Relationships between hormonal contents and the organogenic response in Pinus pinea cotyledons

Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organogenesis in response to treatment with 4.4 μM N6-benzyladenine (BA). In late germination phases, the extent of the response onset decreased and adventitious buds were redistributed to the basal zone of the cotyledons. To understand these two events, analyses of indole-3-acetic acid (IAA) and several cytokinins have been carried out in apical and basal portions of cotyledons taken at four different culture periods of the embryos on germination medium (0, 2, 4 or 6 d). The highest endogenous content of all hormones, except N6-isopentenyladenosine, was found in the most responsive explants, those that were taken from 0-d-old cultured embryos. Apical portions of cotyledons taken from 0 to 4-d-old cultured embryos showed a reduction in the content of IAA and several cytokinins, but basal portions did not show the same pattern. BA dose-response curves were made to explain the loss of caulogenic potential with the age of the explants. The results suggested the combination of two factors, changes in the endogenous hormonal content in original explants and loss of their sensitivity to exogenously applied BA, as the cause for the reduction of the ability of Pinus pinea cotyledons to form adventitious buds.