Morphogenesis of cell lines of plants Lysimachia nummularia L. at in vitro culture promising for the content of biologically active substances

Abstract

Lysimachia nummularia L. is a plant belonging to the Primulaceae family, which is particularly valuable as a medicinal raw material used in folk medicine in many countries. It has excellent antibacterial and antioxidant capacity of metabolites. That is why the microclonal propagation of Lysimachia nummularia L. is a relevant issue. The purpose of this paper is to develop approaches to microclonal propagation of L. nummularia. For microclonal reproduction of L. nummularia, the method of activation of isolated tissues and organs already present in the plant meristem and induction of direct regeneration directly by explant tissues was used. To select highly productive cell lines of representatives of the genus Lysimachia, callus culture was obtained by indirect morphogenesis from stem and leaf explants. It was found that the formation of tissues and organs of L. nummularia into in vitro culture depended on the composition of the nutrient medium and the quantitative and qualitative ratio of growth regulators in it. Active proliferation of L. nummularia microshoots into in vitro culture was noted on the variants of Murashige and Skoog, and Driver and Kuniyuki nutrient medium with 6-benzylaminopurine 4.0 mg∙l -1, indolyl butyric acid 0.03 mg∙l -1, gibberellic acid 0.1 mg∙l -1. It was established that for microclonal reproduction, induction, and proliferation of the root system and obtaining regenerating plants of L. nummularia, the most effective is the use of nutrient media according to Murashige and Skoog with the addition of thidiazuron 0.5 mg∙l -1 and 0.25 mg∙l -1 kinetin. The optimal conditions for the induction of callusogenesis and obtaining the culture of cells and callus tissues of L. nummularia and its passage in vitro were selected. It has been shown that the modified nutrient medium of Murashige and Skoog, with 2.4-dichlorophenacetic acid 1.5 mg∙l -1 and indole-3-acetic acid 0.2 mg∙l -1, is optimal for the accumulation of callus tissue biomass of L. nummularia, which ensured the frequency of callusogenesis for the first and second passages up to 98.0 ± 0.2%. 5 cell lines that actively synthesize stilbenoids and the highly productive LN-EE 02/19 cell line, which is capable of synthesizing and accumulating in callus tissues up to 10-12 mg∙g-1 of myricetrin, were selectively isolated. As a result of the analysis, the callus culture cell line LN-EE 02/19 was obtained, which allows obtaining myricetrin in amounts up to 10.0-12.0 mg∙l -1 of raw biomass. The developed protocol can be used both for L. nummularia plants and other representatives of the Primulaceae family