INHIBITION OF DIRECT SOMATIC EMBRYOGENESIS BY ALPHA-DIFLUOROMETHYLARGININE IN A CICHORIUM HYBRID - EFFECTS ON POLYAMINE CONTENT AND PROTEIN-PATTERNS

Direct somatic embryogenesis was induced in mesophyll cells of leaves of the hybrid Cichorium intybus L. var. sativum x C. endivia L. var. latifolia through a two-step procedure during 5 d in a M17 liquid medium supplemented with 330 mM glycerol. The lack of cell division during the induction step seems to be associated with a relatively stable low free-polyamine content. Synchronised divisions of competent cells occurred after transfer onto a glycerol-free M17 medium. Increased free-polyamine levels, especially putrescine, accompanied embryo differentiation. The addition of 0.1 to 10 mM alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of polyamine biosynthesis in this Cichorium hybrid, to the medium caused a drastic decrease of polyamine content. Putrescine and spermidine rapidly decreased after Id incubation with DFMA and remained at ten times lower concentration than the controls. Embryogenesis was decreased (2 embryos per mm(2) of leaf versus 12 embryos per mm(2)) in DFMA-incubated leaves. The addition of 5 mM putrescine to DFMA-treated cultures restored both the initial spermidine content and the embryogenesis. The effects of somatic-embryogenesis inhibition on protein biosynthesis were analysed using two-dimensional electrophoresis. In control samples, extracellular polypeptides were secreted into the culture medium. Two of these secreted polypeptides exhibited the same features as two tissue somatic-embryogenesis-related polypeptides. In the presence of 5 mM DFMA their contents in the medium were reduced, whereas they reached similar levels as in the control samples in the presence of DFMA and putrescine.