RU2718253C1 - Method for making callus culture of dracocephalum palmatum steph. in vitro conditions - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents a method for obtaining a callus culture of cells of a wild-growing plant of Dracocephalum palmatum Steph. in vitro, involving sterilization of the Dracocephalum palmatum Steph. seeds with solution of 3 % hydrogen peroxide for 5 minutes, 80 % ethyl alcohol for 1 minute, three-time rinsing in sterile distilled water, placing sterile seeds on a solid nutrient medium without hormones of the following composition, mg/l: NHNO33000, KNO38000, CaCl×2HO – 8800, MgSO×7HO – 7400, KHPO– 3400, KI – 166, HBO– 1240, MnSO×4HO – 4460, ZnSO×7HO – 1720, NaMoO×2HO – 50, CuSO×5HO – 5, CoCl×6HO – 5, FeSO×7HO – 5560, Na-EDTA – 7460, mesoinositol – 100, nicotinic acid – 100, pyridoxine – 100, thiamine – 100, saccharose – 2500, water – 1000 ml, agar – 7000. Further, leaf explants obtained from seedlings are placed in a nutrient medium of the following composition, mg/l: NHNO– 33000, KNO– 38000, CaCl×2HO – 8800, MgSO×7HO – 7400, KHPO– 3400, KI – 166, HBO– 1240, MnSO×4HO – 4460, ZnSO×7HO – 1720, NaMoO×2HO – 50, CuSO×5HO – 5, CoCl×6HO – 5, FeSO×7HO – 5560, Na-EDTA – 7460, mesoinositol – 100, nicotinic acid – 100, pyridoxine – 100, thiamine – 100, saccharose – 3000, casein hydrolyzate – 500, kinetin – 1, 2,4-dichlorophenoxy acetic acid – 1, water – 1000 ml, agar – 12000. Cultivation of plants is carried out in the dark, at temperature of 26±1 °C, humidity of room is 70±5 %, the subcultivation cycle is 4 weeks.EFFECT: invention makes it possible to produce callus culture of Dracocephalum palmatum Steph_ under in vitro conditions, which can be used as a source of flavonoids of therapeutic action.1 cl, 3 tbl

Description

The invention relates to the field of plant biotechnology and can be used in the pharmaceutical industry for the production of raw materials containing valuable biologically active compounds (flavonoids, phenolic compounds), as well as the introduction of other representatives of medicinal plants into the culture in vitro, which are difficult to obtain due to the high content in its composition volatile ether compounds.

Dahcoiformes serpent head Dracocephalum palmatum Steph.– a plant of the Lamiaceae family, a perennial evergreen plant with creeping shoots, forms a very dense turf. Leaves and shoots are downy. Peduncles rise 10-15 cm above the turf. The flowers are purple in whorls. Seeds ripen in July. Winters with green leaves. It is found in the north-eastern regions of Yakutia. Grows on dry stony-gravelly slopes, rocks, in stony tundra, racing steppes (see Danilova N.S., Borisova S.Z., Ivanova N.S. Ornamental plants in Yakutia: Atlas-determinant. - M .: ZAO “ Fiton + ", 2012. - 248 s).

It is known that, on the basis of chemical and spectral studies, 23 compounds (phenylpropanoids, coumarins, flavonoids and triterpenes) were isolated from parts of the dahnea head, among which for the first time in the genus Dracocephalum eight compounds were discovered: salvianolic acid B, caftaric acid, cichoric acid, umberbeli esculletin, apigenin-7-O-β-D-glucuronopyranoside, isorohoifolin and luteolin-4'-O-β-D-glucopyranoside (see Olennikov DN, Chirikova NK, Okhlopkova ZM, Zulfugarov IS. Chemical composition and antioxidant activity of Tánara Ótó (Dracocephalum palmatum Stephan), a medicinal plant used by the North-Yakutian nomads. Molecules. 2013 Nov 14; 18 (11): 14105-21.).

The dune-shaped snakehead is used in Tibetan medicine, Yakut folk medicine, the infusion of the aerial parts and flowers is used for peptic ulcer, and in Central Asia - for heart failure, asthenia, stomach diseases and as a diaphoretic.

There is a method of obtaining a callus culture of a representative of the family Lamiaceae of the Scutellaria andrachnoides (Scutellaria andrachnoides) (see EA No. 020503, class C12N 5/04, A01H 4/00, publ. 11/28/2014), which includes obtaining a culture strain of the root of the plant of Scutellaria arachis (Scutellaria andrachnoides), intensively growing in vitro on hormone-free nutrient media, retaining the ability to synthesize phenolic compounds characteristic of the roots of the whole plant, and possessing genetic and biochemical stability.

The authors in open sources, technical solutions for obtaining callus culture of cells of the serpentine duniformis have not been identified.

The objective of the present invention is to obtain a callus culture of cells of the serpentine snakehead (Dracocephalum palmatum Steph.) In vitro.

To solve this problem, a method for producing a callus culture of wild-growing plantarhead dahnea plant cells (Dracocephalum palmatum Steph.) In vitro involves sterilizing the anthropoid seeds with a solution of hydrogen peroxide (3% solution) for 5 minutes, ethanol (80% solution) for 1 min, rinsing three times in sterile distilled water, placing sterile seeds on a solid nutrient medium without hormones of the following composition, in mg / l: NH ₄ NO ₃ - 33000, KNO ₃ - 38000, CaCl ₂ × 2H ₂ O - 8800, MgSO ₄ × 7H ₂ O - 7400, KH ₂ PO ₄ - 3400, KI - 166, H ₃ BO ₃ - 1240, MnSO ₄ × 4H ₂ O - 4460, ZnSO ₄ × 7H ₂ O - 1720, Na ₂ MoO ₄ × 2H ₂ O - 50, CuSO ₄ × 5H ₂ O - 5, CoCl ₂ × 6H ₂ O - 5, FeSO ₄ × 7H ₂ O - 5560, Na-EDTA - 7460, mesoinositol - 100, nicotinic acid - 100, pyridoxine - 100, thiamine - 100, sucrose - 2500, water - 1000 ml, agar - 7000. Next, leaf explants obtained from seedlings are placed in a nutrient medium of the following composition, in mg / l: NH ₄ NO ₃ - 33000, KNO ₃ - 38000, CaCl ₂ × 2H ₂ O - 8800, MgSO ₄ × 7H ₂ O - 7400, KH ₂ PO ₄ - 3400, KI - 166, Н ₃ VO ₃ - 1240, MnSO ₄ × 4H ₂ O - 4460, ZnSO ₄ × 7H ₂ O - 1720, Na ₂ MoO ₄ × 2H ₂ O - 50, CuSO ₄ × 5H ₂ O - 5, CoCl ₂ × 6H ₂ O - 5, FeSO ₄ × 7H ₂ O - 5560, Na-EDTA - 7460, mezoinozit - 100, nicotinic acid - 100, pyridoxine - 100 thiamine - 100 sucrose - 3000, Kasei hydrolyzate a - 500, kinetin - 1, 2,4-Dichlorophenoxyacetic acid - 1, water - 1000 ml, agar - 12000. In this case, the plants are cultivated in the dark, at a temperature of 26 ± 1 ° C, room humidity 70 ± 5%, cycle subculture is 4 weeks.

An analysis of the features of the claimed solution demonstrates the conformity of the claimed solution to the “novelty” criterion, the callus culture of the serpentine duniform (Dracocephalum palmatum Steph.) Was first obtained in vitro.

The set of essential features provides a solution to the stated technical problem, namely, the preparation of a callus culture of a dahl-like dragonhead (Dracocephalum palmatum Steph.).

The proposed solution is based on sterile fresh seedlings cultivated under controlled conditions of a climatic chamber from seeds of an intact plant, the phytomass of which was collected during stationary route work in the Oymyakon Highlands in the Republic of Sakha (Yakutia), known as the “Pole of Cold” - Oymyakon ", in phenophases" the end of flowering, the beginning of fruiting. "

In this case, the selected seeds were sterilized with a solution of 3% hydrogen peroxide for 5 minutes, after which, again, with 80% alcohol for 1 minute. After sterilization, the material was rinsed three times with sterile distilled water. Sterilized seeds were placed on a solid hormone-free culture medium. The seedlings were cultured up to the third to fifth true leaves for three weeks.

Nutrient medium Murashige-Skoog for the cultivation of fresh plant seedlings contains components in the quantitative ratio shown in table 1.

Leaf explants were obtained from the obtained seedling, which were placed in a nutrient medium with the addition of casein hydrolyzate, kinetin, 2,4-dichlorophenoxyacetic acid. It was found that the most intense callus formation occurs when the concentration of phytohormones kinetin 1 mg / l, 2,4-dichlorophenoxyacetic acid 1 mg / l.

After the formation of callus tissue, it was separated and transplanted into culture vessels (Petri dishes, 100 and 250 ml conical flasks) with fresh nutrient medium of the same composition.

Nutrient medium Murashige-Skoog for receiving callus tissue contains components in the quantitative ratio shown in table 2.

Cultivation was carried out in the dark, at 26 ± 1 ° C, room humidity 70 ± 5%, in Petri dishes with a diameter of 90 mm. The subculture cycle was 4 weeks. When reseeding, ¼ callus culture was used.

The transplant was performed every 30 days, thus preserving the obtained callus culture. During the cultivation, morphological and cytological characteristics were determined, and also selected for molecular studies and chemical analyzes.

Description of the method for producing callus culture of Dracocephalum palmatum Steph. currently not registered anywhere.

Received callus culture is characterized by the following features.

Cultural signs: callus cultures are loose and granular, have a yellowish-gray color.

The growth index (the rate of biomass growth per subculture) is determined by the wet and dry weight of the biomass. To determine the weight of the raw biomass, callus cultures are separated from the culture medium on paper filters and weighed. Dry callus biomass is obtained after freeze drying.

In the analysis of the growth curves presented, it should be noted that growth is slowed down on the 20th day of cultivation, which allows the use of a 4-week growth cycle. Growth parameters (except for the growth index) were calculated by the wet weight of biomass. The results are presented in table 3 and indicate the presence of stable growth characteristics.

Thus, a method for producing callus cultures of a dahl-like snakehead (Dracocephalum palmatum Steph.) From explants of seedlings obtained in laboratory conditions was revealed.

Table 1

The composition of the nutrient medium Murashige-Skoog for the cultivation of seedlings to obtain sterile explants, in mg / l

Components Content NH ₄ NO ₃ 33000 Kno ₃ 38000 CaCl ₂ x2H ₂ O 8800 MgSO ₄ x7H ₂ O 7400 KH ₂ PO ₄ 3400 Ki 166 H ₃ BO ₃ 1240 MnSO ₄ x4H ₂ O 4460 ZnSO ₄ x7H ₂ O 1720 Na ₂ MoO ₄ x2H ₂ O fifty CuSO ₄ x5H ₂ O 5 CoCl ₂ x6H ₂ O 5 FeSO ₄ x7H ₂ O 5560 Na-EDTA 7460 Mesoinositis one hundred Thiamine one hundred Pyridoxine one hundred A nicotinic acid one hundred Sucrose 2500 Water 1000 ml Agar 7000

table 2

The composition of the nutrient medium Murashige-Skoog for receiving callus cell culture

Components Content, in mg / l NH ₄ NO ₃ 33000 Kno ₃ 38000 CaCl ₂ x2H ₂ O 8800 MgSO ₄ x7H ₂ O 7400 KH ₂ PO ₄ 3400 Ki 166 H ₃ BO ₃ 1240 MnSO ₄ x4H ₂ O 4460 ZnSO ₄ x7H ₂ O 1720 Na ₂ MoO ₄ x2H ₂ O fifty CuSO ₄ x5H ₂ O 5 CoCl ₂ x6H ₂ O 5 FeSO ₄ x7H ₂ O 5560 Na-EDTA 7460 Mesoinositis one hundred Casein Hydrolyzate 500 Thiamine one hundred Pyridoxine one hundred A nicotinic acid one hundred Sucrose 30000 Kinetin one 2,4-D one Water 1000 ml Agar 12000

Table 3

Growth parameters of callus cultures of the serpentine duniform

Indicators Values, g Growth Indices by: Weight of Raw Biomass 0.0867 Specific growth by weight of crude biomass, day -1 0.0043

Claims (5)

A method of obtaining a callus culture of a dahl-like snakehead (Dracocephalum palmatum Steph.) In vitro, including sterilization of the snakehead seeds with hydrogen peroxide for 10 min and ethyl alcohol for 1 min, rinsing, washing three times for 5 min in sterile distilled water, sterile room seeds on a solid nutrient medium without hormones, of the following composition: NH ₄ NO ₃ 33000 mg Kno ₃ 38000 mg CaCl ₂ x2H ₂ O 8800 mg MgSO ₄ x7H ₂ O 7400 mg KH ₂ PO ₄ 3400 mg Ki 166 mg H ₃ BO ₃ 1240 mg MnSO ₄ x4H ₂ O 4460 mg ZnSO ₄ x7H ₂ O 1720 mg Na ₂ MoO ₄ x2H ₂ O 50 mg CuSO ₄ x5H ₂ O 5 mg CoCl ₂ x6H ₂ O 5 mg FeSO ₄ x7H ₂ O 5560 mg Na-EDTA 7460 mg Mesoinositis 100 mg Thiamine 100 mg Pyridoxine 100 mg A nicotinic acid 100 mg Sucrose 2500 mg Water 1000 ml Agar 7000 mg further placement of plant explants obtained in vitro in a nutrient medium of the following composition: NH ₄ NO ₃ 33000 mg Kno ₃ 38000 mg CaCl ₂ x2H ₂ O 8800 mg MgSO ₄ x7H ₂ O 7400 mg KH ₂ PO ₄ 3400 mg Ki 166 mg H ₃ BO ₃ 1240 mg MnSO ₄ x4H ₂ O 4460 mg ZnSO ₄ x7H ₂ O 1720 mg Na ₂ MoO ₄ x2H ₂ O 50 mg CuSO ₄ x5H ₂ O 5 mg CoCl ₂ x6H ₂ O 5 mg FeSO ₄ x7H ₂ O 5560 mg Na-EDTA 7460 mg Mesoinositis 100 mg Casein Hydrolyzate 500 mg Thiamine 100 mg Pyridoxine 100 mg A nicotinic acid 100 mg Sucrose 30000 mg Kinetin 1 mg 2.4 D 1 mg Water 1000 ml Agar 12000 mg the plants are cultivated in the dark, at a temperature of 26 ± 1 ° С, room humidity 70 ± 5%, the subculture cycle is 4 weeks, when replanting, ¼ callus biomass is used, the obtained callus cultures are preserved by transplantation every 30 days.