Efficient micropropagation protocol and assessment of genetic fidelity of micropropagated plantlets of Pueraria tuberosa

The aim of this study was to develop an efficient micropropagation protocol for Pueraria tuberosa, a medicinally valuable woody liana species. The nodal explants derived from in vitro-grown seedlings were cultured on Murashige and Skoog (MS) medium containing various combinations of 6-benzylaminopurine (BAP), thidiazuron (TDZ), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA). These combinations led to the substantial development of multiple shoots. The combination of TDZ (0.57 mu M/L) and IBA (0.12 mu M/L) resulted in the highest regeneration rate (85%), maximum number of shoots (5.52 +/- 0.67), and best shoot length (4.96 +/- 0.33 cm). The most effective in vitro rooting treatment consisted of half-strength MS medium accompanied by 2.46 mu M/L IBA and a 10-minute pulse treatment with the same concentration of IBA. This treatment resulted in the highest root formation (100%) and maximum roots per shoot (16.72 +/- 1.81), with the highest root length (3.58 +/- 0.54 cm) among all treatments tested. After four weeks under field conditions, hardened in vitro plantlets showed an 88.2% survival rate. The genetic fidelity of regenerated plants was assessed using inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers, which produced monomorphic bands, demonstrating the genetic homogeneity of regeneration and reliability of the in vitro propagation method.