Controlling Factors and Markers for Embryogenic Potential and Regeneration Capacity in Barley (Hordeum Vulgare L.) Cell Cultures

Factors affecting in vitro embryogenesis as well as markers for this developmental process have been characterized in barley cell cultures. Through modifications of the nitrogen composition in culture media of microspore cultures it was possible to manipulate plating efficiency as well as embryogenesis and plant regeneration independently. A certain combination of nitrate, ammonium, and glutamine (N24 A3 G3 medium) e.g. led to the formation of germinating androgenetic embryos in a high frequency, while glutamine as sole nitrogen source (G30 medium) led to the development of embryogenic, but non-regenerating aggregates. This microspore system was used in Northern experiments to analyse the expression of two barley cDNA-clones (B15C, pG22-69) expressed during zygotic embryogenesis. The analysis indicated that these clones are early markers for in vitro embryogenesis and that this development is also induced in non-regenerating aggregates, but is stopped before differentiation in scutellum, shoot and root primordia. These markers could also distinguish embryogenic (regenerable and non-regenerable) from non-embryogenic barley cell suspensions. In these cultures we identified a cellular 85 kDa polypeptide which accumulated during the loss of regenerative capacity. Following the pattern of proteins secreted to the medium polypeptides were characterized correlating with embryogenic capacity (46 kDa), or the loss of regenerative potential (17.4 and 40.5 kDa), respectively. The data indicated that induction of division, prolonged proliferation, induction of embryogenesis and embryo development, respectively, are independently regulated processes. The markers can be used to characterize embryogenie cultures as well as to elucidate the molecular mechanisms of cell differentiation.