Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented.