Effect of Synthetic Auxin Analogs (2.4-D and α-NAA) on Growth and Biosynthetic Characteristics of Suspension Cell Culture of Tribulus terrestris L.

Abstract

Effect of synthetic analogs of auxins—2.4-dichlorophenoxyacetic (2.4-D) and α-naphthylacetic (α-NAA) acids—on growth characteristics and accumulation of steroidal glycosides was investigated in suspension cell culture of Tribulus terrestris L. It was found that the substitution of α-NAA for 2.4-D in the nutrient medium brought about a rise in the content of steroidal glycosides in the cultured cells (up to six times) and broadened their structural diversity (nine identified oligofurostanosides when growing culture on the medium with α-NAA vs. five compounds on the medium with 2.4-D). Positive influence of α-NAA exerted on biosynthetic characteristics of T. terrestris suspension cell culture was accompanied by changes in cell morphology (cytodifferentiation), the extent of their aggregation, and gradual decrease (during 3–4 cycles of growing) in culture viability up to its extinction. Simultaneous presence of both synthetic analogs of auxins (α-NAA and 2.4-D) in the growing medium also caused a rise in the content of steroidal glycosides in the cells of T. terrestris, although it was less pronounced (up to three times); however, the culture showed a relatively steady growth and great viability in this case. It was concluded that these two auxin analogs differently influenced the growth of plants’ cells in vitro and biosynthesis of substances of specialized metabolism, 2.4-D promoted cell proliferation, whereas α-NAA induced cytodifferentiation and activated the production of secondary compounds. This conclusion is corroborated by the data concerning plant cell cultures of other species of medicinal plants accumulating secondary metabolites from other groups. For instance, in the experiments with suspension cell culture of Panax ginseng C.A. Mey., substitution in the growth medium of α-NAA for 2.4-D brought about a rise in the content and diversity of triterpene glycosides (ginsenosides) associated with an elevated cell aggregation and deterioration of culture’s viability. Comparison of production of steroidal glycosides in plant cell cultures of T. terrestris and Dioscorea deltoidea Wall. that is grown for more than 40 years has shown that both cultures accumulated only furostanol (rather than spirostanol) glycosides that promoted cell proliferation. In suspension cell culture of Dioscorea, a high level of oligofurostanosides (up to 12% of dry biomass) was accumulated when growing culture on the medium with 2.4-D. Thus, investigation into the production of furostanol glycosides in plant cultured cells points to a multifactor system of regulation of secondary metabolism in vitro. Different synthetic auxin analogs may exert alternative influences on growth and biosynthetic processes. At the same time, prolonged culturing leads to an autoselection of cells with the properties promoting proliferation, specifically, with a high content of furostanol glycosides.