RU2605912C1 - METHOD OF PRODUCING CULTURE OF ISOLATED ROOT Silene linicola K1601-PRODUCER OF ECDYSTEROIDS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used in pharmaceutical and food industry. Method comprises bacterial transformation of explant root of juvenile plant Silene linicola by agro bacterial strain R-1601 A. Rhizogenes. Transformed roots of explant is separated and cultured. Isolated roots cultured first two passages on Gamborg medium with addition of 250 mg/l cefotaxime, subsequent passages are grown on a Gamborg medium with addition of 500 mg/l casein hydrolysate.

EFFECT: invention allows to ensure permanent high rate of growth of isolated roots of Silene linicola K1601 on non hormonal culture media at high level of biosynthesis of species-specific ecdysteroids.

1 cl, 3 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of biotechnology and genetic engineering and can be used in the pharmaceutical and food industries.

Intensive studies on the production of secondary metabolites from medicinal plant materials and the search for plants producing these substances are carried out due to a wide range of their biological activity, in particular the anabolic, adaptogenic, immunomodulating effects of ecdysteroids [1, 2].

The Caryophyllaceae family is one of the richest ecdysteroid-containing in the world flora and is characterized by a diverse composition of ecdysteroids, the presence of many new compounds discovered so far only in them, and a high content of major components of the ecdysteroid sum. At present, ecdysteroids have been found in more than 150 species of the genus Silene L., 12 in Lychnis L. and a number of species in other genera [1, 3]. An alternative to the known approaches to obtaining raw materials and extracts of medicinal plants is the culture of cells, tissues and organs of plants.

Known strains of cultured cells and isolated roots of ecdysteroid-containing plants are Ajuga reptans L. [4, 5], Ajuga turkestanica () [6], Serratula tinctoria L. [7], Serratula coronata L. [8]. However, in the literature there is no data on obtaining a culture of isolated roots of representatives of this. Caryophyllaceae as sources of ecdysteroids.

Closest to the claimed is a method of obtaining a cell culture of ecdysteroid-containing species of the genus Silene [9], in accordance with which the possibility of obtaining a stably growing culture of calli of representatives of the genus Silene from various types of explants was shown. For this, various types of explants are cultured on Murashige and Skoog medium (MS) with the addition of hormones (growth regulators) - benzylaminopurine 1 μM, 2,4-dichlorophenoxyacetic acid 5 μM and casein hydrolyzate 1 g / L. With this cultivation method, the growth indices of callus Silene linicola of root origin were 4.1 ± 0.6 for raw biomass and 4.0 ± 1.3 for dry.

The disadvantages of this method are the slow growth of biomass, the need to use expensive growth regulators for stable in vitro culture growth, a low level of ecdysteroids in the final product.

The objective of the invention is to obtain a genetically and biochemically stable culture of isolated roots of Silene linicola, characterized by a constant high growth rate on hormone-free nutrient media and a high level of biosynthesis of ecdysteroids characteristic of this plant species.

The problem is solved by creating a new culture of isolated roots of Silene linicola, designated Silene linicola K1601, and optimizing the conditions for their cultivation. A method of obtaining a culture-producer of ecdysteroids includes cultivating an explant of the root of a juvenile plant with the addition of growth regulators, separating the transformed roots from the explant and their subsequent cultivation on a nutrient medium. The difference from the prototype is that they carry out the agrobacterial gene transfer of Agrobacterium rhizogene, plasmid pRi, strain R-1601, to the roots of Silene linicola (hairy root syndrome). After transformation, the bacterial medium is eliminated, and the sterile isolated roots of Silene linicola K1601 are cultured on a liquid non-hormonal Hamburg medium, the first two passages are cultivated with 250 mg / l cefotaxime added to the medium, and all subsequent passages are cultured with 500 mg / l casein hydrolyzate.

The possibility of creating a culture of isolated roots of Silene linicola and biosynthesis of ecdysteroids in it has been experimentally shown, and the created culture of Silene linicola K1601 is an alternative source of ecdysteroids. The enhanced synthesis of ecdysteroids contributes to the optimally selected composition of the nutrient medium.

In FIG. 1 shows a photograph of a culture of isolated roots of Silene linicola K1601.

In FIG. 2 shows HPLC of an ethanol extract of Silene linicola K1601

In FIG. Figure 3 shows the UV spectra of Silene linicola K1601 ecdysteroids.

The culture of Silene linicola K1601 was obtained from the species Silene linicola CCGmelin (family Caryophyllaceae), the seeds of which were obtained from the botanical garden of Göttingen (Germany) and introduced in the Siberian Botanical Garden of Tomsk State University. The origin of the Silene linicola K1601 culture is agrobacterial pRi gene transfer of Agrobacterium rhizogenes, strain R-1601, to the roots of Silene linicola juvenile plants. The characteristic of the resulting culture is thin, intensively branching roots, the growth index for raw biomass is 14.7 + 0.5, and for dry biomass 13.5 + 0.4.

The method according to the invention is as follows.

At the first stage, bacterial transformation of explants of the root of the Silene linicola plant is carried out. To obtain an aseptic culture, Silene linicola seeds were sterilized in a 20% Domestos solution, followed by three times washing with sterile distilled water and placed on a 0.6% germination agar. Seed cultivation conditions: photoperiod light / dark 16/8, T = 22 ± 2 ° C.

The transformation was carried out according to the following scheme: sterile seedlings were divided into a leaf, hypocotyl, root, pricked with an insulin syringe needle and inoculated for 24 hours in a liquid nutrient medium Murashige and Skoog, containing a suspension of agrobacteria A. rhizogenes, strain R-1601, day old. After exposure, the inoculum was washed with sterile Murashige and Skoog medium with a halved content of macro- and micronutrients (½ MS). For the manifestation of transformation, they were placed on an agar medium with the addition of 500 mg / l cefotaxime. After 3 weeks, after the appearance of the rosette, they were separated, transplanted onto the same nutrient medium with cefotaxime (500 mg / L), then the tips of the roots were transplanted twice on an agarized nutrient medium ½ MS with 250 mg / L antibiotic to eliminate bacteria. Subsequently, the liquid nutrient medium of Hamburg was used. The composition of the Hamburg medium is optimally balanced for macro-, micro-salts and vitamins for the biosynthesis of ecdysteroids in Silene linicola K1601. For example, the content of macrosalts, microsalt, and sucrose is 1.5–10 times lower in the Gamborg medium than in MS. The concentration of vitamins in the environment of Hamburg is 2-10 times higher than in MS.

Example 1. Isolated roots were transferred to a liquid Gamborg culture medium with the addition of 250 mg / l cefotaxime and cultivated for 2 passages (the period between passages was 30 days) to achieve complete aseptic culture. After that, the culture of isolated roots was transferred to the Hamburg medium with the addition of 500 mg / l casein hydrolyzate, the period between subsequent passages was 45 days. For transplantation, approximately 200 mg of raw root biomass was taken and placed in 250 ml conical flasks in 100 ml of medium. The pH was adjusted to 5.8 before autoclaving. Cultivation conditions: darkness, temperature 24 ± 2 ° С, orbital shaker 90 rpm. Preservation - on an agarized nutrient medium of Hamburg at a temperature of +17 ° C in low light. The result is positive (Fig. 1 - 3).

Example 2. Similar to example 1, but the first and second passages on the liquid medium of Hamburg were performed without the addition of cefotaxime at a dosage of 250 mg / l. The result is negative. In the absence of an antibiotic in the first passages on liquid media, the appearance of a bacterial infection with a frequency of 75% was observed.

 Example 3. Analogously to example 1, but the cultivation of isolated roots was carried out in a liquid nutrient medium of Hamburg without the addition of casein gyrolyzate. The result is negative. In this nutrient medium, the growth rates of raw and dry biomass sharply decreased. The results are presented in table 1.

Thus, as a result of testing the method according to the invention (example 1), it was shown that the culture of isolated roots of Silene linicola K1601 grows stably, forms a mass of thin tightly bound roots (Fig. 1). The culture is maintained by transplanting to fresh culture media every 45 days. According to the HPLC analysis, during the cultivation process under the stated conditions, an active synthesis of many ecdysteroids occurs (see table 2). The HPLC profile of the ethanol extract of isolated roots of Silene linicola K1601 is very similar to the profile of the corresponding extract of intact plants by the retention time of the detected substances (Fig. 2), which is also confirmed by the characteristic UV absorption spectra (Fig. 3). The extracts identified characteristic ecdysteroids for species of the genus Silene: 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, polypodine B. The content of 20-hydroxyecdysone in Silene linicola K1601 samples is 0.18%. Identification of other ecdysteroids requires additional research.

EFFECT: provision of a constant high growth rate of isolated roots of Silene linicola K1601 on hormone-free nutrient media with a high level of biosynthesis of species-specific ecdysteroids.

Information sources

1. Zibareva L.N. Phytoecdysteroids of plants of the family Caryophyllaceae. Abstract. diss. doc Chem. Sciences, Novosibirsk, 2003.247 s.

2. Volodina S.O. Ecdysteroid-containing plants: resources and biotechnological use. Diss. Cand. biologist. Sciences, Syktyvkar, 2006.166 p.

3. Distribution of phytoecdysteroids in the Caryophyllaceae / L. Zibareva, V. Volodin, Z. Saatov, T. Savchenko, P. Whiting, R. Lafont, L. Dinan // Phytochemistry. - 2004. - V. 64. - No. 2 - P. 499 - 517.

4. Filipova V.N., Volodina S.O., Smolenskaya I.N., Zorinyants S.E., Anufriev E.N., Nosov A.M., Volodin V.V. (2007). The strain of cultured plant cells Ajuga reptans. RF patent No. 2296154 dated 03/27/2007.

5. Matsumoto T., Tanaka N. Production of ecdysteroid by hairy root cultures of Ajuga reptans var autropurpurea // Agric. Biol. Chem. - 1991- V. 55. - P. 1019-1025.

6. Cheng D.M., Yousef G.G., Grace M.H., Rogers R. B., Gorelick-Feldman J., Raskin I., Lila M.A. In vitro production of metabolism-enchancing phytoecdysteroids from Ajuga turkestanica // Plant Cell Tiss. Organ. Cult. - 2008. - V. 93. - P. 73-83.

7. Delbeque J., Beydon P., Chapuis L. In vitro incorporation of radio labelled cholesterol and mevalonic acid into ecdysteron by hairy root cultures of a plant Serratula tinctoria. // Eur J Entomol. - 1995 .-- 92: 301-307.

8. Filipova V.N., Volodina S.O., Smolenskaya I.N., Zorinyants S.E., Kovler L.A., Anufriev E.N., Nosov A.M., Volodin V.V. The strain of cultured plant cells Serratula coronata L. RF patent No. 2296155 from 03/27/2007.

9. Erst A.A., Zibareva L.N. Features of callus formation of ecdysteroid-containing species of the genus Silene L. (Caryophyllaceae Juss.) In an in vitro culture // Siberian Journal of Ecology. - 2013. - No. 4. - S. 603-608.

The method of obtaining culture

isolated roots of Silene linicola

Table 1. Comparative assessment of the growth of raw and dry biomass of the isolated roots of Silene linicola K1601 with different composition of the nutrient medium

Table 2. Identification of ecdysteroids in ethanol extracts of Silene linicola K1601

Примечание: НИДЭ - неидентифицированный экдистероид.

Note: NIDE is an unidentified ecdysteroid.

Claims (1)

A method of obtaining a culture of isolated roots Silene linicola K1601 - producer of ecdysteroids, including bacterial transformation of the explant of the root of the juvenile plant Silene linicola, separation of the transformed roots from the explant and their subsequent cultivation, characterized in that the bacterial transformation is carried out by an agrobacterial strain R-1601 A. rhizogenes, and cultivation isolated roots of Silene linicola K1601 carried out on a liquid hormone-free nutrient medium of Hamburg, and the first two passages are cultivated with the addition of p the final medium was 250 mg / l cefotaxime, and all subsequent passages were cultivated with 500 mg / l casein hydrolyzate added to the nutrient medium.

Images