In vitro propagation of Acacia mangium and A. mangium × A. auriculiformis.

Acacia mangium and A. mangium × A. auriculiformis hybrids have gained an increasing interest in reafforestation programs under the humid tropical conditions, mainly for pulpwood production. This is due to their impressive growth on acid and degraded soils, as well as their capability to restore soil fertility thanks to their natural nitrogen-fixing ability. It is crucial to develop efficient methods for improving the genetic quality and the mass production of the planting stocks of these species. In this regard, in vitro micropropagation is well suited to overcome the limitations of more conventional techniques for mass propagating vegetatively selected juvenile, mature, or even transgenic genotypes. Micropropagation of A. mangium either from seeds or from explants collected from outdoors is initiated on Murashige and Skoog (MS) basal medium supplemented with 4.4 µM BA. Microshoot cultures produced by axillary budding are further developed and maintained by regular subcultures every 60 days onto fresh MS culture medium added with 2.2 µM BA + 0.1 µM NAA. This procedure enhances the organogenic capacity for shoot multiplication by axillary budding, with average multiplication rates of 3-5 every 2 months, as well as for adventitious rooting. The rooting is initiated on Schenk and Hildebrandt culture medium containing 4 µM IAA. The maintenance of shoot cultures in total darkness for 3 weeks increases the rooting rates reaching more than 70%. The hybrid A. mangium × A. auriculiformis genotypes are subcultured at 2-month intervals with an average multiplication rate of 3 and rooting rates of 95-100% on a half-strength MS basal medium containing 1.1 µM NAA. The rooted microshoots are transferred to ex vitro controlled conditions for acclimatization and further growth, prior to transfer to the field, or use as stock plants for cost-effective and true-to-type mass production by rooted cuttings.

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages.

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.

Influences of aging and cloning methods on the capacity for somatic embryogenesis of a mature Hevea brasiliensis genotype.

We compared embryogenic capacities of integument explants excised from three sources of the Hevea brasiliensis (Müll. Arg.) mature genotype PB 260. The three sources were 17-year-old (BT 86) and 7-year-old (BT 96) budded trees and 7-year-old emblings (EM 96). The highest proportions of embryogenic calluses obtained from the total number of integument explants initially used were from trees of EM 96 origin, followed by BT 96 trees, with explants from BT 86 trees producing the lowest number of embryogenic calluses. Further initiation of embryogenic callus lines from the primary somatic embryos derived from the three sources was successful only for EM 96. Somatic embryo cultures from BT 86 and BT 96 sources produced only friable calluses that could not be further amplified. Overall, somatic embryo explants derived from EM 96 responded over a wider range of 3,4-dichlorophenoxyacetic acid and kinetin concentrations than the somatic embryo explants from BT 86 and BT 96 origins. The effects of chronologic, ontogenetic and physiologic aging on explant capacity for somatic embryogenesis and on the overall efficiency of the process in H. brasiliensis are discussed.

Genomic DNA methylation of juvenile and mature Acacia mangium micropropagated in vitro with reference to leaf morphology as a phase change marker.

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.