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 twostep 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 α-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 1 d incubation with DFMA and remained at ten times lower concentration than the controls. Embryogenesis was decreased (2 embryos per mM2 of leaf versus 12 embryos per mM2) 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.