Cryopreservation of embryogenic cultures

Abstract

The first reports on somatic embryogenesis date back to 1958 (Reinert 1958; Steward et al. 1958) and represent one of the most important milestones in plant tissue culture, as they give clear evidence of the concept of 'totipotency' of vegetative cells, a peculiarity which makes possible the regeneration of a whole plant from a single somatic cell (Bajaj 1995). Indeed, thanks to the 'bipolar' nature of the somatic embryo, shoot and root formation are generally induced at the same time from a single regenerative act. This differentiates somatic embryogenesis from organogenesis, where de-novo formed 'unipolar' structures, mainly shoots, need to be stimulated with growth regulators for the induction of adventitious roots, thus producing a complete plant in two steps. Somatic embryos can be induced directly from cells of the explant cultured in vitro ('direct' or 'adventitious' somatic embryogenesis), or more often, from the dedifferentiated cells of a proliferative callus (indirect or induced somatic embryogenesis) after the explant tissue is artificially stimulated (i.e., with growth regulators) to develop embryogenic competence (Hartmann et al. 1990; Krikorian 2000). Embryogenic callus, established in vitro from the original explant, are periodically subcultured to maintain their embryogenic potential. These constitute 'embryogenic cultures' or 'embryogenic callus lines'. The bipolar structure of the somatic embryo, as well as the developmental stages it passes through, makes it similar to the zygotic embryo (Dodeman et al. 1997; Benelli et al. 2001). However, the former evolves into a clonal plant, as it originates directly from a somatic cell without gametic fusion, while the latter produces a new genotype as a result of gametic reproduction. In addition, differently from zygotic embryos, somatic embryos are not protected by a seed coat and they are dependent on the culture medium for nutrition. The synthetic seed technology (i.e., the inclusion of a somatic embryo inside a nutrient-containing alginate bead) was developed to resemble as closely as possible the natural condition of the zygotic embryo.