The effect of L-glutamine on the genetic transformation of embryogenic cell suspensions of gentian species (Gentiana lutea L., Gentiana cruciata L., and Gentiana kurroo Royle) using Agrobacterium tumefaciens

Abstract

In this study, established embryogenic cell suspensions of three gentian species, Gentiana cruciata L., Gentiana kurroo Royle, and Gentiana lutea L., cultured in the presence of two media CMS1 and CMS2, were used to determine the effect of L-glutamine on the efficiency of Agrobacterium tumefaciens-mediated transformation. The presence of 1 g @ l glutamine in the co-cultivation medium and a 48-hr co-cultivation period were found to be optimal for all the cultures investigated. In order to regenerate plants in the post-transformation culture, approximately 100 mg of cell aggregates was plated as a single layer on RM1 and RM2 media. Timentin was used in posttransformation cultures for preventing bacterial contamination and enhancing cell viability. The transformants were selected in the presence of 50 mg @ l kanamycin. Transformation was later confirmed by histochemical analysis of the activity of reporter enzyme (β-glucuronidase) and by polymerase chain reaction for the detection of uidA and nptII genes. Five lines of embryogenic cell suspension cultures of the studied species were selected and grown in the presence of 50 mg @ l kanamycin. Finally, 23 embryos were regenerated, of which only 11 converted into T0 transformants of G. cruciata. These transformants continued to grow in the presence of kanamycin. A solid, dark blue coloration of their leaves confirmed stable integration and expression of the uidA gene. The molecular analysis of T0 plants revealed the absence of bacterial contamination. Thus, the short list of plant species that can be transformed by A. tumefaciens with the help of an embryogenic cell suspension is extended by the three species investigated in this study.