Journal of Plant Physiology 168 (2011) 1598–1601 Contents lists available at ScienceDirect Journal of Plant Physiology j ourna l ho mepage: www.elsevier.de/jplph Short communication CUC2 as an early marker for regeneration competence in Arabidopsis root explants Hans Motte a,b , Inge Verstraeten b , Stefaan Werbrouck a,b , Danny Geelen b,∗ a Department of Plant Production, University College Ghent, member of the Ghent University Association, Schoonmeersstraat, 52, BE-9000 Ghent, Belgium b Department of Plant Production, Ghent University, Coupure links, 653, BE-9000 Ghent, Belgium a r t i c l e i n f o Article history: Received 23 December 2010 Received in revised form 31 January 2011 Accepted 10 February 2011 Keywords: CUC2 Micropropagation Primordium Regeneration Shoot induction a b s t r a c t CUP SHAPED COTELYDON 2 (CUC2) was tested as a marker for shoot induction to monitor and facilitate the optimization of in vitro regeneration of Arabidopsis thaliana. The expression of a pCUC2::3XVENUS-N7 fluorescent marker allowed the observation of early steps in the initiation and development of shoots on root explants. The explants were first incubated on an auxin-rich callus induction medium (CIM) and then transferred to a cytokinin-rich shoot induction medium (SIM). CUC2-expression occurred prior to visible shoot formation during the incubation of the root explant on CIM. Shoot formation was invariably preceded by the accumulation of CUC2 expression at dispersed sites along the root explant. These patches of CUC2-expression also marked the site of lateral root primordium formation in root explants that were transferred to hormone free medium. Thus, CUC2 is a predictive marker for the acquisition of root explant competence for root and shoot organogenesis. © 2011 Elsevier GmbH. All rights reserved. Introduction Shoot regeneration from root explants has been studied in detail in Arabidopsis thaliana. The regeneration involves a two step pro- cess with a pre-incubation on auxin-rich callus induction medium (CIM) to stimulate cell division, followed by transfer to cytokinin- rich shoot induction medium (SIM) to stimulate the formation of shoots (Valvekens et al., 1988; Cary et al., 2002). The use and opti- mization of this technique for other plant species requires testing of different conditions, such as incubation time, medium composi- tion, and the presence of hormones. Shoot formation is a multistep process involving cell dedifferentiation, acquisition of competence, primordium initiation, and the formation and outgrowth of a shoot meristem (Che et al., 2007). In an approach to facilitate the moni- toring of shoot organogenesis, we are testing marker genes that are associated with the shoot induction program. One of the early genes expressed during the induction of shoots from Arabidopsis root explants is CUP-SHAPED COTELYDON 2 (CUC2), which is already expressed during the pre-incubation on CIM (Gordon et al., 2007). Here, CUC2-expression was followed during Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; 2-ip, 2-isopentenyl adenine; BM, basal medium; CIM, callus induction medium; CUC2, CUP-SHAPED COTELYDON 2; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Kin, kinetin; Ler, Landsberg erecta; MES, 2-(4-morpholino-)ethanesulfonic acid; NAA, naphthale- neacetic acid; SIM, shoot induction medium. ∗ Corresponding author. E-mail address: danny.geelen@ugent.be (D. Geelen). shoot regeneration. We found that the localized expression at dis- crete sites along the root explant was correlated with the capacity to form shoots. Moreover, CUC2-expression was also associated with root meristem formation. The expression of CUC2 is therefore infor- mative for the acquisition of organogenesis competence and may be implemented as a marker for the regenerative capacity of root explants. Materials and methods Plant materials and tissue culture procedures We used Arabidopsis thaliana ecotype Landsberg erecta (Ler). The pCUC2::3XVENUS-N7 marker has been described previously (Heisler et al., 2005) and was generously provided by Elliot M. Meyerowitz. Seeds were fumigated for 4 hours in a desiccator jar with chlorine gas by adding 5 mL concentrated HCl to 100 mL 5% (v/v) NaOCl. Sterilized seeds were incubated on basal medium (BM): Gamborg’s B5 medium (Gamborg et al., 1968) supplemented with 0.05% (w/v) 2-(4-morpholino-)ethane sulfonic acid (MES) at pH 5.8, 2% (w/v) glucose and 0.7% (w/v) agar. Before germination, a cold treatment for 4 days at 4 ◦ C was applied to the seeds. Seedlings were grown for 6 days at 20 ◦ C and a 16 h photoperiod with a light irradiance of 45 mol m -2 s -1 provided by cool-white fluorescent tungsten tubes. Shoot formation from root explants was basically as described by Valvekens et al. (1988). Except when stated other- wise, root segments (7 mm) were cut from 6 days old seedlings and explanted onto callus induction medium (CIM; BM supple- 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.02.014