Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus

The possibility to induce direct somatic embryogenesis in Lotus japonicus explants was investigated. Petiol bases, cotyledons, hypocotyls and stem segments were cultivated on Murashige and Skoog medium with different amounts of benzylaminopurine (BAP) and/or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these new structures indicated their nature of somatic embryos. The TDZ was also tested as an agent to improve the kinetic of shoot formation in one of the transformation-regeneration procedure used in Lotus japonicus. A very significant, highly reproducible, speed up of the shoot formation step was observed in independent transformation experiments. Furthermore, we tested the feasibility and reproducibility of an in vitro procedure for the analysis of early and late symbiotic phenotypes.