RU2631927C1 - METHOD FOR OBTAINING OF CALLUS CULTURE OF ACONITUM BARBATUM PATR. ex PERS. - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method for obtaining of a callus culture of Aconitum barbatum Patr. Ex Pers., including seeds sterilisation, placement in a refrigerator at a temperature of 5± 1°C for 2.5 months for stratification and obtaining of a callus culture from etiolated seedlings where the unripened seeds are successively sterilized in a solution of alcohol (70%) for 40 s and 0.1% mercuric cloride for 3-4 minutes before stratification, washed with sterile distilled water, after stratification, they are successively sterilized in a solution of alcohol (70%) for 30 s and mercuric clorid (0.1%) for 1-2 minutes and also washed with sterile distilled water, then seeds are placed in test tubes on the MC agar medium without addition of growth stimulants to obtain etiolated seedlings in the dark for 3-4 weeks, shoots are separated from the resulting etiolated seedlings and placed in test tubes on the MC agar medium by adding 1.8-2.2 mg/l of 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3-0, 8 mg/l of 6-BAP (6-benzylaminopurine) for the production of callus, cultivation is carried out at a temperature of 26±1°C, humidity of 70% in the dark.

EFFECT: allows to get the callus culture of Aconitum barbatum.

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Description

The invention relates to the field of biotechnology, in particular to the cultivation of cell cultures of medicinal plants, and can be used in medicine to obtain raw materials containing biologically active substances, as well as the introduction of other representatives of medicinal plants into the culture in vitro, which are difficult to obtain due to isolation poisonous compounds in the nutrient medium.

The bearded wrestler is a plant of the Ranunculaceae family, is rich in diterpenic alkaloids, such as aconitine, delcozin, lycoctonin, zongorin and batakonin, etc., coumarins, flavonoids, and proazoles. In folk medicine, it is used to treat diseases of rheumatic origin and inflammation. It has analgesic, antitumor properties.

A known method of obtaining callus culture of Pulsatilla taurica (article Sidyakin A.I., Mustafayev U. U. Obtaining callus culture of Pulsatilla taurica and their cytological analysis // Innovations in science / Coll., Based on materials from XLV international scientific and practical conf. No. 5 (42). Novosibirsk: SibAK Publishing House, 2015, pp. 43-53.) From leaf cuttings of seedlings and juvenile plants obtained in vitro from seeds and leaf cuttings from plants during the fruiting phase. The method includes stepwise sterilization of the starting material: 1% KMnO4 (40 minutes); 70% ethanol (1 minute) and 3% H2O2 (5 minutes), and the cultivation of explants in nutrient medium Murashige and Skoog with the following options for adding hormones 0.1 mg / l 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.01 mg / L 6-BAP (6-benzylaminopurin), and 3 mg / L 2,4-D and 1.0 mg / L 6-BAP. Explants are cultivated in the light or in the dark at a temperature of 20-24 ° C. The crop growing cycle is 70–90 days.

The disadvantage of the analogue is the choice of the explant for receiving callus, since it is known that it is rational to use the earliest tissues in which there is no accumulation of alkaloids to obtain a cell culture of alkaloid-bearing plants, such as bearded wrestler. Despite the fact that the plant Pulsatilla taurica also belongs to the ranunculaceae family, like the bearded wrestler, it does not contain the same alkaloids, which means that the above method for producing callus culture is not suitable. In addition, this method uses a higher concentration of growth promoter, which leads to an increase in the cost of the process.

A known method for producing callus culture of hemlock spotted (Conium maculatum L.) (RF Patent 2590586, publ. 07/10/2016, IPC C12N5 / 04), comprising planting seeds in sterile soil, growing intact plants for 1-2 months with an illumination intensity of 150 μM quanta / m ² s, selection of explants of 3-4 weeks of age, sterilization sequentially in 70% alcohol for 30 s and in 0.1% mercuric chloride 4 min, placing pieces of the stem 1.5-2 cm long on a Murasige-Skoog agar medium without the addition of hormones for 7-10 days to control sterility, the allocation of callus and planting in a cult ural vessels with MS nutrient medium with the addition of growth stimulants of NAA and 6-BAP under conditions of laminar boxing, while callus is planted at 30 days of age, and callus tissue is cultured at 26 ± 1 ° C, humidity 70% in the dark, after which seedlings transferred for 6 weeks to Murashige-Skoog agar medium supplemented with 1.5–2.5 mg / L 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3–0.8 mg / L BAP (6-benzylaminopurine ) for callus.

The disadvantages of this method is the more sophisticated technology for obtaining culture, in which the necessary steps are both germination of seeds in the soil and sterility control for 10 days, which requires additional costs for the preparation of nutrient media. Also in this method, plants of 3-4 weeks of age are used, and to obtain a culture of Aconitum barbatum Patr. ex Pers rationalize the use of earlier fabrics.

Closest to the claimed technical solution according to the technical nature and the technical result achieved is a method for producing callus culture of Baikal aconite (Enikeev A.G., Semenov A.A., Gorshkov A.G., Maksimova L.A., Kopytina T.V. , Gamanets L.V., Shvetsov S.G., Permyakov A.V., Shafikova T.N. Cell culture Aconitum baicalense Turcz.ex Rapaics 1907 promising source of biologically active compounds // Scientific statements of BelSU. Series: Natural sciences. 2011 No. 3-1 (98).). The method involves sterilizing seeds in a 3% hydrogen peroxide solution, transferring them to penicillin vials with agar medium (1 / 2MS salts, without sucrose) and placing the vials in a refrigerator (4 ° C) for 2.5 months. After this period, the seed vials are transferred to a dark thermostat at 26 ° C. Callus culture is obtained from etiolated seedlings on B5 salt medium with the addition of thiamine 1 mg / l, pyridoxine 0.5 mg / l, nicotinic acid 0.5 mg / l, inositol 100 mg / l, 2,4-D 1 mg / l, 2% sucrose, agar-agar 0.8%, pH 5.8. The described method is adopted as a prototype of the invention.

The disadvantages of the prototype is a more complex technology for obtaining culture, in which the necessary stage is the germination of seeds in a dark thermostat, which requires certain equipment. Also, inositol is added to the medium for receiving callus culture, which increases its cost.

In the literature of the methods for producing callus tissue Aconitum barbatum Patr. ex Pers. not found.

The present invention is to develop a method of cultivating callus tissue Aconitum barbatum, promising as a possible source of therapeutic alkaloids.

The problem is solved in that the cultivation of callus tissue Aconitum barbatum includes sterilization of seeds, placing them in a refrigerator at a temperature of 5 ± 1 ° C for 2.5 months for stratification and receiving a callus culture from etiolated seedlings. In this case, unripe seeds before stratification are sequentially sterilized in an alcohol solution (70%) for 40 s and mercuric chloride (0.1%) for 3-4 minutes, washed with sterile distilled water, after stratification they are sequentially sterilized in an alcohol solution (70%) for 30 s and mercuric chloride (0.1%) 1-2 minutes and also washed with sterile distilled water, then place the seeds in test tubes on MS agar medium without adding growth stimulators to obtain etiolated seedlings in the dark for 3-4 weeks, shoots are separated from the obtained etiolated seedlings and placed in p test tubes on MS agar medium supplemented with 1.8–2.2 mg / L 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3–0.8 mg / L 6-BAP (6-benzylaminopurine) to obtain callus , cultivation is carried out at a temperature of 26 ± 1ºС, humidity 70% in the dark.

After the formation of callus, it is separated and transplanted into culture vessels with fresh nutrient medium of the same composition.

The nutrient medium Murashige-Skoog contains components in the quantitative ratio shown in table 1.

Table 1 - The composition of the nutrient medium Murashige-Skoog, mg / l

The resulting callus is planted in culture vessels with MS nutrient medium with the addition of growth stimulants - 2,4-D and 6-BAP. Transplantation is carried out every 30 days. The cultivation of callus tissue is carried out at 26 ± 1ºС humidity - 70% in the dark.

EFFECT: obtaining a callus culture of a bearded wrestler (Aconitum barbatum Patr. Ex Pers.).

Example 1

To prepare the nutrient medium, a macro-salt concentrate is prepared (KNO3 - 1900 mg / L, NH4NO3 - 1650 mg / L, MgSO4 × 7H2O 370 mg / L, KH2PO4 - 170 mg / L), while each of the macro-salts is dissolved successively in a small amount of water, and then the volume was adjusted to 1 liter. Concentrates of micro salts are prepared in the same way (H3BO3 - 6.2 mg / L, MnSO4 × 4H2O - 32.3 mg / L, ZnSO4 × 7H2O - 8.6 mg / L, Na2MoO4 × 2H2O - 0.25 mg / L, KI - 0 , 83 mg / L, CuSO4 × 5H2O - 0.025 mg / L, CoCl2 × 6H2O - 0.025 mg / L), Fe chelate (FeSO4 × 7H2O - 37.3 mg / L, Na2EDTA × 2H2O - 27.8 mg / L ), CaCl2 × 2H2O, vitamins (pyridoxine-HCl - 0.4-0.6 mg / l, thiamine-HCl - 0.5-1.5 mg / l, nicotinic acid-HCl - 0.4-0.6 mg / l). Macro and micro salts, Fe-chelate, CaCl2 × 2H2O, vitamins in the form of concentrates are mixed in a small amount of water. The solution was adjusted with distilled water to 1 liter. In a 500 ml flask, pour 3 g of agar, 9 g of sucrose, pour medium into 300 ml, cover with foil and sterilize in an autoclave for 20 minutes at 1.0 atm. Petri dishes, culture vessels, tweezers and a scalpel are sterilized for 2.5 hours in a dry oven at 160 ° C. Then, sterile culture medium under conditions of a laminar box is added 2.4 - D (1.8 mg / l) and 6-BAP (0.3 mg / l), and place it on the culture vessels.

Immature seeds of a bearded fighter in a laminar box are sterilized sequentially in a solution of alcohol (70%) for 40 s and mercuric chloride (0.1%) for 3-4 minutes, then washed with sterile distilled water. Next, the seeds are placed in Petri dishes with sterile, moistened sand, and put in the refrigerator for 2-2.5 months at a temperature of 5 ± 1ºС for stratification. After 2-2.5 months, the seeds are removed from the Petri dishes, sequentially sterilized in a solution of alcohol (70%) for 30 s and mercuric chloride (0.1%) for 1-2 minutes. Next, the seeds are washed with sterile distilled water and placed in tubes on MS agar medium without the addition of growth stimulators to obtain seedlings and placed in the dark for 3-4 weeks. Shoots are separated from the obtained etiolated seedlings and placed in test tubes on MS agar medium by adding 1.8–2.2 mg / L 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3–0.8 mg / L 6- BAP (6-benzylaminopurine) for callus formation. Test tubes are placed on racks at a temperature of 26 ± 1ºС, humidity - 70% in the dark. After the formation of callus, its separation is carried out, and planting in culture vessels with fresh nutrient medium of the same composition.

The tissue is planted at the age of 30 days. The cultivation of callus culture is carried out at 26 ± 1 ° C, humidity - 70%, in the dark.

Example 2

To prepare the nutrient medium, a macro-salt concentrate is prepared (KNO3 - 1900 mg / L, NH4NO3 - 1650 mg / L, MgSO4 × 7H2O 370 mg / L, KH2PO4 - 170 mg / L), while each of the macro-salts is dissolved successively in a small amount of water, and then the volume was adjusted to 1 liter. Concentrates of micro salts are prepared in the same way (H3BO3 - 6.2 mg / L, MnSO4 × 4H2O - 32.3 mg / L, ZnSO4 × 7H2O - 8.6 mg / L, Na2MoO4 × 2H2O - 0.25 mg / L, KI - 0 , 83 mg / L, CuSO4 × 5H2O - 0.025 mg / L, CoCl2 × 6H2O - 0.025 mg / L), Fe chelate (FeSO4 × 7H2O - 37.3 mg / L, Na2EDTA × 2H2O - 27.8 mg / L ), CaCl2 × 2H2O, vitamins (pyridoxine-HCl - 0.4-0.6 mg / l, thiamine-HCl - 0.5 - 1.5 mg / l, nicotinic acid-HCl - 0.4-0.6 mg / l). Macro and micro salts, Fe-chelate, CaCl2 × 2H2O, vitamins in the form of concentrates are mixed in a small amount of water. The solution was adjusted with distilled water to 1 liter. In a 500 ml flask, pour 3 g of agar, 9 g of sucrose, pour medium into 300 ml, cover with foil and sterilize in an autoclave for 20 minutes at 1.0 atm. Petri dishes, culture vessels, tweezers and a scalpel are sterilized for 2.5 hours in a dry oven at 160 ° C. Then, sterile culture medium under conditions of a laminar box is added 2.4 - D (2.2 mg / l) and 6-BAP (0.8 mg / l), and place it on the culture vessels.

Unripe seeds of a bearded fighter, in a laminar box, are sterilized sequentially in a solution of alcohol (70%) for 40 s and mercuric chloride (0.1%) for 3-4 minutes, then washed with sterile distilled water. Next, the seeds are placed in Petri dishes with sterile, moistened sand, and put in the refrigerator for 2-2.5 months at a temperature of 5 ± 1ºС for stratification. After 2-2.5 months, the seeds are removed from the Petri dishes, sequentially sterilized in a solution of alcohol (70%) for 30 s and mercuric chloride (0.1%) for 1-2 minutes. Next, the seeds are washed with sterile distilled water and placed in tubes on MS agar medium without the addition of growth stimulators to obtain seedlings and placed in the dark for 3-4 weeks. Shoots are separated from the obtained etiolated seedlings and placed in test tubes on MS agar medium by adding 1.8–2.2 mg / L 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3–0.8 mg / L 6- BAP (6-benzylaminopurine) for callus formation. Test tubes are placed on racks at a temperature of 26 ± 1ºС, humidity - 70% in the dark. After the formation of callus, its separation is carried out, and planting in culture vessels with fresh nutrient medium of the same composition.

The tissue is planted at the age of 30 days. The cultivation of callus culture is carried out at 26 ± 1 ° C, humidity - 70%, in the dark.

Claims (1)

A method of obtaining a callus culture of a bearded fighter (Aconitum barbatum Patr. Ex Pers.), Comprising sterilizing the seeds, placing them in the refrigerator at a temperature of 5 ± 1 ^° C for 2.5 months for stratification and receiving a callus culture from etiolated seedlings, characterized in that unripe seeds before stratification are sequentially sterilized in an alcohol solution (70%) for 40 s and mercuric chloride (0.1%) for 3-4 minutes, washed with sterile distilled water, after stratification they are sequentially sterilized in an alcohol solution (70%) for 30 s and mercuric chloride (0 , 1%) 1-2 minutes and also washed erased distilled water, then seeds are placed in test tubes on MS agar medium without the addition of growth stimulants to obtain etiolated seedlings in the dark for 3-4 weeks, shoots are separated from the obtained etiolated seedlings and placed in test tubes on MS agar medium by adding 1.8–2. 2 mg / l 2-4 D (2,4-dichlorophenoxyacetic acid) and 0.3–0.8 mg / l 6-BAP (6-benzylaminopurine) for callus, cultivation is carried out at a temperature of 26 ± 1 ^° C, humidity 70% in the dark.