The mechanisms underlying the de novo regeneration of adventitious roots are still poorly understood, particularly in trees. In this study, we developed a system for studying different modulators of adventitious rooting (AR) at physiological and molecular levels by using for the first time leaves excised from chestnut microshoots. The rooting response of leaves was ontogenetic-stage dependent, and similar to that of the original microshoots. Initiation of root primordia and root development were inhibited by application of N-1-naphthyl-phthalamic acid (NPA), although the effect of this inhibitor of auxin polar transport (PAT) depended on the timing of application. Expression of CsSCL1, an auxin-inducible gene in juvenile and mature shoots during AR, was upregulated by auxin only in rooting-competent leaves during the novo root organogenesis and the expression was reduced by the NPA treatment. This inhibitory effect on gene expression was detected during reprograming of rooting competent cells towards root initials in response to auxin, indicating that PAT-mediated upregulation of CsSCL1 is required in the initial steps of AR in chestnut leaves. The localized expression of CsSCL1 in the quiescent center (QC) also suggests a role for this gene in the maintenance of meristematic competence and root radial patterning.

Phase change refers to the process of maturation and transition from the juvenile to the adult stage. In response to this shift, certain species like chestnut lose the ability to form adventitious roots, thereby hindering the successful micropropagation of adult plants. While auxin is the main hormone involved in adventitious root formation, other hormones, such as ethylene, are also thought to play a role in its induction and development. In this study, experiments were carried out to determine the effects of ethylene on the induction and growth of adventitious roots. The analysis was performed in two types of chestnut microshoots derived from the same tree, a juvenile-like line with high rooting ability derived from basal shoots (P2BS) and a line derived from crown branches (P2CR) with low rooting responses. By means of the application of compounds to modify ethylene content or inhibit its signalling, the potential involvement of this hormone in the in-duction of adventitious roots was analyzed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterize the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. Findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modu-lation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour.

Wounding and exogenous auxin are needed to induce adventitious roots in Chestnut microshoots. However, the specific inductive role of wounding has not been characterized in this species. In the present work two main goals were established: First, we prompted to optimize exogenous auxin treatments to improve overall health status of the shoots at the end of the rooting cycle. Second, we developed a time-series transcriptomic analysis to compare gene expression in response to wounding alone and wounding plus auxin, focusing in the early events within the first days after treatments. Results suggest that the expression of many genes involved in the rooting process is under direct or indirect control of both stimuli. However, specific levels of expression of relevant genes are only attained when both treatments are applied simultaneously, leading to the successful development of roots. In this sense, we have identified four transcription factors upregulated by auxin (CsLBD16, CsERF113, Cs22D, CsIAA6), with some of them being also induced by wounding. The highest expression levels of these genes occurred when wounding and auxin treatments were applied simultaneously, correlating with the rooting response of the shoots. Results in this work clarify the genetic nature of the wounding response in chestnut, its relation to adventitious rooting and might be helpful in the development of more specific protocols for the vegetative propagation of this species.