Improved in vitro propagation, solasodine accumulation and assessment of clonal fidelity in regenerants of Solanum trilobatum L. by flow cytometry and SPAR methods

Abstract

An efficient protocol for the development of genetically uniform clones of a valuable medicinal plant Solanum trilobatum L. has been established. An optimal shoot regeneration response was observed in a modified Murashige and Skoog medium (M-MS) containing 25 mM ammonium nitrate, 2 mg l−1 6-benzyl adenine and 0.1 mg l−1 indole-3-acetic acid using in vitro derived node and shoot tip explants. Consequently, the multiple shoot buds were elongated in MS medium supplemented with 0.5 mg l−1 Gibberellic acid. The in vitro regenerated shoots were rooted best in MS medium containing 1.5 mg l−1 indole-3-butyric acid and successfully acclimatized in the field. The single primer amplification reaction (SPAR) approach, including random amplified polymorphic DNA, inter simple sequence repeats and directed amplification of minisatellite DNA regions markers did not identify any genetic polymorphism among in vitro regenerants. Similarly flow cytometry analysis illustrated that the DNA content and genome size of micropropagated plants were equivalent to that of intact plants from field. In addition, the accumulation of solasodine in micropropagated plants was confirmed by thin layer chromatography and further quantified by high performance liquid chromatography analysis as 2.47 mg g−1 DW which is comparable to field grown plants. Thus the protocol can be effectively exploited for commercial propagation of this species to obtain solasodine and also in genetic transformation studies.