Культура генетически трансформированных корней (hairy roots) Silene roemeri Friv. – источник получения фитоэкдистероидов

Abstract

The hairy roots culture attracts increasing attention of researchers as the system of obtaining valuable secondary metabolites. Investigation of the features of biosynthesis of ecdysteroids by this biotechnological system and evaluation of the parameters of its productivity are important problems in the studies of the directed biosynthesis of secondary metabolites of plants and selection of the lines that are superproducers of biologically active substances. The aim of the research was to obtain hairy roots Silene roemeri culture and study the content of phytoecdysteroids in this culture. We obtained hairy roots culture of ecdysteroid-containing species S. roemeri by means of Agrobacterium-mediated transformation (strain A4 RT). The transformation was carried out according to the following scheme: sterile seedlings were divided into cotyledons, hypocotyls, and roots, pricked with a needle of an insulin syringe and inoculated for 24 hours in the liquid nutrient medium MS containing a suspension of a day-old A. rhizogenes agrobacterium. After exposure, the inoculates were washed with the sterile V MS medium and placed, for the development of the transformation, in the agar-containing V MS medium with antibiotic added (500 mg l -1 cefotaxime). Two weeks later, after the appearance of root rosettes, they were separated and relocated in the same nutrient medium with cefotaxime. We cultivated hairy roots in Gamborg's В 5 medium with an addition of 500 mg l -1 casein hydrolyzate. The growth index was 8.3-9.6. We carried out biochemical analysis of thus obtained culture for the concentrations of ecdysteroids by means of high-performance liquid chromatography. We discovered a number of nonpolar and medium-polar ecdysteroids. We established that cultivation is accompanied by the biosynthesis of over 20 ecdysteroids, including integristerone A, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. Estimation of the levels of the major ecdysteroid 20-hydroxyecdysone showed that its content was 0.1%. It allows considering hairy roots S. roemeri as a culture with a high biosynthetic activity. Our results show that the use of the hairy roots cultures of S. roemeri are a potential source of phytoecdysteroids and a promising system to study biosynthesis routes, localization of phytoecdysteroids, and methods to improve their products.