Cellular and Molecular Tools for the Investigation of Somatic Embryogenesis in Medicago Species

The chapter presents the knowledge accumulated on the recent investigation of somatic embryogenesis (SE) in genera Medicago. The role of 2,4-D in the process of induction of embryogenic potential in diploid Medicago and its transport by the combined action of auxin transporters or diffusion of dissociated molecules is discussed. Among the many methods for studying the process, this chapter is focused on cellular and molecular tools – flow cytometry, assessment of expression level of SE related transcripts of key genes of auxin inducible process and different PCR techniques. Our recent studies on the process of SE in M. truncatula are focused on the role of the two genes MtLAX3 (an auxin transmembrane transporter) and a transcriptional factor MtARF-B3 (an auxin response factor, containing a B3-binding domain). The transcription profiles of these genes are evaluated and their expression patterns are assessed during indirect somatic embryogenesis – steps of callus formation, embryogenic zone formation and the stages of globular, torpedo and cotyledonary embryos. The localization of expression during the process of SE is traced by the β-glucuronidase reporter gene (GUS) under the control of the promoters of these genes. Inverse PCR (IPCR) and Transposon display (TD) are techniques which evaluate transposition and new retrotransposon copies in the investigated mutant lines, and we used these methods as markers for the efficiency of the induction phase of the process of SE. The use of all these methods turns light on a better understanding of the process of somatic embryogenesis in the model species Medicago truncatula and other annual medics.

Keywords: Somatic embryogenesis, genus Medicago, Flowcytometry, Transcript profiles, PCR-techniques.