Immunobiochemical Analysis of a Nuclear Protein Marker for Regeneration Potential in Higher Plants

A differential hybridoma screening was applied to select monoclonal antibodies directed against marker proteins for somatic embryogenesis in pea (Pisum sativum L.). We identified and isolated two different hybridoma lines that produce monoclonals recognizing proteins specific for early somatic embryos (7C5) and later stages of development (12A2). A 50 kDa protein recognized by antibodies from hybridoma clone 7C5 was further characterized. This early embryogenic protein (EEP) was also detected in somatic embryos from other species, dicots as well as monocots, and in tips of both shoots and roots from pea. Depending on seedling age, its concentration decreased continuously. Its absence in rapidly dividing nonembryogenic soybean (Glycine max.) suspension cells indicated the correlation of its occurrence with embryogenic potential, but not with cell division. EEP was shown to be localized in the nucleus. Due to its high molecular weight and its acidic pI of 5.9 EEP must belong to the non-histone proteins. The expression of EEP was inducible in isolated pea protoplasts within 6 h after application of 10 μM 2,4-D or Picloram. Using auxin concentrations in this range applied to protoplast derived calli cause induction of somatic embryogenesis. Our results showed that this 50 kDa-protein is likely a marker for embryogenic potential of plant cells with a yet unknown function.