Comparison of the morphogenic potential of five Gentiana species in leaf mesophyll protoplast culture and ploidy stability of regenerated calli and plants

The morphogenic potential of five Gentiana species of medicinal and ornamental value, i.e.G. cruciata, G. kurroo, G. lutea, G. septemfida, and G. tibetica, was compared using agarose-bead leaf mesophyll protoplast culture. Modified Murashige and Skoog medium containing 2.0 mg l(-1) 1-naphthaleneacetic acid and 0.1 mg l(-1) thidiazuron ensured the highest cell division frequency during both protoplast and protoplast-derived cell culture or callus formation. Indirect plant regeneration mainly via the somatic embryogenesis pathway was achieved for G. kurroo and G. tibetica with the greatest efficiency occurring with MS medium supplemented with 1.0 mg l(-1) kinetin, 0.5 mg l(-1) gibberellic acid, and 80 mg l(-1) adenine sulfate. A considerable percentage of autopolyploid and aneuploid cells was detected in callus lines obtained from protoplasts using flow cytometry. The presence of polyploid cells in calli resulted in the regeneration of 85 % polyploid plants of G. kurroo and 14 % polyploids of G. tibetica. Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.