Effect of nutrient media enhanced with plant growth regulators on genetic stability in sub-cultures of Digitalis purpurea callus

Abstract

This study investigates the effects of nutrient media on the genetic stability of cell lines of callus produced by Digitalis purpurea leaves explants. Four cell lines of D. purpurea callus were selected after inducing 80 sub-cultures of callus in four different nutrient media: Vollosovich (5C01) medium, Murashige and Skoog (MS), Linsmaier and Skoog (LS), and Gamborg (B5) were compared with control samples of seeds and in vitro grown plant (micropropagation). DNA was isolated from the callus by cetyltrimethylammonium bromide, and molecular biomarkers were quantified by inter-simple sequence repeat (ISSR)-polymerase chain reaction (PCR). The purity of the extracted DNA from cell lines and the in vitro has grown plant ranged from 1.94 to 1.98 compared to seeds (1.74) at wavelength 260/280 nm. Out of 360 ISSR-PCR reactions conducted for amplifying 18 samples with 20 primers, 19 primers provided 232 bands with polymorphism and 25 were unique bands with band sizes ranging from 75 to 3500 Bas pair. Cluster analysis and the Jaccard similarity coefficient showed that the medium 5C01 was the most stable compared to the MS, B5, and LS media. The genetic stability of D. purpurea callus was affected by the type and concentration of electrolyte and cytokinin in the nutrient media.