Plant Science 190 (2012) 40–51 Contents lists available at SciVerse ScienceDirect Plant Science jou rn al hom epa ge: www.elsevier.com/locate/plantsci In vitro shoot organogenesis and hormone response are affected by the altered levels of Brassica napus meristem genes Mohamed Elhiti 1 , Claudio Stasolla ∗ Department of Plant Science, University of Manitoba, Winnipeg, R3T 2N2 Manitoba, Canada a r t i c l e i n f o Article history: Received 16 February 2012 Received in revised form 28 March 2012 Accepted 1 April 2012 Available online 7 April 2012 Keywords: Auxin Arabidopsis Brassica napus Cytokinin Meristem Shoot organogenesis a b s t r a c t Arabidopsis shoot meristem activity is regulated by a molecular network involving the participation of several components, including SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), and ZWILLE (ZLL). In an effort to identify the role of these genes during in vitro shoot formation Brassica and Arabidopsis plants were transformed with the Brassica napus (Bn) STM, CLV1, ZLL1 and ZLL2 identified in previous work [1]. In both systems shoot organogenesis was promoted by the over-expression of BnSTM, BnZLL1, and BnZLL2, and repressed by the over-expression of BnCLV1. This distinct regulation, analogous to that occurring during in vivo meristem formation where STM and ZLL encourage stem cell formation while CLV1 accelerates transition to differentiation, suggests similar regulatory mechanisms governing shoot formation in vivo and in vitro. While the BnZLL1 and BnZLL2 induction of shoot organogenesis corre- lated only to changes in auxin signaling, BnSTM and BnCLV1 evoked major transcriptional alterations in cytokinin response. Besides increasing the transcript levels of two cytokinin receptors, ARABIDOPSIS HIS- TIDINE KINASE4 (AHK4) and CYTOKININ INDEPENDENT KINASE (CKI1), ectopic expression of BnSTM induced Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) and repressed Type-A ARRs. Opposite transcriptional patterns occurred in explants over-expressing BnCLV1, characterized by a decreased ability to produce shoots. The role played by Type-A and Type-B ARRs during shoot organogenesis was further examined using a genetic approach which revealed the requirement of ARR12 for the BnSTM positive regulation of shoot organogenesis. Collectively these results expand our knowledge on the function of meristem genes, and provide new tools for enhancing in vitro propagation systems. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Shoot organogenesis refers to that process whereby de-novo shoots are initiated from somatic cells of the explants. In culture, shoot formation can be executed directly, without an intervening callus phase, or indirectly, with a callus step. Examples of both have been documented [2]. Morphological, physiological, and genetic characterizations of shoot organogenesis have identified distinct phases: competence acquisition, canalization, and morphogenesis [3]. In the first phase, cells within the explant acquire the ability or competence to responds to inductive signals. These competent Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2iP, 6-(,- dimethylallylamino)-purine; AAR, ARABIDOPSIS RESPONSE REGULATOR; AHK, ARABIDOPSIS HISTIDINE KINASE; CIM, callus induction medium; CLV1, CLAVATA1; CKI1, CYTOKININ INDEPENDENT KINASE1; MDE, microspore-derived embryos; SIM, shoot induction medium; SAM, shoot apical meristem; STM, SHOOTMERISTEMLESS; ZLL, ZWILLE. ∗ Corresponding author. Fax: +1 204 474 7528. E-mail address: stasolla@ms.umanitoba.ca (C. Stasolla). 1 Permanent address: Department of Botany, Faculty of Science, Tanta University, Tanta 31527, Egypt. cells are then “canalized” into the shoot developmental program which culminates in “morphogenesis”, that is the formation of shoots. In Arabidopsis, viable shoots can be produced from several explants, including petals and sepal [4], hypocotyls [5] and roots [6]. In the latter case young roots are induced to form shoots by a procedure consisting of a preculture onto an auxin-containing medium (callus induction medium, CIM), followed by a transfer onto a cytokinin-containing medium (shoot induction medium, SIM) [6,7]. This efficient system has been very useful in examining events related to competence acquisition [7] and the requirement of specific factors needed for proper shoot formation [7–9]. Studies on primary hormone response during Arabidopsis organogenesis revealed precise changes in the expression of genes involved in auxin and cytokinin signaling. Competence acquisition requires auxin which activates three distinct classes of gene fami- lies: Aux/IAA, GH3, and SAUR [10,11]. Specific Aux/IAA factors were in fact up-regulated during preincubation on the auxin-rich CIM [7]. Initiation of shoot formation on SIM is associated with profound changes in cytokinin perception and signaling. Genetic dissection of the cytokinin pathway has identified several components, includ- ing sensor histidine kinases (AHKs), histidine phosphotransmitters (AHPs) and response regulators (ARRs). The Arabidopsis genome 0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2012.04.002