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

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of 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 structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation-regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.