Involvement of the ARRO-1 gene in adventitious root formation in apple Anders Smolka *, Margareta Welander, Peter Olsson, Anna Holefors, Li-Hua Zhu Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, Box 101, SE-230 53 Alnarp, Sweden 1. Introduction Vegetative propagation is one of the most important methods for commercial production of horticultural crops throughout the world [1]. For many woody species, adventitious root formation is a limiting factor for vegetative propagation. However, the molecular mechanisms underlying adventitious rooting are still largely unknown. Since cuttings of many apple (Malus domestica) rootstocks do not root easily, a system of propagation using layering of mother plants in stool beds is often used, with high production costs. Therefore, it is important to further elucidate the molecular mechanisms underlying root formation in woody species in order to effectively improve the rooting ability. Auxin plays a central role in root formation including both lateral [2–4] and adventitious roots [5–7]. It is well known that auxin concentrations favourable for root initiation inhibit root growth [8,9]. In apple, the process of adventitious root formation can be divided into three developmental phases: dedifferentiation, induction and differentiation. Each phase has its specific auxin requirement. During the initial 24 h after the microcuttings have been taken, the cells are not yet very sensitive to auxin. In this period, dedifferentiation occurs and the cells become competent to respond to the rhizogenic action of auxin. During the following induction phase, cells committed to the formation of root primordia begin to divide and form meristemoids. The first cell divisions occur after 48 h, and by 96 h meristemoids of approxi- mately 30 cells are present. After 96 h the differentiation phase starts, where the meristemoids first develop into root primordia and thereafter into roots [10]. At the differentiation phase, auxin is no longer required and becomes inhibitory [11]. Even though the central role of auxin in adventitious root initiation is well characterised, the signalling pathways and gene expression during the whole rooting process are still poorly understood, especially in woody species. To better understand and possibly manipulate this process, the rooting related genes must be identified and their functions charaterised. So far, a number of auxin-induced and rooting related genes have been isolated in several species. In Arabidopsis thaliana, a number of genes involved in the formation of adventitious roots from the hypcotyl have been identified. These genes include AUXIN RESPONSE FACTOR 17 (ARF17) which, when overexpressed, reduces the number of adventitous roots [6]. ARF17 belongs to the ARF protein family which binds to auxin response elements present in the promoter region of auxin-regulated genes, thereby acting as a transcriptional repressor or activator [12]. ARF17 is believed to negatively regulate the expression of the three GH3-like proteins GH3-3, GH3-5 and GH3-6, which have been shown to be auxin-conjugating enzymes [13,14]. The expression of these GH3-like proteins are all positively correlated with the number of adventitious roots [15]. Many of the genes involved in development of the primary root, such as HOBBIT Plant Science 177 (2009) 710–715 ARTICLE INFO Article history: Received 31 July 2009 Received in revised form 7 September 2009 Accepted 11 September 2009 Available online 18 September 2009 Keywords: Adventitious rooting Agrobacterium transformation Apple ARRO-1 RNAi ABSTRACT Adventitious root formation is a limiting factor for vegetative propagation in apple. The molecular mechanisms underlying the rooting process are still largely unknown and need to be extensively investigated. The Adventitious Rooting Related Oxygenase (ARRO-1) has previously been isolated from apple and has been shown to be upregulated during adventitious root induction. However, the function of this gene is still unclear. The aim of this study was to downregulate the ARRO-1 gene expression, using RNAi technique, to study the function of ARRO-1 in adventitious root formation in apple. The apple rootstock M26 was transformed with the RNAi-ARRO-1 construct. The transgenic clones, confirmed by PCR and Southern blot analysis, showed significantly reduced adventitious root formation both with microcuttings and stem discs, indicating the involvement of ARRO-1 in adventitious root formation. The transgenic clones also appeared to be more sensitive to exogenous hormones compared to the untransformed control plants, suggesting that ARRO-1 may be involved in regulating hormone homeostasis. Relative quantification of the ARRO-1 mRNA showed no obvious difference in transcript levels between untransformed control and the transformants. A possible explanation for this is that the downregulation of the ARRO-1 gene might be translational rather than transcriptional. ß 2009 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +46 40 415348. E-mail address: Anders.Smolka@ltj.slu.se (A. Smolka). Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.09.009