An efficient adventitious shoot regeneration system for potato (Solanum tuberosum L.) using leaf discs

Adenine-type cytokinins, such as zeatin (Z) and its riboside (ZR) are usually used for in vitro plant regeneration in potato (Solanum tuberosum L.), with the latter being more potent for both organogenesis and somatic embryogenesis. Here, we report an efficient ZR-free adventitious shoot regeneration (ASR) system for potato. In this two-step ASR system, callus was induced from leaf explants on Murashige and Skoog medium supplemented with 1.0 mg l(-1) N-6-benzyladenine (BA), 1.0 mg l(-1) alpha-naphthaleneacetic acid (NAA), 1.0 mg l(-1) calcium D-pantothenate, 100.0 mg l(-1) polyvinylpyrrolidone (PVP) and 30.0 g l(-1) sucrose followed by multiple ASR on the second medium containing 3.0 mg l(-1) BA, 1.0 mg l(-1) thidiazuron (TDZ) and 1.0 mg l(-1) gibberellic acid (GA(3)), with the other additives remaining the same. The efficiency of this ZR-free ASR system was compared with that of the ZR-based one where the growth regulators in the second medium were replaced by 5.0 mg l(-1) ZR. There was no significant difference in mean number of days to regeneration between the two ASR systems. Depending on the genotypes, mean number of days to regenerate were 12.6-23.6 days and 13.0-23.0 days for ZR-based and ZR-free ASR systems, respectively. However, in two out of four genotypes tested, significantly higher number of shoots (17.0-19.2) developed per callus in ZR-free ASR system. In vitro regenerants derived from the ZR-free ASR system did not show any morphological variation having comparable competence for tuberization in vitro and maintained genetic fidelity as revealed by inter-simple sequence repeat (ISSR) analysis. In this study, we have developed a leaf-based ZR-free ASR system which can reliably be used for potato transformation.