Auxin Control of Root Organogenesis from Callus in Tissue Culture

摘要

Introduction to direct and indirect de novo root regeneration During post-embryonic development, roots can be initiated by a programmed developmental order or by environmental and wound stimulation (Bellini et al., 2014 ; Xu and Huang, 2014 ; Birnbaum, 2016 ; Ikeuchi et al., 2016 ; Kareem et al., 2016 ; Lup et al., 2016 ; Rellan-Alvarez et al., 2016 ; Steffens and Rasmussen, 2016 ). De novo root regeneration (DNRR) is a type of plant regeneration to produce adventitious roots upon wounding or stress (Liu et al., 2014 ; Xu and Huang, 2014 ). For example, using leaf explants of Arabidopsis ( Arabidopsis thaliana ), adventitious roots could usually be regenerated by two ways: adventitious roots could be formed directly from detached leaf explants when cultured on B5 medium without added hormones (Chen et al., 2014 ; Liu et al., 2014 ), hereafter called direct DNRR (Figure 1A ); or adventitious roots could be formed from callus in tissue culture, hereafter called indirect DNRR. In indirect DNRR, leaf explants are first cultured on callus-inducing medium (CIM) with high auxin levels to induce callus formation, and then the callus is transferred to root-inducing medium (RIM) with low auxin levels or even on B5 medium without auxin supplement to allow root formation (Figure 1B ). Figure 1 Direct and indirect DNRR. (A) A system to study direct DNRR. Leaf explants were cultured on B5 medium without added hormones (Chen et al., 2014 ). (B) A system to study indirect DNRR. Leaf explants were cultured on CIM to induce callus and were then transferred to RIM or B5 medium to produce roots. (C,D) Indirect DNRR from rice (C) and Arabidopsis (D) . Leaf explants were first cultured on CIM for 11 d (C) or 6 d (D) and then transferred to B5 medium for another 6 d (C) or 5 d (D) . (E,F) WOX5 _( pro ) :GUS (He et al., 2012 ) in callus on CIM (E) and in roots after transferred to B5 medium (F) during indirect DNRR. Leaf explants were first cultured on CIM for 4 d before being transferred to B5 medium for another 2 d. Notably, the GUS signal was strong in newly formed callus cells on CIM (E) and was gradually restricted to the stem cell niche in root tips after transferred to B5 medium (F) . (G,H) Proposed cell lineage in direct DNRR (G) and indirect DNRR (H) . Scale bars, 1 mm in (C,D) and 100 μm in (E,F) . Many plants such as Arabidopsis and rice ( Oryza sativa ) can form adventitious roots from callus (Figures 1C,D ). While the cell fate transition during direct DNRR from Arabidopsis leaf explants has been carefully studied in Arabidopsis (Liu et al., 2014 ; Chen et al., 2016b , c ; Hu and Xu, 2016 ; Sheng et al., 2017 ), in indirect DNRR the cell fate transition is still not clear. How roots are formed from callus remains unanswered. In this paper, we present our analyses of the cell lineage of indirect DNRR and discuss the similarities and differences between direct and indirect DNRR. Cell fate transition during direct DNRR Here, we summarize the four steps of cell fate transition involved in direct DNRR from leaf explants together with adventitious rooting in other systems (Figure 1G ). In the first step “priming,” the endogenous auxin is transported into regeneration-competent cells (i.e., procambium and vascular parenchyma cells) in the vasculature near the wound and activates WUSCHEL-RELATED HOMEOBOX11 ( WOX11 ) expression for the fate transition from regeneration-competent cells to root founder cells (Liu et al., 2014 ; Chen et al., 2016a , b ). In the second step “initiation,” WOX11 and auxin coordinately activate WOX5 and LATERAL ORGAN BOUNDARIES DOMAIN16 ( LBD16 ) expression for the fate transition from root founder cells to root primordium (Liu et al., 2014 ; Hu and Xu, 2016 ; Sheng et al., 2017 ). WOX11 expression then decreases in this step (Liu et al., 2014 ; Hu and Xu, 2016 ). Auxin keeps a high level in the root primordium. In the third step “patterning,” cell division continues in the root primordium, which begins to differentiate into a root apical meristem (RAM). The auxin level is tuned down and auxin distribution is restricted to the tip of the meristem to confine the region of the stem cell niche (De Klerk et al., 1999 ; Della Rovere et al., 2013 ; Druege et al., 2016 ). WOX5 is gradually restricted into the stem cell niche and LBD16 expression decreases (Hu and Xu, 2016 ). In the fourth step “emergence,” the mature root tip and stem cell niche are formed and the root tip grows out of the leaf explant (Chen et al., 2016c ; Hu and Xu, 2016 ). Cell fate transition during indirect DNRR In tissue culture, adventitious roots could be obtained via indirect DNRR (Figure 1H ). On CIM, callus is induced from leaf explants by a high level of auxin. Recent theory suggests that callus formation is via the rooting pathway (Che et al., 2007 ; Atta et al., 2009 ; Sugimoto et al., 2010 ; Fan et al., 2012 ; He et al., 2012 ; Liu et al., 2014 ) and also involves two cell fate transition steps in Arabidopsis (Liu et al., 20