The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30-196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 micromol (g DW)(-1), on average] were similar in both genotypes. In 1 mM NO3(-)-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO3- (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO3-. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO3-.
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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