Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum |
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Authors: | Konieczny Robert Obert Bohuš Bleho Juraj Novák Ondřej Heym Claudia Tuleja Monika Müller Jens Strnad Miroslav Menzel Diedrik Samaj Jozef |
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Affiliation: | 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, Palacký University, Šlechtitel? 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, Palacký University & Institute of Experimental Botany ASCR, Šlechtitel? 11, CZ-783 71 Olomouc, Czech Republic |
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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 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 levels 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. |
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Keywords: | 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&prime MP, cis-zeatin riboside-5&prime -monophosphate DHZ, dihydrozeatin DHZ9G, dihydrozeatin-9-glucoside DHZOG, dihydrozeatin-O-glucoside DHZR5&prime MP, dihydrozeatin riboside-5&prime -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, isopentenyladenosine iPR-5&prime MP, isopentenyladenosine-5&prime -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&prime MP, trans-zeatin riboside-5&prime -monophosphate |
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