A simple method suitable to study de novo root organogenesis

Abstract

De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of β-glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis

“…We found that excised whole leaves of Arabidopsis can root without hormone supplementation, similarly to leaf blades as previously described (Chen et al, 2014) and at similar percentages (Supplemental Fig. S1, A and B).…”

“…Moreover, callus formation, which is a prerequisite for hormonal-induced regeneration, does not appear to occur during endogenous organogenesis, although both processes share regulation (Sugimoto et al, 2010;Liu et al, 2014;Perianez-Rodriguez et al, 2014;Ramirez-Parra et al, 2017). Using a simple method to study de novo root organogenesis without hormone supplementation (Chen et al, 2014;Liu et al, 2014) but applied to whole leaves (Fig. 8A), we found that formation of an endogenous callus is a required step for de novo root organogenesis, and thus we established a direct connection between both regenerative processes.…”

“…Pericycle-like cells (those expressing the J0121 reporter) have been associated to regenerative and morphogenic processes as the source of reprogrammable cells (Sugimoto et al, 2010;Chen et al, 2014). Sections of petioles at the time of excision revealed that the rootpericycle line J0661-GFP marks cells around xylem and procambium cells (Fig.…”

“…Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved.Plants have striking regeneration capacities, and can produce new organs from postembryonic tissues (Hartmann et al, 2010;Chen et al, 2014;Liu et al, 2014) as well as reconstitute damaged organs upon wounding (Xu et al, 2006;Heyman et al, 2013; PerianezRodriguez et al, 2014;Melnyk et al, 2015;Efroni et al, 2016). Intriguingly, root regeneration upon stem cell damage recruits embryonic pathways (Hayashi et al, 2006;Efroni et al, 2016), whereas in contrast, postembryonic formation of whole new organs, such as lateral roots, appears to use specific postembryonic pathways (Lavenus et al, 2013).…”

“…Recently, a hormone-free method to study de novo root organogenesis in excised leaf blades has been described (Chen et al, 2014). YUCCA-mediated auxin biosynthesis was shown to be ubiquitously enhanced in the leaf mesophyll and indirectly contribute to auxin accumulation near the excision site to trigger localized auxin signaling in the vasculature Chen et al, 2016).…”