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Isolated cotyledons from mature Pinus pinea L. embryos were cultured in vitro in a factorial combination of 4.4, 10 and 44.4 microM N6-benzyladenine (BA) for 2, 4, 8, 16 and 35 days to optimize shoot regeneration. Incubation of explants in 44.4 microM BA for 4 days, in place of the standard incubation in 4.4 microM BA for 35 days, reduced the entire culture period to 4 weeks. Shortening the culture period had no significant effect on the caulogenic response or the number of buds formed per cotyledon. To establish the relationship between key moments in the caulogenic process induced by 4.4 microM BA and the endogenous concentrations of the active forms of BA and other isoprenoid-type cytokinins (CKs), we examined uptake, metabolism and amount of BA, as well as the amounts of zeatin, dihydrozeatin and their ribosides in P. pinea cotyledons after 1, 2, 6, 12 and 24 h, and 2, 4, 8, 16 and 35 days of exposure to 8-[14C]BA. Uptake and release of BA were associated with water movement between explants and the medium during the first 8 days of culture. The interconvertible forms of BA were the main metabolites formed in the tissues. Inactivation of BA as a result of conjugation or oxidation was insignificant. The endogenous concentration of BA + N6-benzyladenosine was 20-fold higher than the exogenously applied BA during the competence acquisition phase (Days 0-3). The concentration of isoprenoid-type CKs also increased 16-fold and then decreased during this time. Induction of shoot buds (Days 4-8) was characterized by a second peak of BA uptake by explants that triggered the synthesis of N6-benzyladenosine-5 -monophosphate and by the maintenance of isoprenoid-type CKs. Reestablishment of CK homeostasis marked the shift from the induction phase to the shoot development phase in this organogenic process (Days 8-12).

Organogenic responses of Pinus pinea cotyledons to hormonal treatments: BA metabolism and cytokinin content