Ethylene Action Inhibition Improves Adventitious Root Induction in Adult Chestnut Tissues.

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 a 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 induction of adventitious roots was analysed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterise the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. The findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modulation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour.

Recent Advances in Adventitious Root Formation in Chestnut.

The genus Castanea includes several tree species that are relevant because of their geographical extension and their multipurpose character, that includes nut and timber production. However, commercial exploitation of the trees is hindered by several factors, particularly by their limited regeneration ability. Regardless of recent advances, there exists a serious limitation for the propagation of elite genotypes of chestnut due to decline of rooting ability as the tree ages. In the present review, we summarize the research developed in this genus during the last three decades concerning the formation of adventitious roots (ARs). Focusing on cuttings and in vitro microshoots, we gather the information available on several species, particularly C. sativa, C. dentata and the hybrid C.sativa × C. crenata, and analyze the influence of several factors on the achievements of the applied protocols, including genotype, auxin treatment, light regime and rooting media. We also pay attention to the acclimation phase, as well as compile the information available about biochemical and molecular related aspects. Furthermore, we considerate promising biotechnological approaches that might enable the improvement of the current protocols.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells.

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene.

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species.

We characterized SCARECROW-LIKE genes induced by auxin in rooting-competent cuttings of two distantly related forest species (Pinus radiata D. Don and Castanea sativa Mill.) before the activation of cell division that results in adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family and show a strong similarity to the SCARECROW-LIKE proteins, indicating conserved functions of these proteins. Quantitative RT-PCR analysis showed that these genes are expressed at relatively high levels in roots. Induction of increased mRNA levels in rooting-competent cuttings of both species in response to exogenous auxin was observed within the first 24 h of the root induction process, a time when cell reorganization takes place, but before the resumption of cell division and the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes play a role during the earliest stages of adventitious root formation.