SU1707073A1 - Train of cultured shikonin-producing cells of plant lithospermum crythrorhiron sieb et zucc - Google Patents

Abstract

The invention relates to biotechnology and can be used in the medical, food and perfume industry. The purpose of the invention is to obtain a red-rooted sparrowing callus strain producing shikonin. The callus strain of the korseinik of crznose root VK-39 was obtained as a result of the selection of calusses of root origin. Growth index of culture 15, the content of shikonin-5-6% of the dry mass of cells. 1 tabl,

Description

The invention relates to biotechnology and can be used in the medical, food and perfume industry.

Shikonin has a bactericidal, antitumor, early healing and anti-oxidative effect; abroad it is used in medicine, as well as in the perfumery and food industry. In Japan, the red-headed sparrow is grown on plantations, while up to 2% of shikonin accumulates in its roots for 5-7 years. Japan has now mastered the biotechnological production of shikonin.

one.

It is known that callus cultures of red-root sparrow plants, producing shikonin, have been obtained abroad. In these cultures, the content of shikonin varies between 1.7 and 2.5% of the dry weight of calluses 2.

Description of callus cultures, their characteristics are absent in the literature, also

There are no references to the deposition of shikonin-producing strains in the available collections.

The purpose of the invention is to obtain a cultured culture of the red-rooted sparrow's cells, producing shikonin.

Such a strain was obtained at the Biology and Soil Institute of the Far East Branch of the Academy of Sciences of the USSR and deposited with the All-Union Collection of Higher Plant Cells at the Institute of Plant Physiology of the USSR, USSR Academy of Sciences. KA Timir zev under the number VSKK (BP) N ° 36. The name of the strain VK-39.

The strain VK-39 is characterized by the following features.

Starting material: the root of a wild-growing plant is a red-root pigeon duster (Lithospermum crythrorhizon Sieb. Et Zucc.), Harvested in Partizansky district of Primorsky Krai in 1986. An intensely red root with a diameter of 20 was selected for in vitro planting.

(L

WITH

vj o vi

about VI

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mm The explants were isolated from root discs 2-3 mm thick. Cultural properties. The growing callus culture is a fabric of a loose red consistency. Growth index 15 for calluses with an initial weight of 150 kg, dry matter content of 5.8 - 6.0% of the raw biomass. Cultivation method - growing in accumulative mode in the absence of illumination at 25 ± 1 ° C, relative air humidity 70 ± 10% on a nutrient medium of the following composition, mg / l of water:

MN4Moz350-450 KMOz 1500-2300 KN2P04 150-190 CaClt 6H20 590-680 Md504-7N20 350-400 NzVOZ 4.2-8-8.0 MnS04 4H20 15.6-26.7 CoCl2 2H20 0.02-0.03 CuS04 5H20 0.07-0.08 ZnS04-7H20 6.0-10.3 Na2MoOv2H: 0 0.2-0.3 Kt 0.53 - 0.90 FeS047H20 25.0-30.5 Thiamine hydrochloride 0.15-0.25 Pyridoxine hydrochloride 0.4-0.6 Nicotinic acid 0.4-0.6 Meso-Inositol 80-120 Indolyl acetic acid 0.15-0.25 Kinetin 1.5 - 2.5 Sucrose 25 - 35 g Agar 5.8-6 , 2 g Dinaitric salt of EDTA is 33.6-41.0 and the pH of the media before autoclaving is 5.6 - 5.8.

The passage number is the 35th, passivation of calli is carried out with an interval of 25 days, the collection of calli to obtain shikonin from them - after 35 days of cultivation.

Cytological and karyological characteristics.

The fabric contains oval cells of medium size microns. The maximum mitotic activity was observed on the 6th day of cultivation. The distribution of cells according to the number of chromosomes is as follows:

Number of chromosomes 28 36 42 48 56

% cells 22 20 14 13 31

Ability to morphogenesis.

Spontaneous rhizoge is rarely observed. Characterization of shikonin biosynthesis. The content of shikonin in calluses increases in parallel with the growth of culture and reaches 5–6% of the dry biomass on the 35th day of cultivation. The achieved level of productivity is consistently maintained when the culture is passivated for 2 years. Shikonin accumulates only in cells and is not released into the medium.

PRI me R. In Erlenmeyer flasks with a capacity of 100 ml poured 30 ml of nutrient medium of the following composition, mg / l of water:

MN4MOZ400

CMOZ 1900

КН2Р04170

CaCl2- 6НгО655

fogS04-7H20370

NZOZ6.2

MnSCM 4H2022,3

CoCl2-2H200,025

CuSCM-5H200,075

ZnS04-7H208,6

Pa2Mo04-2N200.25

K 0.83

FeS04-7H2027.8

EDTA37.3 disodium salt

Thiamine Hydride Loride02

Pyridoxine hydrochloride 0.5

Nicotinic acid 0.5

meso-inositol100

Indu-Lipacetic Acid 0.2

Kinetin2

Sucrose200QO

Agar6000

pH 5.6-5.8. . .

The flasks with medium were autoclaved at 117 ° C for 20 min, then cooled. Calluses of the strain VK-39 were placed on the surface of the agar — three inoculums weighing 150 mg in each flask. Cultures were incubated in the dark at 25 ° С and relative air humidity of 70% for 35 days. Then the calli were removed from the flasks and weighed (data in the table). After drying the calli for 6 h under a stream of hot (50 ° C) air, the content of shikonin was determined in them. For this, the calluses from each flask were separately ground in a mortar. A precise sample (within 30 mg) was extracted with hexane in a ratio of 1: 100. An aliquot of the extract in an amount of 25 µl was diluted with hexane to 900 µm, then 100 µl of KOH solution was added. The optical density of the solutions was measured at a wavelength of 622 nm on a spectrophotometer, and the concentration of shikonin was determined according to a calibration graph. The content of shikonin in the samples was 5.5-5.8%.

The table presents the data of studies on the content of shikonin in calli of strain VK-39.

Claims (1)

The obtained VK-39 strain of the red-rooted sparrow's culture allows, when grown on the same nutrient medium, to obtain a high shikonin content simultaneously with good growth. The content of shiksnin in cells of the strain VK-39 is 2 to 3 times its content in the roots of an intact plant. Claims of Invention Strain of cultured plant cells Lithospermum erythrorhizcn Sieb. et zucc. - BCKK (BP) No. 36 - producing shikonin.