Abstract
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.
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Abbreviations
- DFMA:
-
d,l α-difluoromethylarginine
- SER:
-
somatic embryogenesis related
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We thank Marion Merrell Dow Inc. (Cincinnati, Ohio, USA) for providing the DFMA. We also thank Professor J.D. Bewley (University of Guelph, Canada) and Dr. F. Martin (INRA, Nancy, France) for critical reading of the manuscript, and our colleagues from the laboratoire de Physiologie Cellulaire et Morphogenèse Végétale for helpful discussion and encouragements. This research was supported by a grant “Contrat Plan-Etat-Région” to the Laboratoire de Physiologie Cellulaire et Morphogenèse Végétale.
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Helleboid, S., Couillerot, JP., Hilbert, JL. et al. Inhibition of direct somatic embryogenesis by α-difluoromethylarginine in a Cichorium hybrid: effects on polyamine content and protein patterns. Planta 196, 571–576 (1995). https://doi.org/10.1007/BF00203658
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DOI: https://doi.org/10.1007/BF00203658