table transformation of Mesembryanthemum crystallinum ( L . ) with grobacterium rhizogenes harboring the green fluorescent protein targeted to the ndoplasmic reticulum

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 capacity 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 levPlease cite this article in press as: Konieczny R, et al. Stable transformation o harboring the green fluorescent protein targeted to the endoplasmic reticu els of the free bases of isopentenyladenine, dihydrozeatin, cisand 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 p callus-derived roots showed a Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; cZ, cis-zeatin; cZ9G, is-zeatin-9-glucoside; cZOG, cis-zeatin-O-glucoside; cZR, cis-zeatin riboside; ZR5′MP, cis-zeatin riboside-5′-monophosphate; DHZ, dihydrozeatin; DHZ9G, ihydrozeatin-9-glucoside; DHZOG, dihydrozeatin-O-glucoside; DHZR5′MP, dihyrozeatin riboside-5′-monophosphate; DHZR, dihydrozeatin riboside; GFP, Green uorescent protein gene; GFP, green fluorescent protein; IAA, indole-3-acetic cid; iP, isopentenyladenine; iP9G, isopentenyladenine-9-glucoside; iPR, isopenenyladenosine; iPR-5′MP, isopentenyladenosine-5′-monophosphate; Kinetin, 6urfurylaminopurine; MS, Murashige and Skoog medium (1962); NAA, 1aphthaleneacetic acid; OD600, optical density at 600 nm; PAGE, polyacrylamide el electrophoresis; PCR, polymerase chain reaction; PGR, plant growth regulator; DS, sodium dodecyl sulfate; tZ, trans-zeatin; tZ9G, trans-zeatin-9-glucoside; tZR, rans-zeatin riboside; tZR5′MP, trans-zeatin riboside-5′-monophosphate. ∗ Corresponding author at: Department of Plant Cytology and Embryology, Jagielonian University, ul. Grodzka 52, 31-044 Kraków, Poland. Tel. : +48 124228107; fax: 48 124228107. E-mail address: robert.konieczny@uj.edu. pl (R. Konieczny). 176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. oi:10.1016/j.jplph. 2010.10. 013 otential depending on the auxin/cytokinin ratio in the medium. Calli and strong GFP signal for over 2 years. © 2010 Elsevier GmbH. All rights reserved.