SU1036053A1 - Method of cultivating ginseng callus tissue - producent of biologically active substances - Google Patents

Abstract

THE METHOD OF CULTIVATING THE CALFUS TISSUE OF THE GIFT - A PRODUCER OF BIOLOGICALLY ACTIVE SUBSTANCES ways of growing seed material on the nutrient medium with kinetine, and then, aspirin the seed is subcultured on a nutrient medium containing (mg / l of water) sucrose 25000-35000, thiamine-chloride 0.3 0, 6, meso-inosite 70-90, f-naphthylacetic acid 1.5-2.5, nicotinic acid from 1.5-2.5, agar-agar 5500-6500, chelate 4.5-5.5, macronutrients according to Murashige and Scuta 4.5-4.57-10, trace elements according to Murashige and Skoog 38-38,22 , a part of the biomass is taken out, and the remaining part of the biomass is used (it is used at the cultivation stage of the seed.

Description

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The invention relates to the microbiological industry and relates to the technology of obtaining and strengthening the transplantation of plant tissue cultures used as producers of biologically active substances in various industries.

There is a known method of cultivating Gin-Sheng callus tissue - a producer of biologically active substances by growing seed on a nutrient medium with kinetin, followed by subculture on a nutrient medium and selecting the grown biomass to isolate biologically active substances.

However, the known method does not provide a high yield of biologically active substances. The aim of the invention is to increase the yield of biologically active substances. The goal is achieved by the fact that in the method of cultivating callus tissue, ginseng - a producer of biologically active substances by growing seed on a nutrient medium with kinetin, followed by subculture on a nutrient medium and selecting grown biomass for the release of biologically active substances a distinctive feature is the fact that seeding material is subcultured on a nutrient medium containing (mg / l of water sucrose 25000-35000, thiamine-chloride 0.3-0.6, meso-inosite 70-90, C-naphthylacetic acid 1.5-2.5, nicotinic acid 1.5-2.5, ag p-agar 5500-6500 chelate 4.5-5.5, macronutrients according to Murashige and Skoogu 4.5-4.57 -10, trace elements according to Murashige and Skoogu 38 38, 22, select part of the biomass grown, and the remaining part of biomass is used at the stage of inoculation of the inoculum. The method is carried out as follows: Nutrient medium is prepared. Then the nutrient medium is poured into the culture vessels. The nutrient medium containers are sterilized .The callus tissue is then cultured, a part of the grown tissue is transplanted (1/10 of the total volume). on fresh nutrient medium addition same composition. Then transplanted grown tissue (9/10 of the crop) on a modified nutrient medium.

The modified nutrient medium contains trace elements according to Murasige and Skoog, trace elements according to Murassige and Skoog, sucrose, thiamine-chloride, meso-inositol, casein hydrolyzate, 2-naphthylacetic acid, nicotinic acid, agar-agar, iron-chelate.

The transplanted tissue is cultured on a modified nutrient medium, with the possibility of multiple growth on the modified medium until the biological activity of the biomass is minimal. After that, the biomass is transplanted to the full nutrient medium Murashige and Skoogu and used in the future as an inoculum.

Then callus tissue growing on the modified medium is collected and dried, extracted and biologically active substances are obtained from the callus tissue cultured on the modified medium. Example 1 (control) The process for the preparation of biologically active substances involves a number of operations. Prepare a nutrient medium. Murashige and Skoogu medium are used in the following composition: Macroelements according to Murashige and Skoogu Microelements according to Murashige and Skogu Saccharose 30000 mg / l Thiamine-chloride 0.4 mg / l Mesoinosite 80 mg / l Casein hydrolyzate 500 mg / l Kinetin 1 mg / l -Oil / l acid 6000 mg / l Agar-agar 5 mg / l Fe-chelate B thoroughly washed cultivation bottles are poured into 250 ml of nutrient medium. The vials with medium are sealed and autoclaved to destroy microorganisms, Boxing is sterilized with bactericidal lamps, tools in a dry-air cabinet at 150-200 with Kraft paper is wrapped. Then the material is removed from the vial with sterile tools and transplanted into prepared vials with a medium of 1 g each. transferred to a cultivation room, where the temperature and humidity of 70% is kept stable, there is no cultivation of light. 30 days Crop is harvested to obtain biologically active substances. 1/10 hour of the grown biomass is used as an inoculum, transplanted to the ground nutrient medium of the same composition. 9/10 is dried and the biologically active substances are extracted with ethyl alcohol at a ratio of raw materials: extractant 1:10. The biomass growth of biologically active substances and productivity are determined. To control the growth of the culture and the release of biologically active substances, the dry weight (at least five replications), the sum of glycosides (biologically active substances) are determined by the gravimetric method and productivity. Dry weight of biomass is 10.44 g / l. Amount of glycosides is 4.2%. Biomass productivity is 438.5 mg / Example 2. Production of biologically active substances from callus tissue growing on the modified Murashige and Skoog medium containing: Macro salts of Murashige and Skohu Trace Elements Murashige and Skougou 25000 mg / l Saccharose 0.3 mg / l Thiamine chloride 70 mg / l Meso-inositum 450 mg / l Casein hydrolyzate t-Naphthyloacetic 1.5 mg / l Nicotinic acid 1.5 mg / l Agar-agar5500 mg / l Fe-chelate4.5 mg / l. The operations of cultivation and preparation of biologically active substances are described in example 1, dan Noah biomass dry weight of 10.3 g / l, biologically active substances (the sum of glycosides) of 4.15%, productivity 427.5 mg / l. Example 3, Preparation of biologically active substances from callus tissue growing on modified Murashige and Skoog medium: Macrosols according to Murashige and Skoog Microelements according to Murashige and Skoogu Sucrose 30000 mg / l Thiamine-chloride 0.4 mg / l Mezoinosit 80 mg / l Casein hydrolyzate 500 mg / l 1 3 P-Naphthylacetic acid 2 mg / l Nicotinic acid 2 mg / l Agar-Agar 6000 mg / l Fe-Chelate 5 mg / l Cultivation operations and the preparation of biologically active substances are described in example 1. Dry biomass yield -schen is 11.8 g / l, biologically active substances tv - 4.18%, productivity - 493.3 mg / l. The medium provides the highest biomass yield and, accordingly, the highest yield of biologically active substances. Example 4. Obtaining biologically active substances from callus tissue growing on a nutrient medium of the following composition: Macrosols according to Murashige and Skogu Saccharose 35000 mg / l Thiamine chloride 0.6 mg / l Meso-inosit 90 mg / l Casein hydrolyzate 550 mg / l E-Naphthylacetic acid2.5 mg / l Nicotinic acid 2.5 mg / l Agar-agar 6500 mg / l Fe-chelate5.5 mg / l Description of operations for the cultivation and production of biologically active substances is given in example 1. On this medium, the biomass yield is dry weight 11.65 g / l, the amount of glycosides - 4.05%, productivity 471, 8 mg / l. Example 5. Cultivation of callus tissue and the production of biologically active substances from it with repeated use of the modified nutrient medium specified in Example 3. Detailed operations for the cultivation and production of biologically active substances are described in Example 1. With one-time cultivation (1 passage) dry biomass output 11 , 80 g / l, sum of glygosides - 4.18%, productivity - 493.3 mg / l. With the double cultivation of the second passage) VELHOD dry biomass of 3.15 g / l, the amount of glycosides -3.25%, deductibility - 427.4 mg / l. With a threefold cultivation of passage III), the yield of dry biomass is 3.97 g / l, the sum of glycosides is 2.9%, and the yield strength is 405.1 mg / l.

510360536

With four-fold cultivar-12.53 g / l, the amount of glycosides is 2.5%

Scientific research institute (IV passage) dry biomass productivity of 313.2 mg / l. Synthes 13.56 g / L, the amount of glycosides. The proposed method allows to increase 2.5%, productivity - 339.0 mg / l

With repeated cultivation, and also reduce the consumption of kinetig

{V passages) dry biomass yield 10 times.

5 substances by 35% compared with the known

Claims (1)

METHOD FOR CULTIVATION OF THE GALLEN-SENIUS CALLUS TISSUE - PRODUCER OF BIOLOGICALLY ACTIVE SUBSTANCES by growing seed on a nutrient medium with kinetin with ~ subsequent reseeding on the nutrient medium and selecting grown biomass to isolate biologically active substances, characterized in that in order to increase biologically active substances, which substances, inoculum is subcultured on a nutrient medium containing (mg / l of water) sucrose 25000-35000, thiamine chloride 0.3 0.6, mesoinositol 70-90, f-naphthylacetic acid 1.5-2.5, nicotinic acid 1.5-2.5 , agar-agar 5500-6500, chelate 4.5-5.5, macrocells according to Murasig and Skoogoo 4.5-4.57 10 ⁵ , microelements according to Murasig and Skoogoo 38-38.22, part of the grown biomass is selected, and the remaining part of the biomass is used at the stage of growing seed. 1036053