Protocol: a method to study the direct reprogramming of lateral root primordia to fertile shoots

Abstract

BackgroundPlants have the remarkable property to elaborate entire body plan from any tissue part. The conversion of lateral root primordium (LRP) to shoot is an ideal method for plant propagation and for plant researchers to understand the mechanism underlying trans-differentiation. Until now, however, a robust method that allows the efficient conversion of LRP to shoot is lacking. This has limited our ability to study the dynamic phases of reprogramming at cellular and molecular levels.ResultsHere we present an efficient protocol for the direct conversion of LRP to a complete fertile shoot system. This protocol can be readily applied to the various ecotypes of Arabidopsis. We show that, the conversion process is highly responsive to developmental stages of LRP and changes in external environmental stimuli such as temperature. The entire conversion process can be adequately analyzed by histological and imaging techniques. As a demonstration, using a battery of cell fate specific markers, we show that confocal time-lapse imaging can be employed to uncover the early molecular events, intermediate developmental phases and relative abundance of stem cell regulators during the conversion of LRP to shoot.ConclusionOur method is highly efficient, independent of genotypes tested and suitable to study the reprogramming of LRP to shoot in intact plants as well as in excised roots.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0127-5) contains supplementary material, which is available to authorized users.

De novo assembly of plant body plan: a step ahead of Deadpool

“…These hormonal signals activate certain key regulators of shoot regeneration. Various studies have shown that the key shoot stem cell regulator WUSCHEL ( WUS ) is one of the earliest shoot determinants induced in cytokinin‐rich medium (Gordon et al., ; Chatfield et al., ; Kareem et al., , ). Cytokinin induces WU S through the activation of cytokinin receptors ARABIDOPSIS HISTIDINE KINASE 2 ( AHK2 ) and AHK4 (Gordon, Chickarmane, Ohno, & Meyerowitz, ).…”

“…An ideal example of direct reprogramming is the induction of shoot from root. Culturing of root explants on cytokinin‐rich medium promotes the direct conversion of LRP into shoot (Atta et al, ; Chatfield et al., ; Kareem et al., , ). Real‐time monitoring of dynamic cellular events using live imaging enables us to understand the early molecular events during LRP to shoot conversion (Fig.…”

“…High cytokinin induces the expression of shoot‐specific genes such as WUS in LRP which can lead to reprogramming of root cells to shoot fate. During the early stages of cell fate transition, cells pass through a transient developmental phase in which both root‐specific and shoot‐specific genes are expressed (Kareem et al, ). Subsequently, there is an elevation in the expression of shoot‐specific genes and disappearance of root‐specific genes.…”

“…These hormonal signals activate certain key regulators of shoot regeneration. Various studies have shown that the key shoot stem cell regulator WUSCHEL (WUS) is one of the earliest shoot determinants induced in cytokininrich medium (Gordon et al, 2007;Chatfield et al, 2013;Kareem et al, 2015Kareem et al, , 2016 (Leibfried et al, 2005;Buechel et al, 2009). Thus cytokinin and WUS act in a positive feedback loop.…”

“…1); however, cellular reprogramming is inevitable in both the modes. During direct regeneration, the cells undergo transdifferentiation wherein root cells are reprogrammed to shoot and vice versa (Chatfield et al, 2013;Liu et al, 2014;Kareem et al, 2016). The indirect mode involves the formation of an intermediate regenerative mass of cells called callus from adult stem cells that are distributed throughout the plant body (Sugimoto et al, 2010(Sugimoto et al, , 2011Duclercq, Sangwan-Norreel, Catterou, & Sangwan, 2011).…”