SU440814A1 - - Google Patents

Description

(54) METHOD FOR OBTAINING CHEMICAL METABOLITES

PLANTS in an aerobic environment in the submerged state to obtain fully matured mass. This growth leads to the accumulation of the necessary quantities of metabolites, which are isolated and collected. The chemical metabolites of plants that can be obtained by the method described are alkaloids, such as strychnine and brucine from samples of Strychnes; Rauwolfia alkaloids from Rauwolfia alstonia samples; vinblastine from Catharanthus samples (mainly Vinca), anthropic alkaloids from a number of members of the Solanaceae family, gum from Beta viilgaris samples and L,; lta, dyes from Indigofera, Rhus, Beta and Zawsane samples, etc. a medium containing one or more carbon sources, such as carbohydrates or fatty acid derivatives, a source of assimilated nitrogen, such as nitrate-ion or ammonium-ion, glycine, urea. It is preferable to use substances such as glucose, sucrose, corn syrup, starch, dextrin and other similar substances as a carbon source. In addition, the nutrient medium contains mineral salts, such as phosphates, chlorides, sulfates, metals (sodium, potassium, calcium, magnesium) and trace elements. Trace elements are usually present as impurities even in well-purified inorganic compounds. In addition, one or more common vitamins are introduced into the nutrient medium: thiaM .IN, riboflavin, pantothenic acid, and naicin. This mixture ensures the normal growth of plant tissue. Auxin-active substances are photo hormonal substances that regulate plant growth. Natural auxins, i.e., those that exhibit activity in regulating plant growth and are found in the whole living plant, are 3-indole derivatives, for acid and some of it, for example, esters, glycosides. Auxin active substances include both natural compounds and synthetically prepared substances that detect activity, as phytohormonal substances that promote growth. Auxin actives are sometimes preferred as germline stimulants. Typical auxinactive substances used in the invention are such as 3-ipdolucetic acid, 3-indol-butyl acid, 1-naphthalene acid, p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and etc. The method also uses anti-auxin compounds, i.e., such compounds that inhibit or neutralize the phytohormonal activity of auxin-active substances. As a typical anti-auxins take. for example, 1-naphthoxyacetic acid, cinnamic acid, tropic acid, 2,3,5triodobenzoic acid and 2,4,6-trichlorophenoxyacetic acid. Example 1. Inducing growth of Beta vulgaris on normal plant material while maintaining a cured environment. Grains of ordinary red beets. Beta vulgaris varieties Detroit Selest clobe, germinated on moist filter paper and then completely dried, germinated grains sterilized by immersion in 1% hypochlorite solution for 5 minutes. From now on, all operations with living tissues are carried out under the condition of complete asepsis. This seedling is transferred to a wet sterilized filter paper, cut into pieces in the form of circles: radial segments, hypocotul segments and seed segments, and each of these segments is transferred into separate I-inch Petri dishes containing 30 ml of medium each (Table 1). one). Table 1 Medium Concentration, Component mg / l Potassium nitrate Ammonium nitrate Magnesium sulfate heptahydrate Calcium chloride dihydrate Potassium phosphate Iron sulfide heptahydrate Manganese sulfate hydrate Zinc sulfate heptahydrate Anhydrous boric acid 0.1 Ammonium molybdate, calcium iodide Calcium 0.12 I used in the same mode. Sucrose 10000 0.1 Agar Biotin 1 1 Cholinechloride Nicotinic acid 1000 Inositol Riboflavin Thiamine 2,4-Dichlorophenoxyacetic acid Kinetin Distilled water This medium is sterilized in an autoclave for 20 minutes at a vapor pressure of 15 lb / d ym2 (1 lb / in2 0.070307 kgf / cm2). The inoculated plates are incubated at 25 ° C and under reduced light: after three weeks of incubation, large growths are visible at the ends of several of the original inoculated samples. Some cups are damaged by growing molds or bacteria, they are thrown away. The overgrowths that occur on the inoculated thumps are transferred to 100 ml containers containing 20 ml of the same agar medium and incubate,

Biro continue. five weeks under the same conditions. After this time has elapsed, sufficient growth appears, so that the subculture can be transferred several times to other containers.

Example 2. Induction of unorganized growth in a liquid medium and selection of the type having the desired growth properties.

From each subculture in agar (see example 1) a piece of about 8 mm in diameter is taken and transferred to an Erlenmeyer flask containing 60 ml of liquid medium (its composition is given in example 1, with the exception of agar). These flasks, containing an ioculated liquid medium, are placed on a rota-ion booster having 120 rpm and a shaking circle diameter of 25 mm, and incubated with shaking under diffuse light for four weeks at 25 ° C. In the fourth week conpe, different results were observed in different flasks, reflecting the degree of carelessness in selecting cells at the previous stage. Some flasks contain only one large piece of outgrowth, while others contain one large and several small, apparently related to nature, forming, apparently, like outgrowths on the original piece and detached from it during the incubation process; Some flasks contain broken fragments that themselves represent further cell secretion.

Those flasks that contain small fragments, and those that contain fragments with various features, are selected for further subculture, and after these types of selection, transfer and cultivation operations are obtained, cells have the desired growth rate in agitated flasks and grow to form simple, unpigmented cells or small aggregates less than 0.5 mm in diameter. Microscopic examination of these aggregates does not reveal any fine structure, i.e., other than cell components. Their growth rate is such that when less than 1% of the cells are introduced into the flask during two weeks of incubation on a rotary shaker, the total percentage of cells formed is 20 vol. % These properties are retained during subsequent mass transfers to the same medium.

Example 3. Repeated differential growth of the primary plant culture immersed in the aerated liquid.

The content of one of the flasks with dispersed unorganized, non-pigmented cell culture of Beta vulgaris is allowed to stand, and the clear liquid is decanted and discarded. The deposited cells and groups of cells are washed twice with 60 ml for 1 time with an auxin-free liquid medium (the medium composition is shown in Example 1, except for 2,4-dichlorophenoxyacetic acid and agar), and the liquid is removed by decantadium after sedimentation of the cellular material.

table 2

Wednesday

Concentration

Component

mg / l

Potassium nitrate

950

Magnesium sulfate heptahydrate 185

Calcium chloride dihydrophosphate 220

Equal portions of the washed cells are then used to inoculate 10 Erlenmeyer flasks, each containing 60 ml of auxin-free liquid medium, after which these flasks are incubated on a rotary shaker, as described further, for three weeks at 25 ° C. During this incubation period, the inoculated cells and cell groups grow vigorously and at the end redifferentiation is observed with the formation of a paste of dark orange or red particles, which, when a small increase in the microscopic appearance appears to be almost entirely made up of the fine roots of the original plant (primary), each one they are abundant in normal pigment used for cultivation.

Example 4. The allocation of pigment. The paste of the roots of the primary of the 10 flasks of the previous example is filtered, and the entire pigment is contained in the cellular material and is not present in the filtered liquid, which is discarded. The whole mass of cells is distributed in 500 ml of water and the mixture is boiled, and the pigment goes into liquid. Filtered and discarded cellular

Table 3

Wednesday

Concentration

45

Component

mg / l

950

Potassium nitrate

185

Su.pfat magnesium heptahydrate

0

220

Calcium Chloride Dihydrate

68

Potassium dihydrophosphate

thirty

Ferrous sulfate heptahydrate

7

Manganese Sulphate Hydrate

four

Zinc sulfate heptahydrate

2

Borna acid

five

0.1

Ammonium molybdate 0.4

Potassium iodide 0.12

Copper sulphate pentahydrate 20000

Sucrose

OD

Biotin 1000 1.0

Inositol (Meso)

Nicotinic acid 1.0

Pantothenic Acid 1.0

Pyridoxine 1.0

Riboflavin 1.0

Thiamine 0.06

Kinetin 1.0

Choline chloride

mass, and the resulting red filtrate is passed through a 6-inch column with a cation exchanger Dowex 50X2. which retains almost all of the pigment contained in the liquid in the 2-inch layer at the top of the column. The pigment was eluted with a 1% sodium bicarbonate solution, and then the pigment was separated. When acidified to pHH5, the dry solution was frozen and the pigment was extracted from the frozen solution with methanol. Betanidin is used as a food paint of plant origin.

Example 5. Growth of undifferentiated cells as a dispersed culture in an agitated flask.

Groups of cells approximately 8 mm in diameter from the agar surface are used to inoculate each of the five Erlenmeyer flasks, containing 25 ml of liquid medium of the following composition (Table 2).

In addition, the medium contains trace elements, sucrose, vitamins, auxin-active compounds and cytokines.

After incubation for 18 days, the cell material is allowed to settle, the liquid is separated by decantation, and washed

sediment 50 ml of fresh, free from auxin, medium, of the following composition (table. 3).

This slurry is shaken for 5 minutes, then

again give her the opportunity to settle down and separate and discard the clean sedate

liquid. The cell mass is washed alone.

times the medium MM P by the same method.

Subject invention

Claims (2)

1. A method of producing chemical metabolites of plants, including in vitro culture, of isolated undifferentiated tissues or cells under controlled conditions, characterized in that, in order to improve conditions for accumulation of metabolites, plant cells are redifferentiated by reducing the level of auxin activity of plant cells. 2. The method according to claim 1, characterized in that the decrease in the level of auxin activity is carried out by diluting in a 1: 1 ratio of working nutrient medium fresh, of the same formulate or administer an anti-auxin-active compound.