The ability for adventitious rooting of micropropagated shoots from juvenile and mature Pinuspinaster~dt. explants was assessed in vitro on a rooting expression medium. The different rooting traits observed, namely the rooting rate, the number and the length of the adventitious roots, and the root score, were greatly influenced by the age of the donor plant: 98% of juvenile explants rooted, while only 49% of mature explants did. Addition of activated charcoal in the rooting expression medium improved the overall rooting capacity of the mature explants to an average of 78%. Whatever the plant material, the number and the length of the adventitious roots, as well as the root score, fluctuated according to the sampling date.

Genomic DNA methylation was analyzed in Acacia mangium Willd. microshoots micropropagated in vitro from juvenile and mature explants, and in relation to leaf morphology of the microshoots, which is considered a phase change indicator. Based on high performance liquid chromatography (HPLC) analyses, we found more DNA methylation in microshoots exhibiting juvenile leaf morphology (22.4%) than in microshoots of the mature phyllode morphological type (20.7%), irrespective of the age of the source material. Overall, the degree of DNA methylation in A. mangium microshoots was consistent with values reported for other angiosperms. Complementary investigations based on methylation sensitive amplification polymorphism (MSAP) techniques established that, of 1204 fragments revealed by the different primer pairs used, 49 (i.e., 4.08%) were derived from C(5m)CGG methylated sites. Three of these C(5m)CGG sites were exclusive to the juvenile plant material, and three sites were exclusive to the mature source. No fragments were associated specifically with leaf morphology, rather than with plant age. Thus, although the two age classes could not be distinguished based on a quantitative HPLC measure of DNA methylation, qualitative differences existed, as demonstrated by the six age-specific markers identified by MSAP. The reliability of the MSAP data was confirmed on a larger sample of juvenile plant material, which suggested that the total of six methylation markers detected is probably an underestimation of the age-related differences in DNA methylation that may exist between juvenile and mature plant materials.

In vitro responsiveness of microshoots derived from three genetically related and different age Eucalyptus urophylla 9 Eucalyptus grandis clones kept cultivated by monthly subcultures was assessed on two rooting media in relation to the time spent in culture. Significant differences of rooting capacity were found between the two 22-yearold half sibling genotypes 147 and 149 according to the concentration of BA added to the media and also to light intensity. The contrast was even more salient with the 1-year-old clone S, which displayed the poorest rooting ability despite being full sibling with clone 147 and much younger. The various criteria observed, i.e. rooting rate, number of roots produced, root length, microshoot height and shoot tip necrosis varied greatly and differently for the three origins according to the successive time replicates of the same rooting protocols, with rooting rates of more than 80% for clone 147 at certain dates. These results were discussed, suggesting the influence of endogenous rhythms as the most rationale cause of the time-related fluctuations of responses observed in steady in vitro environment, notwithstanding possible interferences of non-optimal tissue culture conditions. Such unexpected variations of rootability between closely related genotypes, and the notable interactions pointed out between the time spent in culture and the rooting conditions deserve to be taken into consideration before stating definitive judgment on the rooting capacities of a given genotype for arborescent species.

DNA methylation was analyzed in Acacia mangiumWilld. buds collected from in vitro and field growing shoots from the same clone and its juvenile offspring. Shoot tips from in vitro microshoots showed higher levels of DNA methylation for juvenile (23.4 %) than for mature (21.8 %) plant material, irrespective of their morphology. Likewise, under the same in vitro conditions, the apical buds collected from microshoots exhibiting the juvenile morphology displayed higher DNA methylation rates (23.2 %) than those characterized by the mature phyllode morphological type (22.1%), regardless of the age of the plant material source. In outdoor conditions, apical buds collected from mature tree growing shoots were significantly less methylated (20.9 %) than the resting axillary buds located just underneath (25.2 %) and than the apical buds from the in vitro microshoots of the same clone and mature-like phyllode morphology (22.6 %). Overall, the DNA methylation levels found for our Acacia mangium plant materials were consistent with those reported for other Angiosperms, but showed, similarly to other recent findings in different species, that DNA methylation in plants does not always increase with ageing. This was discussed in relation to other phase-related traits and more particularly foliage characteristics, which are conspicuous markers of ageing in this heteroblastic species.

A meristem-issued rejuvenated line was obtained in 1986 from a 100-year-old Sequoiadendron giganteum tree and has been since then micropropagated in tissue culture conditions maintaining its juvenile-like characteristics. By contrast, grafts and rooted microcuttings from the same genotype planted in outdoor conditions for several years exhibited mature foliage traits and the grafts started to produce cones, which are obvious indicators of physiological aging. These three different clonal lines were compared with regard to global DNA methylation assessed by HPLC. The in vitro rejuvenated line showed a much higher level of DNA methylation (23% as average value) than the two other outdoor origins from the same clone which displayed similar degrees of global methylation (average values of 13.4% for the grafts and 13.8% for the cuttings). Overall these DNA global methylation values obtained for the first time in S. giganteum are consistent with the level of methylation reported for many plants using the same HPLC protocols. The fact that shoots exhibiting a juvenile-like leaf morphology can be characterized by higher DNA methylation than mature-like ones is discussed in relation to physiological aging, referring to other studies on the same topic.