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).

-A protocol for micropropagation from isolated cotyledons from Pinus pinea L. has been developed. Major improvements are the increase in rooting rates and the description for the first time of a successful continuous multiplication procedure. For bud induction, isolated cotyledons were cultured on 1 /2 LP with 44.4 µM BA during 4 days. Shoot development was obtained by transfer to 1 /2 LP hormone-free medium with activated charcoal. Since an additional 20 weeks were required for shoot elongation and rooting, at least 70 plantlets can be expected per seed after 29 weeks based on a 70% success rate. Besides, shoots can be micropropagated successively by subculturing on 1 /2 LPC. conifère / micropropagation / organogenèse / enracinement / culture de tissus

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A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.

-This paper describes an in vitro micrografting method for selected mature Pinus pinea L. trees. Needle fascicles of five selected clones were micrografted onto hypocotyls of two weeks-old germinated isolated embryos. Fascicle meristems outgrowth was recorded after one month of culture and the performance of the clones assayed was evaluated. Clones behave statistically different in establishment and development rates. Overall success of our protocol reached 43% of the graftings made.