RU2458121C1 - METHOD FOR CULTIVATION OF TYLOSIS TISSUE Centaurea scabiosa l - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for cultivation of tylosis tissue Centaurea Scabiosa l. involves preparing a tylosis culture of intact plant explants by preparing the Murashige and Skoog nutrient medium to be dispensed for tylosis culture cultivation. The tylosis culture is prepared on the Murashige and Skoog medium with added 0.5-1 mg/l of α-naphthyl acetic acid and 0.2-0.5 mg/l of 6-benzylaminopurine; the Murashige and Skoog medium is despensed in plastic Petri dishes; the tylosis culture is transferred into said dishes that is followed by cultivation of the tylosis culture in the plastic Petri dish in blue light (380-560 nm).

EFFECT: invention provides higher growth index of the tylosis culture.

1 dwg, 1 tbl, 2 ex

Description

The invention relates to the field of biotechnology, in particular to the cultivation of cell cultures of medicinal plants. The scope is the medical industry, obtaining a new type of raw material of biologically active substances of plant origin.

A known method of growing callus culture Cistanche deserticola (article Jie Ouyang, Xiaodong Wang, Bing Zhao, Yuchun Wang. Light intensity and spectral quality influencing the callus growth of Cistanche deserticola and biosynthesis of phenylethanoid glycosides // Plant Science - 2003 - Vol. 165, pp. .657-661). Cistanche deserticola callus culture was obtained from intact plants grown from pre-sterilized seeds in Murashige-Skoog medium containing an additional 1 mg / l 2,4-D, 0.5 mg / l kinetin and 1 mg / l gibberellic acid. The resulting calli were transferred onto B5 medium in Erlenmeyer flasks and grown under conditions of illumination with selective light with an intensity of 24 μmol / m ² s. It was established that blue light stimulated the accumulation of biomass of callus culture and phenylethanoid glycosides in it compared with a culture grown in white light.

The disadvantage of this method is that it was used to cultivate only Cistanche deserticola, belonging to the family Orobanchaceae (Zarazikhovyh), while Centaurea scabiosa belongs to the family Asteraceae or Compositae (Astro or Compositae). For the cultivation of callus culture, B5 medium is used, which has an altered composition of the main macro and micro salts compared to the MS medium. Additionally, casein hydrolyzate is added to the medium, which complicates and increases the cost of preparing a nutrient medium, especially in large volumes.

A known method of in vitro culturing a cell culture of transgenic tobacco Nicotiana tabacum l. Containing the human interleukin-18 gene (RF patent No. 2354692, C12N 5/04, publ. 05/10/2009) The invention can be used in pharmacology for growing cell culture biomass transgenic tobacco Nicotiana tabacum L., containing the human interleukin-18 gene, as well as for research purposes. The seeds of transgenic plants Nicotiana tabacum L. are sterilized, transferred to a hormone-free culture medium and germinated in vitro in the light for 28 days. The obtained seedlings are cut into segments 0.2-0.3 cm ² in size, transferred to a modified nutrient medium, including 2,4-dichlorophenoxyacetic acid, indolylacetic acid and kinetin. For seed sterilization, a solution is used consisting of 96% ethanol, 37% hydrogen peroxide and water in a volume ratio of 7.5: 1: 1. The resulting callus containing the human interleukin-18 gene is cultured in the dark for 21-28 days and propagated by passivation to the required amount.

The disadvantage of this method is that the cultivation uses a cell culture of transgenic tobacco Nicotiana tabacum, which prevents the application of the data to cell cultures of plants of other genera and families. Also, the disadvantage is that a transgenic plant is used, the introduction of a foreign gene can adversely affect the functioning of the genome and subsequently lead to gene and chromosomal abnormalities, which will interfere with the stable cultivation of this culture. Obtaining transgenic plants for cultivation is associated with a large amount of preliminary work on the creation of a genetic construct and its introduction into the plant genome.

A known method of cultivating Artemisia annua L. (article Yuchun Wang, Haoxian Zhang, Bing Zhao, Xiaofan Yuan. Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions // Biotechnology Letters - 2001 - Vol.23, pp. 1971 -1973). In the present invention, a culture of genetically modified roots (hairy-root) is grown in a liquid nutrient medium Murasige-Skoog, with the addition of sucrose 30 g / l on an orbital shaker (120 rpm) at 25 ± 1 ° C in the light with an intensity of 25 μmol / m ² s and a photoperiod of 16 hours. To accelerate growth, the culture was illuminated with red, blue, green and yellow light, an intensity of 25 μmol / m ² s and a photoperiod of 16 hours. It was found that red light stimulates the growth of hairy-root culture and the accumulation of sesquiterpene artemisinin lactone, in contrast to other parts of the spectrum.

The disadvantages of this method is that this method of cultivation is applicable to a certain type of annual plant - Artemisia annua L., while the authors established the optimal light mode of cultivation for a perennial plant Centaurea scabiosa L. In addition, this method of cultivation was studied only on a hairy culture -root Artemisia annua L., little used in practical activities, its use for growing callus and suspension cultures, which form the basis of industrial plant biotechnology, has not been studied. To obtain a hairy-root culture, genetic modification of cells with a Ti plasmid by the bacterial culture of Agrobacterium rhizogenes is used, which can lead to disruptions in the genetic apparatus of plant cells, reduced viability, growth parameters and productivity of primary and secondary metabolism.

The objective of the present invention is to develop a method for cultivating callus tissue Centaurea scabiosa L., promising as a possible source of sesquiterpene lactones of medicinal action, in order to accelerate its growth and high yield of biomass, by applying a certain lighting regime.

The problem is solved in that the cultivation of callus tissue Centaurea scabiosa L. includes the production of callus culture from explants of intact plants by growing explants on nutrient medium Murashige-Skoog with further spilling of the medium and growing callus culture, but in contrast to the prototype callus culture is obtained on Murashige-Skoog medium with the addition of 0.5-1 mg / L NAA (α-naphthylacetic acid) and 0.2-0.5 mg / L BAP (6-benzylaminopurine). The resulting callus culture is then transplanted into plastic Petri dishes with Murashige-Skoog medium without changing its composition and further cultivated in blue light (380-560 nm).

The modified nutrient medium Murashige-Skoog contains components in the following quantitative ratio, mg / l:

Kno ₃ 1900 NH ₄ NO ₃ 1650 CaCl ₂ 332 MgSO ₄ 7H ₂ O 370 KH ₂ PO ₄ 170 FeSO ₄ 7H ₂ O 27.85 Na ₂ EDTA 37.25 H ₃ IN ₃ 6.2 MnSO ₄ 5H ₂ O 24.1 ZnSO ₄ 7H ₂ O 8.6 Na ₂ MoO ₄ 0.25 Ki 0.83 CuSO ₄ 5H ₂ O 0,025 CoCl ₂ 6H ₂ O 0,025 Pyridoxine 1,0 Thiamine chloride 1,0 Nicotinamide 2.0 NUK 1-0.5 6-BAP 0.5-0.2 Sucrose 15,000 Glucose 15,000 Agar 9000 Water Up to 1 liter

To prepare a nutrient medium, a macro-salt concentrate is prepared, with each of the macro-salts being sequentially dissolved in a small amount of water, and then the volume is brought up to 1 liter. Concentrates of micro salts, Fe-chelate, and vitamins are prepared in a similar way. Macro and micro salts, Fe-chelate, vitamins in the form of concentrates are mixed in a small amount of water. Then, 6-BAP (6-benzylaminopurine), NAA (α-naphthylacetic acid), sucrose are added to the resulting mixture and everything is thoroughly mixed. The solution was adjusted with distilled water to 1 liter. 1.35 g of agar is poured into 250 ml flasks, 150 ml medium is poured, covered with foil and sterilized in an autoclave for 20 minutes at 1.2-1.4 atm. Sterilized culture medium under aseptic conditions is poured into sterile plastic Petri dishes with a diameter of 90 mm, 15-20 ml per cup. The tissue is planted at the age of 25-30 days, inoculum 200-300 mg. The cultivation of callus tissue is carried out at 26 ± 1ºС under blue light conditions with an intensity of 160 μM m ² / with Philips TL-D 36W / 18 fluorescent lamps. The luminous flux is aligned with the incident quanta.

Examples of the invention are given below.

Example 1

To prepare the nutrient medium, a macro-salt concentrate is prepared (KNO ₃ 1900 mg / L, NH ₄ NO ₃ 1650 mg / L, MgSO ₄ 7H ₂ O 370 mg / L, KH ₂ PO ₄ 170 mg / L), with each of the macro salts dissolving sequentially in a small amount of water, and then the volume is brought up to 1 liter. Concentrates of microsalts are prepared in the same way (H ₃ BO ₃ 6.2 mg / L, MnSO ₄ 5H ₂ O 24.1 mg / L, ZnSO ₄ 7H ₂ O 8.6 mg / L, Na ₂ MoO ₄ 2H ₂ O 0.25 mg / L, KI 0.83 mg / L, CuSO ₄ 5H ₂ O 0.025 mg / L, CoCl ₂ 6H ₂ O 0.025 mg / L), Fe chelate (FeSO ₄ 7H ₂ O 27.85 mg / L, Na ₂ EDTA 37.25 mg / l), CaCl ₂ (CaCl ₂ 6H ₂ O 332 mg / l), vitamins (Pyridoxine 1.0 mg / l, Thiamine chloride 1.0 mg / l, Nicotinamide 1.0 mg / l ) Macro and micro salts, Fe-chelate, CaCl ₂ , vitamins in the form of concentrates are mixed in a small amount of water. Then, 6-BAP (0.5 mg / L), NAA (0.2 mg / L), sucrose (30,000 mg / L) are added to the resulting mixture and everything is thoroughly mixed. The solution was adjusted with distilled water to 1 liter. 1.35 g of agar is poured into 250 ml flasks, 150 ml of medium is poured, covered with foil and sterilized in an autoclave for 20 minutes at 1.2-1.4 atm. Then, in a laminar, the sterilized growth medium is poured into sterile plastic Petri dishes with a diameter of 90 mm, 15-20 ml per cup. The tissue is planted at the age of 25-30 days, inoculum 200-300 mg. The cultivation of callus tissue is carried out at 26 ± 1ºС under blue light conditions with an intensity of 160 μM m ² / with Philips TL-D 36W / 18 fluorescent lamps. The luminous flux is aligned with the incident quanta.

Example 2

To prepare the nutrient medium, a macro-salt concentrate is prepared (KNO ₃ - 1900 mg / l, NH ₄ NO ₃ - 1650 mg / l, MgSO ₄ 7H ₂ O - 370 mg / l, KH ₂ PO ₄ - 170 mg / l), while each of the macrosalts is dissolved sequentially in a small amount of water, and then the volume is brought up to 1 liter. Concentrates of microsalts are prepared in the same way (H ₃ BO ₃ - 6.2 mg / L, MnSO ₄ 5H ₂ O - 24.1 mg / L, ZnSO ₄ 7H ₂ O - 8.6 mg / L, Na ₂ MoO ₄ 2H ₂ O - 0.25 mg / l, KI - 0.83 mg / l, CuSO ₄ 5H ₂ O - 0.025 mg / l, CoCl ₂ 6H ₂ O - 0.025 mg / l), Fe-chelate (FeSO ₄ 7H ₂ O - 27.85 mg / l, Na ₂ EDTA - 37.25 mg / l), CaCl ₂ (CaCl ₂ 6H ₂ O - 332 mg / l), vitamins (Pyridoxine - 1.0 mg / l, Thiamine chloride - 1, 0 mg / l, Nicotinamide - 1.0 mg / l). Macro and micro salts, Fe-chelate, CaCl ₂ , vitamins in the form of concentrates are mixed in a small amount of water. Then, 6-BAP (1 mg / L), NAA (0.5 mg / L), sucrose (30,000 mg / L) are added to the resulting mixture and everything is thoroughly mixed. The solution was adjusted with distilled water to 1 liter. 1.35 g of agar is poured into 250 ml flasks, 150 ml of medium is poured, covered with foil and sterilized in an autoclave for 20 minutes at 1.2-1.4 atm. Then, in a laminar, the sterilized growth medium is poured into sterile plastic Petri dishes with a diameter of 90 mm, 15-20 ml per cup. The tissue is planted at the age of 25-30 days, inoculum 200-300 mg. The cultivation of callus tissue is carried out at 26 ± 1ºС under blue light conditions with an intensity of 160 μM m ² / with Philips TL-D 36W / 18 fluorescent lamps. The luminous flux is aligned with the incident quanta. Table 1 shows the growth index of the callus culture Centaurea scabiosa L. grown in the light of different spectral composition.

Table 1 The growth index of the callus culture Centaurea scabiosa L. grown in the light of different spectral composition Option Growth index Darkness 2.1 ± 0.2 White light 1.8 ± 0.3 Red light 2.6 ± 0.2 Blue light 4.0 ± 0.4 Green light 2.6 ± 0.6

Claims (1)

A method of cultivating callus tissue Centaurea Scabiosa l., Comprising obtaining a callus culture from explants of intact plants by preparing a culture medium of Murashige-Skoog with further spilling of the medium and growing a callus culture, characterized in that the callus culture is obtained on Murashige-Skoog medium with the addition of 0.5 -1 mg / l NAA (α-naphthylacetic acid) and 0.2-0.5 mg / l BAP (6-benzylaminopurin), pouring Murashige-Skoog medium in plastic Petri dishes, transplanting callus culture into these cups and further growing callusculture in a plastic Petri dish in blue light (380-560 nm).

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