameters in all cases were constant monitoring of biomass growth in terms of wet and air-dry tissue mass in an in vitro culture in an SPHFA bank for one growing cycle - 35 days.

The content of water-soluble polysaccharides (GRP) and the amount of glycosides (GHF) as one of the main active substances at the end of the cultivation cycle were also determined.

It can be noted that in this variant of the experiments, the infected vessels were completely absent. The growth of biomass in all variants passed along a characteristic "S" -shaped curve with a reduction in the "lag" period from 5 days in the control to 3 days in the experimental variants.

Morphologically, the biomass of ginseng cells in all cases was more yellow than in the control, remaining in the lower part of the light, coarse-grained and often biomass cells grew into the nutrient medium. This indicated a good consumption of salts from agar-agar.

At the end of the cultivation cycle, on the 35th day (observation period), the biomass was separated from the remains of the nutrient medium and dried in an infrared dryer. In the air-dry biomass, the sum of water-soluble polysaccharides-GRP and the sum of glycosides-SGF were determined as the main biologically active substances with which a wide spectrum of pharmacological activity of ginseng preparations is associated.

Growth parameters (wet and air-dry mass) in the process of optimizing the environment and strain growth were taken into account by the generally accepted method. In all cases, data from five vessels of the same type were taken into account; all data were processed statistically.

The following are indicators of the wet and dry mass of the tissues of the ginseng strain when grown on experimental media (Table 1).

Table 1
Indicators of wet and dry weight of the tissues of the ginseng strain in experimental environments Environment option Strain Wet mass, g / 50 ml of medium Dry weight, g / 50ml medium The control D 31.40 ± 1.2 1.00 ± 0.01 AVA - 1 D 20.76 ± 2.7 0.95 ± 0.02 AVA + GUM D 16.80 ± 2.8 0.81 ± 0.02 AVA + M D 19.30 ± 1.5 0.96 ± 0.05 The control Selective D 36.10 ± 03 1.59 ± 0.03 AVA - 1 Selective D 20.48 ± 0.5 1.10 ± 0.02 AVA + M Selective D 15.6 ± 0.6 0.91 ± 0.01 AVA + GUM Selective D 16.80 ± 0.4 0.94 ± 0.01

From the above data it is seen that on all variants of media with AVA, the accumulation of fresh cell mass was significantly reduced by 40-50%. At the same time, the air-dry cell mass practically did not differ from the control variant for the ginseng strain and was slightly inferior in the case of the selective strain (without ammonium nitrate and kinetin). It is possible that in the first case (strain D), the absence of a kinetin growth stimulator in the medium affected the biomass growth, which caused less stretching of the cells and their water cut and slowed down growth, as well as stress, obviously associated with the replacement of some components of the nutrient medium. The decrease in the fresh cell mass of the selective strain (D-) is also possibly associated with stress exposure.

In this regard, it was of interest to compare the accumulation of protein in cells during cultivation on media with glassy AVA metaphosphates (Table 2).

table 2
Indicators of protein accumulation in the biomass of cells of the ginseng strain when cultured on media with a mixture of glassy metaphosphates Strain Option The protein content, mg / g biomass Ginseng - D The control 2,30 ± 0,05 Ginseng - D AVA - 1 3.01 ± 0.03 Ginseng - D AVA + GUM 2.85 ± 0.02 Ginseng - D AVA + M 2.62 ± 0.01 Selective Ginseng - The control 3.60 ± 0.05 Selective Ginseng - AVA - 1 3.25 ± 0.03 Selective Ginseng - AVA + GUM 3.10 ± 0.02 Selective Ginseng - AVA + M 3.20 ± 0.01

The above indicators indicate that the protein content in the cells of ginseng (strain-D) not only did not yield to the control option, but even exceeded the latter, despite the absence of a growth stimulator. The selective strain also showed a high level of protein accumulation in biomass cells in all variants of nutrient media with AVA. This indicates that the macro- and microelement composition of the glassy AVA metaphosphates in all cases was sufficient to undergo all metabolic processes of protein synthesis.

The activity of antioxidant defense enzymes in ginseng strains was determined at the final stage of crop growth on media with different contents of glassy AVA metaphosphates (Table 3).

Table 3
The specific activity of antioxidant enzymes in the biomass of ginseng strains with growth on nutrient media with a mixture of glassy metaphosphates AVA Strain Environment option Catalase, E / g protein SOD, E / g protein Ginseng (D) The control 75.5 ± 10.5 56.7 ± 5.1 Ginseng (D) AVA - 1 15.2 ± 3.5 31.8 ± 2.0 Ginseng (D) AVA + M 15.2 ± 3.5 31.8 ± 2.0 Ginseng (D) AVA + GUM 12.4 ± 1.5 29.0 ± 1.5 Selective D - The control 28.0 ± 2.0 12.1 ± 0.2 Selective - D- AVA - 1 30.0 ± 2.0 13.5 ± 0.5 Selective - D- AVA + M 27.5 ± 3.0 9.45 ± 0.5 Selective - D- AVA + GUM 28.6 ± 1.0 13.3 ± 0.2

The obtained data show that the level of activity of both antioxidant enzymes in the biomass of the ginseng strain in all variants of culture media with glass AVA metaphosphates was 1.5-2 times lower than in the control. At the same time, in a selective strain adapted for growth without ammonium nitrate and kinetin, both the level of activity of catalase and superoxide dismutase (SOD) practically did not differ from the level of activity of these enzymes in the control variant.

This can be explained by the fact that there are fewer toxic peroxide radicals when using these fertilizers in all cases, which is possibly associated with a decrease in respiration rate.

Given that ginseng glycosides and polysaccharides are one of the main biologically active metabolic products, a comparative analysis of these substances in the biomass of ginseng strains was carried out (Table 4).

Table 4
The total glycoside fraction (GFS) and water-soluble polysaccharides (GRP) in the biomass of the ginseng strain Strain Environment option GHF,% GRP,% Ginseng - D The control 4.10 ± 0.2 8.00 ± 0.51 Ginseng - D AVA + GUM 4.30 ± 0.3 4.15 ± 0.20 Ginseng - D AVA + M 4.50 ± 0.2 3,50 ± 0,10 Ginseng - D AVA - 1 3.85 ± 0.1 5.21 ± 0.11 Selective - D The control 4.17 ± 0.5 10.00 ± 0.60 Selective - D- AVA - 1 3.69 ± 0.3 8.85 ± 0.40 Selective - D AVA + M 4.20 ± 0.2 5.45 ± 0.25 Selective - D- AVA + GUM 4.53 ± 0.4 6.75 ± 0.30

Thus, analyzing the data obtained, we can conclude that the content of the total glycoside fraction (GFS) in the ginseng strain in all cases is practically independent of the introduced AVA.

The content of water-soluble polysaccharides decreased almost twofold compared with the control, but still it remained quite high. Perhaps with increasing doses of "AVA + GUM" and "AVA + M", the content of GRP may increase. As our previous studies with "AVA - 1" showed, at a concentration of 3 g / l, there was an increase in the content of GRP in the ginseng strain to 10.7%.

Summing up the studies, we can conclude that the use of new stacked AVA metaphosphates in modifications with humate and urea carbide instead of a complex of macro and micro salts according to the prescription Murashige and Skoog in the control medium did not affect the accumulation of air-dry biomass of ginseng strains even during the first passage in the absence of one of the growth stimulants - kinetin.

The biological productivity of protein accumulation in the biomass of ginseng strains was not inferior to the control options, and in some cases was even higher. The content of the main active substances: the sum of glycosides and water-soluble polysaccharides also remained quite high.

Example 2

The five-leaf panax strain, as well as the ginseng strain, was first obtained by L.I. Slepyan and R.G. Butenko from a 6-year-old greenhouse root from the collection of the Botanical Institute of the USSR Academy of Sciences. Since 1969, the strain has been in the collection of the SPHFA bank (Am code).

The strain is cultivated on the same nutrient medium as the ginseng strain, with a cycle of 35 days in 250 ml vessels per 50 ml of culture medium at a temperature of 25-26 ° C. The biomass is light yellow, loose, easily separated from the nutrient medium at the end of the cultivation cycle. It has been proven that it contains the main glycosides close to ginseng glycosides - these are Rb ₁ , Re, Rd and Re. In contrast to ginseng, the glycosides Rb ₁ and Rg ₁ , which have a stimulating effect, are practically absent in American ginseng. Therefore, American ginseng exhibits a mild sedative effect and, like ginseng, has an anti-carcinogenic effect. It was found that it increases the protection of blood cells against the damaging effect of the effect of O-LDL. In addition, American ginseng identified ginsenosides F ₂ , pseudoginsenoside F ₁₁ and quinqueloside R ₁ .

Along with ginsenosides, the plant contains active peptides and polysaccharides, with which immunological and radioprotective activity is associated. Essential oils, vitamins and minerals and essential amino acids that determine the antidepressant effect and antioxidant and other properties of this type of ginseng. The lipophilic fraction represented by fatty acids, lipids, β-sitosterol and panacene. The latter has a painkiller and sedative effect. This set of substances in the complex defines the wide market of drugs and products that the root of American ginseng is conquering today. Therefore, tissue culture in vitro of the five-leaf panax strain is no less promising and was chosen as the second object of research.

In this regard, experiments were carried out to study the effect of glassy AVA metaphosphates on the growth and biological productivity of the five-leaf panax strain (code Am) and a selective strain of American ginseng adapted to growth on a nutrient medium without ammonia nitrogen and a kinetin growth stimulator (code Am-)

All variants of culture medium with AVA were taken in the same concentrations as in previous experiments for the ginseng strain.

At the end of the cultivation period, the biomass was separated from agar-agar, dried in an infrared dryer, and the raw and air-dry mass, total glycoside fraction and the content of water-soluble polysaccharides were determined in it. In crude biomass, protein content and antioxidant enzyme activity were determined.

In all cases, data from five vessels of the same type were also taken into account. All data are processed statistically.

Below are the indicators of raw and dry biomass of the five-leaf panax strain when grown on experimental media (Table 5).

Table 5
Indicators of raw and dry biomass of the five-leaf panax strain Strain Environment option Wet Weight, g / 50 ml Dry weight, g / 50 ml Panax - Am - The control 43.5 ± 2.5 1,5 ± 03 Panax - Am - AVA + GUM 16.4 ± 4.2 0.84 ± 0.5 Panax - Am - AVA + M 18.5 ± 2.1 1.1 ± 0.3 Panax - Am - AVA - 1 18.1 ± 1.8 0.8 ± 0.2 Panax - Am The control 41.3 ± 1.0 1.5 ± 0.2 Panax - Am AVA + GUM 20.6 ± 1.2 1.2 ± 0.1 Panax - Am AVA + M 19.8 ± 1.6 1.2 ± 0.2 Panax - Am AVA - 1 20.2 ± 1.0 1.2 ± 0.1

It can be seen from the data presented that, as in the case of the ginseng strain, in this case, a significant decrease in the fresh weight was also observed, while the air-dry mass remained practically at the control level in all experimental variants.

This testified to the fact that the main metabolic processes in Panax American biomass cells in these experiments proceed at the same level as in the case of strain growth on a control medium, even in cases when the kinetin growth stimulator was excluded from the medium.

Table 6
Indicators of protein accumulation in the biomass of cells of the five-leaf panax strain when cultured on media with AVA fertilizers Strain Environment option The protein content, mg / g Panax - Am - The control 3.65 ± 0.2 Panax - Am - AVA + GUM 5.2 ± 0.2 Panax - Am - AVA + M 4.35 ± 0.05 Panax - Am - AVA - 1 4.3 ± 0.15 Panax Am The control 3.1 ± 0.1 Panax Am AVA + GUM 3.95 ± 0.05 Panax Am AVA + M 4.25 ± 0.2 Panax Am AVA - 1 4.7 ± 0.1

It can be assumed that the mineral composition of AVA, in combination with urea humate and carbide, completely replaced the main mineral components of the Murasige and Skoog medium, even enhancing protein metabolism. Data on the protein content in the biomass of the five-leaf panax strain are presented in Table 6.

Table 7
The specific activity of antioxidant enzymes in the biomass of the five-leaf panax strain when grown on culture media with a mixture of glassy metaphosphates AVA Strain Environment option The specific activity of catalase, E / g protein The specific activity of SOD, E / g protein Panax - Am The control 65.0 ± 4.5 56.3 ± 2.4 Panax - Am AVA + GUM 30.1 ± 1.2 35.4 ± 0.5 Panax - Am AVA + M 42.0 ± 2.1 22.5 ± 0.5 Panax - Am AVA - 1 46.2 ± 4.0 15.0 ± 6.5 Selective Am The control 40.0 ± 3.5 20.0 ± 2.5 Selective - Am AVA + GUM 21.0 ± 1.5 14.0 ± 1.0 Selective - Am - AVA + M 22.5 ± 2.5 13.0 ± 0.5 Selective - Am AVA - 1 25.2 ± 3.0 15.0 ± 0.7

The data on the level of specific activity of antioxidant defense enzymes had the same orientation as in the case of the cultivation of ginseng strain. On all experimental versions of experimental nutrient media with AVA metaphosphates, a decrease was observed in the activity of both enzymes in the biomass cells of the five-leaf panax strain while maintaining a fairly high, in this case, control, protein metabolism and active synthesis of other biologically active substances, the sum of glycosides and water-soluble polysaccharides.

Table 8
The total glycoside fraction (GFS) and water-soluble polysaccharides (GRP) in the biomass of the five-leaf panax strain Strain Environment option The content of GFS,% GRP content,% Panax - Am The control 3.5 ± 0.3 15.0 ± 5.0 AVA + GUM 4.3 ± 1.2 7.0 ± 1.2 AVA + M 3.6 ± 0.5 4.0 ± 0.5 AVA - 1 3.8 ± 0.2 5.8 ± 0.2 Selective Am - The control 3.0 ± 0.7 17.0 ± 3.6 AVA + GUM 5.5 ± 0.4 6.7 ± 2.4 AVA + M 3.5 ± 0.2 4,5 ± 1,0 AVA - 1 4.0 ± 0.2 4.0 ± 0.5

The results obtained indicate that the accumulation of the sum of glycosides on all variants of nutrient media with AVA fertilizers in the biomass of the five-leaf panax strain was either at the level of the control strain, or even exceeded this indicator in the variant with humate. For the selective strain, the same pattern was noted. It can be assumed that the higher than in the trace elements Murashige and Skoog in the control, the content of trace elements such as Cu, Zn and Fe in these metaphosphates activated the synthesis of glycosides.

Summing up the experiments on replacing the macro and micro salts according to the prescription Murashige and Skoog in a standard nutrient medium for strains of ginseng and panax five-leaved and their selective lines on glassy AVA metaphosphates in three versions with humate and urea carbide, we can conclude that these metaphosphates are completely can be used in biotechnology for the cultivation of these strains. In this case, both general and individual characteristics of the response of biological activity to the accumulation of dry mass, protein, GHF and GRP depending on the strain were established.

The strains grew along the usual “S ″ -shaped curve with a reduction in the“ lag ”period from 5 days in the control to 3 in the experiment. Morphologically, the biomass of the strains did not change, remaining coarse-grained, crumbly, but both strains acquired a darker yellow coloration compared with the control option.

For both strains in the first passage, a decrease in the fresh cell mass was observed in all variants of nutrient media with AVA. At the same time, the indices of dry cell biomass in all variants remained at a rather high level, which indicated that the synthesis processes of the main metabolites are within the standard (control) environment.

Thus, the protein content in ginseng cells (strain-D) not only did not yield to the control option, but even exceeded the latter, despite the absence of a growth stimulator. Selective strains of both ginseng and five-leaf panax also showed a high level of protein accumulation in biomass cells on all variants of nutrient media with AVA fertilizers. This indicates that the macro- and microelement composition of the glassy fertilizers AVA in all variants was sufficient for all metabolic processes of protein synthesis to undergo.

The content of the total glycoside fraction (GHF) in the ginseng strain in all cases was practically independent of the introduced AVA.

The content of water-soluble polysaccharides in both strains was almost halved compared with the control, but still it remained quite high. Perhaps with increasing doses of "AVA + GUM" and "AVA + M" the content of GRP can be increased.

The data on the level of specific activity of antioxidant enzymes (SOD and catalase) both in the case of cultivation of the ginseng strain and five-leaf panax were of the same type. On experimental nutrient media with AVA, in all modifications, a decrease in the activity of both enzymes in the cells was observed. It can be assumed that in these experiments there is no accumulation of an aggressive peroxide radical and therefore antioxidant defense enzymes are not activated.

The accumulation of the sum of glycosides in the biomass of the five-leaf panax strain in all variants of nutrient media with AVA was either at the level of the control strain, or even exceeded this indicator in the variant with humate. For the American panax selective strain, the same pattern was noted.

Obviously, the higher than in the trace elements Murashige and Skoog in the control, the content of trace elements such as Cu, Zn and Fe in these fertilizers activated the synthesis of glycosides.

The use of glassy metaphosphates implies their gradual dissolution and entry into plants, so it is possible to use them in biotechnology when cultivated in a liquid nutrient medium in fermenters would also be promising and economical.

Example 3

The strain of tropical aralic Polyscias filicifolia Bailey was introduced into the culture of isolated tissues in vitro in 1972 at the Leningrad Physiological Institute (now SPCP) I. Grushvitsky, N. N. Arnautov. and Slepyan L.I. from the root of an intact greenhouse plant.

A preliminary study of the native plant revealed the localization of triterpene glycosides in the parenchymal cells of the root cortex. Explants isolated from the root bark zone, after treatment under sterile conditions, were planted on nutrient media of different compositions. Three variants of media with a mineral base according to the prescription Murashige and Skoog, differing in the composition of vitamins and growth stimulants, were studied.

For further studies, samples of the biomass of the strain were selected, characterized by intensive growth in cell mass already on the 5-7th day. By the 60th day, tissue growth in this case was 15.3 g of dry weight per 1 liter of culture medium and contained triterpene glycosides characteristic of the native plant. This strain was transferred to VSCKKVR at number 24 as a strain with adaptogenic and immunomodulating effects, but later it was lost there. Currently, the strain Polyscias filicifolia Bailey is preserved as the initial strain, in which the chemical composition and pharmacological properties were studied, in the bank of strains of medicinal plants of SPHFA, is available in the CBW at the Russian Academy of Sciences in the company "VITA" (St. Petersburg) and the research and production company " Biopharmtox "(St. Petersburg).

As a result of phytochemical studies conducted at the LHFI, N.V. Mikhaylova, two triterpene glycosides (A and B) were identified and identified, the aglycon of which is hederagenin - an oleanan derivative].

Chromatographic analysis of the carbohydrate part of glycosides showed that for glycoside A it is represented by galactose and ramnose, and for glycoside B it is represented by galactose, ramnose and glucose.

The biomass of the strain also contains β-sitosterol (up to 2%), campesterol, fructose, amino acids (aspartic acid, lysine, arginine, cysteine, histidine, tryptophan, etc.), saturated and unsaturated fatty acids (lauric, myristic, palmitic, oleic) .

The strain of polysacias fern is one of the promising biotechnological strains, the preparations of which have valuable pharmacological properties and which is in the collection of several manufacturing companies.

The process of cultivating a strain associated with its multiple reclamation on new nutrient media (in a liquid or agarized medium) is very laborious and multi-stage. Therefore, its simplification at the stage of preparation of nutrient medium concentrates reduces the risk of errors and improves production control, increasing its reliability.

Summing up the above, it was of interest to study the possibility of culturing in biotechnology a strain of poliscias as a raw material for drugs or dietary supplements using a new type of environmentally friendly glassy glass metaphosphates AVA, developed by AGROVIT CJSC.

The strain has been cultivated in SPhFA since 1972 on a modified agarized nutrient medium with macro and micro salts according to the prescription Murashige and Skoog under stationary cultivation conditions at a temperature of 25 + 1 ° C in 250 ml vessels per 50 ml of nutrient medium. The cultivation cycle is 40 days and in a dark room.

The biomass at the end of the cycle, as a product of reclamation and raw materials, is represented by a loose mass of white cells, without organogenesis, with a characteristic smell of fresh herbs (with tissue mixing) and a specific aromatic odor after drying.

To this end, to cultivate the polyscias strain (code Pol.) (SPHFA collection) on a standard nutrient medium, macro and micro salts according to the prescription Murashige and Skoog were replaced with new glassy metaphosphates of three AVA variants:

1. AVA - 1 (code AVA - 1, Wagon);

2. AVA with humic acids (97% AVA + 3% humate) (code-AVA + GUM);

3. AVA with urea carbide (85% AVA + 15% urea carbide) (code AVA + M).

The remaining components of the control culture medium corresponded to the standard medium for poliscias, excluding the kinetin growth stimulator. The strains were cultured under standard conditions on nutrient media prepared in the following options:

No. 1 Control medium - macro salts according to Murashiga and Skoogoo - 750.0 ml / l;

microsalt according to Murashige and Skoogoo - 1.5 ml / l,

sugar - 3%, agar-agar - 0.6%,

inositol - 0.100 g / l, casein hydrolyzate - 0.5 g / l,

iron chelate - 5 ml / l,

α-naphthylacetic acid - 3 ml / l,

kinetin - 1 ml / l.

No. 2. Strain poliscias (Pol) medium: instead of macro- and micro-salts, AVA - 1 - 1 g / l is given, the remaining components are described according to the control medium without a kinetin growth stimulator.

Number 3. Strain poliscias (Pol) medium: instead of macro- and micro-salts, AVA + GUM is given - 1 g / l, the remaining components according to the prescription of the control medium without a kinetin growth stimulator.

Number 4. Strain poliscias (Pol) medium: instead of macro and micro salts AVA + M is given - 1 g / l, other components according to the recipe of the control medium without a kinetin growth stimulator.

The cultivation cycle in all cases was 40 days.

At the end of the cycle, wet and dry tissue masses, Lowry protein, water-soluble polysaccharides — GRP, and total glycoside fraction — GHF were determined.

Controlled parameters in all cases were constant monitoring of biomass growth in terms of wet and air-dry tissue mass in an in vitro culture in an SPHFA bank for one growing cycle - 40 days. After that, part of the biomass cells of the strain was again recultivated in the second passage to the same nutrient media as in the first passage.

Morphologically, the biomass of poliscias cells in all experimental variants in the first passage remained light, as loose as in the control, and practically did not differ from the control variant. This indicated that the main metabolic processes associated with growth took place in cells with the same activity as in the control medium, although kinetin growth stimulant was excluded from it and macro- and micro-salts were replaced.

At the end of the cultivation cycle on day 40 (observation period), the biomass was separated from the remains of the nutrient medium and dried in an infrared dryer. In air-dry biomass, the sum of water-soluble polysaccharides-GRP and the sum of glycosides-GFS were determined.

Growth parameters (wet and air-dry mass) during the growth of the strain on experimental media were taken into account by the generally accepted method. In all cases, data from five vessels of the same type were taken into account; all data were processed statistically.

The following are indicators of the wet and dry mass of tissues of the polyscias strain when grown on experimental media (Table 9).

It can be seen from the above data that when replacing the macro and micro salts of Murashige and Skoog on AVA-1 metaphosphates, the wet weight of the cells decreased, but the dry weight of the cells of this strain practically did not differ from the control variant.

Table 9
Indicators of wet and dry mass of tissues of the polyscias strain in experimental environments Environment option Strain Wet mass, g / 50 ml of medium Dry weight, g / 50ml medium The control Pol 44.43 ± 2.0 1.54 ± 0.1 AVA - 1 Pol 39.6 ± 1.5 1.65 ± 0.2 AVA - GUM Pol 28.7 ± 2.3 1.40 ± 0.1 AVA - M Pol 30.78 ± 1.0 1,50 ± 0,2

It decreased slightly in terms of wet weight (by 10%) in the AVA variant with urea carbide, but remained almost the same in air-dry mass. In the humate version of AVA, the decrease in the fresh cell mass was more significant, although the dry mass was also close to the control.

Thus, it can be concluded that in the first passage, the replacement of the traditional Murashige and Skoog salts in the nutrient medium with new glassy AVA metaphosphates in various modifications did not practically affect the morphology of cells and the increase in the biomass of the polyscias strain even in the absence of a kinetin growth stimulator.

In this regard, it was of interest to compare the accumulation of protein in polyscias cells during cultivation on media with glassy AVA metaphosphates (Table 10).

Table 10
Indicators of protein accumulation in the biomass of polyscias strain cells upon cultivation on media with a mixture of glassy AVA metaphosphates Strain Environment option The protein content, mg / g biomass Poliscias - (Pol) The control 2.80 ± 0.01 Poliscias - (Pol) AVA - 1 3.45 ± 0.02 Poliscias - (Pol) AVA + GUM 2.80 ± 0.01 Poliscias - (Pol) AVA + M 2.65 ± 0.01

The above indicators indicate that the protein content in polyscias cells on experimental media with AVA was not inferior to the control variant and even exceeded the latter (in the variant with AVA - 1). This indicates that the macro- and microelement composition of glassy AVA metaphosphates in all cases was sufficient for metabolic processes of protein synthesis. Obviously, a high content of humus (up to 7% in AVA + GUM), potassium (K ₂ O), phosphorus (P ₂ O ₅ ) and magnesium (MgO), as well as trace elements such as Cu, S, B, Fe, and others sufficiently activate protein synthesis enzymes, affecting its activation.

We observed a similar response in the case of using AVA fertilizers to cultivate strains of ginseng and panax five-leaved.

Considering that glycosides (SGF) and water-soluble polysaccharides (GRP) are one of the main biologically active substances in polysiac cells, a comparative analysis of these substances in the biomass of this strain on experimental media was performed (Table 11).

Table 11
The total glycoside fraction (GFS) and water-soluble polysaccharides (GRP) in the biomass of the polyscias strain Strain Environment option GHF,% GRP,% Policias- (Pol) The control 2.36 ± 0.2 6.0 ± 0.55 Policias- (Pol) AVA + GUM 4.21 ± 0.3 4.50 ± 0.25 Policias- (Pol) AVA + M 3.38 ± 0.1 4.10 ± 0.15 Policias- (Pol) AVA - 1 2,50 ± 0,2 4.11 ± 0.05

Analyzing the obtained data, we can conclude that culturing the polyscias strain on experimental media with AVA fertilizers led to an increase in glycoside synthesis by almost 1.5 times when using AVA with humate, but the GRP content decreased by almost 1 as compared to the control, 5 times.

This is of particular interest, since the trace elements Cu and Zn, which are present in higher concentrations in AVA, were primarily used to activate the synthesis of glycosides as endogenous hormones under stress, while GRP synthesis was not activated and some of them were used for adaptation cells.

Next, the activity of antioxidant defense enzymes was determined in the biomass of the poliscias strain. The data are presented in table.12.

Table 12
The specific activity of antioxidant enzymes in the biomass of the polyscias strain during growth on nutrient media with glassy metaphosphates AVA Strain Environment option The specific activity of catalase, E / g protein The specific activity of SOD, E / g protein Poliscias - Pol The control 63.0 ± 3.5 50.3 ± 5.4 Poliscias - Pol AVA + GUM 37.1 ± 5.2 45.4 ± 1.5 Poliscias - Pol AVA + M 47.0 ± 3.5 32.5 ± 2.5 Poliscias - Pol AVA - 1 43.0 ± 4.0 27.0 ± 4.5

From the presented data it can be seen that in all experimental nutrient media with AVA, a decrease in the activity of antioxidant defense enzymes was observed at the end of the cultivation cycle. Given that these are substrate-dependent enzymes, it can be assumed that trace elements AVA did not affect the accumulation of peroxide radicals in cells.

Thus, the conducted studies allow us to conclude that cultivation of the polyscias strain on experimental nutrient media with AVA metaphosphates in various modifications in the first passage practically did not affect the morphological and biochemical parameters of the biomass of this strain.

Therefore, at the end of the cultivation period after the first passage, part of the biomass of the polyscias strain was recultivated in the second passage to the same variants of culture media with glassy AVA metaphosphates.

In the process of biomass growth in the second passage, a slowdown in biomass growth, an increase in the lag period to 7-10 days, and a significant browning of cells were noted. By the end of the cultivation period, a significant decrease in the wet and air-dry cell mass was noted (Table 13).

Table 13
Indicators of wet and air-dry mass of polyscias cells on experimental media in the second passage Environment option Strain Wet mass, g / 50 ml of medium Dry weight, g / 50ml medium The control Pol 44.43 ± 2.0 1.54 ± 0.1 AVA - 1 Pol 22.1 ± 2.5 0.55 ± 0.1 AVA - GUM Pol 18.2 ± 5.3 0.50 ± 0.3 AVA - M Pol 15.78 ± 1.0 0.51 ± 0.2

Perhaps a decrease in growth in the second passage on experimental media was associated not so much with AVA metaphosphates as with the fact that one of the growth stimulants, kinetin, was absent in the nutrient medium. It is known that kinetin is involved in the activation of cell divisions, therefore the absence of this stimulant in the second passage could affect cell division and, as a result, a decrease in wet and dry mass.

To determine the activity of metabolic processes, we determined the amount of protein at the end of the cell growth cycle at the second passage. The data are presented in table.14.

Table 14 Indicators of protein accumulation in the biomass of polyscias strain cells upon cultivation on media with glassy AVA metaphosphates in the second passage Strain Environment option The protein content, mg / g Poliscias (Pol) The control 3.5 ± 0.2 Poliscias (Pol) AVA + GUM 2.54 ± 0.1 Poliscias (Pol) AVA + M 2.74 ± 0.3 Poliscias (Pol) AVA - 1 2.81 ± 0.2

From the above data, it can be concluded that protein synthesis in polyscias cells in the second passage in all cases decreased. This is possibly due to a decrease in cell nutrition due to drying of agar-agar and a decrease in the intake of AVA salts, as well as due to the absence of kinetin.

Table 15
The specific activity of antioxidant enzymes in the biomass of the polyscias strain when growing on nutrient media with AVA metaphosphates in the second passage Strain Environment option The specific activity of catalase, E / g protein The specific activity of SOD, E / g protein Poliscias (Pol) The control 59.0 ± 2.5 53.0 ± 1.4 Poliscias (Pol) AVA + GUM 70.1 ± 3.5 61.4 ± 0.5 Poliscias (Pol) AVA + M 89.0 ± 2.1 70.5 ± 1.5 Poliscias (Pol) AVA - 1 82.2 ± 4.0 91.0 ± 2.5

As can be seen from the obtained data, there was an increase in the activity of antioxidant enzymes in all variants of nutrient media with AVA. The general browning of the cell mass in the second passage says that there is an activation of polyphenol oxidase and the formation of secondary metabolites associated with stress and the restructuring of the general metabolism of cells. At the same time, some areas of normal white biomass were noted, and by successive selection of these cells, it will be possible to obtain a line that is further resistant to growth without kinetin and on AVA metaphosphates.

Table 16
The total glycoside fraction (GFS) and water-soluble polysaccharides (GRP) in the biomass of the polyscias strain in the second passage Strain Environment option The content of GFS,% GRP content,% Poliscias (Pol) The control 2.0 ± 0.2 5.0 ± 0.5 Poliscias (Pol) AVA + GUM 2.3 ± 0.3 3.6 ± 0.2 Poliscias (Pol) AVA + M 1.6 ± 0.5 3.0 ± 0.5 Poliscias (Pol) AVA - 1 1.8 ± 0.2 3.8 ± 0.2

The content of the main biologically active substances in the cells of the polyscias strain at the second passage decreased compared to the first passage, although the content of GHF remained at the control level or slightly higher, in particular with AVA - 1.

For greater clarity, all the results obtained in the examples given in the application are summarized in one table 17.

In this table, as a control nutrient medium, a standard nutrient medium with macro and micro salts according to the recipe Murashige and Skoog was used. And as the claimed variants of the nutrient medium, three variants of the medium were considered:

1. AVA - 1

(cipher AVA - 1, Universal), the composition is the same as in example 1;

2. AVA with humic acids (97% AVA + 3% humate) (code AVA + GUM), the composition is the same as in example 1;

3. AVA with urea carbide (85% AVA + 15% urea carbide) (code AVA + M), the composition is the same as in example 1.

Table 17 Indicators Strain Environment option Wet mass, g / 50 ml of medium Dry weight, g / 50 ml of medium Sod. Protein, mg / g biomass Catalase specific activity, E / g protein The specific activity of SOD, E / g protein GHF,% GRP,% Ginseng (D) The control 31.4 ± 1.2 1.00 ± 0.01 2.3 ± 0.05 75.5 ± 10.5 56.7 ± 5.1 4.1 ± 0.2 8.00 ± 0.51 The inventive option one 20.76 ± 2.7 0.95 ± 0.02 3.01 ± 0.03 17.5 ± 1.0 30.1 ± 1.0 3.85 ± 0.1 5.21 ± 0.11 2 16.80 ± 2.8 0.81 ± 0.02 2.85 ± 0.02 12.4 ± 1.5 29.0 ± 1.5 4.30 ± 0.3 4.15 ± 0.20 3 19.30 ± 1.5 0.96 ± 0.05 2.62 ± 0.01 15.2 ± 3.5 31.8 ± 2.0 4.50 ± 0.2 3.5 ± 0.10 Selective Ginseng (D-) The control 36.10 ± 0.3 1.59 ± 0.03 3.6 ± 0.05 28.0 ± 2.0 12.1 ± 0.2 4.17 ± 0.5 10.00 ± 0.60 The inventive option one 20.48 ± 0.5 1.1 ± 0.02 3.25 ± 0.03 30.0 ± 2.0 13.5 ± 0.5 3.69 ± 0.3 8.85 ± 0.40 2 16.80 ± 0.4 0.94 ± 0.01 3.1 ± 0.02 28.6 ± 1.0 13.3 ± 0.2 4.53 ± 0.4 6.75 ± 0.30 3 15.6 ± 0.6 0.91 ± 0.01 3.2 ± 0.01 27.5 ± 3.0 9.45 ± 0.5 4.20 ± 0.2 5.45 ± 0.25 Panax (A _m ) The control 41.3 ± 1.0 1.5 ± 0.2 3.1 ± 0.1 65.0 ± 4.5 56.3 ± 2.4 3.5 ± 0.3 15.0 ± 5.0 The inventive option one 20.2 ± 1.0 1.2 ± 0.1 4.7 ± 0.1 46.2 ± 4.0 15.0 ± 6.5 3.8 ± 0.2 5.8 ± 0.2 2 20.6 ± 1.2 1.2 ± 0.1 3.95 ± 0.05 30.1 ± 1.2 35.4 ± 0.5 4.3 ± 1.2 7.0 ± 1.2 3 19.8 ± 1.6 1.2 ± 0.2 4.25 ± 0.2 42.0 ± 2.1 22.5 ± 0.5 3.6 ± 0.5 4.0 ± 0.5 Selective Panax (A _m -) The control 43.5 ± 2.5 1.5 ± 0.3 3.65 ± 0.2 40.0 ± 3.5 20.0 ± 2.5 3.0 ± 0.7 17.0 ± 3.6 The inventive option one 18.1 ± 1.8 0.8 ± 0.2 4.3 ± 0.15 25.2 ± 3.0 15.0 ± 0.7 4.0 ± 0.2 4.0 ± 0.5 2 16.4 ± 4.2 0.84 ± 0.5 5.2 ± 0.2 21.0 ± 1.5 14.0 ± 1.0 5.5 ± 0.4 6.7 ± 2.4 3 18.5 ± 2.1 1.1 ± 0.3 4.35 ± 0.05 22.5 ± 2.5 13.0 ± 0.5 3.5 ± 0.2 4,5 ± 1,0 Poliscias (Pol) The control 44.43 ± 2.0 1.54 ± 0.1 2.8 ± 0.01 63.0 ± 3.5 50.3 ± 5.4 2.36 ± 0.2 6.0 ± 0.55 The inventive option one 39.6 ± 1.5 1.65 ± 0.2 3.45 ± 0.02 43.0 ± 4.0 27.0 ± 4.5 2.5 ± 0.2 4.11 ± 0.05 2 28.7 ± 2.3 1.40 ± 0.1 2.8 ± 0.01 37.1 ± 5.2 45.4 ± 1.5 4.21 ± 0.3 4.5 ± 0.25 3 30.78 ± 1.0 1,50 ± 0,2 2.65 ± 0.01 47.0 ± 3.5 32.5 ± 2.5 3.38 ± 0.1 4.1 ± 0.15

The following conclusions can be drawn from the data in the table:

1. Despite the decrease in crude cell biomass, the air-dry mass in all cases exceeded or was equal to the dry cell mass in the control sample.

2. The protein content in all cases exceeded the control samples.

3. The content of the main active substances (humates, glycosides, etc.) corresponded to control samples.

4. The activity of antioxidant enzymes (SOD, catalase, etc.) fluctuated due to changing cultivation conditions, but remained sufficient for the normal level of plant cell metabolism.

Thus, the use of the proposed nutrient mixture leads to a decrease in the number of operations during the preparation of the mixture, since a number of salts are replaced by one substance - vitreous fertilizer - AVA, while reducing the risk of errors in the preparation of the mixture, increasing the reliability of the biosystem.

The proposed nutrient mixture is economically viable and physiologically acceptable for widespread use in biotechnology for the cultivation of medicinal plants with multiple transfers.

The use of the proposed nutrient mixture significantly reduces labor costs, increases the efficiency and reliability of this method of plant cultivation.

Comparative economic indicators of the use of traditional methods of preparing a nutrient medium for growing plant biomass and methods using the AVA complex are shown in table 18.

Table 18
The cost of using traditional methods of preparing the nutrient medium and new methods using the AVA complex Culture medium The number of operations to prepare 1 liter of medium Labor time, min Cost, rub./kg Cost, rub / liter Cooking frequency Environmental harm Control nutritious MS environment 5-6 1 time / month no - macro fourteen 90 10-15 - micro 3-4 - stimulants - carbohydrates 1-2 - vitamins Nutritious Wednesday on basis complex 8 twenty 60-80 2-4 ^* 1 time / month no "AVA" - macro - micro - stimulants - carbohydrates - vitamins ^* The concentration of AVA in the nutrient medium 1 g / liter

Claims (1)

A method of producing plant cell biomass by cultivating the cell culture biomass on a nutrient medium containing inorganic components, a substrate, carbohydrates, vitamins and growth stimulants, characterized in that a mixture of glassy metaphosphates of variable composition of the following content, wt. %: phosphorus oxide 40-60 potassium oxide 10-30 calcium oxide 5-20 magnesium oxide 3-13 silica 1-6 boron oxide 1-6 oxides of elements of the periodic table (Co, Fe, Cu, Mo, Mn, Cr, Zn, Ge, Se, V) 0.05-2