MOLECULAR AND BIOCHEMICAL MARKERS FOR EMBRYOGENIC POTENTIAL AND REGENERATIVE CAPACITY OF BARLEY (HORDEUM-VULGARE L) CELL-CULTURES

The embryogenic potential and regenerative capacity of cereal cell cultures was examined using the expression of two embryo-specific genes (the dormancy-related cDNA-clone B15C, R. Aalen, Norway; the aldose reductase cDNA-clone pG22-69, D. Bartels, Germany) in barley cell cultures. By Northern blotting the expression of both clones was limited to embryogenic cultures and not to non-embryogenic cultures. In situ hybridization showed high gene expression in the shoot and root apex of embryos and in subepidermal cell layers of embryogenic barley cultures. In androgenetic embryos the expression of the embryo-specific clones was detected as early as 2 weeks after isolation of microspores, indicating their usefulness as early markers for embryogenesis. Nevertheless, cell aggregates which were embryogenic but no longer able to regenerate plants, expressed both genes. Cell cultures consisting of embryogenic aggregates unable to regenerate plantlets were used to verify that an embryo-specific marker alone is not sufficient to characterize regenerable cultures. Comparing the protein pattern of embryogenic/regenerable and embryogenic/non-regenerable suspension cultures of barley, we identified a 85-kDa polypeptide (pi 5.8) accumulating only in non-regenerable cultures. Moreover, following the electrophoretic pattern of secreted proteins, two glycoproteins were found correlating with the embryogenic capacity (46 kDa, pi 6.1) and the loss of regenerative potential (17.4 kDa), respectively. The data indicate that at least two levels of control exist in somatic embryogenesis. One is the induction of bipolar embryos and the second the germination of somatic embryos into plantlets.