Histological investigation of the multiplication step in secondary somatic embryogenesis of Quercus robur L.

Abstract

Standardized explants composed of hypocotyl and root-tip were prepared from embryonic structures obtained from one embryogenic line of Quercus robur L. maintained by regular transfer onto a solidified reference medium composed of the MS mineral solutions, glucose (0.12 M), casamino acid (0.1%), and NAA (10.74 μM). The regeneration capacity from these explants were tested on the reference medium and on 2 alternative media in which the NAA (10.74 μM) was omitted or substituted for a combination of IBA (9.80 μM)/BAP (8.90 μM). Within 30 days, 4 types of responses were observed including direct and indirect secondary embryogenesis. In the direct pathway, somatic embryos arose from 3-4 epidermal cells following two different modes, depending on whether or not the formation of a meristematic mass preceded the initiation of the embryogenic process. In the indirect pathway the embryos were formed from clumps of mitotically active cells included in callus developed within the cortical tissues. Depending on their histological origin, the embryos exhibited differences in their structural organization which could influence their potential for further maturation and conversion into viable plantlets. Explants prepared from small translucent embryonic structures were more embryogenic and expressed the direct pathway of secondary embryogenesis at higher frequency than explants prepared from more advanced embryonic structures. On the culture medium without growth regulator, direct secondary embryogenesis was the exclusive response whereas on the culture medium with growth regulators added both direct and indirect secondary embryogenesis occurred. NAA favoured the direct secondary embryogenesis, while conversely, the IBA/BAP combination stimulated the indirect secondary embryogenesis. The results are discussed in reference to the PEDC concept (pre-embryogenic determined cells).