Optimisation of Immunohistochemical Detection of Cytokinins in Transgenic Arabidopsis Seedlings Expressing Activated ipt, a Cytokinin Biosynthesis Gene

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Authors

DUBOVÁ Jaroslava ROTKOVSKÁ Julie Olegivna REKOVÁ Alena BRZOBOHATÝ Břetislav

Year of publication 2002
Type Article in Proceedings
Conference Progress in Basic, Applied and Diagnostic Histochemistry
MU Faculty or unit

Faculty of Science

Citation
Field Morphological specializations and cytology
Keywords cytokinins; immunohistochemistry; Arabidopsis
Description Aseptically grown Arabidopsis seedlings were collected, fixed in PFA - GA mixture. Indirect immunohistochemistry on cryostat sections was used. Primary antibodies against zeatin-riboside and isopentenyladenine-riboside were used respectively. Goat anti-rabbit IgG conjugated with AP was used as a secondary antibody. AP activity was detected by reaction with BCIP and NBT. The best results of immunostaining were obtained by higher dilution of primary antibodies and lowering the concentration of BCIP and NBT. Arabidopsis seedlings with activated ipt showed higher the amount of reaction product in comparison with the wild type plants. Accumulation of cytokinins was established immunohistochemically during culture period in transgenic Arabidopsis seedlings, which is in accordance with the biochemical analyses. Modified immunohistochemical detection of free bases of cytokinins can be used for the semi quantitative evaluation of cytokinin content in Arabidopsis seedlings.
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