Please cite this article in press as: Konieczny R, et al. Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum. J Plant Physiol (2011), doi:10.1016/j.jplph.2010.10.013 ARTICLE IN PRESS G Model JPLPH-51143; No. of Pages 8 Journal of Plant Physiology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.de/jplph Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum Robert Konieczny a,c,∗,1 , Bohuˇ s Obert c,d,1 , Juraj Bleho c,d , Ondˇ rej Novák e , Claudia Heym c , Monika Tuleja a , Jens Müller c , Miroslav Strnad b,e , Diedrik Menzel c , Jozef ˇ Samaj b a Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Kraków, Poland b Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palack´ y University, ˇ Slechtitel˚ u 11, 783 71 Olomouc, Czech Republic c Institute of Cellular and Molecular Botany, Department of Cell Biology, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany d Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-950 07 Nitra, Slovakia e Laboratory of Growth Regulators, Palack´ y University & Institute of Experimental Botany ASCR, ˇ Slechtitel˚ u 11, CZ-783 71 Olomouc, Czech Republic article info Article history: Received 9 March 2010 Received in revised form 28 October 2010 Accepted 29 October 2010 Keywords: Agrobacterium Callus Common ice plant Cytokinin content GFP Hairy roots abstract Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capac- ity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower lev- els of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years. © 2010 Elsevier GmbH. All rights reserved. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; cZ, cis-zeatin; cZ9G, cis-zeatin-9-glucoside; cZOG, cis-zeatin-O-glucoside; cZR, cis-zeatin riboside; cZR5 ′ MP, cis-zeatin riboside-5 ′ -monophosphate; DHZ, dihydrozeatin; DHZ9G, dihydrozeatin-9-glucoside; DHZOG, dihydrozeatin-O-glucoside; DHZR5 ′ MP, dihy- drozeatin riboside-5 ′ -monophosphate; DHZR, dihydrozeatin riboside; GFP, Green fluorescent protein gene; GFP, green fluorescent protein; IAA, indole-3-acetic acid; iP, isopentenyladenine; iP9G, isopentenyladenine-9-glucoside; iPR, isopen- tenyladenosine; iPR-5 ′ MP, isopentenyladenosine-5 ′ -monophosphate; Kinetin, 6- furfurylaminopurine; MS, Murashige and Skoog medium (1962); NAA, 1- naphthaleneacetic acid; OD600, optical density at 600 nm; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PGR, plant growth regulator; SDS, sodium dodecyl sulfate; tZ, trans-zeatin; tZ9G, trans-zeatin-9-glucoside; tZR, trans-zeatin riboside; tZR5 ′ MP, trans-zeatin riboside-5 ′ -monophosphate. ∗ Corresponding author at: Department of Plant Cytology and Embryology, Jagiel- lonian University, ul. Grodzka 52, 31-044 Kraków, Poland. Tel.: +48 124228107; fax: +48 124228107. E-mail address: robert.konieczny@uj.edu.pl (R. Konieczny). Introduction Mesembryanthemum crystallinum L. (common ice plant) is a slow-growing, annual halophyte that displays facultative Crassu- lacean acid metabolism (CAM) (Adams et al., 1998). The specific features of ice plant biology, such as extreme tolerance to high salinity, stress-inducible switching from C 3 photosynthesis to CAM as well as accumulation of betalains under enhanced irradiation make this organism a useful experimental model to study molecu- lar aspects of plant responses to different environmental stimuli (Bohnert and Cushman, 2000). Recently, the common ice plant has also emerged as a suitable plant for studying mechanisms underlying organogenesis, particularly the role of hydrogen per- 1 These authors contributed equally to this study. 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.10.013