The present study investigated how auxin concentration and the method of application affected the formation of adventitious roots in microshoots of chestnut (Castanea sativa) and oak (Quercus robur). The activity of two urea derivatives (2, 3-MDPU and 3, 4-MDPU) was also evaluated. Microshoots were derived from basal sprouts of two mature chestnut trees (P1 and P2) and one adult oak genotype (Sainza). In chestnut, rooting percentage was positively affected by auxin in a dose-dependent manner, particularly in shoots treated with the hormone for 24 h. The effect of auxin on rooting also differed depending on the application method. In shoots treated for 24 h, the highest concentration of auxin produced the healthiest rooted plantlets, in terms of the root system and shoot quality. By contrast, in shoots treated by the basal quick-dip method, the shoot quality was best at the lowest auxin concentration. The effect of urea derivatives on the root system depended on the species as well as on the auxin concentration and application period. Use of the MDPUs improved the root system architecture of auxin-treated shoots by promoting lateral root development and triggering the synchronous initiation of root primordia at the base of the shoot. Shoot quality was also improved by MDPUs, which promoted resumption of growth and reduced shoot-tip necrosis.
Maturation imposes several changes in plants, which are particularly drastic in the case of trees. In recalcitrant woody species, such as chestnut (Castanea sativa Mill.), one of the major maturation-related shifts is the loss of the ability to form adventitious roots in response to auxin treatment as the plant ages. To analyze the molecular mechanisms underlying this phenomenon, an in vitro model system of two different lines of microshoots derived from the same field-grown tree was established. While juvenile-like shoots root readily when treated with exogenous auxin, microshoots established from the crown of the tree rarely form roots. In the present study, a transcriptomic analysis was developed to compare the gene expression patterns in both types of shoots 24 h after hormone and wounding treatment, matching the induction phase of the process. Our results support the hypothesis that the inability of adult chestnut tissues to respond to the inductive treatment relies in a deep change of gene expression imposed by maturation that results in a significant transcriptome modification. Differences in phytohormone signaling seem to be the main cause for the recalcitrant behavior of mature shoots, with abscisic acid and ethylene negatively influencing the rooting ability of the chestnut plants. We have identified a set of related MADS-box genes whose expression is modified but not suppressed by the inductive treatment in mature shoots, suggesting a putative link of their activity with the rooting-recalcitrant behavior of this material. Overall, distinct maturation-derived auxin sensibility and homeostasis, and the related modifications in the balance with other phytohormones, seem to govern the outcome of the process in each type of shoots.
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